Phagocytic Stimulation Research Articles

Bayesian 13C-Metabolic Flux Analysis of Parallel Tracer Experiments in Granulocytes: A Directional Shift within the Non-Oxidative Pentose Phosphate Pathway Supports Phagocytosis.

The pentose phosphate pathway (PPP) plays a key role in the cellular regulation of immune function; however, little is known about the interplay of metabolic adjustments in granulocytes, especially regarding the non-oxidative PPP. For the determination of metabolic mechanisms within glucose metabolism, we propose a novel set of measures for 13C-metabolic flux analysis based on ex vivo parallel tracer experiments ([1,2-13C]glucose, [U-13C]glucose, [4,5,6-13C]glucose) and gas chromatography-mass spectrometry labeling measurements of intracellular metabolites, such as sugar phosphates and their fragments. A detailed constraint analysis showed that the permission range for net and irreversible fluxes was limited to a three-dimensional space. The overall workflow, including its Bayesian flux estimation, resulted in precise flux distributions and pairwise confidence intervals, some of which could be represented as a line due to the strength of their correlation. The principal component analysis that was enabled by these behaviors comprised three components that explained 99.6% of the data variance. It showed that phagocytic stimulation reversed the direction of non-oxidative PPP net fluxes from ribose-5-phosphate biosynthesis toward glycolytic pathways. This process was closely associated with the up-regulation of the oxidative PPP to promote the oxidative burst.

Evaluation of Immunomodulatory Activity of Extract and Fractions of Sida acuta Burm. f. Leaves in Mice and GC-MS Analysis of n- Hexane Fraction

The leaves of Sida acuta Burm. f. (Malvaceae) has been reported to possess potent anti- inflammatory, anti- plasmodial and anti-microbial activities. The relationship of these bioactivities and immune responses lead to the evaluation of the immunomodulatory activity of Sida acuta Burm. f. leave extract and fractions. This our study was done to determine the immunomodulatory activity and chemical study of methanol leave extract and fractions of Sida acuta Burm. f. The immunomodulatory evaluation was done by invivo Delay Type Hypersensitivity reaction (DTHR) in the body and in vitro measurement of phagocytosis of killed Candida albicans by the phagocyte polymorphonuclear leucocytes using slide method. Acute toxicity, phytochemical and GC-MS analysis were also performed. The DTHR tested in the blood with T-cells in mice showed that the extract and its fractions caused a delayed hypersensitivity response in 24hrs which was very significant (P ? 0.05) in the n- hexane fraction of the extract when compared to the control group at the dose of 100mg/kg. The in vitro studies showed a very significant difference (P ? 0.05) in the positive control group (LEVA) at concentration of 50, 100 and 200µg/ml, in crude extract (SrE) at concentrations of 50, 100 and 200µg/ml, n- hexane fraction 50, 100 and 200µg/ml, Ethyl acetate fraction at 200µg/ml and Absolute methanol fraction at 100µg/ml and also have high percentage phagocytic stimulation (PPS). The acute toxicity test did not cause clinical signs or death within 24hours post treatment in all doses tested and highest dose of 5000mg/kg. Phytochemical analysis revealed the presence of flavonoids, saponins, alkaloids, triterpenoids, tannins, steroids and cardiac glycosides. GC-MS analysis of fraction with highest activity was carried out on n-hexane fraction which showed the presence of some compounds like hexadecanoic acid, 2-hydroxy-1 (hydroxymethyl) ethyl ester, 3,4-seco-5alpha-cholestan-3-oic acid,4-hydroxy-4-methyl epsilon-lacto

Modeling the immune response to Salmonella during typhoid.

