Chemiluminescence response of beta-glucan stimulated leukocytes isolated from different tissues and peritoneal cavity of Dicentrarchus labrax

The respiratory burst of leukocytes isolated from sea bass (Dicentrarchus labrax) pronephros, peritoneal cavity (P.C.), spleen and blood, was measured by a chemiluminescence (CL) assay after stimulation with beta-glucan. The CL response by P.C. and pronephros leukocytes was significantly higher than that expressed by a similar number of cells separated from spleen and blood. This probably reflects the observation that the proportion of macrophages and neutrophils was highest in the populations of leukocytes from peritoneal cavity and pronephros. Comparative observations showed a higher degree of yeast phagocytosis by leukocytes taken from peritoneal cavity than the pronephros. Moreover phagocytic index evaluated by microscopical observations, indicated that peritoneal macrophages internalised more yeast cells than neutrophils (identified by the peroxidase reaction). Scanning electron microscopy observations were also carried out.Inhibition experiments by a myeloperoxidase inhibitor sodium azide, iodonium-diphenyl-chloride which inhibits NADPH-oxidase, and exogenous superoxide dismutase, which catalyses O-2 dismutation to H(2)O(2), supported the correlation between CL and respiratory burst. Treatment with ouabain and DNP suggested that in this response, Ca(++) pump channels and calmodulin are involved in a metabolic energy-dependent pathway.     

Electron and light microscopy of neutrophil responses in mice vaccinated and challenged with third-stage infective hookworm (Ancylostoma caninum) larvae

The role of neutrophils in mediating host inflammation was examined in mice vaccinated with living third-stage infective hookworm larvae (L₃). Mice were vaccinated by oral immunization with 500 L₃ (Ancylostoma caninum) once every 2 weeks for a total of three immunizations. The vaccinated mice were then challenged intraperitoneally with 2000 L_3, 1 week after the final immunization. To stimulate peritoneal production of neutrophils, 2 ml of 2% glycogen were injected intraperitoneally at 16 h prior to the challenge infection. Neutrophils were found to comprise 85% of the peritoneal cell population. L₃ from the challenge infection were collected and then examined at timed intervals by inverted light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Greater than a fivefold increase in the total numbers of peritoneal cells was noted in the vaccinated mice as compared to unvaccinated mice. In the peritoneal cavity of vaccinated mice, the neutrophils adhered to the L₃ within 2 h, and over 55% of the L₃ were surround by clusters of neutrophils to form a sausage-like sheath 4 h later. At 24–72 h after challenge, almost all of the L₃ recovered from the vaccinated mice were covered with thick clusters of cells. Both SEM and TEM demonstrated extensive ultrastructural damage to the L_3. In contrast, the L₃ recovered from the unvaccinated mice appeared to be unaffected by neutrophils. These studies suggest that neutrophils, like macrophages, can have an important role as effector cells in L₃-vaccinated mice.     

The efficacy of a commercial β-glucan preparation, EcoActiva™, on stimulating respiratory burst activity of head-kidney macrophages from pink snapper (Pagrus auratus), Sparidae

This study investigated the in vitro effects of a commercial β-glucan preparation, EcoActiva™, on the respiratory burst activity of head-kidney macrophages isolated from pink snapper (Pagrus auratus), a marine fish cultured in Australia. Macrophages incubated with EcoActiva™ displayed morphological characteristics of activation, and were stimulated to produce superoxide. Pre-incubation with low levels of EcoActiva™ significantly increased the response to phorbol myristate acetate (PMA) and lipopolysaccharide (LPS), indicating that EcoActiva™ could prime these macrophages. Co-culturing macrophages with both LPS and PMA, or EcoActiva™ and PMA, increased burst activity compared with the response to PMA alone, however, this increase was additive and not synergistic. These results suggest that EcoActiva™ is able to stimulate non-specific immunity in snapper through increased respiratory burst activity of macrophages, an important component of the host defence network.     

Cadmium-induced depression of the respiratory burst in mouse pulmonary alveolar macrophages, peritoneal macrophages and polymorphonuclear neutrophils.

