Mouse Peritoneal Exudate Research Articles

Brugia malayi cystatin therapeutically ameliorates dextran sulfate sodium-induced colitis in mice.

Helminth immunomodulation in the host has been shown to have therapeutic implications in inflammatory bowel diseases. In this study we aimed to evaluate the therapeutic effect of Brugia malayi recombinant cystatin (rBmCys) in a dose-dependent manner on dextran sulfate sodium (DSS)-induced colitis in mice. The anti-inflammatory activity of rBmCys on mice peritoneal exudate cells was initially analyzed in vitro. BALB/c mice were fed with 5% DSS for 7 days to induce colitis. The colitis mice were treated intraperitoneally with rBmCys (10, 25 or 50 µg for the three different groups of mice) on days 1, 3 and 5 of the DSS administration. Disease severity was assessed by the disease activity index (DAI) and macroscopic and histopathological scores of colon and myeloperoxidase activity in colonic mucosa. Cytokine profiles were measured in sera and cultured splenocytes of treated mice followed by stimulation with rBmCys. rBmCys showed anti-inflammatory activity in vitro. Treatment of DSS-induced colitis with rBmCys in mice ameliorated the overall disease severity as reflected by a significant reduction in weight loss, the DAI, mucosal edema, colon damage and myeloperoxidase activity of the colonic mucosa. While the mRNA expressions of IFN-γ, TNF-α, interleukin (IL)-5, IL-6 and IL-17 were downregulated, IL-10 expression was upregulated in the splenocytes of colitis mice treated with rBmCys. The amelioration of DSS-induced colitis occurred in a dose-dependent manner. The results of this study indicate an anti-inflammatory potential of rBmCys and provide evidence for using this protein as a promising therapeutic agent in ulcerative colitis.

The alum-processing mechanism attenuating toxicity of Araceae Pinellia ternata and Pinellia pedatisecta.

The present study aimed at investigating the alum-processing mechanism attenuating toxicity of Araceae Pinellia ternata and Pinellia pedatisecta. Animal retroperitoneal inflammatory model in vivo and macrophagocyte release inflammatory factor model in vitro were used to detect the effect of alum processing on raphides and lectin. Scanning electron microscopy was used to observe the change in raphides during processing; HPLC method was used to determine the correlation between the dissolution and corrosion of raphides and ion in the alum solution; (27)Al-NMR technology was used to detect the relationship between aluminum oxalate complex formation and the dissolved and corrosion of raphides. The change in protein peptide sequence of lectin during the processing of alum solution was determined by Shotgun LC-MS assay. Raphides induced severe rabbit conjunctival edema and an intraperitoneal injection of lectin increased PGE2 and protein in mice peritoneal exudate, while decreased after treatment with alum solution processing. During the processing raphides was dissolved and corroded, then its structure was damaged. Raphides was soaked in the alum solution and significantly decreased the oxalate content, and the effect was related with Al(3+) in the alum. Al(3+) in the alum combined with C2O4(2-) of raphides into a stable complex compound promoted the dissolution of calcium oxalate. Raphides soaked in the alum made lectin proteins dissolve, whereas protein peptide sequence of lectin was changed and the protein structure was damaged. Alum solution could decrease the toxicity of P. ternata (Thunb.) Breit. and P. pedatisecta Schott. Since it made a special crystal structure of raphides damage and the protein of lectin dissolve. The structure of toxic substances significantly changed, which decreased the inflammatory effect.

Resistance to P. brasiliensis Experimental Infection of Inbred Mice Is Associated with an Efficient Neutrophil Mobilization and Activation by Mediators of Inflammation.