Several facets of the host immune response to Salmonella infection have been studied independently at great depths to understand the progress and pathogenesis of Salmonella infection. The circumstances under which a Salmonella-infected individual succumbs to an active disease, evolves as a persister or clears the infection are not understood in detail. We have adopted a system-level approach to develop a continuous-time mechanistic model. We considered key interactions of the immune system state variables with Salmonella in the mesenteric lymph node to determine the final disease outcome deterministically and exclusively temporally. The model accurately predicts the disease outcomes and immune response trajectories operational during typhoid. The results of the simulation confirm the role of anti-inflammatory (M2) macrophages as a site for persistence and relapsing infection. Global sensitivity analysis highlights the importance of both bacterial and host attributes in influencing the disease outcome. It also illustrates the importance of robust phagocytic and anti-microbial potential of M1 macrophages and dendritic cells (DCs) in controlling the disease. Finally, we propose therapeutic strategies for both antibiotic-sensitive and antibiotic-resistant strains (such as IFN-γ therapy, DC transfer and phagocytic potential stimulation). We also suggest prevention strategies such as improving the humoral response and macrophage carrying capacity, which could complement current vaccination schemes for enhanced efficiency.

Absolute Neutropenia and Infection Development in Premature Infants in Early Neonatal Period: Cross-Sectional Study

Background. Premature infants have high risk of developing of neutropenia and infections in the early neonatal period. The correlation of these events requires further studies.Objective. The aim of the study was to investigate the frequency of absolute neutropenia and infectious complications cases in premature infants in the early neonatal period with estimation of phenotypical and functional features of cord blood neutrophils.Methods. The study included premature infants (gestational age 25–36 weeks) with APGAR score < 8 on the 1st and 5th minutes of life. The frequency of absolute neutropenia (at least once < 1.5109/l) and infectious complications (localized infections of bacterial etiology, early neonatal sepsis) cases in the first 14 day of life was analysed. Additionally, we have determined the expression of CD64, CD16, CD32 by cord blood neutrophils in premature (n = 102) and mature infants (n = 30) via method of flow cytofluorometry. We have used FITC labeled Escherichia coli to estimate their phagocytic activity, and stimulation of E. coli neutrophils in the presence of 5 mM of dihydrorhodamine 123 to estimate their stimulation index (ratio of mean fluorescent intensity (MFI) of activated neutrophils in stimulated samples and in negative controls, E. coli free samples).Results. The episodes of absolute neutropenia in the first 14 days of life were recorded in 17 cases, infectious complications — in 87 children (in 24 cases — sepsis) in the group of premature infants. The frequency of infectious complications in premature children did not correlate with the frequency of absolute neutropenia episodes. Cord blood neutrophils in premature infants had higher CD64 expression and, on the contrary, lower CD16 expression, as well as low phagocytic activity and stimulation index value (in all cases p < 0.001).Conclusion. Absolute neutropenia in premature infants in early neonatal period does not correlate with high risk of bacterial infections. However, cord blood neutrophils in premature infants had lower functional activity.

Laser irradiation of blood as a method of prevention of oral mucositis

Aim. Study the effectiveness of oral mucositis (OM) prevention by the stimulation of white cells phagocytic activity and activation of endogenous keratinocyte growth factor production by laser blood irradiation method (LBI). Materials and methods. This study covers 44 patients with malignant tumors. 29 patients have already passed the high-dose polychemotherapy complicated by OM development. 15 patients have not passed the high-dose polychemotherapy yet before this admission. In the study we applied the method of identification of the white cells phagocytic activity state using the test sheet of latex microparticles. The study was performed before high-dose polychemotherapy, after LBI and after chemotherapy. Blood exposure to laser irradiation was made by placing the emitter on skin above the large vessels: areas of carotid arteries and veins, cubital, subclavian or popliteal. Results. LBI technique was used in 92 courses of high-dose polychemotherapy. Stimulation of phagocytes by LBI method showed that application of this method before chemotherapy leads to the positive result in all cases. All children (n=44), that were subject to white cells phagocytic activity stimulation, had not OM. Conclusion. Blood test for white cells phagocytic activity gives a chance to determine the actual readiness of the body to resist infections. If it was impossible to predict oral mucositis development before, now implementation of white cells phagocytic activity determination method improves the effectiveness of such prognosis. Thus, first in the world, all results that we obtained became possible only due to implementation of the white cells phagocytic activity test and stimulation of the white cells phagocytic activity and activation of the endogenous keratinocyte growth factor production by laser blood irradiation method.