No profound alteration in the resting O₂ consumption of mouse pulmonary alveolar macrophages, polymorphonuclear neutrophils or peritoneal macrophages incubated in media containing either cadmium chloride or cadmium acetate was observed. However, when heat-killed $\text{P. aeruginosa}$, opsonized in autologous serum, were added to the cell suspension a significant depression in the respiratory burst accompanying the phagocytic event was manifested. The suppression of the respiratory burst appeared to be related to the concentration of cadmium. The possible alteration in the relationship between macrophage microtubule assembly and endocytosis is discussed.     

In vivo studies of the early, peritoneal, cellular and free radical response in rats infected with Fasciola hepatica by flow cytometric analysis

Early recruitment of the peritoneal cell population was observed during migration of newly excysted juvenile flukes. The peritoneal lavages were examined for T cells, cytotoxic NK cells (CNK) and free radicals production of rats at an early stage of infection by Fasciola hepatica. Male Sprague–Dawley rats were infected with 50 metacercariae of F. hepatica and non-infected controls were euthanized 2, 4 and 7 days post infection (d.p.i.), respectively. The peritoneal fluid of experimental animals was analyzed by flow cytometry to estimate cell phenotypes. The peritoneal areas were infiltrated by inflammatory cells, particularly from numerous neutrophils, eosinophils and CD4+ lymphocytes, which were significantly higher for infected rats than non-infected. CNK cells dominated in the peritoneal fluid of infected rats as early as 2 d.p.i. However, after 4 d.p.i. there was a decreased level of CNK cells which may indicate a change from a cytotoxic natural killer (NK) to a regulatory NK response. The challenged group generated very high in vivo levels of inducible nitric oxide (NO) from eosinophils. Superoxide expression was very high in macrophages and neutrophils compared to the uninfected control. In conclusion, our studies suggest that early F. hepatica infection could directly affect lymphoid cells and generate a high in vivo NO production by eosinophils in the peritoneal cavity. Moreover juvenile flukes could stimulate the macrophages and neutrophils to generate H₂O₂ radicals. The host parasite interactions resulting from immune response regulation by effector cells and immune evasion are discussed.     

Rat Macrophage C-Type Lectin Is an Activating Receptor Expressed by Phagocytic Cells

Macrophage C-type lectin (MCL) is a membrane surface receptor encoded by the Antigen Presenting Lectin-like gene Complex (APLEC). We generated a mouse monoclonal antibody for the study of this receptor in the rat. We demonstrate that rat MCL is expressed on blood monocytes and neutrophils, as well as on several tissue macrophage populations, including alveolar and peritoneal cavity macrophages. We also demonstrate MCL expression on a subset of resident spleen macrophages. Immunohistochemistry analysis of the spleen showed staining specifically in the marginal zone and red pulp. Exposure to pro-inflammatory mediators or to yeast cell wall extract (zymosan) increased surface MCL expression on peritoneal macrophages. We characterized a rat myeloid cell line, RMW, which expresses high levels of MCL. We found that MCL co-immunoprecipitated with the activating adaptor protein FcεRIγ in these cells. Moreover, beads coated with anti-MCL antibody increased phagocytosis in the RMW cells. Together, these observations indicate that rat MCL is a receptor that activates phagocytosis in myeloid cells under inflammatory conditions.     

cDNA sequence and tissue expression of an antimicrobial peptide, dicentracin; a new component of the moronecidin family isolated from head kidney leukocytes of sea bass, Dicentrarchus labrax

A 483-bp cDNA was isolated from sea bass (Dicentrarchus labrax) head kidney leukocytes, dicentracin, using PCR primers designed from conserved moronecidin domains. Gene bank analysis revealed that dicentracin cDNA belongs to the moronecidin family. As deduced from alignment with Morone chrysops moronecidin, the precursor of 79 aa appeared to be composed of a signal peptide of 22 aa, followed by the mature AMP (antimicrobial peptide) of 22 aa named dicentracin, and a C-terminal extension of 35 aa. Dicentracin precursor displayed 3 aa substitutions with other moronecidin sequence but none in the mature peptide sequence. Using in situ hybridization assay, dicentracin gene expression was observed in 68–71% of peripheral blood leukocytes, kidney leukocytes or peritoneal cavity leukocytes without significant statistical differences. Dicentracin mRNA was observed in most of the granulocytes, as well as in monocytes from both peripheral blood and head kidney, and in macrophages from peritoneal cavity. No expression was observed in thrombocytes or in lymphocytes.     

