Ingestion Of Zymosan Research Articles

Mechanosensing of Substrate Stiffness Regulates Effector Functions of Human Neutrophils

During a response to injury or infection, neutrophils (PMNs) navigate through a tissue microenvironment that is densely packed with parenchymal cells and extracellular matrix. Within these tissues, PMNs sense the physical property of elasticity as a regulatory element as it pertains to motility and effector functions. Historically, rigid tissue culture surfaces like glass and plastic have been used to study many key anti-microbial effector functions of PMNs; however, these fail to mimic the elasticity of peripheral tissues. We hypothesize that elasticity plays a key regulatory role for PMN anti-microbial effector functions associated with pathogen clearance such as phagocytosis, NETosis, respiratory burst, and degranulation. When comparing phagocytosis by neutrophils on soft substrates and stiff substrates coated with fibronectin, we found that stiffness is selectively regulatory. While Zymosan and Whole Glucan Particles were more readily phagocytosed on soft substrates, IgG-opsonized latex beads were more efficiently taken up on stiff substrates. Further, C3bi-opsonized latex beads and apoptotic bodies were phagocytosed equivalently on all stiffnesses. In addition to a mechanosensitive response to phagocytosed particles, we also saw differential mechanosensitive responses in their mechanism. For example, on stiff substrates, inhibiting Rho-associated protein kinase reduced phagocytosis of Zymosan, while blocking CD45 reduced phagocytosis of IgG-opsonized latex beads. Further, Zymosan uptake was found to increase on soft substrates when actin-related protein Arp2/3 complex was blocked, but have no effect on stiff substrates, further exaggerating the soft/stiff phenotype; conversely, blocking formin-mediated actin had the opposite effect and lowered phagocytosis of Zymosan on soft substrates, attenuating the soft/stiff phenotype. Finally, not all blocking targets showed mechanosensitive responses; while inhibition of Spleen Tyrosine Kinase had no effect on Zymosan uptake on any stiffness, blocking total actin polymerization reduced uptake on all stiffnesses. Taken together, these findings show a highly complex mechanosensitive response to phagocytosis in regards to both particle type and mechanism of uptake. Further, PMNs seeded on Candida albicans hyphae were mechanosensitive in regards to oxidative burst and NETosis; both ROS production and NETosis increased on stiffer substrates. Finally, in addition to phagocytosis and hyphae, we also looked at cytokine release to secondary stimulation by fMLP. We found both Lactoferrin and IL-8 release to increase on stiffer substrates, while elastase release was not mechanosensitive. Taken together, these findings indicate a strong regulatory role of substrate stiffness during PMN effector responses. While extensive work has been done to elucidate PMN biochemical interactions, understanding important mechanosensitive mechanisms underlying phagocytosis, NETosis, respiratory burst, and cytokine release may provide further therapeutic targets to benefit patients in the future by reducing harmful PMN behaviors and/or promoting behaviors crucial to pathogen clearance.

Tspan2 is involved in anti-infectious immune responses and CNS autoimmunity

Abstract Tetraspanin proteins regulate critical cellular processes in CNS development and immune defence against pathogens. Through lateral association within membrane domains or binding of interaction partners these proteins are involved in pathogen recognition, proliferation and trafficking of immune cells, as well as cell differentiation and motility in the CNS. The tetraspanin family member Tspan2 was originally identified in oligodendrocyte lineage cells. It has been implicated in immunoregulation in the CNS as Tspan2−/− mice show increased microglia and astrocyte activation in the absence of inflammatory stimuli. A role of Tspan2 in CNS inflammation and anti-infectious immune responses remains to be elucidated. In newly generated Tspan2/DTR/EGFP reporter mice, we detected Tspan2 expression predominantly in mature oligodendrocytes in the CNS and an enhanced clinical severity of experimental autoimmune encephalomyelitis upon immunization with MOG35–55 peptide in Tspan2−/− mice. In peripheral lymphoid organs, Tspan2 expression was found in neutrophils and the majority of dendritic cells (DCs). Tspan2−/− DCs showed reduced uptake of zymosan as compared to wild-type (WT) DCs indicating that Tspan2 affects fungal recognition by DCs. In the absence of Tspan2 neutrophils exhibited a reduced accumulation of reactive oxygen species (ROS) and enhanced migration to inflammatory sites in thioglycollate-induced peritonitis as compared to WT neutrophils. LPS challenge in vivo induced increased production of the proinflammatory cytokines TNF, IL-1β, and IL-6 in the spleens of Tspan2−/− as compared to WT mice. These data provide first evidence that Tspan2 regulates anti-infectious immune responses as well as CNS autoimmunity.