In Vitro Activation of Epinephelus Malabaricus Head-Kidney Leukocytes by Angelica Sinensis Polysaccharide

Phagocytic, respiratory burst, bactericidal, and proliferative activity stimulation of Epinephelus malabaricus head-kidney leukocytes by Angelica sinensis polysaccharide (ASP) were evaluated in vitro. Leukocytes were incubated for 2, 6, 12 and 24 h in the presence of 0, 10, 100 and 1000 μg/mlASP or 100 μg/ml lipopolysaccharide (LPS) before phagocytic and respiratory burst activity assay, and for 24 h with 0, 100, 1000, 10000 μg ASP/ml before bactericidal activity detection. Meanwhile, leukocytes were incubated for 24 and 48 h followed by proliferative activity evaluation. The results showed that ASP boosted leukocytes phagocytic, respiratory burst, bactericidal, and proliferative activity significantly in a dose-dependent manner. Leukocytes incubated with 100 μg/ml ASP exhibited the highest phagocytic and respiratory burst activity. Leukocytes incubated with 1000 μg/ml ASP showed the highest bactericidal activity, which was followed by leukocytes incubated with 100μg/ml ASP. Leukocyte proliferation was significantly enhanced with increasing concentration of ASP, and reached the highest value with 10000 μg/ml ASP. Compared with LPS in the same concentration (100 μg/ml), ASP exerted a stronger stimulating effect on respiratory burst but was weaker on proliferation in E. malabaricus leukocytes. These findings indicate that ASP prototype enhanced cellular immunity of E. malabaricus, and potentially contributed to the pursuit of environmentally friendly prophylactic measures to enhance the immunity of E. malabaricus

In Vitro Activation of Epinephelus Malabaricus Head-Kidney Leukocytes by Angelica Sinensis Polysaccharide

Phagocytic, respiratory burst, bactericidal, and proliferative activity stimulation of Epinephelus malabaricus head-kidney leukocytes by Angelica sinensis polysaccharide (ASP) were evaluated in vitro. Leukocytes were incubated for 2, 6, 12 and 24 h in the presence of 0, 10, 100 and 1000 μg/mlASP or 100 μg/ml lipopolysaccharide (LPS) before phagocytic and respiratory burst activity assay, and for 24 h with 0, 100, 1000, 10000 μg ASP/ml before bactericidal activity detection. Meanwhile, leukocytes were incubated for 24 and 48 h followed by proliferative activity evaluation. The results showed that ASP boosted leukocytes phagocytic, respiratory burst, bactericidal, and proliferative activity significantly in a dose-dependent manner. Leukocytes incubated with 100 μg/ml ASP exhibited the highest phagocytic and respiratory burst activity. Leukocytes incubated with 1000 μg/ml ASP showed the highest bactericidal activity, which was followed by leukocytes incubated with 100μg/ml ASP. Leukocyte proliferation was significantly enhanced with increasing concentration of ASP, and reached the highest value with 10000 μg/ml ASP. Compared with LPS in the same concentration (100 μg/ml), ASP exerted a stronger stimulating effect on respiratory burst but was weaker on proliferation in E. malabaricus leukocytes. These findings indicate that ASP prototype enhanced cellular immunity of E. malabaricus, and potentially contributed to the pursuit of environmentally friendly prophylactic measures to enhance the immunity of E. malabaricus

Immunomodulatory effects of Stachytarpheta cayennensis leaf extract and its synergistic effect with artesunate.

