Lipopolysaccharide (LPS)-induced intra-uterine fetal death (IUFD) in mice is principally due to maternal cause but not fetal sensitivity to LPS

The present study deals with whether lipopolysaccharide (LPS)-induced intra-uterine fetal death (IUFD) is related to LPS-susceptibility of either mother or fetus and how LPS or LPS-induced TNF causes IUFD. LPS-susceptible C3H/HeN or -hypo-susceptible C3H/HeJ pregnant mice and the mice mated reciprocally with these mice were used on days 14 to 16 of gestation for experiments. All of fetuses in pregnant C3H/HeN mice mated with either C3H/HeN males [HeN(HeN)] or C3H/HeJ males [HeN(HeJ)] were killed within 24 hr when injected intravenously (i.v.) with 50 or 100 microg of LPS. On the other hand, the majority of fetuses in C3H/HeJ females mated with either C3H/HeJ males [HeJ(HeJ)] or C3H/HeN males [HeJ(HeN)] survived when injected i.v. with even 400 microg of LPS. These findings indicate that LPS-induced IUFD depends on the maternal LPS-responsiveness. LPS injected into mothers could pass through placenta to fetuses, since an injection with 125I-labeled LPS or IgG into pregnant mice resulted in considerable levels of radioactivity in fetuses as well as placenta. Cultured peritoneal macrophages derived from F1 mice of HeJ(HeN) or HeN(HeJ) mice, produced nitric oxide (NO) and tumor necrosis factor (TNF) in response to LPS, although the levels of NO and TNF were lower in comparison with those of C3H/HeN macrophage cultures, suggesting a possibility that the fetus as well as F1 cells might be responsible to LPS. LPS-induced IUFD was not blocked by treatment with anti-TNF antibody which inhibited LPS-induced TNF production in pregnant females, although an injection of recombinant TNFalpha instead of LPS could induce IUFD, suggesting that the cause of IUFD cannot be attributed to mother-derived TNF alone. The roles of LPS passed through placenta and LPS-induced mediators on IUFD were discussed.     

Defective helper T-cell activity in LPS-unresponsive C3H/HeJ mice

C3H/HeJ mice, unresponsive to LPS, exhibit a defective ability to mount antibody responses to T-dependent immunogens. The anti-TNP antibody response to TNP-HRBC, a T-dependent immunogen, was found to be lower in these mice as compared to LPS-responsive C3H/HeN mice, whereas the anti-TNP antibody response to TNP-Ficoll, a T-independent immunogen, was of the same magnitude in C3H/HeJ and C3H/HeN mice. An impaired helper activity of C3H/HeJ HRBC-primed spleen cells was demonstrated in a titration assay in which graded numbers of C3H/HeJ or C3H/HeN HRBC-primed spleen cells were added to cultures containing a constant number of unprimed spleen cells from either C3H/HeJ or C3H/HeN mice and the immunogen TNP-HRBC. The reduced helper T-cell activity of C3H/HeJ HRBC-primed spleen cells appears to be independent of macrophage defects, since C3H/HeJ and C3H/HeN macrophages were found equally effective in antigen presentation as evaluated by an in vitro antigen-specific T-cell proliferation assay. The difference in helper T-cell activity between these two substrains probably reflects a lower number and/or proliferation rate of antigen-responsive T cells in C3H/HeJ mice.     

Modulation of Immune Response by Bacterial Lipopolysaccharide (LPS): Roles of Macrophages and T Cells in In Vitro Adjuvant Effect of LPS on Antibody Response to T Cell-Dependent and T Cell-Independent Antigens

The roles of macrophages and T cells in the adjuvant effect of lipopolysaccharide (LPS) were studied. In vitro anti-trinitrophenyl (anti-TNP) antibody responses to TNP-Ficoll and TNP-keyhole limpet hemocyanine (TNP-KLH) in spleen cells of C57BL/6 mice showed the most enhancement, when LPS was added to cultures at 1 μg/ml 48 hr after culture was started. The responses to these antigens were enhanced markedly by LPS in whole and macrophage-depleted spleen cells. The enhancement was greater in the latter group than in the former. The adjuvant effect among whole, T cell-depleted, macrophage-depleted and both macrophage- and T cell-depleted spleen cells was compared. The response to TNP-Ficoll was enhanced markedly by LPS in all groups. The enhancement was greater in the latter two groups than in the first two groups. The response to TNP-KLH was enhanced by LPS strongly in macrophage-depleted spleen cells, moderately in whole and both macrophage- and T cell-depleted spleen cells, and only slightly in T cell-depleted spleen cells. Enhancement was restored to T cell-depleted spleen cells by adding T cells. The response to TNP-KLH of macrophage-depleted spleen cells of LPS-responsive C3H/HeN mice which was enhanced by LPS was suppressed by adding splenic macrophages of C3H/HeN mice, but not of LPS-nonresponsive C3H/HeJ mice. The response to TNP-KLH of macrophage-depleted spleen cells of C3H/HeJ mice was not enhanced by LPS, irrespective of the addition of macrophages of C3H/HeN mice. The results indicate that B cells are activated directly by LPS, and T cells enhance and macrophages suppress the adjuvant effect of LPS.     