In Vitro Analysis of Nanoparticle Effects on the Zymosan Uptake by Phagocytic Cells.

This chapter provides a protocol for analysis of nanoparticle effects on the function of phagocytic cells. The protocol relies on luminol chemiluminescence to detect zymosan uptake. Zymosan is an yeast particle which is typically eliminated by phagocytic cells via the complement receptor pathway. The luminol, co-internalized with zymosan, is processed inside the phagosome to generate a chemiluminescent signal. If a test nanoparticle affects the phagocytic function of the cell, the amount of phagocytosed zymosan and, proportionally, the level of generated chemiluminescent signal change. Comparing the zymosan uptake of untreated cells with that of cells exposed to a nanoparticle provides information about the nanoparticle's effects on the normal phagocytic function. This method has been described previously and is presented herein with several changes. The revised method includes details about nanoparticle concentration selection, updated experimental procedure, and examples of the method performance.

Multiple roles of filopodial dynamics in particle capture and phagocytosis and phenotypes of Cdc42 and Myo10 deletion

Macrophage filopodia, finger-like membrane protrusions, were first implicated in phagocytosis more than 100 years ago, but little is still known about the involvement of these actin-dependent structures in particle clearance. Using spinning disk confocal microscopy to image filopodial dynamics in mouse resident Lifeact-EGFP macrophages, we show that filopodia, or filopodia-like structures, support pathogen clearance by multiple means. Filopodia supported the phagocytic uptake of bacterial (Escherichia coli) particles by (i) capturing along the filopodial shaft and surfing toward the cell body, the most common mode of capture; (ii) capturing via the tip followed by retraction; (iii) combinations of surfing and retraction; or (iv) sweeping actions. In addition, filopodia supported the uptake of zymosan (Saccharomyces cerevisiae) particles by (i) providing fixation, (ii) capturing at the tip and filopodia-guided actin anterograde flow with phagocytic cup formation, and (iii) the rapid growth of new protrusions. To explore the role of filopodia-inducing Cdc42, we generated myeloid-restricted Cdc42 knock-out mice. Cdc42-deficient macrophages exhibited rapid phagocytic cup kinetics, but reduced particle clearance, which could be explained by the marked rounded-up morphology of these cells. Macrophages lacking Myo10, thought to act downstream of Cdc42, had normal morphology, motility, and phagocytic cup formation, but displayed markedly reduced filopodia formation. In conclusion, live-cell imaging revealed multiple mechanisms involving macrophage filopodia in particle capture and engulfment. Cdc42 is not critical for filopodia or phagocytic cup formation, but plays a key role in driving macrophage lamellipodial spreading.

Effects of Toll-like receptor ligands on RAW 264.7 macrophage morphology and zymosan phagocytosis.