BackgroundThe leaves of Stachytarpheta cayennensis C. Rich. (Verbenaceae) have been reported to possess potent anti-inflammatory and anti-malarial activities. Due to close association between inflammatory and immune responses, we evaluated the immunomodulatory activity of leaves extract of S. cayennensis. The combined effects of the leaves extract and artesunate, a standard antimalarial agent with immunomodulatory effects, were also evaluated.MethodsThe immunomodulatory activity of the methanol extract of the leaves of S. cayennensis (MESC) was evaluated in mice using the Delayed-Type hypersensitivity response (DTHR), primary and secondary humoral immune responses and the in vivo leucocyte mobilization tests. The immunomodulatory effect of artesunate and the combined effects of MESC and artesunate were evaluated using the phagocytic activity of polymorphonuclear neutrophils. Acute toxicity and lethality test in addition to the preliminary phytochemical studies of MESC were also performed.ResultsThe MESC exhibited 64.21% inhibition of DTHR at 500 mg/kg dose and evoked 139.64% of phagocytic stimulation at 100 μg/ml dose. Also MESC significantly (p < 0.05) showed dose related stimulation of humoral immunity and a highest percentage leucocyte mobilization of 10.15% at 250 mg/kg dose. Artesunate offered a non-significant (p > 0.05) percentage phagocytic stimulation (PPS) while the combined effect of artesunate and MESC exhibited a significant (p < 0.05) dose dependent PPS with highest PPS of 393.77% at 100 μg/ml. The LD50 of the MESC was estimated to be greater than 5000 mg/kg since there were no lethality and signs of acute intoxication after 48 h observation. Preliminary phytochemical analysis revealed the presence of carbohydrates, glycosides, flavonoids, saponins, alkaloids, terpenoids and steroids.ConclusionsThe results of this study indicated that MESC possesses immunostimulatory action with significant synergistic effects with artesunate, and can therefore, offer immune boosting activities in disorders of immune suppression.

Palmitoylethanolamide stimulates phagocytosis of Escherichia coli K1 by macrophages and increases the resistance of mice against infections.

BackgroundPalmitoylethanolamide (PEA), an endogenous lipid and a congener of anandamide, possesses a wide range of effects related to metabolic and cellular homeostasis including anti-inflammatory and neuroprotective properties.MethodsIn vitro, we studied the ability of macrophages to phagocytose Escherichia coli K1 after stimulation with increasing doses of PEA. In vivo, wild-type mice were treated with PEA intraperitoneally 12 hours and 30 minutes before infection. Meningoencephalitis or sepsis was induced by intracerebral or intraperitoneal infection with E. coli K1.ResultsStimulation of macrophages with PEA for 30 minutes increased the phagocytosis of E. coli K1 without inducing the release of TNFα or CXCL1. Intracellular killing of E. coli K1 was higher in PEA-stimulated than in unstimulated peritoneal macrophages and microglial cells. Pre-treatment with PEA significantly increased survival of mice challenged intracerebrally or intraperitoneally with E. coli K1. This effect was associated with a decreased production of CXCL1, IL-1β and IL-6 in homogenates of spleen and cerebellum in mice treated with PEA.ConclusionsOur observations suggest that these protective effects of PEA in mice can increase the resistance to bacterial infections without the hazard of collateral damage by excessive stimulation of phagocytes.

Increased Production of Nitric Oxide by Phagocytic Stimulated Neutrophils in Patients With Chronic Granulomatous Disease

Chronic granulomatous disease (CGD) is an inherited disorder in which phagocytic leukocytes fail to generate superoxide (O(2)(-)) and antimicrobial oxidants. Patients with CGD develop recurrent and often life-threatening infections due to catalase-positive microorganisms. We attempted to measure the production of nitric oxide and reactive oxygen species in polymorphonuclear neutrophils (PMNs) from patients with CGD using a flow cytometry technique. Venous blood was obtained from 5 male patients with X-linked CGD and from 10 healthy volunteers. The nitric oxide production by PMNs after phagocytosis was measured using diaminofluorescein-2 diacetate, a fluorescent indicator of intracellular nitric oxide production. After erythrocytes were hypotonically lysed, the cell pellet was applied to a cytofluorometer. Although the production of hydrogen peroxide by PMNs from patients with CGD was almost absent, nitric oxide production was detected at nearly half the level as was seen in the PMNs from healthy volunteers (CGD vs. healthy volunteers=140.1±28.6 vs. 256.4±10.3, mean fluorescence intensity; P<0.01). In conclusion, we demonstrated that human PMNs produces nitric oxide after phagocytic stimulation. Also nitric oxide production after phagocytic stimulation by PMNs of patients with CGD is observed although its amount is lower than that observed on healthy control. Despite the fact that CGD patients cannot produce H(2)O(2) which is essential for intracellular bacteriocidal process after phagocytosis, our data suggested that the phylactic effect in PMNs induced by nitric oxide could be at least partially related to the survival of patients with CGD.