Paracoccidioidomycosis (PCM) is a systemic fungal infection, endemic in Brazil, that leads to severe morbidity and even mortality if not correctly treated. Patients may respond differently to PCM depending on the pattern of the acquired immune response developed. The onset of protective immune response is notably mediated by neutrophils (PMN) that play an important role through directly killing the fungi and also by interacting with other cell types to modulate the acquired protective immune response that may follow. In that way, this study aimed to present and compare different experimental models of PCM (intraperitoneal and subcutaneous) regarding PMN production and maturation inside femoral bone marrow and also PMN infiltration in peritoneal and subcutaneous exudates of resistant and susceptible mice. We also assessed the fungal colony forming units and the levels of soluble inflammatory mediators (LTB4, KC, IFN-γ, GM-CSF, and IL-10) inside subcutaneous air-pouches to compare the efficiency of the PMN present at this site in relation to the two main neutrophil functions: initial lysis of the invading pathogen and modulation of the acquired immune response. P. brasiliensis inoculated intraperitoneally was able to disseminate to the bone marrow of susceptible mice, causing a more marked alteration of PMN production and maturation than that observed after resistant mice infection by the same route. Subcutaneous air-pouch inoculation of P. brasiliensis elicited a controlled and limited infection that produced a PMN-rich exudate, thus favoring the study of the interaction between the fungus and the neutrophils. Susceptible mice produced higher numbers of PMN; however, these cells were less effective in killing the fungi. Inflammatory cytokines were more pronounced in resistant mice, which supports their PCM raised resistance.

Development of Immunonutrient from Pumpkin (Cucurbita Moschata Duchense Ex. Lamk.) Seed

Pumpkin seeds has long been used as a source of nutrition. Based on its content, it is possible that part of this plant used as immunonutrient. The purpose of this research is to study the immunomodulatory activity of pumpkin seeds. It effects on the non-specific immune response was determined through carbon clearance test, organ indexes (liver, spleen and thymic gland) and mice peritoneal exudate activity (PEA). The effects on specific immune response were determined through total antibody titre, delayed type of hypersensitivity (DTH) reaction and number of lymphocytes. On non specific immune response test, pumpkin seed at a low dose (PLD-3.8g/kg bw) and at a high dose (PHD-7.6g/kg bw) showed immunostimulanting effect with phagocytic index of 1.219 and 1.347, respectively. PHD increased PEA activity (p<0.01) in lysing microbes. On specific immune response test, PLD and PHD showed immunostimulation effect indicated through 50% haemaglutination activity (HA titre) i.e., 1:512 and 1:2048, respectively compared to than that of control (1:256) for secondary antibody titre. Through cellular immunity test, 48hours after challenged, PHD showed significant (p<0.05) increased in footpad thickness. After 7 days administration, both doses of pumpkin increased lymphocytes number significantly (p<0.05). Based on the data it can be concluded that, pumpkin seeds have immunomodulatory effects, and can therefore be used as immunonutrient

Comparative study on pro-inflammatory toxicity of Pinellia pedatiecta before and after being processed with alum

To explore the pro-inflammatory toxicity of Pinellia pedatiecta, as well as the alum processing method on its pro-inflammatory effect. Raphide and agglutinin (PPA) proteins were isolated from fresh P. pedatiecta. The overall animal and cellular level models were applied to investigate the pro-inflammatory effect of raphide and PPA in P. pedatiecta, as well as the impact of the alum processing method on the pro-inflammatory effect, with inflammatory mediators as the index. Intraperitoneal injection with P. pedatiecta raphide suspension could significantly increase the content of inflammatory mediators PGE2 and NO. After the alum processing method was adopted, fresh P. pedatiecta and raphide-induced PGE2 and NO release significantly reduced. The stimulation of mice macrophages with P. pedatiecta agglutinin protein could cause the content of dose-dependent inflammatory mediators TNF-alpha and IL-6. After the alum processing method was adopted, PGE2 content in P. pedatiecta agglutinin protein-induced mice peritoneal exudate notably decreased. The irritation and toxicity of P. pedatiecta were inflammatory responses in organisms. Its raphide and agglutinin proteins were toxic components, both could cause significant the release of inflammatory medium. The alum processing method could help significantly reduce the pro-inflammatory toxicity of P. pedatiecta.

Abstract 328: Viral Anti-Atherogenic Proteins Alter Monocyte and Neutrophil Invasion in Mice and Bag3 Gene Expression