In this study we compared the effects of the Toll-like receptor (TLR) ligands lipopolysaccharide (LPS), flagellin, the synthetic bacterial triacylated lipopeptide Pam3-Cys-Ser-Lys4 (Pam3CSK4), Polyinosinic:polycytidylic acid (Poly I:C), and macrophage-activating lipopeptide (MALP-2), which are TLR4, TLR5, TLR1/2, TLR3, and TLR2/6 agonists, respectively, on cell morphology and phagocytosis of zymosan particles, derived from Saccharomyces cerevisiae, and rich in fungal PAMPs including beta-glucan, mannose, and chitin. LPS, Pam3CSK4, and MALP-2 induced an activated macrophage phenotype and enhanced zymosan phagocytosis. In contrast, flagellin and Poly I:C, respectively, had little effect on cell morphology and phagocytosis. We examined the role of scavenger receptor A (SR-A) on zymosan phagocytosis. Cells cultured in medium alone expressed SR-A, and LPS induced further expression of the receptor. We also observed inhibitory effects of scavenger receptor antagonists fucoidan, dextran sulphate, and Polyinosinic (Poly I), respectively, on zymosan phagocytosis of cells in medium alone and those pre-treated with LPS. We conclude that exposure to specific TLR ligands impacts both cellular morphology and phagocytic capacity, and that scavenger receptors contribute to zymosan ingestion as well as LPS-induced augmentation of phagocytosis.

Activation-Inactivation Cycling of Rab35 and ARF6 Is Required for Phagocytosis of Zymosan in RAW264 Macrophages.

Phagocytosis of zymosan by phagocytes is a widely used model of microbial recognition by the innate immune system. Live-cell imaging showed that fluorescent protein-fused Rab35 accumulated in the membranes of phagocytic cups and then dissociated from the membranes of newly formed phagosomes. By our novel pull-down assay for Rab35 activity, we found that Rab35 is deactivated immediately after zymosan internalization into the cells. Phagosome formation was inhibited in cells expressing the GDP- or GTP-locked Rab35 mutant. Moreover, the simultaneous expression of ACAP2—a Rab35 effector protein—with GTP-locked Rab35 or the expression of plasma membrane-targeted ACAP2 showed a marked inhibitory effect on phagocytosis through ARF6 inactivation by the GAP activity of ACAP2. ARF6, a substrate for ACAP2, was also localized on the phagocytic cups and dissociated from the membranes of internalized phagosomes. In support of the microscopic observations, ARF6-GTP pull-down experiments showed that ARF6 is transiently activated during phagosome formation. Furthermore, the expression of GDP- or GTP-locked ARF6 mutants also suppresses the uptake of zymosan. These data suggest that the activation-inactivation cycles of Rab35 and ARF6 are required for the uptake of zymosan and that ACAP2 is an important component that links Rab35/ARF6 signaling during phagocytosis of zymosan.

Innate Immune Response of Human Alveolar Macrophages during Influenza A Infection

Alveolar macrophages (AM) are one of the key cell types for initiating inflammatory and immune responses to influenza virus in the lung. However, the genome-wide changes in response to influenza infection in AM have not been defined. We performed gene profiling of human AM in response to H1N1 influenza A virus PR/8 using Affymetrix HG-U133 Plus 2.0 chips and verified the changes at both mRNA and protein levels by real-time RT-PCR and ELISA. We confirmed the response with a contemporary H3N2 influenza virus A/New York/238/2005 (NY/238). To understand the local cellular response, we also evaluated the impact of paracrine factors on virus-induced chemokine and cytokine secretion. In addition, we investigated the changes in the expression of macrophage receptors and uptake of pathogens after PR/8 infection. Although macrophages fail to release a large amount of infectious virus, we observed a robust induction of type I and type III interferons and several cytokines and chemokines following influenza infection. CXCL9, 10, and 11 were the most highly induced chemokines by influenza infection. UV-inactivation abolished virus-induced cytokine and chemokine response, with the exception of CXCL10. The contemporary influenza virus NY/238 infection of AM induced a similar response as PR/8. Inhibition of TNF and/or IL-1β activity significantly decreased the secretion of the proinflammatory chemokines CCL5 and CXCL8 by over 50%. PR/8 infection also significantly decreased mRNA levels of macrophage receptors including C-type lectin domain family 7 member A (CLEC7A), macrophage scavenger receptor 1 (MSR1), and CD36, and reduced uptake of zymosan. In conclusion, influenza infection induced an extensive proinflammatory response in human AM. Targeting local components of innate immune response might provide a strategy for controlling influenza A infection-induced proinflammatory response in vivo.