The Anti-Tumorigenic Mushroom Agaricus blazei Murill Enhances IL-1β Production and Activates the NLRP3 Inflammasome in Human Macrophages

Agaricus blazei Murill (AbM) has been reported to possess immune activity against tumors and infections through stimulation of mononuclear phagocytes. Recently, AbM extract was shown to induce the production of the pro-inflammatory cytokine, interleukin-1β (IL-1β), in human monocytes. IL-1β is a key pro-inflammatory cytokine produced by activated macrophages and monocytes and its secretion is strictly controlled by the inflammasome. The purpose of this study is to investigate the effect of AbM water extracts on the regulation of IL-1β production and activation of the NLRP3 inflammasome in human THP-1 macrophages. The NLRP3 inflammasome consists of an NLRP3 receptor, an adaptor protein called ASC, and the inflammatory protease, caspase-1. Typically, stimulation of immune cells with microbial products results in production of pro-IL-1β, but a second stress-related signal activates the inflammasome and caspase-1, leading to processing and secretion of IL-1β. Our results show that AbM enhances transcription of IL-1β and triggers NLRP3 inflammasome-mediated IL-1β secretion in human THP-1 macrophages. AbM-mediated IL-1β secretion was markedly reduced in macrophages deficient in NLRP3 and ASC, demonstrating that the NLRP3 inflammasome is essential for AbM-induced IL-1β secretion. In addition, caspase-1 was activated and involved in proteolytic cleavage and secretion of IL-1β in AbM-treated macrophages. AbM-mediated IL-1β secretion also decreased in cells treated with cathepsin B inhibitor, suggesting that AbM can induce the release of cathepsin B. Furthermore, our data show that AbM-induced inflammasome activation requires the release of ATP, binding of extracellular ATP to the purinergic receptor P2X7, the generation of reactive oxygen species, and efflux of potassium. Taken together, these findings reveal that AbM activates the NLRP3 inflammasome via multiple mechanisms, resulting in the secretion of IL-1β.

Reaction of CD117+ Cells to Renal Lesion under Conditions of Phagocytic Mononuclear System Stimulation

The content of CD117(+)cells in the kidneys and CD45(low)CD117(+)cells in the bone marrow and blood of mice were studied after partial nephrectomy and under conditions of macrophage stimulation with 3-aminophthalhydrazide. The counts of tubular CD117(+)epitheliocytes sharply increased and the content of CD45(low)CD117(+)cells in the bone marrow decreased after renal damage. Injection of 3-aminophthalhydrazide stimulated the expression of CD117 by renal epitheliocytes and led to reduction of CD45(low)CD117(+)cell counts in the bone marrow and blood. Macrophages stimulated proliferative processes in the kidney and differentiation of stem cells in the bone marrow due to synergic effects of their cytokines and stem cell factor.