Aim Atherosclerosis is characterized by chronic inflammation and cell death (apoptosis). Serine protease inhibitors, or serpins, regulate inflammatory, thrombotic and apoptotic pathways. Poxviruses encode cross-class serpins that prevent host cell apoptosis. Serp-2, from Myxoma, reduces plaque, inflammation and apoptosis in animal models; CrmA, from Vaccinia, does not. Both serpins target Caspase 1 and Granzyme B, but the reasons for the differing effects in vivo are unknown. In prior research three Myxoma viral proteins, including Serp-2, reduced monocyte invasion, plaque growth, and aneurysm formation in ApoE-/- mice after angioplasty. These same proteins alter expression of a shared cohort of 48 apoptosis-related genes in human monocytes treated with camptothecin. This study assesses the effects of Serp-2 and CrmA on apoptotic gene expression in a mouse peritoneal inflammation model. Methods Mouse strains deficient for Granzyme B (GzmB, N=13) and Caspase 1 (Casp1, N=15) were compared to their respective background mice (C57Bl/6 and Nod, N=15 each) 18 hours after treatment with PMA and either Serp-2 or CrmA. RNA was isolated and analyzed by RT-PCR and normalized to GAPDH, then to the PMA-only treated control. Results Compared to human monocytes, mouse peritoneal exudates from knockout mice displayed differential alteration of BCL2-associated athanogene 3 (BAG3) in response to treatment with Serp-2 or CrmA. Serp-2 treatment reduced expression levels compared to CrmA treatment in GzmB (p=0.0267) and C57Bl/6 mice (p=0.0280). Casp1-/- mice treated with Serp-2 downregulate BAG3, expressing 9.7-fold less than Nod mice (p=0.0006), but CrmA had no significant effect in either strain. An associated differential migration of Ly6Chi and Ly6Ghi cells was also discovered in knockout mice with serpin treatments. Discussion One candidate gene found in human monocytes, BAG3, has reduced gene expression after Serp-2 treatment in mouse peritoneal exudates but increased by CrmA. BAG3 is known to alter cell migration and apoptosis, important to atherosclerotic progression. BAG3 is regulated by 3 myxomaviral proteins in human and mouse cells, underscoring a potential role as a lynchpin in viral protein anti-inflammatory and anti-apoptotic pathways.

Annexin A5 inhibits atherogenic and pro-inflammatory effects of lysophosphatidylcholine

ObjectiveAtherosclerosis is an inflammatory condition, and rupture of atherosclerotic plaques is a major cause of cardiovascular disease (CVD). Lysophosphatidylcholine (LPC) is generated in low-density lipoprotein (LDL) during oxidation and/or enzymatic modification and has been implicated in atherosclerosis. Annexin A5 (ANXA5) is an antithrombotic and atheroprotective plasma protein. Here, we demonstrate novel pro-inflammatory and atherogenic properties of LPC, and inhibitory effects of ANXA5. MethodsEndothelial cells and macrophages (differentiated from, THP-1 a monocytic cell line) were co-cultured. Expression of MMP-9 and OxLDL uptake by macrophages were studied by flow cytometry. The effect of LPC on leukotriene B4 (LTB4) synthesis in macrophages was studied by enzyme immunoassay (EIA). Chemotactic properties of LPC were investigated using a mouse intra-peritoneal recruitment model. ResultsCo-culture of macrophages and endothelial cells enhanced MMP-9 expression in both cell types. This effect was increased by LPC and diminished by ANXA5. Likewise, LPC induced LTB4 production by macrophages, whereas native LDL or phosphatidylcholine (PTC) had no effect. ANXA5 inhibited uptake of OxLDL in macrophages. LPC induced cell infiltration in vivo, as determined by increased cell count in mouse peritoneal exudates, and this effect was inhibited by ANXA5. ConclusionsANXA5 could potentially play an important protective role in both atherogenesis and atherosclerotic plaque rupture by reducing pro-inflammatory effects of OxLDL and LPC as well as inhibiting OxLDL binding and uptake by macrophages. The possibility that ANXA5 could be developed into a novel therapy against CVD deserves further study.

Changes in Activity of Free Radical Oxidation Processes in the Early Stages of BCG Granulomatosis

We studied the intensity of free radical oxidation in the liver and activity of oxidative metabolism in mouse peritoneal exudate phagocytes at the early stages of chronic generalized BCG-induced granulomatosis (days 3 and 30 after a single intraperitoneal or intravenous administration of 0.5 mg of BCG vaccine). It was found that both methods of injection did not change the intensity of free radical lipid peroxidation in the liver in comparison with the control, but activity of free radical oxidation mediated by production of hydrogen peroxide was increased in the liver and peritoneal exudate at the stages of mature granuloma formation (day 30). At the same time, intraperitoneal injection contributed to more pronounced activation of lipid peroxidation and synthesis of hydrogen peroxide in the liver.