Effect of conglutinin on phagocytic activity of bovine granulocytes

In the present study we investigated the effect of bovine conglutinin on the phagocytic activity of leukocytes. We measured both the chemotactic activity of conglutinin and its effect on the internalization of zymosan particles and E. coli by granulocytes. We also assessed the binding of conglutinin to various microorganisms isolated from clinical cases in cattle. We showed that conglutinin binds strongly to the surface of yeast cells and to mannan-rich zymosan particles, while weak binding was observed in the case of the bacterial strains tested, including those whose O antigen is composed of mannan. Conglutinin (1-10 microg/ml) neither acts as a chemotactic factor for peripheral blood leukocytes nor affects ingestion of E. coli by granulocytes. However, as flow cytometry based assay showed, conglutinin (0.1-1 microg/ml) increased ingestion of zymosan expressed as mean fluorescence intensity (MFI) of positive cells.

Scavenger receptors and β-glucan receptors participate in the recognition of yeasts by murine macrophages

ObjectivesNumerous receptors have been implicated in recognition of pathogenic fungi by macrophages, including the β-glucan receptor dectin-1. The role of scavenger receptors (SRs) in anti-fungal immunity is not well characterized.MethodsWe studied uptake of unopsonized Saccharomycetes cerevisiae (zymosan) and live Candida albicans yeasts as well as zymosan-stimulated H2O2 production in J774 macrophage-like cells and peritoneal exudate macrophages (PEMs). The role of different receptors was assessed with the use of competitive ligands, transfected cells and receptor-deficient macrophages.ResultsThe uptake of zymosan by untreated J774 cells was mediated approximately half by SRs and half by a β-glucan receptor which was distinct from dectin-1 and not linked to stimulation of H2O2 production. Ligands of β-glucan receptors and of SRs also inhibited uptake of C. albicans by macrophages (J774 cells and PEMs). In macrophages pretreated with a CpG motif-containing oligodeoxynucleotide (CpG-ODN) the relative contribution of SRs to yeast uptake increased and that of β-glucan receptors decreased. Whereas the class A SR MARCO participated in the uptake of both zymosan and C. albicans by CpG-ODN-pretreated, but not untreated macrophages, the related receptor SR-A/CD204 was involved in the uptake of zymosan, but not of C. albicans. The reduction of zymosan-stimulated H2O2 production observed in DS-pretreated J774 cells and in class A SRs-deficient PEMs suggest that class A SRs mediate part of this process.ConclusionsOur results revealed that SRs belong to a redundant system of receptors for yeasts. Binding of yeasts to different receptors in resting versus CpG-ODN-pre-exposed macrophages may differentially affect polarization of adaptive immune responses.

Laminarin, a soluble beta-glucan, inhibits macrophage phagocytosis of zymosan but has no effect on lipopolysaccharide mediated augmentation of phagocytosis

Phagocytosis is a fundamental aspect of innate resistance against microbes, including fungi. In this study we investigated the significance of beta-glucan on the surfaces of zymosan particles, derived from Saccharomyces cerevisiae, during phagocytosis by RAW 264.7 macrophages. Phagocytosis was assessed in vitro by macrophage exposure to zymosan particles followed by cell staining and light microscopy. Macrophage ingestion of zymosan was dependent on cellular recognition of the particles' beta-glucans since laminarin, a soluble beta-glucan, inhibited phagocytosis in a concentration dependent manner when added to cell cultures. In contrast, the presence of another carbohydrate, mannan, had no effect on zymosan phagocytosis by cells. In addition we showed that LPS and dexamethasone had opposing effects on phagocytosis of zymosan. LPS significantly augmented ingestion while in contrast dexamethasone, like laminarin, suppressed it. The LPS-enhanced ingestion of zymosan was insensitive to the presence of laminarin in cell cultures, however dexamethasone partially ameliorated the effects of LPS on phagocytosis. Our findings confirm beta-glucan as an important ligand identified by macrophages and required for zymosan phagocytosis in naïve cells, but not in cells previously exposed to LPS.