Immunology of tuberculosis and cytokines

One of the unique features characterizing human tuberculosis (TB) is its pathogenesis. The pathogenesis of TB involves cell-mediated immune responses against Mycobacterium tuberculosis. Concisely, macrophages activated by various soluble mediators or cytokines released through the cellular interactions after infection with M. tuberculosis play a pivotal role in the pathogenesis of human TB. In fact, very complex cellular interactions are going on within the host after infection with or endogenous reactivation of M. tuberculosis. Cells communicate by cell-cell contact and by the release of mediators which may originate locally, called cytokines. In TB infection, macrophages can be activated by two ways; directly with mycobacterial organisms or lipid fractions of their cell walls at the earlier phase of infection, and indirectly with cytokines produced by CD4+ T cells specifically activated by mycobacterial peptide antigens at the later phase of infection. The various clinical features of TB are the summarized outcome of cell to cell interactions mediated by diverse cytokines produced by various immune cells which are initially triggered by M. tuberculosis infection. CD4+ T cells can be classified into two subsets according to the patterns of cytokines they produce; Th1 cells give rise to cell-mediated immunity and are characterized by the production of IL-2 and IFN-gamma, whereas Th2 cells are more efficient in mediating antibody production and secrete IL-4, IL-5, IL-6 and IL-10. Th2 cells can control Th1 cells and vice versa. Th2 cells therefore inhibit the production of cytokines by Th1 cells by releasing IL-4 and IL-10. Infection with mycobacteria stimulates macrophage IL-12 production which appears to act directly on naive CD4+ T cells to induce Th1 development and initiation of cell-mediated immunity. IL-12 is a critical component in the development of cell-mediated immunity. In addition, IL-12 also activates NK cells and gamma/delta T cells, both of which secrete various macrophage-activating factors to kill M. tuberculosis. One of the structural characteristics of M. tuberculosis is the cell wall rich in lipid components. Of importance among various biological activities of the cell wall lipids is the stimulation of mononuclear phagocytes to produce a certain number of cytokines or monokines including IL-12 and IL-10, both of which play important roles in regulation of immune responses in mycobacterial infection and in pathogenesis of TB.(ABSTRACT TRUNCATED AT 400 WORDS)

Ral Isoforms Are Implicated in FcγR-Mediated Phagocytosis: Activation of Phospholipase D by RalA

Phagocytosis is an essential element of the immune response permitting the elimination of pathogens, cellular debris, apoptotic cells, and tumor cells. Recently, both phospholipase D (PLD) isoforms, PLD1 and PLD2, were shown to be necessary for efficient FcgammaR-mediated phagocytosis. In this study, we investigated the role of a potential PLD regulator, the Ral GTPases RalA and RalB, in murine RAW 264.7 macrophages. Both Ral isoforms are expressed in macrophages and are transiently activated following FcgammaR stimulation. When Ral expression levels were varied using Ral mutants or interference RNA, phagocytosis assays revealed that Ral isoforms have antagonistic effects; RalA is a positive modulator, whereas RalB plays a negative role. We then focused on RalA and its possible relationship with PLD. The increase in PLD activity that occurs when phagocytosis is stimulated was inhibited in cells with reduced RalA protein, but it was unaffected by reduced levels of RalB. Furthermore, in macrophages transfected with dsRed-RalA and GFP-PLD1 or GFP-PLD2, RalA colocalized with PLD1 and PLD2 at the phagocytic cup during phagosome formation. Additional results obtained from immunoprecipitation of PLD from macrophages transfected with myc-RalA and hemagglutinin-tagged PLD1 or PLD2 indicated an enhanced interaction of RalA with both PLD isoforms during phagocytic stimulation. The increase in RalA and PLD1 interaction was transient and correlated with the time course of RalA activation. These findings reveal a novel pathway involving RalA and PLD in the regulation of FcgammaR-mediated phagocytosis.