γ-Ray-associated XBP-1+ macrophages as an inducer of M2 macrophages (117.3)

Abstract The susceptibility of mice to infections with opportunistic pathogens is strongly influenced by M2MΦ appeared in association with whole body γ-irradiation. Therefore, in this study, we investigated why M2MΦ were predominantly generated in mice exposed to whole body γ-irradiation. F4/80+ MΦ were isolated from peritoneal exudates of mice 6-24 h after 5 Gy of γ-irradiation. The expressions of XBP-1 in these MΦ preparations were detected by Western Blot. The production and intracellular CCL2 expression by XBP-1+ MΦ were assayed by ELISA and flow cytometry. In some experiments, resident MΦ were cultured with media supplemented with culture fluids of XBP-1+ MΦ (33%, v/v). MΦ with the ability to produce IL-10 and not produce IL-12 were considered as M2MΦ. In the results, XBP-1+ MΦ were demonstrated in peritoneal MΦ preparations isolated from mice irradiated with 5 Gy of γ-rays. CCL2 was detected in culture fluids of XBP-1+ MΦ. Also, intracellular CCL2 was detected in XBP-1+ MΦ by flow cytometry. The conversion of MΦ from resident MΦ to M2MΦ was demonstrated when resident MΦ were cultured with media supplemented with the culture fluids of XBP-1+ MΦ. Resident MΦ did not change to M2MΦ when they were cultured with XBP-1+ MΦ culture fluids previously depleted of CCL2. These results indicate that the MΦ conversion from resident MΦ to M2MΦ is stimulated by CCL2 released from XBP-1+ MΦ that appear in association with whole body γ-irradiation.

Up-regulation of IL-33 expression in various types of murine cells by IL-3 and IL-4

The crucial roles of the novel cytokine IL-33 in allergic, inflammatory, infectious and autoimmune diseases are becoming characterized. However, the cytokines which regulate IL-33 expression and secretion are still largely unknown. In this study, IL-3 and IL-4 were found to up-regulate IL-33 mRNA expression in mouse peritoneal exudate cells by a two-color DNA microarray and further confirmed by real time PCR and ELISA. IL-3 and IL-4 synergistically promote IL-33 mRNA expression and IL-33 intracrine in the heterogeneous cell populations as peritoneal exudates cells, bone marrow cells and splenic cells. IL-3 and IL-4 also induced IL-33 introcrine in the peritoneal exudate cells from the macrophage-deficient op/op mice, suggesting that macrophage is not the only target of IL-3 and IL-4 in the heterogeneous peritoneal exudate cells. Furthermore, IL-3 and IL-4 were verified to promote the IL-33 intracrine in the homogeneous cell population as fibroblasts and mast cells. These results indicate that up-regulation of IL-33 expression by IL-3 and IL-4 is not a feature particular to a specific type of cells. Up to 100 cytokines were screened, but none of them stimulated the secretion or release of IL-33 in the culture system. In summary, we confirm for the first time that IL-3 and IL-4 are critical for IL-33 intracrine in murine cells of various types, indicating that IL-3 and IL-4 may play an important role in the constitutive expression of IL-33 in vivo.

Human beta-defensin 2 and 3 and their mouse orthologs induce chemotaxis through interaction with CCR2.

Beta-defensins play a dual role during immune response. Their direct antimicrobial properties contribute to the local innate immune response by combating microbial invasions. Furthermore, previous studies revealed the capacity of certain beta-defensin family members to chemoattract immature dendritic cells and CD45RO+ CD4+ T cells through chemokine receptor CCR6. However, because beta-defensins also chemoattract macrophages and monocytes, which do not express CCR6, efforts have been made to identify other receptors for these polypeptides. In this study, we demonstrate the capacity of human beta-defensin (hBD)2 and 3 and their mouse orthologs, beta-defensin 4 and 14, to interact with CCR2, a chemokine receptor expressed on monocytes, macrophages, and neutrophils. These beta-defensins, fused to the Fc region of human IgG1, showed binding to CCR2-transfected HEK293 cells, as revealed by flow cytometry. The beta-defensin fusion proteins also induced CCR2-specific chemotaxis of transfected HEK293 cells, human peripheral blood monocytes, and mouse peritoneal exudate cells in a dose-dependent manner. Preincubation of human monocytes with CCL2/MCP-1, the chemokine ligand for CCR2, abolished migration induced by beta-defensins. Conversely, preincubation with hBD2:Ig or hBD3:Ig inhibited MCP-1 induced migration. Peritoneal exudate cells from CCR2-deficient mice failed to migrate toward these fusion proteins. In conclusion, the beta-defensins used in this study contribute to the innate and adaptive immune response in their role as chemoattractants. Our data indicate that hBD2 and hBD3, together with their mouse orthologs (beta-defensin 4 and 14), are chemotactic for a broad spectrum of leukocytes in a CCR6- and CCR2-dependent manner.

