Macrophages rapidly internalize their tumor necrosis factor receptors in response to bacterial lipopolysaccharide

The effect of bacterial lipopolysaccharide (LPS) on macrophage receptors for tumor necrosis factor/cachectin (TNF-R) was studied. At equilibrium, iodinated recombinant human TNF alpha (rTNF alpha) bound to 1100 +/- 200 sites/cell on macrophage-like RAW 264.7 cells with a Kd of 1.3 +/- 0.1 x 10(-9) M. Preexposure of RAW 264.7 cells to 10 ng/ml LPS for 1 h at 37 degrees C resulted in complete loss of cell surface TNF alpha binding sites. 50% loss ensued after 1 h with 0.6 ng/ml LPS, or after 15 min with 10 ng/ml LPS. Complete loss of TNF alpha binding sites occurred without change in numbers of complement receptor type 3. No decrease in TNF-R followed preexposure to LPS at 4 degrees C, nor could LPS displace 125I-rTNF alpha from its binding sites. Although TNF-R disappeared from the surface of intact macrophages following exposure to LPS, specific TNF alpha binding sites were unchanged in permeabilized macrophages, indicating that TNF-R were rapidly internalized. Conditioned media from LPS-treated RAW 264.7 cells induced 30% down-regulation of TNF-R on macrophages from LPS-hyporesponsive mice (C3H/HeJ), suggesting that a soluble macrophage product may be responsible for a minor portion of the LPS effect. Additional evidence against endogenous TNF alpha being the major cause of TNF-R internalization was the rapid onset of the effect of LPS on TNF-R compared to the reported onset of TNF alpha production, the relatively high concentrations of exogenous rTNF alpha required to mimic the effect of LPS, and the inability of TNF alpha-neutralizing antibody to block the effect of LPS. LPS-induced down-regulation of TNF-R was complete or nearly complete not only in RAW 264.7 cells, but also in primary macrophages of both human and murine origin, was less marked in human endothelial cells, and was absent in human granulocytes and melanoma cells and mouse L929 cells. Thus, in situ, macrophages and some other host cells may be resistant to the actions of TNF alpha produced during endotoxinemia, because such cells may internalize their TNF-R in response to LPS before TNF alpha is produced.     

The antimicrobial peptide, tilapia hepcidin 2-3, and PMA differentially regulate the protein kinase C isoforms, TNF-α and COX-2, in mouse RAW264.7 macrophages

The antimicrobial and immunomodulatory functions of the antimicrobial peptide, tilapia hepcidin (TH)2-3, were previously studied. Herein, we report the differential modulation of protein kinase C (PKC)-associated proteins by TH2-3, and the PKC activator, phorbol 12-myristate 13-acetate (PMA), in RAW264.7 macrophages. Treatment with TH2-3 at 40 or 80 μg/ml did not affect the cell morphology, but TH2-3 at 120 μg/ml produced morphological changes similar to those after treatment with PMA in RAW264.7 cells. The coexistence of the PKC inhibitor, Ro-31-8220, prevented morphological changes induced by either PMA or 120 μg/ml TH2-3 in RAW264.7 cells. Since PMA is known to induce expression of the proinflammatory cytokine, tumor necrosis factor (TNF)-α, activation of the TNF-α promoter in response to TH2-3 and PMA treatments in lipopolysaccharide (LPS)-stimulated cells was compared. In LPS-stimulated RAW264.7 cells, TNF-α promoter activity was significantly suppressed by TH2-3, but not by PMA. In addition, PMA activated prostaglandin synthase-associated cyclooxygenase (COX)-2 proteins on the cell surface, while the presence of TH2-3 inhibited its expression. Western blotting demonstrated that the expressions of PKC-μ, phosphorylated (p)-PKCμ at serine (S)-744, and p-PKCδ were activated by PMA, but were suppressed by TH2-3. In addition, p-PKC at S-916 was activated by TH2-3 and inhibited by PMA. In conclusion, the differential regulation of PKC isoforms by PMA and TH2-3 may influence morphological changes and regulation of TNF-α in RAW264.7 cells.     