The Potency of Hyaluronan of Different Molecular Weights in the Stimulation of Blood Phagocytes

The regulatory functions of glycosaminoglycan hyaluronan (HA) are suggested to be dependent on its molecular weight (MW). Proinflammatory and stimulatory effects are proposed mainly for the low MW HA. However, the complex response of blood phagocytes to HA of different MW is unclear. Herein, the effects of highly purified HA of precisely defined MW (52, 250, and 970 kDa) on human blood phagocytes were tested. All MW HA activated blood phagocytes, including the spontaneous production of ROS, degranulation, and the production of tumor necrosis factor alpha, with low MW HA 52 kDa having the highest potency and high MW HA 970 kDa having the lowest potency. Interestingly, HA inhibited ROS production stimulated by opsonized zymosan particles and, in contrast, potentiated starch-activated ROS production, mostly independent of MW. Data showed a significant effect of HA of different MW on blood phagocytes, including high MW HA.

Internalization-dependent recognition ofMycobacterium aviumssp.paratuberculosisby intestinal epithelial cells

Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of Johne's disease, a highly prevalent chronic intestinal infection in domestic and wildlife ruminants. The microbial pathogenesis of MAP infection has attracted additional attention due to an association with the human enteric inflammatory Crohn's disease. MAP is acquired by the faecal-oral route prompting us to study the interaction with differentiated intestinal epithelial cells. MAP was rapidly internalized and accumulated in a late endosomal compartment. In contrast to other opportunistic mycobacteria or M. bovis, MAP induced significant epithelial activation as indicated by a NF-kappaB-independent but Erk-dependent chemokine secretion. Surprisingly, MAP-induced chemokine production was completely internalization-dependent as inhibition of Rac-dependent bacterial uptake abolished epithelial activation. In accordance, innate immune recognition of MAP by differentiated intestinal epithelial cells occurred through the intracellularly localized pattern recognition receptors toll-like receptor 9 and NOD1 with signal transduction via the adaptor molecules MyD88 and RIP2. The internalization-dependent innate immune activation of intestinal epithelial cells is in contrast to the stimulation of professional phagocytes by extracellular bacterial constituents and might significantly contribute to the histopathological changes observed during enteric MAP infection.

Macrophage fusion leading to foreign body giant cell formation persists under phagocytic stimulation by microspheres in vitro and in vivo in mouse models

The formation of surface-damaging foreign body giant cells (FBGC) from the fusion of macrophages is considered a hallmark of the foreign body response. Experimental evidence indicates that when macrophages are unable to internalize foreign bodies via phagocytosis due to their large size, they acquire a fusogenic phenotype. The mechanism behind this transformation is unclear, and questions, such as which phenotype takes precedence for co-stimulated macrophages engaged in the foreign body response and whether or not such phenotypic alteration is graded, remain unanswered. By recapitulating fusion in vitro using cell lines and primary mouse bone marrow-derived macrophages, we investigated whether concurrent exposure of macrophages to phagocytic and fusogenic stimuli would limit fusion. Induction of phagocytosis by addition of 3.0 mum-diameter polystyrene microspheres to cells under fusogenic conditions, at ratios of 1:10, 1:1, and 10:1 did not prevent fusion. To determine the effect of microsphere phagocytosis on fusion in vivo, we first determined the kinetics of monocyte recruitment, surface adhesion, and fusion following intraperitoneal implantation of a foreign body in a mouse model. Concomitant or subsequent injection of microspheres resulted in their significant accumulation at the biomaterial surface at 2 weeks, but FBGC were still detected. Our findings indicate that despite increasing the abundance of a phagocytic stimulus (microspheres), significant FBGC formation occurs.

Expression profiles of matrix metalloproteinase 9 in teleost fish provide evidence for its active role in initiation and resolution of inflammation