Epigallocatechin gallate inhibits intracellular survival of Listeria monocytogenes in macrophages

Epigallocatechin gallate (EGCg), the major tea catechin, is known as a potent anti-microbial and anti-tumor compound. The effects of EGCg on host defense mechanisms against Listeria monocytogenes infection were examined in vitro using mouse peritoneal exudate cells. The study showed that EGCg inhibited the intracellular growth of L. monocytogenes in macrophages. The enhancement of in vitro anti- L. monocytogenes activity by EGCg is not due to the modulation of reactive oxygen intermediates or the production of reactive nitrogen intermediates but due to the inhibition of its escaping from the phagosome into cytosolic space. Anti- L. monocytogenes of EGCg is through the inhibition of hemolytic and cholesterol-binding activity of listeriolysin O, which usually disrupts the phagosomal membrane in the escaping phase of L. monocytogenes.

IL-17B and IL-17C Are Associated with TNF-α Production and Contribute to the Exacerbation of Inflammatory Arthritis

IL-17A is a T cell-derived proinflammatory cytokine that contributes to the pathogenesis of rheumatoid arthritis. Recently, six related molecules have been identified to form the IL-17 family, as follows: IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F. Whereas IL-17A and IL-17F up-regulate IL-6 in synovial fibroblasts, IL-17B and IL-17C are reported to stimulate the release of TNF-alpha and IL-1beta from the monocytic cell line, THP-1 cell. However, their detailed function remains to be elucidated. We report in this study the effects of IL-17 family on the collagen-induced arthritis (CIA) progression by T cell gene transfer and bone marrow chimeric mice. The mRNA expressions of IL-17 family (IL-17A, IL-17B, IL-17C, and IL-17F) and their receptor (IL-17R and IL-17Rh1) genes in the arthritic paws of CIA mice were elevated compared with controls. Although IL-17A and IL-17F were expressed in CD4(+) T cells, IL-17B and IL-17C were expressed in the cartilage and in various cell populations in the CIA arthritic paws, respectively. In vitro, IL-17A, IL-17B, IL-17C, and IL-17F induced TNF-alpha production in mouse peritoneal exudate cells. In vivo, adoptive transfer of IL-17B- and IL-17C-transduced CD4(+) T cells evidently exacerbated arthritis. Bone marrow chimeric mice of IL-17B and IL-17C exhibited elevated serum TNF-alpha concentration and the high arthritis score upon CIA induction. Moreover, neutralization of IL-17B significantly suppressed the progression of arthritis and bone destruction in CIA mice. Therefore, not only IL-17A, but also IL-17B and IL-17C play an important role in the pathogenesis of inflammatory arthritis.

Immunomodulatory Effects of theLutzomyia longipalpisSalivary Gland Protein Maxadilan on Mouse Macrophages

Infection with Leishmania major is enhanced when the sand fly Lutzomyia longipalpis salivary peptide maxadilan (MAX) is injected along with the parasite. Here we determined the effect that MAX has on the secretion of cytokines and nitric oxide (NO) and on parasite survival in macrophages (MPhis). The cytokines produced by MPhis can enhance a type 1 response, which will increase NO and the killing of intracellular pathogens such as L. major, or a type 2 response, leading to antibody production that is ineffective against intracellular pathogens such as L. major. A mouse macrophage cell line (RAW 264.7) was stimulated with various concentrations of MAX and lipopolysaccharide (LPS), and the supernatants were collected after 1, 2, and 3 days. Supernatants were assayed for interleukin-12p70 (IL-12p70), IL-10, IL-6, transforming growth factor beta (TGF-beta), NO, and tumor necrosis factor alpha (TNF-alpha). Our results indicate that the addition of MAX upregulates the cytokines associated with a type 2 response (IL-10, IL-6, and TGF-beta) but downregulates type 1 cytokines (IL-12p70 and TNF-alpha) and NO. MAX was also added to L. major-infected mouse peritoneal exudate cells (PECs), and the parasite load increased significantly. The enhanced parasite load correlated with decreased NO production by PECs that were stimulated with LPS and gamma interferon in the presence of MAX. The ability of MAX to foster a type 2 response, to enhance parasite survival, and to decrease NO argues that MAX may be crucial for the early survival of Leishmania in the vertebrate host, and therefore, MAX holds considerable promise as an antigenic component for a vaccine against Leishmania.