Quantitative aspects of lipopolysaccharide and cytokine requirements to generate nitric oxide in macrophages from LPS-hyporesponsive (<Emphasis Type="Italic">Lps</Emphasis><Superscript>d</Superscript>) C3H/HeJ mice

Due to a gene defect (Lps(d)), C3H/HeJ mice are known to be hyporesponsive to the immunobiological potential of lipopolysaccharide (LPS). We studied dose requirements for LPS, IFN-gamma, and cytokines TNF-alpha and IL-10 to produce nitric oxide (NO) in peritoneal macrophages (Mphi) from these animals. In contrast to the Lps(n) C3H/HeN mice, high concentrations of LPS (up to 5 microg/mL) or IFN-gamma (up to 5 ng/mL) by themselves were unable to activate NO production in C3H/HeJ Mphi. The failure to produce NO could not be overcome by addition of L-arginine or tetrahydropterin. The high-output NO biosynthesis was dose-dependently stimulated by combined administration of varying concentrations of IFN-gamma (50-5000 pg/mL) and LPS (approximately 1 ng/mL) or to a lesser extent by IFN-gamma plus TNF-alpha or TNF-alpha/IL-10. Formation of NO in C3H/HeJ MCO triggered by high concentration of LPS (approximately 1 microg/mL) given together with IFN-gamma (0.2-5 ng/mL) reached the values typical for Lps(n) C3H/HeN mice. While Mphi from C3H/HeN mice secreted TNF-alpha, IL-10, and IL-10 upon contact with a low dose of LPS (1 ng/mL), C3H/HeJ Mphi required high concentration of LPS (5 microg/mL) to enhance the secretion of the cytokines. Yet, this dose remained ineffective to stimulate IFN-gamma in Mphi from C3H/HeJ mice. It can be presumed that one of the important factors influencing their deficient ability to form NO is a failure of Mphi to produce IFN-gamma upon LPS contact.     

Defective tumoricidal capacity of macrophages from C3H/HeJ mice

Peritoneal macrophages from C3H/HeN mice treated i.p. with T cell mitogens or viable BCG organisms were cytotoxic to syngeneic tumor cells in vitro. Macrophages from endotoxin-unresponsive C3H/HeJ mice treated with BCG or T cell mitogens, however, were not tumoricidal. Furthermore, unlike cells from C3H/HeN mice, macrophages from C3H/HeJ mice could not be activated for tumor cytotoxicity after in vitro treatment with bacterial endotoxins or with lymphokine-rich supernatants. The subnormal induction of cytotoxic macrophages after in vitro or in vivo treatments in C3H/HeJ mice appears to be a highly selective defect. Macrophage responses (yield, phagocytosis, or peroxidase staining) in inflammatory exudates induced by BCG, T cell mitogens, or heterologous serum in C3H/HeJ or C3H/HeN mice were identical. C3H/HeJ macrophages also responded normally in vitor to chemotactic lymphokines. Thus, C3H/HeJ macrophages possess a profound and selective defect in tumoricidal capacity. This defect was not dependent upon exogenous endotoxins. Defective macrophage cytotoxic responses may reflect non-LPS related functions regulated by the LPS gene.     

Soluble production and function of vascular endothelial growth factor/basic fibroblast growth factor complex peptide

Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are important proangiogenic factors in tumor procession. The autocrine and paracrine bFGF and the VEGF in tumor tissue can promote tumor angiogenesis, tumor growth, and metastasis. A VEGF/bFGF Complex Peptide (VBP3) was designed on the basis of epitope peptides from both VEGF and bFGF to elicit in vivo production of anti‐bFGF and anti‐VEGF antibodies. In this study, we reported on the production of recombinant VBP3 using high cell density fermentation. Fed‐batch fermentation for recombinant VBP3 production was conducted, and the production procedure was optimized in a 10‐L fermentor. The fraction of soluble VBP3 protein obtained reached 78% of total recombinant protein output under fed‐batch fermentation. Purified recombinant VBP3 could inhibit tumor cell proliferation in vitro and stimulate C57BL/6 mice to produce high titer anti‐VEGF and anti‐bFGF antibodies in vivo. A melanoma‐grafted mouse model and an immunohistochemistry assay showed that tumor growth and tumor angiogenesis were significantly inhibited in VBP3‐vaccinated mice. These results demonstrated that soluble recombinant VBP3 could be produced by large‐scale fermentation, and the product, with good immunogenicity, elicited production of high‐titer anti‐bFGF and anti‐VEGF antibodies, which could be used as a therapeutic tumor vaccine to inhibit tumor angiogenesis and tumor growth. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:194–203, 2015     