Recombinant form of human wild type mannan-binding lectin (MBL/A) but not its structural variant (MBL/C) promotes phagocytosis of zymosan by activating complement

Mannan-binding lectin (MBL) mediates innate immune responses, such as activation of the complement lectin pathway and phagocytosis, to help fight infections. In the present study, employing recombinant forms of human MBL (rMBL), the role of wild type MBL (rMBL/A) and its structural variant rMBL/C in mediating THP-1 phagocytosis of fluorescent-labeled zymosan was examined and compared to MBL purified from human plasma (pMBL/A). Flow cytometric analyses revealed that opsonization of zymosan with rMBL/A and pMBL/A (0.5–30 μg/ml) resulted in a 1.9- and 2.7-fold enhancement in its uptake by THP-1 cells in the presence of serum that was depleted of both MBL and the classical pathway component, C1q (MBL/C1q Dpl serum). In contrast, no enhancement in phagocytosis was observed when zymosan was opsonized with rMBL/C. Addition of MBL monoclonal antibody, EDTA, or mannan to the opsonization reaction mixture inhibited THP-1 phagocytosis of pMBL/A opsonized zymosan. Heat inactivation of MBL/C1q Dpl serum abolished the 2-fold increase in phagocytosis and in the absence of serum the direct opsonic activity of MBL did not contribute significantly to the uptake of zymosan into THP-1 cells. Activation products of complement components C3 and C4 were deposited on zymosan opsonized with pMBL/A and rMBL/A but not rMBL/C indicating that MBL-mediated phagocytosis of zymosan requires activation of the complement lectin pathway. The findings imply that impaired MBL-mediated phagocytosis may put individuals homozygous for the mutant allele MBL/C but not wild type MBL/A at increased risk to infections such as yeast.

Abstract 5571: Induction of Macrophage Activation and Foam Cell Formation by High Glucose is Mediated by Rho-associated Coiled-coil Forming Kinase

Background: Vascular complications are major causes of morbidity and mortality in patients with diabetes mellitus. Although the precise mechanism by which diabetes leads to vascular disease remains unknown, hyperglycemia has been shown to accelerate atherogenesis, a process involving the uptake of modified low-density lipoproteins (LDL) by macrophages and formation of macrophage-derived foam cells. We have recently shown that deletion of Rho-associated coiled-coil forming kinase (ROCK)-1 in macrophages leads to decreased LDL uptake, foam cell formation, and atherosclerosis. Therefore, we hypothesize that high glucose may enhance atherogenesis by inducing uptake of LDL by macrophages and increasing foam cell formation through activation of ROCK. Methods & Results: To investigate the effects of high glucose on ROCK and macrophage function, we isolated bone marrow-derived macrophage (BMDM). Treatment with high glucose but not mannitol increased ROCK activity in BMDM in a dose-dependent manner, with initial increase observed after 8 hrs. Downstream metabolites of glucose such as sorbitol and L-glutamine, as well as intracellular oxidative stress and protein kinase C all induced ROCK activation in BMDM. Indeed, co-treatment with the ROCK inhibitor, fasudil (30 uM), decreased glucose-induced oxidative stress. High glucose increased BMDM adhesion and migration, which was inhibited by co-treatment with fasudil. Proliferation and phagocytic activity of BMDM as determined by [ 3 H]-tritium incorporation and zymosan uptake, respectively, were increased in high glucose culture medium, but these effects of high glucose were not affected by fasudil. High glucose (4.5 g/L) increased the size of BMDM compared to low glucose (1.0 g/L), and led to more foam cell formation in the presence of 50 ug/ml of cholesterol. In addition, the expression of TNFalpha in BMDM was increased by 2.7-fold in high glucose medium, which was suppressed by treatment with fasudil. Conclusion: These findings indicate that high glucose induces macrophage activation and foam cell formation is mediated by increased ROCK activity. These results suggest that targeting ROCK may have therapeutic benefits in ameliorating atherosclerosis in diabetic patients.