Degradation of IkappaBalpha in activated RAW264.7 cells is blocked by the phosphatidylinositol 3-kinase inhibitor LY294002

The mechanism by which lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA) induces production of proinflammatory cytokines in murine macrophages, and the role of phosphatidylinositol 3-kinase (PI3-kinase) have not been well investigated. Activation of nuclear factor κB (NF-κB) is initiated by the phosphorylation of the inhibitory subunit, IκB, which targets IκB for degradation and leads to the release of active NF-κB. In this study we demonstrate that 2- (4-morpholinyl)-8-phenylchromone (LY294002), which inhibits PI3-kinase, specifically inhibited degradation of IκBα in RAW264.7 cells stimulated with interferon-γ (IFN-γ) plus LPS or IFN-γ plus PMA. To elucidate the importance of this activity in RAW264.7 cells, we examined tumor necrosis factor-α (TNF-α) and interleukin IL)-6 production in the activated cells. Pretreatment of the cells with LY294002 resulted in the inhibition of TNF-α and IL-6 production in RAW264.7 cells stimulated with IFN-γ plus LPS or IFN-γ plus PMA. Furthermore, LY294002 inhibited the production of nitric oxide NO) in RAW264.7 cells stimulated with IFN-γ plus LPS or IFN-γ plus PMA. LY294002 also inhibited inducible NO synthase (iNOS) mRNA expression in the activated RAW264.7 cells. In conclusion, the present results suggest that PI3-kinase is involved in the signal transduction pathway responsible for LPS- or PMA-mediated TNF-α and IL-6 production, and that LY294002 inhibits NO generation through blocking the degradation of IκBα in activated RAW264.7 cells. This revised version was published online in August 2006 with corrections to the Cover Date.     

Cortisol and IGF-1 synergistically up-regulate taurine transport by the rat skeletal muscle cell line, L6

This study was undertaken to evaluate effects of exercise-induced hormones, cortisol, IGF-1, and beta-endorphin, on the regulation of taurine transport activity in rat skeletal myoblasts, L6 cells. Challenge of L6 cells with cortisol (100 nM) for 24 hrs resulted in a 165% increase in taurine transport activity, 220% increase in Vmax of the taurine transporter, and 55% increase in taurine transporter/ beta-actin mRNA level compared with untreated control cells. Neither IGF-1 (1 approximately 100 nM) nor beta-endorphin (1 approximately 20 nM), added in the incubation medium separately for 24 hrs, affected taurine uptake by L6 cells. However, when cells were co-treated with IGF-1 (10 nM) plus cortisol (100 nM), taurine transport activity (37% increase, p < 0.05), Vmax of the transporter (54%, p < 0.05), and taurine transporter/ beta-actin mRNA level were further increased compared to the value for cells treated with cortisol alone. These results suggest that taurine transport by skeletal muscle cells appear to be synergistically up-regulated during a prolonged exercise via elevated levels of cortisol and IGF-1 in muscle.     

Sphingosine kinase protects lipopolysaccharide-activated macrophages from apoptosis

Lipopolysaccharide (LPS) signaling is critical for the innate immune response to gram-negative bacteria. Here, evidence is presented for LPS stimulation of sphingosine kinase (SPK) in the RAW 264.7 murine macrophage cell line and rat primary hepatic macrophages (HMs). LPS treatment of RAW 264.7 cells resulted in a time- and dose-dependent activation of SPK and membrane translocation of SPK1. Further, LPS-induced SPK activation was blocked by SPK1-specific small interfering RNA (siRNA). Overexpression of Toll-like receptor 4 and MD2, the receptor and coreceptor of LPS, in HEK 293 cells activated SPK activity in the absence of LPS treatment. Inhibition of SPK by the pharmacological inhibitor N,N-dimethylsphingosine (DMS) or SPK1-specific siRNA blocked LPS stimulation of extracellular signal-regulated kinase 1/2 and p38 but enhanced LPS-induced c-Jun N-terminal kinase activation. The SPK inhibitor DMS and dominant-negative SPK1 also blocked LPS activation of Elk-1 and NF-kappaB reporters in RAW 264.7 cells. Inhibition of SPK sensitized RAW 264.7 cells and HMs to LPS-induced apoptosis. These data demonstrate the critical role of SPK1 in LPS signaling in macrophages and suggest that SPK1 is a potential therapeutic target to block hyperimmune responses induced by gram-negative bacteria.     