Resistance of Histoplasma capsulatum to killing by human neutrophils

The basis for resistance of yeast form of Histoplasma capsulatum to antifungal activity of human neutrophils was studied. In limiting dilution assays and short term coculture assays human neutrophils were ineffective in killing H. capsulatum whereas Candida albicans was readily killed. By contrast, in a cell free hydrogen peroxide-peroxidase-halide system H. capsulatum was as sensitive to killing as C. albicans. Moreover, lysate of human neutrophils effectively substituted for horse-radish peroxidase in a cell free system for killing H. capsulatum. H. capsulatum elicited significant products of the oxidative burst in human neutrophils as detected by luminol-enhanced chemiluminescence. However, the response was two-fold less (p<0.05) than that induced by C. albicans. Transmission electron microscopy studies showed that phagosome-lysosome fusion took place when neutrophils phagocytosed C. albicans or H. capsulatum. Taken together, these findings indicate that, even though H. capsulatum elicits an oxidative burst and phagosome-lysosome fusion within the phagosome, it is capable of evading damage in short term assays.Abbreviations CFU colony forming units - PMN polymorphonuclear neutrophil - CTCM complete tissue culture medium - CL chemiluminescence - HPO horseradish peroxidase - P-L lysosomal peroxidase positive material     

A distinct subset of podoplanin (gp38) expressing F4/80+ macrophages mediate phagocytosis and are induced following zymosan peritonitis

Macrophages are important tissue resident cells that regulate the dynamics of inflammation. However, they are strikingly heterogeneous. During studies looking at podoplanin (gp38) expression on stromal cells in the murine spleen and peritoneal cavity we unexpectedly discovered that podoplanin was expressed on a subset of F4/80⁺ macrophages; a subset which we have termed fibroblastic macrophages (FM). These cells function as phagocytes in vitro as measured by bead mediated phagocytosis assays. FM also exist at high frequency in the peritoneal cavity and in zymosan induced peritonitis in vivo. These FM represent a unique subgroup of F4/80⁺ macrophages and their presence in the inflamed peritoneum suggests that they play a role in zymosan induced peritonitis.     

Enhancement of Host Resistance against Listeria Infection by Lactobacillus casei: Activation of Liver Macrophages and Peritoneal Macrophages by Lactobacillus casei

The functions of liver macrophages and peritoneal macrophages obtained after injection of Lactobacillus casei were examined. Listericidal activity in vivo was enhanced in liver macrophages 13 days after L. casei injection but was somewhat suppressed in the macrophages 2 days after the injection. The listericidal activity in vitro was enhanced in peritoneal macrophages obtained 13 days after L. casei injection but was suppressed in cells obtained 2 days later. The PMA-triggered respiratory burst in the liver macrophages elicited by L. casei was higher than that of resident macrophages. Alkaline phosphodiesterase activity in the liver macrophages was decreased by L. casei injection, as was also the case with peritoneal macrophages. These observations indicate that L. casei augmented cellular functions of both liver and peritoneal macrophages.     

Respiratory burst responses of rat macrophages to microsporidian spores.

This study investigated the respiratory burst responses of rat resident peritoneal macrophages and of peritoneal macrophages stimulated 5 days previously with viable spores of the fish infecting microsporidian Microgemma caulleryi. Nitric oxide production by resident macrophages and prestimulated macrophages in response to viable microsporidian spores was significantly lower than in response to Escherichia coli lipopolysaccharide (LPS) (nitrite concentration in medium 57 +/- 1 microM for resident macrophages stimulated with LPS versus 31 +/- 1 microM for resident macrophages stimulated with microsporidian spores and 36 +/- 4 microM for M. caulleryi prestimulated macrophages; P < 0.05). Extracellular release of reactive oxygen species (ROS) by resident macrophages in response to microsporidian spores was similar to that in response to Kluyveromyces lactis yeast cells and to that in response to phorbol myristate (a stimulator of protein C kinase). Intracellular ROS production by resident macrophages in response to microsporidian spores was similar to that produced in response to yeast cells. Both extracellular ROS production and intracellular ROS production (in response to all stimuli) were significantly lower after in vivo prestimulation of macrophages with microsporidian spores. These results demonstrate that microsporidian spores of species other than those that habitually infect mammals are capable of modulating the respiratory burst of rat peritoneal macrophages. Such modulation may contribute to avoidance by the microsporidian of cytotoxic responses associated with the respiratory burst.     