Scavenger Receptor Collectin Placenta 1 (CL-P1) Predominantly Mediates Zymosan Phagocytosis by Human Vascular Endothelial Cells

Collectin placenta 1 (CL-P1), a recently discovered scavenger receptor, mediates the uptake of oxidized low density lipoprotein and microbes. In this study, we investigated CL-P1-mediated binding and ingestion of yeast-derived zymosan bioparticles using Chinese hamster ovary (CHO) cells stably expressing human CL-P1 (CHO/CL-P1) and human vascular endothelial cells constitutively expressed CL-P1. The uptake of zymosan by CHO/CL-P1 was dependent upon the level of CL-P1 expressed on the membrane and was inhibited by cytochalasin D and wortmannin. The binding of zymosan was also inhibited by ligands of other scavenger receptors such as poly(I) and dextran sulfate. Real time reverse transcription-PCR analyses showed that other scavenger receptors, namely LOX-1, Stabilin-2, or macrophage receptor with collagenous structure (MARCO), were not expressed in human umbilical vein endothelial cells isolated from different individuals. Nonopsonic zymosan ingestion was inhibited in three primary cultured vascular endothelial cells, including different human umbilical vein endothelial cells from nine individuals treated with CL-P1 small interfering RNAs, although small interfering RNAs of other scavenger receptors had no effect on zymosan uptake in these cells. Furthermore, we confirmed that CL-P1 is expressed in human and murine vascular endothelial layers. Our results demonstrated that CL-P1 predominantly mediated phagocytosis for fungi in vascular endothelia.

Cystic Fibrosis Transmembrane Conductance Regulator-independent Phagosomal Acidification in Macrophages

It was reported recently that the cystic fibrosis transmembrane conductance regulator (CFTR) is required for acidification of phagosomes in alveolar macrophages (Di, A., Brown, M. E., Deriy, L. V., Li, C., Szeto, F. L., Chen, Y., Huang, P., Tong, J., Naren, A. P., Bindokas, V., Palfrey, H. C., and Nelson, D. J. (2006) Nat. Cell Biol. 8, 933-944). Here we determined whether the CFTR chloride channel is a generalized pathway for chloride entry into phagosomes in macrophages and whether mutations in CFTR could contribute to alveolar macrophage dysfunction. The pH of mature phagolysosomes in macrophages was measured by fluorescence ratio imaging using a zymosan conjugate containing Oregon Green(R) 488 and tetramethylrhodamine. Acidification of phagolysosomes in J774A.1 macrophages (pH approximately 5.1 at 45 min), murine alveolar macrophages (pH approximately 5.3), and human alveolar macrophages (pH approximately 5.3) was insensitive to CFTR inhibition by the thiazolidinone CFTR(inh)-172. Acidification of phagolysosomes in alveolar macrophages isolated from mice homozygous for DeltaF508-CFTR, the most common mutation in cystic fibrosis, was not different compared with that in alveolar macrophages isolated from wild-type mice. We also measured the kinetics of phagosomal acidification in J774A.1 and murine alveolar macrophages using a zymosan conjugate containing fluorescein and tetramethylrhodamine. Phagosomal acidification began within 3 min of zymosan binding and was complete within approximately 15 min of internalization. The rate of phagosomal acidification in J774A.1 cells was not slowed by CFTR(inh)-172 and was not different in alveolar macrophages from wild-type versus DeltaF508-CFTR mice. Our data indicate that phagolysosomal acidification in macrophages is not dependent on CFTR channel activity and do not support a proposed mechanism for cystic fibrosis lung disease involving defective phagosomal acidification and bacterial killing in alveolar macrophages.

Impaired phagocytic mechanism in annexin 1 null macrophages.