The role of endoplasmic reticular Ca2+ stores in cell viability and tumor necrosis factor-α production of the murine macrophage RAW 264.7 cell line

Thapsigargin (TG), an endoplasmic reticular (ER) Ca(2+)-ATPase inhibitor, can increase the intracellular calcium concentration and then deplete the TG-sensitive intracellular Ca(2+) pool. In this study, we investigated the effects of TG on cell viability and tumor necrosis factor-alpha (TNF-alpha) production in the murine macrophage RAW 264.7 cell line. We found that treatment with TG (10-800 nM) induced apoptosis in RAW 264.7 cells in a dose-dependent manner (IC(50), 200 nM). Lipopolysaccharide (LPS, 1 microg/ml) markedly potentiated low concentrations of TG (10-75 nM) in inducing apoptosis (IC(50), 20 nM) as revealed by the DNA ladder. Polymycin B (an LPS receptor antagonist) inhibited the cytotoxic effect induced by LPS plus TG. Although TG, A23187 and ionomycin all definitely increased intracellular Ca(2+) concentrations, neither A23187 nor ionomycin mimicked TG in inducing apoptotic events in LPS-activated RAW 264.7 cells. Moreover, the production of TNF-alpha induced by LPS was profoundly potentiated by TG but not by A23187 or by ionomycin. We conclude from these combined results that TG-sensitive ER Ca(2+) stores play a pivotal role in modulating cell viability and TNF-alpha production. The mutual potentiation between the LPS receptor signaling pathway and the depletion of ER Ca(2+) stores implies the existence of cross-talk between these multiregulatory mechanisms in this murine macrophage RAW 264.7 cell line.     

Hydrogen peroxide induces the production of tumor necrosis factor-alpha in RAW 264.7 macrophage cells via activation of p38 and stress-activated protein kinase

The effect of hydrogen peroxide (H(2)O(2)) on production of tumor necrosis factor (TNF)-alpha was examined in RAW 264.7 murine macrophage cells. H(2)O( 2) led to production of TNF-alpha up to 24 h after the treatment, but not nitric oxide in RAW 264.7 cells. H(2)O(2) induced TNF-alpha production in mouse peritoneal macrophages as well as RAW 264.7 cells. The H(2)O(2)induced TNF-alpha production was prevented by inhibitors of p38 and stress-activated protein kinase (SAPK/JNK), and H(2)O( 2) induced the phosphorylation of p38 and SAPK. Further, H(2)O( 2) significantly augmented the AP-1 activity, but not nuclear factor (NF)-kappaB activity in RAW 264.7 cells. A high level of intracellular reactive oxygen radicals (ROS) was detected in H(2)O(2)-exposed RAW 264.7 cells. Ebselen, a cell permeable antioxidant, prevented the H( 2)O(2)-induced TNFalpha production. H(2)O(2) significantly enhanced lipopolysaccharide (LPS)-induced TNF-alpha production. Therefore, H( 2) O(2) was suggested to induce TNF-alpha production in macrophages via activating p38 and SAPK/JNK as oxidative stress-related signal pathways.     

Cathelicidin family of antibacterial peptides CAP18 and CAP11 inhibit the expression of TNF-alpha by blocking the binding of LPS to CD14(+) cells.

Mammalian myeloid and epithelial cells express several kinds of antibacterial peptides (alpha-/beta-defensins and cathelicidins) that contribute to the innate host defense by killing invading micro-organisms. In this study we evaluated the LPS-neutralizing activities of cathelicidin peptides human CAP18 (cationic antibacterial proteins of 18 kDa) and guinea pig CAP11 using the CD14(+) murine macrophage cell line RAW264.7 and the murine endotoxin shock model. Flow cytometric analysis revealed that CAP18 and CAP11 inhibited the binding of FITC-conjugated LPS to RAW264.7 cells. Likewise, Northern and Western blot analyses indicated that CAP18 and CAP11 suppressed LPS-induced TNF-alpha mRNA and protein expression by RAW264.7 cells. Interestingly, CAP18 and CAP11 possessed LPS-binding activities, and they strongly suppressed the interaction of LPS with LPS binding protein that mediates the transport of LPS to CD14 to facilitate the activation of CD14(+) cells by LPS. Moreover, when CAP18 and CAP11 were preincubated with RAW264.7 cells, they bound to the cell surface CD14 and inhibited the binding of FITC-LPS to the cells. Furthermore, in the murine endotoxin shock model, CAP18 or CAP11 administration inhibited the binding of LPS to CD14(+) cells (peritoneal macrophages) and suppressed LPS-induced TNF-alpha expression by these cells. Together these observations indicate that cathelicidin peptides CAP18 and CAP11 probably exert protective actions against endotoxin shock by blocking the binding of LPS to CD14(+) cells, thereby suppressing the production of cytokines by these cells via their potent binding activities for LPS and CD14.     