A simple preparation method for mouse eosinophils and their responses to anti-allergic drugs

A simple method for preparing mouse eosinophils was established, and the characteristics of the eosinophils were assessed including their responses to anti-allergic drugs. Mouse eosinophils were prepared from peritoneal exudate cells of BALB/c mice primed and boosted with antigen ovalbumin (OVA). Surface phenotypes, migration activities and leukotriene C(4) (LTC(4)) production abilities of these eosinophils were examined. In addition, the effects of anti-allergic drugs, oxatomide and tranilast, on generation of LTC(4) from mouse eosinophils were examined. Eosinophils of mice boosted with OVA were phenotypically and functionally identical with human eosinophils. Around 1 x 10(7) eosinophils were obtained from mouse peritoneal exudate. It was found that these mouse eosinophils enabled to migrate in response to eotaxin as well as platelet-activating factor (PAF), and generated LTC(4) by IL-5 stimulation. Moreover, it was revealed that clinically used anti-allergic drugs inhibited LTC(4)-production dose-dependently. The present study provides a convenient method to obtain fully functional mouse eosinophils that are useful for drug screening and for evaluating implications of eosinophils in allergic responses.

Inflammatory Immune Responses by Water-insoluble α-glucans

Water-insoluble alpha-glucans are synthesized from sucrose by glucosyltransferase-I of mutans streptococci and play an important role in the development of dental plaque. Several types of beta-glucans in fungal cell wall components and water-soluble alpha-glucans from Streptococcus mutans are known to modulate innate immunity. In the present study, we investigated whether water-insoluble alpha-glucans also induced inflammatory innate immune responses. Our results showed that water-insoluble alpha-glucans synthesized by Streptococcus sobrinus activated mouse peritoneal exudate macrophages to produce pro-inflammatory cytokines. The immunological responses were not due to contamination by sucrose, water-soluble alpha-glucan, lipopolysaccharide, or peptidoglycan. Furthermore, human monocytes stimulated by water-insoluble alpha-glucans produced TNF-alpha and IL-8, while human polymorphonuclear cells were activated by water-insoluble alpha-glucans, resulting in chemotaxis and hydrogen peroxide production. The results demonstrated that water-soluble alpha-glucans modulate macrophage- and granulocyte-induced inflammatory immune responses, and suggest that inflammation induced by those alpha-glucans is associated with the development of periodontal diseases.

Expression and function of cystine/glutamate transporter in neutrophils

Reactive oxygen species (ROS) produced by neutrophils are essential in the host defense against infections but may be harmful to neutrophils themselves. Glutathione (GSH) plays a pivotal role in protecting cells against ROS-mediated oxidant injury. Cystine/glutamate transporter, designated as system xc- and consisting of two proteins, xCT and 4F2hc, is important to maintain GSH levels in mammalian-cultured cells. In the present paper, we have investigated system xc- in neutrophils. In human peripheral blood neutrophils, neither the activity of system xc- nor xCT mRNA was detected. The activity was induced, and xCT mRNA was expressed when they were cultured in vitro. The mRNA expression was much enhanced in the presence of opsonized zymosan or PMA. In contrast, mouse peritoneal exudate neutrophils, immediately after preparation, exhibited system xc- activity and expressed xCT mRNA. The activity and the expression were heightened further when they were cultured. Peritoneal exudate cells (mostly neutrophils) from xCT-deficient (xCT-/-) mice had lower cysteine content than those from the wild-type mice. GSH levels in the xCT-/-cells decreased rapidly when they were cultured, whereas those in the wild-type cells were maintained during the culture. Apoptosis induced in culture was enhanced in the xCT-/-cells compared with the wild-type cells. These results suggest that system xc- plays an important role in neutrophils when they are activated, and their GSH consumption is accelerated.