The role of the anti-inflammatory protein annexin-A1 (Anx-A1) in the phagocytic process has been investigated using a murine bone marrow culture-derived macrophage model from Anx-A1(+/+) and Anx-A1(-/-) mice. Macrophages prepared from Anx-A1(-/-) mice exhibited a reduced ingestion of zymosan, Neisseria meningitidis or sheep red blood cells, when compared to Anx-A1(+/+) cells and in the case of zymosan this effect was also mirrored by a reduced clearance in vivo when particles were injected into the peritoneal cavity of Anx-A1(-/-) mice. The ablation of the Anx-A1 gene did not cause any apparent cytoskeletal defects associated with particle ingestion but the cell surface expression of the key adhesion molecule CD11b was depressed in the Anx-A1(-/-) cells providing a possible explanation for the attenuated phagocytic potential of these cells. The production of the cytokines TNFalpha and IL-6 was increased in Anx-A1(-/-) macrophages following phagocytosis of all types of particle.

Group V Secretory Phospholipase A2 Translocates to the Phagosome after Zymosan Stimulation of Mouse Peritoneal Macrophages and Regulates Phagocytosis

We have previously reported that group V secretory phospholipase A2 (sPLA2) amplifies the action of cytosolic phospholipase A2(cPLA2) alpha in regulating eicosanoid biosynthesis by mouse peritoneal macrophages stimulated with zymosan (Satake, Y., Diaz, B. L., Balestrieri, B., Lam, B. K., Kanaoka, Y., Grusby, M. J., and Arm, J. P. (2004) J. Biol. Chem. 279, 16488-16494). To further understand the role of group V sPLA2, we studied its localization in resting mouse peritoneal macrophages before and after stimulation with zymosan and the effect of deletion of the gene encoding group V sPLA2 on phagocytosis of zymosan. We report that group V sPLA2 is present in the Golgi apparatus and recycling endosome in the juxtanuclear region of resting peritoneal macrophages. Upon ingestion of zymosan by mouse peritoneal macrophages, group V sPLA2 is recruited to the phagosome. There it co-localizes with cPLA2alpha, 5-lipoxygenase, 5-lipoxygenase-activating protein, and leukotriene C4 synthase. Using immunostaining for the cysteinyl leukotrienes in carbodiimide-fixed cells, we show, for the first time, that the phagosome is a site of cysteinyl leukotriene formation. Furthermore, peritoneal macrophages from group V sPLA2-null mice demonstrated a >50% attenuation in phagocytosis of zymosan particles, which was restored by adenoviral expression of group V sPLA2 but IIA not group sPLA2. These data demonstrate that group V sPLA2 contributes to the innate immune response both through regulation of eicosanoid generation in response to a phagocytic stimulus and also as a component of the phagocytic machinery.

TLR2 modulates inflammation in zymosan-induced arthritis in mice

The interplay between the innate and acquired immune systems in chronic inflammation is not well documented. We have investigated the mechanisms of inflammation in murine zymosan-induced arthritis (ZIA) in the light of recent data on the roles of Toll-like receptor 2 (TLR2) and Dectin-1 in the activation of monocyte/macrophages by zymosan. The severity of inflammation, joint histology, lymphocyte proliferation and antibody production in response to zymosan were analyzed in mice deficient in TLR2 and complement C3, and the effects of Dectin-1 inhibition by laminarin were studied. In comparison with wild-type animals, TLR2-deficient mice showed a significant decrease in the early (day 1) and late phases (day 24) of joint inflammation. C3-deficient mice showed no differences in technetium uptake or histological scoring. TLR2-deficient mice also showed a significant decrease in lymph node cell proliferation in response to zymosan and a lower IgG antibody response to zymosan at day 25 in comparison with wild-type controls, indicating that TLR2 signalling has a role in the development of acquired immune responses to zymosan. Although laminarin, a soluble β-glucan, was able to significantly inhibit zymosan uptake by macrophages in vitro, it had no effect on ZIA in vivo. These results show that ZIA is more prolonged than was originally described and involves both the innate and acquired immune pathways. C3 does not seem to have a major role in this model of joint inflammation.