Taurine inhibits osteoclastogenesis through the taurine transporter

Several studies have suggested a direct link between taurine and bone homeostasis. However, the mechanisms of taurine on the regulation of bone metabolism have not been elucidated. Using a coculture of osteoblasts and bone marrow cells as a model for the study of osteoclastogenesis, RANKL-stimulated RAW264.7 cells and M-CSF- and RANKL-induced bone marrow macrophages were investigated to elucidate the possible roles of taurine in osteoclastogenesis. Taurine inhibited osteoclastogenesis in the coculture of osteoblasts and bone marrow cells, but did not influence the expression of OPG and RANKL in osteoblasts. The taurine transporter (TAUT) expressed by RAW264.7 and bone marrow macrophages exhibited typical taurine uptake activity. Taurine directly reduced osteoclastogenesis in RANKL-stimulated RAW264.7 cells and M-CSF- and RANKL-induced bone marrow macrophages, while TAUT siRNA relieved this effect. Our study demonstrated that taurine directly inhibited osteoclastogenesis through the taurine transporter. Taken together, these data suggest that taurine plays a direct role in bone homeostasis by inhibiting osteoclastogenesis.     

Lipopolysaccharide increases glucocorticoid receptor expression in murine macrophages. A possible mechanism for glucocorticoid-mediated suppression of endotoxicity.

In a previous study we demonstrated that IFN-gamma induced an increase in the number of glucocorticoid receptors (GR) in murine macrophages. To examine further the environmental signals involved in regulation of macrophage GR availability, we asked whether another classical macrophage-activating factor, LPS, would induce an increase in GR number in the macrophage cell line, RAW 264.7, and in primary macrophages from C3H mice. We report that treatment of RAW 264.7 cells and peritoneal exudate macrophages from C3H/OuJ mice with protein-free, phenol water-extracted LPS (PW-LPS) induced an increase in the number of GR. A significant increase in GR number was observed as early as 4 h after PW-LPS treatment, was maximal at 12 h, and remained heightened through 48 h. Optimal induction of the GR by PW-LPS was observed when murine macrophages were treated with 10 ng/ml of PW-LPS. The LPS-induced increase in macrophage GR number could be inhibited by polymyxin B. Macrophages obtained from the LPS hyporesponsive C3H/HeJ strain did not respond to PW-LPS, but did respond to protein-rich, butanol-extracted LPS with a modest increase in GR number after treatment with 2 micrograms/ml. Moreover, taxol, an antineoplastic agent with LPS mimetic activity, also increased GR number in murine macrophages. These results suggest that LPS is not only an important macrophage-activating signal, but may also be important in sensitizing the cell for negative regulatory events such as feedback inhibition by glucocorticoids.     

Down-regulation of mannose receptor activity in macrophages after treatment with lipopolysaccharide and phorbol esters

We have investigated the effects of LPS and PMA on the expression of functional mannose receptors in rat bone marrow-derived macrophages. After 48 h of treatment with LPS (10 ng/ml) and PMA (100 nM), mannose receptor activity was reduced by 70 to 80%. The effect of these agents on receptor activity was not reversible, and activity continued to decline after the agents were removed. Pretreatment of cells with dexamethasone was effective in blocking the LPS/PMA-induced down-regulation. Serine protease inhibitors did not block the reduction in receptor activity, suggesting that proteolysis is not involved in receptor down-regulation. LPS/PMA treatment did not increase turnover of the receptor. Ligand uptake studies showed that the total capacity of the uptake system was reduced by 80%, although the Kuptake was unaffected. Binding of 125I-mannose-BSA to intact macrophages showed a 70% decrease in surface receptor activity after treatment with LPS/PMA. LPS/PMA treatment had no effect on total receptor synthesis as quantitated by immunoprecipitation of metabolically labeled receptor. However, binding of metabolically labeled receptor to mannose-Sepharose, and binding of 125I-mannose-BSA to immunoprecipitated receptor revealed that intracellular plus surface binding sites were reduced to approximately 30% after LPS/PMA treatment. These results suggest that LPS/PMA treatment of macrophages results in an inactivation of mannose receptors with no effect on receptor turnover or biosynthesis.     