Effective inhibition of Candida albicans growth by the combination of murine peritoneal neutrophils and activated macrophages.

The effect of leukocytes on the anti-Candida activity of neutrophils was examined. Murine neutrophils which were purified from casein-induced peritoneal cells inhibited the mycelial growth of Candida albicans. This anti-Candida activity of neutrophils was augmented by the addition of spleen cells prepared from mice pretreated with bacterial lipopolysaccharide 3 hr before, but not from non-treated mice. The population in the spleen cells, which enhanced the anti-Candida activity of neutrophils, was plastic-plate adherent, nylon-fiber columns adherent and anti-Mac-1 antigen-positive. These immunological profiles suggested that the enhancing cells are classified to splenic macrophages. Peritoneal-exudated macrophages from mice treated with lipopolysaccharide also augmented the anti-Candida activity of neutrophils. These results suggest that the anti-Candida activity of neutrophils may be upregulated by activated macrophages.     

Lutzomyia longipalpis Saliva Triggers Lipid Body Formation and Prostaglandin E2 Production in Murine Macrophages

Background

Sand fly saliva contains molecules that modify the host''s hemostasis and immune responses. Nevertheless, the role played by this saliva in the induction of key elements of inflammatory responses, such as lipid bodies (LB, also known as lipid droplets) and eicosanoids, has been poorly investigated. LBs are cytoplasmic organelles involved in arachidonic acid metabolism that form eicosanoids in response to inflammatory stimuli. In this study, we assessed the role of salivary gland sonicate (SGS) from Lutzomyia (L.) longipalpis, a Leishmania infantum chagasi vector, in the induction of LBs and eicosanoid production by macrophages in vitro and ex vivo.

Methodology/Principal Findings

Different doses of L. longipalpis SGS were injected into peritoneal cavities of C57BL/6 mice. SGS induced increased macrophage and neutrophil recruitment into the peritoneal cavity at different time points. Sand fly saliva enhanced PGE₂ and LTB₄ production by harvested peritoneal leukocytes after ex vivo stimulation with a calcium ionophore. At three and six hours post-injection, L. longipalpis SGS induced more intense LB staining in macrophages, but not in neutrophils, compared with mice injected with saline. Moreover, macrophages harvested by peritoneal lavage and stimulated with SGS in vitro presented a dose- and time-dependent increase in LB numbers, which was correlated with increased PGE₂ production. Furthermore, COX-2 and PGE-synthase co-localized within the LBs induced by L. longipalpis saliva. PGE₂ production by macrophages induced by SGS was abrogated by treatment with NS-398, a COX-2 inhibitor. Strikingly, SGS triggered ERK-1/2 and PKC-α phosphorylation, and blockage of the ERK-1/2 and PKC-α pathways inhibited the SGS effect on PGE₂ production by macrophages.

Conclusion

In sum, our results show that L. longipalpis saliva induces lipid body formation and PGE₂ production by macrophages ex vivo and in vitro via the ERK-1/2 and PKC-α signaling pathways. This study provides new insights regarding the pharmacological mechanisms whereby L. longipalpis saliva influences the early steps of the host''s inflammatory response.     

Isolation of the respiratory burst oxidase: The role of a flavoprotein component

The article reviews the enzymatic and electron transfer properties of a low-potential FAD-dependent flavoprotein that is a component of the NADPH-dependent O ₂ ^– · -generating respiratory burst oxidase of phagocytes. Current methods available for isolation of the respiratory burst oxidase and the flavo-protein component of the complex are also reviewed. These studies and data obtained from affinity-labeling of respiratory burst oxidase components, suggest that the flavoprotein has a molecular weight of 65–67 kD. The prevailing evidence suggests that the flavoprotein functions as a dehydrogenase/electron transferase and can directly catalyse NADPH-dependent O ₂ ^– · formation when isolated. However, in neutrophil plasma membranes, the prevailing evidence suggests that the flavoprotein functions primarily to transfer electrons from NADPH to cytochromeb _–245 and that this latter redox component is the catalytic side of O ₂ ^– · formation. A working model for the arrangement of the flavorprotein and cytochromeb _–245 components of the respiratory burst oxidase in neutrophil membranes is proposed.     