LPS regulation of the immune response: Bacteroides endotoxin induces mitogenic, polyclonal, and antibody responses in classical LPS responsive but not C3H/HeJ mice

Recent studies have suggested that lipopolysaccharide (LPS) derived from gram-negative organisms such as Bacteroides, which are not members of the Enterobacteriaceae, stimulate B cells from the classic LPS-hyporesponsive C3H/HeJ mouse. In the present study, purified, phenol-water-extracted LPS from Bacteroides fragilis ATCC 25285 (B-LPS) was tested for its ability to induce in vivo and in vitro responses in classic LPS-responsive C3H/HeN, LPS-hyporesponsive C3H/HeJ, and (C3H/HeN X C3H/HeJ)F1 hybrid mice. B-LPS induced mitogenic responses in both C3H/HeN and C3H/HeJ spleen cell cultures when cells were cultured under standard conditions, i.e., 8 X 10(5) cells/well. Interestingly, when lower spleen cell numbers were tested with B-LPS, a typical responsive-nonresponsive pattern developed in which good mitogenic responses were induced by B-LPS in C3H/HeN cultures and in which low responses in C3H/HeJ spleen cell cultures were evident. In vivo immunization of mice with B-LPS resulted in high antibody responses in C3H/HeN, intermediate responses in F1, and low responses in C3H/HeJ mice. When purified splenic B cells were incubated with B-LPS, both mitogenic responses and polyclonal immunoglobulin M (IgM) synthesis occurred in C3H/HeN cultures, whereas intermediate responses were noted in F1 cultures and no response was seen in B cell cultures from C3H/HeJ mice. Furthermore, in vitro TNP-B-LPS responses were induced in C3H/HeN spleen cells or purified B cell cultures, and intermediate anti-TNP PFC responses occurred in F1 spleen cells or purified B cell cultures. The toxicity of B-LPS was tested in galactosamine-sensitized mice. The LD50 values for B-LPS in classic LPS-responsive C3H/HeN and C57BL/6J mice were 0.6 microgram and 1.1 microgram, respectively; F1 hybrid mice were approximately 15-fold more resistant, whereas C3H/HeJ mice gave an LD50 of 1650 micrograms. This study shows that phenol-water preparations of B-LPS are biologically active and induce responses in the classic LPS-responsive but not in the LPS-hyporesponsive C3H/HeJ mouse strain.     

Lipopolysaccharide-induced suppression of antibody-directed cell-mediated cytotoxicity to chicken red blood cells

Prior treatment with commercially prepared and acetone-extracted lipopolysaccharide (LPS) was found to suppress the expression of antibody-directed, cell-mediated cytotoxicity (ADCC) to chicken red blood cells (CRBC) by spleen cells from C57/BL10, C3H, and BALB/c mice. The in vitro incubation with commercial LPS suppressed ADCC-CRBC activity of spleen cells from both C3H/HeJ and C3H/HeN mice. Only the C3H/HeN strain was suppressed when treated with purified LPS. ADCC-CRBC activity of neonatal spleen cells could be suppressed after a 3-hr in vivo incubation with LPS while adult spleen cells required a minimum of 15 hr preincubation.     

Immunomodulatory Activity of 3β,6β‐Dihydroxyolean‐12‐en‐27‐oic Acid in Tumor‐Bearing Mice

3β,6β‐Dihydroxyolean‐12‐en‐27‐oic acid ( 1 ) is a pentacyclic triterpenoid isolated from the rhizomes of Astilbe chinensis. To evaluate the in vivo antitumor potential and to elucidate its immunological mechanisms, effect of 1 on the growth of mouse‐transplantable tumors, and the immune response in naive and tumor‐bearing mice were investigated. The mice inoculated with mouse tumor cell lines were orally treated with 1 at the doses of 40, 60, and 80 mg/kg for 10 days. The effects of 1 on the growth of mouse‐transplantable S180 sarcoma and H22 hepatoma, splenocyte proliferation, cytotoxic T lymphocyte (CTL) activity, natural killer (NK) cell activity, and production of interleukin‐2 (IL‐2) from splenocytes in S180‐bearing mice were measured. Furthermore, the effect of 1 on 2,4‐dinitrofluorobenzene (DNFB)‐induced delayed‐type hypersensitivity (DTH) reactions and the sheep red blood cell (SRBC)‐induced antibody response in naive mice were also studied. Compound 1 could not only significantly inhibit the growth of mouse transplantable S180 sarcoma and H22 hepatoma, increase splenocytes proliferation, CTL and NK cell activity, and the level of IL‐2 secreted by splenocytes in tumor‐bearing mice, but also remarkably promote the DTH reaction and enhance anti‐SRBC antibody titers in naive mice. These results suggested that 1 could improve both cellular and humoral immune response, and could act as antitumor agent with immunomodulatory activity.     