Serotonin upregulates the activity of phagocytosis through 5-HT1A receptors.

1 In this study, we investigated whether serotonin could regulate the in vitro activity of phagocytosis through 5-hydroxytryptamine or serotonin (5-HT(1A)) receptors. 2 Mouse peritoneal macrophages were cultured with serotonin and the activity of phagocytosis was assessed by the uptake of zymosan and latex particles added to the culture media. Specific binding of [(3)H]8-OH-DPAT and immunohistochemistry using an affinity-purified anti-5-HT(1A)-receptor antibody were assayed in the macrophages. In addition, we took advantage of the availability of pharmacological inhibitors of nuclear factor-kappaB (NF-kappaB) to explore its role in the regulation of the 5-HT(1A) receptor. 3 Serotonin increased the in vitro activity of phagocytosis in a dose-dependent manner. The 5-HT(1A) receptor agonist (+/-)-8-hydroxy-2-(di-n-propyl-amino)-tetralin (R(+)-8-OH-DPAT) reproduced these effects. Serotonin- or R(+)-8-OH-DPAT-induced increases in phagocytosis were blocked by the 5-HT(1A) receptor antagonist WAY100635 and the NF-kappaB inhibitor pyrrolidinedithiocarbamate. Moreover, mouse peritoneal macrophages expressed specific binding sites for [(3)H]8-OH-DPAT when cultivated in the presence of zymosan or latex beads. Immunohistochemistry confirmed the expression of the 5-HT(1A) receptor protein in the macrophages. 4 These results show that serotonin can upregulate the activity of peritoneal macrophages through 5-HT(1A) receptors.

HUVEC take up opsonized zymosan particles and secrete cytokines IL-6 and IL-8 in vitro

Uptake of zymosan A particles by human umbilical vein endothelial cells (HUVEC) and its effect on cellular cytokine and oxygen radical production was examined. HUVEC took up more serum-opsonized than -unopsonized zymosan as demonstrated by flow cytometry with fluorescence-labeled particles. The former uptake was inhibited in the presence of anti-C3c antibodies and thus complement-mediated. It probably occurred via CR1 (CD35), although participation of other receptors cannot be ruled out. Scanning electron microscopy indicated that HUVEC with fully internalized zymosan particles were damaged. Prolonged incubation of both serum-opsonized and -unopsonized zymosan particles with HUVEC induced increased secretion of the proinflammatory cytokines IL-6 and IL-8 to the cell culture supernatants, but had no effect on production of oxygen radicals. The results confirm previous reports that EC can internalize yeast and other pathogens and points to complement as a mechanism of uptake, but illustrates that the cells may be damaged in the process. Moreover, EC may participate in the anti-infection defense effort by secreting proinflammatory and chemotactic cytokines in response to the contact with pathogens.

The phagocytosis by polymorphonuclear neutrophils in patients with systemic lupus erythematosus

To evaluate the presence of immune complexes and the phagocytes by polymorphonuclear neutrophils in patients with systemic lupus erythematosus, with and without disease activity. The peripheral blood of 55 subjects was analyzed. Ten of those subjects had disease activity, 15 had not disease activity, and 30 were healthy. We used radial immune diffusion to detect immune complexes. The phagocytic function was estimated by the ingestion of zymosan by polymorphonuclear neutrophils. In this study we found the presence of immune complexes formatted of IgM, IgG, IgA, and complement component C3 and C4 in LES patients. The arithmetic average of zymosan particles ingested by the neutrophils incubated with homologous human serum and autologous human serum was significantly decreased (p<0.05) in the LES activity patients when we compare with the group without activity, and the control group. We conclude that there are immune complexes in the LES patients with and without disease activity, and there is a reduction in the digestive step of the phagocytes by polymorphonuclear neutrophils in patients with disease activity. The conclusions of the present study are according with the pathogenesis of the disease and with the high mortality in these patients.