Induction of interferon-γ by Taenia crassiceps glycans and Lewis sugars in naive BALB/c spleen and peritoneal exudate cells

Helminth parasites are known to alter host immune responses and the responsible molecules are a potential source of biological immunoadjuvants. Previously, we have reported strong Th-2 type immunomodulatory properties of Taenia crassiceps glycans. In this study, we report interferon-γ (IFN-γ) stimulatory activity of fractionated Taenia glycans and Lewis sugars with comparable glycan composition. Our data show that Taenia glycans and Lewis X pentasaccharide are potent stimulators of the Th-1 type cytokine IFN-γ. We postulate that the terminal β-(1-4)-galactose residue in Lewis X is associated with IFN-γ stimulation from naive BALB/c mouse spleen and peritoneal exudate cells. Antibodies to toll-like receptors (TLRs) inhibited the Lewis X-induced IFN-γ secretion. Lewis X up-regulated the expression of NF-κB p65 from naive spleen cells and IFN-γ transcription in peritoneal exudate cells. These data demonstrate the ability of Lewis type helminth glycans to modulate host responses in a Th-1 direction via NF-κB p65, IFN-γ and macrophage TLRs.

The macrophage heterogeneity: Difference between mouse peritoneal exudate and splenic F4/80+ macrophages

Macrophages isolated from various tissues manifest differences in cell shape, the expression of surface markers, as well as metabolic and functional activities. However, the heterogeneity of macrophages expressing the same marker in different tissues has not been fully addressed. In the present study, mouse F4/80+ peritoneal exudate macrophages (PEMs) and splenic macrophages (SPMs) appeared similar in most respects. But the percentages of cells expressing CD80, CD40, MHC-II, TLR2, or TLR4, but not CD11c, CD54, or CD23, in freshly isolated F4/80+ SPMs were significantly higher than those in PEMs, whereas the levels of CD86+ cells in F4/80+ SPMs were markedly lower than those in PEMs. After lipopolysaccharide (LPS) stimulation, F4/80+ SPMs expressed significantly higher levels of CD86, CD40, or MHC-II than F4/80+ PEMs, but not CD11c, CD80, CD54, or CD23. F4/80+ SPMs had remarkably lower non-opsonic phagocytotic capacity against chicken RBCs or allo-T cells than PEMs as determined by two-photon microscopes and flow cytometry. SPMs produced markedly more NO than PEMs when cultured with LPS or allo-T cells. Furthermore, SPMs exhibited stronger immunogenicity than PEMs, as determined by the ability to stimulate T cell proliferation, delayed type hypersensitivity, and IFN-gamma production. The data showed the differences between SPMs and PEMs with regard to the phenotypes, phagocytosis, and immunogenicity, which may offer important information for us to better understand the distinguished immune responses of macrophages in spleens and the peritoneal cavity.

LPS-INDUCED MACROPHAGE (M??) ACTIVATION AUGMENTS PHAGOCYTOSIS OF CANDIDA GLABRATA

Candida glabrata is now the 2nd or 3rd most frequent cause of candidemia (after C. albicans) in trauma patients, but little is known about its pathogenesis. Others have reported that systemic injection with Escherichia coli (or its LPS) augments systemic C. albicans infection, but the opposite may be true for C. glabrata. Fourteen days after 108 intravenous C. glabrata, mice were injected intraperitoneally (ip) with 108E. coli (live or heat-killed), 100 μg E. coli LPS, or saline, and sacrificed 16 hr later. Numbers of C. glabrata recovered from the kidneys were consistently decreased in mice given E. coli or its LPS.TableTo clarify the role of LPS-induced MØ activation in clearance of C. glabrata, glass-adherent mouse peritoneal exudate cells (>90% positive for the F4/80 MØ antigen) were incubated with 0 or 100 μg/ml LPS for 24 hr, with MØ activation confirmed by increased fluorescent intensity to rat-anti-mouse MARCO (MØ receptor with collagenous structure) antibody (687 versus 2664 fluorescent intensity units, Metamorph software, P < 0.01, t-test). After 30 min incubation with 106C. glabrata, LPS-activated MØs had increased numbers of cell-associated C. glabrata, measured by light microscopy of Wright-Giemsa stained cultures (avg of 6.4 versus 3.7 C. glabrata per MØ incubated with and without LPS, P < 0.01, t-test, three experiments containing observations from 450 MØs). Thus, LPS-induced MØ activation was associated with a nearly 2-fold increase in C. glabrata phagocytosis. These data indicate that clinical conditions, or therapeutic agents, that increase MØ activation may help ameliorate systemic infection with C. glabrata.