Matrix metalloproteinase 9 (MMP-9) belongs to a family of zinc-dependent endopeptidases. As a consequence of its ability to cleave structural extracellular matrix molecules, mammalian MMP-9 is associated with vital inflammatory processes such as leucocyte migration and tissue remodelling and regeneration. Interestingly, MMP-9 genes have been identified in fish, but functional data are still limited and focus on the involvement of MMP-9 in embryonic development, reproduction and post-mortem tenderization. Here, we describe the involvement of MMP-9 in the innate immunity of carp. In carp, MMP-9 was most notably expressed in classical fish immune organs and in peritoneal and peripheral blood leucocytes, indicating a role of MMP-9 in immune responses. In our well-characterized zymosan-induced peritonitis model for carp, we analysed expression of the MMP-9 gene and the gelatinolytic levels of both pro- and activated forms of MMP-9. The biphasic profile of MMP-9 mRNA expression indicated involvement during the initial phase of inflammation and during the later phase of tissue remodelling. Also, in vitro stimulation of carp phagocytes with lipopolysaccharide or concanavalin A increased MMP-9 gene expression, with a peak at 24 hr. The increase of MMP-9 mRNA correlated with the peak of MMP-9 gelatinolytic level in culture supernatants. These results provide evidence for an evolutionarily conserved and relevant role of MMP-9 in the innate immune response.

IL-4 induces neutrophilic maturation of HL-60 cells and activation of human peripheral blood neutrophils.

IL-4 is a T-helper cell derived cytokine that has effects on myelomonocytic cell maturation and activation. We have studied the effect of IL-4 on neutrophilic maturation using the cell line HL-60 and found that it has a profound effect on the maturation and activation of the cell line. The treatment of HL-60 cells with recombinant hu IL-4 (0.15 to 15.0 ng/ml) induced a shift in the percentage of HL-60 cells staining positive for chloroacetate esterase enzyme activity (indicating commitment to the neutrophilic lineage). IL-4 increased surface expression of the neutrophil-lineage antigen WEM G11, the complement receptors CR3 (CD11b) and CR1 (CD35), but not for the monocyte differentiation antigen CD14. IL-4 treated HL-60 cells demonstrated enhanced Fc- and complement-mediated phagocytic capacity and increased hexose-monophosphate shunt activity. In addition, IL-4 was capable of sustaining the neutrophil maturation of HL-60 cells that had been pre-treated for 24 h with DMSO. To investigate the effect of IL-4 on the mature neutrophil, we studied freshly isolated and rested human peripheral blood neutrophils. In the absence of other stimuli, neutrophils were induced by IL-4 to have significantly elevated phagocytic responses. The response was specific since treatment with anti-human IL-4 abolished phagocytic stimulation. Finally, IL-4 treatment also stimulated resting neutrophils to migrate toward zymosan-activated serum (ZAS) and human IL-5. The results demonstrate that IL-4 is a potent maturation factor for myelocytes to become neutrophils and that IL-4 can stimulate resting mature neutrophils.

Molecular and functional characterization of gilthead seabream Sparus aurata caspase-1: The first identification of an inflammatory caspase in fish

Caspases are a family of cysteine proteases that fulfil critical roles in mammalian apoptosis and in the proteolytic activation of cytokines. In humans, the caspase family includes 13 members whose functions seem to correlate with their phylogenetic relationship. They are classified into two main groups, the cell death (apoptotic) and the inflammatory caspases. Caspase-1 is the best characterized inflammatory caspase and is responsible for the processing of interleukin-1β (IL-1β), IL-18 and IL-33. Despite the importance of caspase-1 in inflammation, no information is available on the presence and activity of this enzyme in fish. In this study, we cloned a caspase-1-like gene from the bony fish gilthead seabream (Sparus aurata L.) which shows a conserved N-terminal caspase-recruitment domain (CARD) and a C-terminal caspase catalytic domain. The seabream caspase-1 gene was expressed in 1 day post-hatching larvae and its mRNA levels increased throughout development. In adult fish, caspase-1 was found to be constitutively expressed in all immune tissues analyzed and, unexpectedly, infection of fish and stimulation of professional phagocytes in vitro decreased its mRNA levels. It was also demonstrated that the recombinant seabream caspase-1 ectopically expressed in HEK293 cells was able to cleave a caspase-1 specific substrate, this activity being enhanced upon activation of the rat P2X7 receptor with BzATP. Finally, seabream fibroblast cell line SAF-1 and primary leukocytes showed endogenous caspase-1 activity, which was almost completely inhibited by a caspase-1 specific inhibitor.