Linear and branched ß(1–3) d-glucans activate but do not prime teleost macrophages in vitro and are inactivated by dilute acid: Implications for dietary immunostimulation

ß(1–3) glucans are a diverse range of carbohydrate polymers of differing lengths and structures that make up the cell walls of yeast, fungi, algae and some plants and activate innate immune responses in plants, invertebrates and higher animals. Consequently glucans are often used as dietary immunostimulants in commercial feeds for aquacultured fish species. The present study investigates the capability of purified glucans of differing structures and configurations, including curdlan, paramylon, laminarin and purified yeast ß glucan to activate innate immunity in vitro using barramundi pronephros macrophages as a model, and compares them to Zymosan, a complex mixture derived from yeast cell walls, and lipopolysaccharide from Gram negative bacteria. All of the glucans were able to stimulate respiratory burst in barramundi macrophages at concentrations of 100 μg/mL and 1000 μg/mL, with curdlan eliciting the highest respiratory burst response at 1000 μg/mL. LPS and Zymosan were the only immunostimulants tested that could prime barramundi macrophages by incubating with low concentrations (0.1 and 1 μg/mL) for 24 h before triggering respiratory burst with PMA, suggesting teleost macrophages may not prime through the glucan receptor. As glucans are used as dietary immunostimulants, the pH of the barramundi stomach was assayed for 6 h following feeding and indicated that pH was as low as 2 for up to 6 h. Treating the glucans with dilute HCl at pH 2 completely neutralised their macrophage-activating capability. These results are important as they indicate that glucans do not prime barramundi macrophages but will activate them at high concentrations. However, it is debatable whether glucans will have any effect on macrophages if administered in the diet due to the combination of high concentration required and probable hydrolysis of the polymer structures as they pass through the acid environment of the stomach.     

Molecular cloning of rock bream (Oplegnathus fasciatus) tumor necrosis factor-α and its effect on the respiratory burst activity of phagocytes

Rock bream (Oplegnathus fasciatus) tumor necrosis factor-α (rbTNF-α) gene was cloned, recombinantly produced, and the effect of the recombinant rbTNF-α on the respiratory burst activity of rock bream phagocytes was analyzed. Structurally, genomic DNA of rbTNF-α was comprised with four exons and three introns, and deduced amino acid sequence of its cDNA possessed the TNF family signature, a transmembrane domain, a protease cleavage site, and two cysteine residues, which are the typical characteristics of TNF-α gene in mammals and fish. The chemiluminescent (CL) response of rock bream phagocytes was significantly enhanced by pre-incubation with recombinant rbTNF-α, when opsonized zymosan was used as a stimulant of the respiratory burst. However, CL enhancing effect of the recombinant rbTNF-α was very weak when the respiratory burst activity of phagocytes was triggered with phorbol-12-myristate-13-acetate (PMA) instead of zymosan. These results suggest that rock bream TNF-α might have an ability to prime the respiratory burst activity of phagocytes against receptor-mediated phagocytosis inducing stimulants, such as zymosan, but have little ability against stimulants not accompanying receptor-mediated phagocytosis.     

Effects of cyclooxygenase inhibitors and prostaglandin E2 on macrophage activation in vitro

The effect of the cyclooxygenase inhibitors, indomethacin and diclofenac, and of PGE₂ on either resting or stimulated macrophages was investigated. Peritoneal macrophages were obtained from untreated mice and cultured for 10 days. Macrophage activation was induced by zymosan phagocytosis and was monitored by testing for plasminogen activator secretion and the cellular levels of lactate dehydrogenase, β-glucoronidase and alkaline phosphodiesterase I.It was found that cyclooxygenase inhibitors activate resting macrophages and enhance the degree of activation obtained after zymosan phagocytosis. Addition of exogenous PGE₂, on the other hand, had the opposite effect, it suppressed activation induced either by cyclooxygenase inhibitors, phagocytosis or a combination of both. Cyclooxygenase inhibitors and PGE₂ did not affect the hexose monophosphate shunt activity of resting macrophages and had only a minor effect on the respiratory on the respiratory macrophages and had only a minor effect on the respiratory burst occuring during zymosan phagocytosis. It appears, therefore, that the observed changes in the state of activation of the machrophages are not related to hexose monophosphate shunt activity.The described effects suggest that PGE₂ and possibly other cyclooxygenase products may function as inhibitory feed-back regulators of macrophage activation.     