Peroxiredoxin-1, a possible target in modulating inflammatory cytokine production in macrophage like cell line RAW264.7

Peroxiredoxin (PRX), a scavenger of H₂O₂ and alkyl hydroperoxides in living organisms, protects cells from oxidative stress. Contrary to its known anti‐oxidant roles, the involvement of PRX‐1 in the regulation of lipopolysaccharide (LPS) signaling is poorly understood, possible immunological functions of PRX‐1 having been uncovered only recently. In the present study, it was discovered that the PRX‐1 deficient macrophage like cell line (RAW264.7) has anti‐inflammatory activity when stimulated by LPS. Treatment with LPS for 3 hrs resulted in increased gene expression of an anti‐inflammatory cytokine, interleukin‐10 (IL‐10), in PRX‐1 knock down RAW264.7 cells. Gene expression of pro‐inflammatory cytokines IL‐1β and tumor necrosis factor‐ α (TNF‐α) did not show notable changes under the same conditions. However, production of these cytokines significantly decreased in PRX‐1 knock down RAW264.7 cells with 12 hrs of stimulation. Production of IL‐10 was also increased in PRX‐1 knock down RAW264.7 cells with 12 hrs of stimulation. We predicted that higher concentrations of IL‐10 would result in decreased expression of IL‐1β and TNF‐α in PRX‐1 knock‐down cells. This was confirmed by blocking IL‐10, which reestablished IL‐1β and TNF‐α secretion. We also observed that increased concentrations of IL‐10 do not affect the NF‐κB pathway. Interestingly, STAT3 phosphorylation by LPS stimulation was significantly increased in PRX‐1 knockdown RAW264.7 cells. Up‐regulation of IL‐10 in PRX‐1 knockdown cells and the resulting downregulation of proinflammatory cytokine production seem to involve the STAT3 pathway in macrophages. Thus, down‐regulation of PRX‐1 may contribute to the suppression of adverse effects caused by excessive activation of macrophages through affecting the STAT3 signaling pathway.     

Contribution of the exosome-associated form of secreted endoplasmic reticulum aminopeptidase 1 to exosome-mediated macrophage activation

Macrophages secrete endoplasmic reticulum aminopeptidase 1 (ERAP1) in response to lipopolysaccharide (LPS) and interferon (IFN)-γ to enhance their phagocytic and nitric oxide (NO) synthetic activities. In this study, we found that a subset of secreted ERAP1 bound to exosomes released from LPS/IFN-γ-treated murine RAW264.7 macrophages compared to untreated cells. ERAP1-bound exosomes enhanced phagocytic and NO synthetic activities of macrophages more efficiently than free ERAP1 and exosomes derived from untreated cells. Deletion of the exon 10 coding sequence in ERAP1 gene resulted in loss of binding to exosomes. By comparing the activities of exosomes derived from wild-type and ERAP1 gene-deficient RAW264.7 cells, we observed that ERAP1 contributed to the exosome-dependent phagocytosis and NO synthesis of the cells. Upon stimulation of RAW264.7 cells with LPS/IFN-γ, TNF-α, IFN-γ, and CCL3 were also associated with the released exosomes. Analyses of cytokine function revealed that while CCL3 in the exosomes was crucial to the phagocytic activity of RAW264.7 cells, TNF-α and IFN-γ primarily contributed to the enhancement of NO synthesis. These results suggest that treatment with LPS/IFN-γ alters the physicochemical properties of exosomes released from macrophages in order to facilitate association with ERAP1 and several cytokines/chemokines. This leads to exosome-mediated enhancement of macrophage functions. It is possible that packaging effector molecules into exosomes upon inflammatory stimuli, facilitates the exertion of effective pathophysiological functions on macrophages. Our data provide the first evidence that ERAP1 associated with exosomes plays important roles in inflammatory processes via activation of macrophages.     

Ganglioside GM3 suppresses lipopolysaccharide‐induced inflammatory responses in rAW 264.7 macrophage cells through NF‐κB,AP‐1, and MAPKs signaling

Gangliosides are known to specifically inhibit vascular leukocyte recruitment and consequent interaction with the injured endothelium, the basic inflammatory process. In this study, we have found that the production of nitric oxide (NO), a main regulator of inflammation, is suppressed by GM3 on murine macrophage RAW 264.7 cells, when induced by LPS. In addition, GM3 attenuated the increase in cyclooxyenase‐2 (COX‐2) protein and mRNA levels in lipopolysaccharide (LPS)‐activated RAW 264.7 cells in a dose‐dependent manner. Moreover, GM3 inhibited the expression and release of pro‐inflammatory cytokines of tumor necrosis factor‐alpha (TNF‐α), interleukin‐6 (IL‐6), and interleukin‐1β (IL‐1β) in RAW 264.7 macrophages. At the intracellular level, GM3 inhibited LPS‐induced nuclear translocation of nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) and activator protein (AP)‐1 in RAW 264.7 macrophages. We, therefore, investigated whether GM3 affects mitogen‐activated protein kinase (MAPK) phosphorylation, a process known as the upstream signaling regulator. GM3 dramatically reduced the expression levels of the phosphorylated forms of ERK, JNK, and p38 in LPS‐activated RAW 264.7 cells. These results indicate that GM3 is a promising suppressor of the vascular inflammatory responses and ganglioside GM3 suppresses the LPS‐induced inflammatory response in RAW 264.7 macrophages by suppression of NF‐κB, AP‐1, and MAPKs signaling. Accordingly, GM3 is suggested as a beneficial agent for the treatment of diseases that are associated with inflammation.     

A differential role for the mitogen-activated protein kinases in lipopolysaccharide signaling: the MEK/ERK pathway is not essential for nitric oxide and interleukin 1beta production.

Endotoxin (lipopolysaccharide, LPS) is a component of the outer membrane of Gram-negative bacteria and promotes the activation of macrophages and microglia. Although these cells are highly LPS-responsive, they serve unique tissue-specific functions and exhibit different LPS sensitivities. Accordingly, it was of interest to evaluate whether these biological differences reside in variations within LPS signaling pathways between these two cell types. Because the mitogen-activated protein kinases ERK-1 and ERK-2 have been implicated in the control of many immune responses, we tested the concept that they are a key indicator for differences in cellular LPS sensitivity. We observed that murine RAW 264.7 macrophages and murine BV-2 microglial cells both respond to LPS by exhibiting increased IkappaBalpha degradation, enhanced NF-kappaB DNA binding activity, and elevated nitric oxide and interleukin-1beta production. Although LPS potently stimulates ERK activation in RAW 264.7 macrophages, it does not activate ERK-1/-2 in BV-2 microglia. Moreover, antagonism of the MEK/ERK pathway potentiates LPS-stimulated nitric oxide production, suggesting that LPS-stimulated ERK activation can exert inhibitory effects in macrophage-like cells. These data support the idea that ERK activation is not a required function of LPS-mediated signaling events and illustrate that alternative/additional pathways for LPS action exist in these cell types.     

The in vitro anti-platelet,antioxidant and cellular immunity activity of <Emphasis Type="Italic">Phellinus gilvus</Emphasis> fractional extracts

The in vitro anti-platelet and antioxidant activities of various solvent extracts from Phellinus gilvus (PG), and the effects of hot water extract from PG (PGW) on murine cellular immunity were investigated. Chloroform extract (CE), methanol extract (ME) and butanol extract (BE) from PG could significantly inhibit platelet aggregation induced by thrombin. Ethyl acetate extract (EAE), BE, ME from PG had significant 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity compared with the control, and the EAE showed the highest effect with IC₅₀ values of 13.34 μg/ml, which is higher than that of ascorbic acid (40 μg/ml). In addition, EAE displayed the inhibition of xanthine oxidase (XO) activity with IC₅₀ value of 2.45 μg/ml. As to the cellular immunity activity, PGW could enhance both the lipopolysaccharide (LPS)-induced B lymphocyte proliferation and concanavalin A (Con A)-induced T lymphocyte proliferation in vitro. The phagocytosis of both peritoneal macrophages and RAW264.7 macrophage cells were also increased by the addition of PGW. Moreover, PGW was found to inhibit the nitric oxide (NO) production of RAW264.7 macrophages induced by LPS in a concentration-dependant manner.