Macrophages as effector cells of protective immunity in murine schistosomiasis

Mice infected with Schistosoma mansoni develop a dramatic (five- to eightfold) increase in numbers of peritoneal leukocytes, and approximately 65% of these cells are macrophages. By several biochemical and cytochemical criteria, these cells were comparable to resident peritoneal macrophages of normal mice. However, macrophages from schistosome-infected mice exhibited significant nonspecific tumoricidal activity in vitro, a function associated with immunologically activated cells. The time course for development of activated macrophages in the peritoneal cavity was dependent upon the route of infection. Cytotoxic cells were present in the peritoneal cavity by 3 weeks after intraperitoneal infection, but were not evident until several weeks later in animals infected percutaneously, subcutaneously, or intravenously. However, by 3 weeks after subcutaneous infection, tumoricidal macrophages appeared in the peritoneal cavity after intraperitoneal challenge with soluble schistosome antigens. Macrophage activation was independent of the development of egg granulomas, since tumoricidal cells could be found prior to the onset of egg production and were also present in mice infected with only male worms. Development of activated macrophages in these instances is thus consistent with previous observations on induction of T lymphocyte reactivity toward schistosomula. Since other manipulations known to activate macrophages have been shown to induce partial resistance to schistosome infection, the finding that macrophage activation results from primary S. mansoni infection itself suggests that these cells may play a major role in acquired immunity to this parasite.     

Macrophage depletion in the rat after intraperitoneal administration of liposome-encapsulated clodronate: Depletion kinetics and accelerated repopulation of peritoneal and omental macrophages by administration of freund's adjuvant

The purpose of this study was to develop a method for the depletion of macrophages from the peritoneal cavity and the omentum of the rat. Rats received two intraperitoneal injections (at days 0 and 3) with liposome-encapsulated clodronate (dichloromethylene bisphosphonate: Cl₂MBP-liposomes). This treatment resulted in complete elimination of mature tissue macrophages (ED2-positive macrophages) from the peritoneal cavity and the omentum within 2 days. The elimination included the strongly ED2-positive spindle-shaped cells of the omental membrane. Repopulation of the omental ED2-positive macrophages was not seen within the next 23 days. Whereas ED2-positive macrophages were completely depleted, few ED1-positive cells remained and repopulation of ED1-positive cells was faster. The treatment further depleted macrophages from the spleen, especially from the red pulp, parathymic lymph nodes and liver. Freund's incomplete adjuvant administered one day after the last injection of Cl₂MBP-liposomes considerably accelerated repopulation in the omentum. The protocol described might be used to investigate the contribution of mature tissue macrophages to the induction of immune responses, drug metabolism and the elimination of intestinal tumours.     

Regulations of macrophage-mediated tumor cell killing by prostaglandins: Comparison of the effects of PGE2 and PGI2

Mouse resident peritoneal macrophages stimulated by purified bacterial lipopolysaccharide (LPS) produced both prostaglandin E₂ (PGE₂) and prostaglandin I₂ (PGI₂), the latter detected as its stable metabolite, 6-keto PGF_1α. Maximum production, induced in each case by 1 ng/ml purified LPS, was in the range of 10⁻⁷M for PGI₂ and 3 × 10⁻⁸M for PGE₂. A quantitatively similar increase in intracellular levels of macrophage cyclic AMP (measured on a whole cell basis), with a similar duration of effect, was stimulated by PGE₂ and PGI₂; however, only PGE₂ had a negative regulatory effect on macrophage activation for tumor cell killing. These data confirm that more than a whole cell increase in the concentration of cyclic AMP is needed to shut off nonspecific tumor cell killing mediated by LPS-activated resident peritoneal macrophages.