Restoration of LPS responsiveness of C3H/HeJ mouse lymphocytes by microinjection of cytoplasmic factor(s) from LPS-stimulated normal lymphocytes

Cytosol fractions of lymphocytes from LPS-responder mice (C3H/HeN) were prepared and injected into B lymphocytes of LPS-nonresponder mice (C3H/HeJ) by a microinjection technique utilizing polyethyleneglycol-mediated cell fusion. The B lymphocytes of C3H/HeJ mice microinjected with cytosol prepared from LPS-stimulated C3H/HeN cells became normally responsive to LPS. Microinjection of cytosol itself did not stimulate C3H/HeJ cells to proliferate or differentiate into immunoglobulin-producing cells, and the cells injected with cytosol had to be restimulated with LPS in order to proliferate and differentiate. These data suggested that C3H/HeJ B cells acquired LPS responsiveness by microinjection of cytoplasmic factor(s) from LPS-stimulated C3H/HeN cells and that these factor(s) may be one of the components involved in normal signal transmission from cell surface to nucleus in the early stages of the LPS response. The apparent m.w. of the cytoplasmic factor(s) is 100,000 by gel filtration. Chromatofocusing analysis suggested that these factors may consist of two components with the same m.w.     

LPS regulation of the immune response: separate mechanisms for murine B cell activation by lipid A (direct) and polysaccharide (macrophage-dependent) derived from Bacteroides LPS

Lipopolysaccharide (LPS) derived from Bacteroides fragilis has been reported to stimulate mitogenic responses in spleen cell cultures from the classical LPS-hyporesponsive C3H/HeJ mouse strain; however, we have shown that purified splenic B cells from C3H/HeJ mice are hyporesponsive to phenol-water extracted LPS from B. fragilis ATCC 25285 (B-LPS). In the present study, B-LPS and its purified lipid A and polysaccharide components were tested for their ability to induce mitogenic and polyclonal IgM synthesis in spleen cell and purified splenic B cell cultures from classical LPS-responsive and -hyporesponsive mice. Mitogenic responses to B-LPS and E. coli K235 LPS(Ph) of whole spleen cells (2 X 10(5) cells/culture) or purified B cells (5 X 10(5) cells/culture) from classical LPS-responsive mouse strains (C3H/HeN, BALB/c, C57BL/6J, C57BL/10Sn, and DBA/2), F1 mice (derived from crosses between LPS responsive and C3H/HeJ mice), and classical LPS-hyporesponsive mice (C3H/HeJ and C57BL/10ScN) were high, intermediate, and low, respectively. When a higher number of whole spleen cells (5 X 10(5) cells/well) were cultured, B-LPS induced high mitogenic responses in C3H/HeN, intermediate responses in F1, and lower but significant responses in C3H/HeJ cultures. Similar results were obtained when polyclonal IgM synthesis was assessed in cultures containing 1 X 10(6) cells/culture. In contrast, the purified lipid A component of B-LPS failed to induce mitogenic responses in either whole spleen or purified B cell cultures. The addition of purified splenic B cells from C3H/HeJ mice to C3H/HeN or C3H/HeJ splenic adherent cells resulted in mitogenic responses to B-LPS, implying that the hyporesponsiveness to B-LPS seen in whole spleen cell cultures from C3H/HeJ mice at the lower cell concentration was due to limiting numbers of M phi. When splenic B cells and M phi from either C3H/HeN or C3H/HeJ mice were incubated with the lipid A or the polysaccharide moiety of B-LPS, lipid A induced mitogenic responses only in C3H/HeN cultures, whereas the polysaccharide moiety induced similar responses in both C3H/HeN and C3H/HeJ cultures. These results suggest that Bacteroides lipid A does not stimulate B cells from the classical LPS-hyporesponsive C3H/HeJ mouse strain, whereas the polysaccharide moiety of B-LPS is biologically active and mediates B cell stimulation via M phi.     

Anti‐inflammatory function of Withangulatin A by targeted inhibiting COX‐2 expression via MAPK and NF‐κB pathways

Withangulatin A (WA), an active component isolated from Physalis angulata L., has been reported to possess anti‐tumor and trypanocidal activities in model systems via multiple biochemical mechanisms. The aim of this study is to investigate its anti‐inflammatory potential and the possible underlying mechanisms. In the current study, WA significantly suppressed mice T lymphocytes proliferation stimulated with LPS in a dose‐ and time‐dependent manner and inhibited pro‐inflammation cytokines (IL‐2, IFN‐γ, and IL‐6) dramatically. Moreover, WA targeted inhibited COX‐2 expression mediated by MAPKs and NF‐κB nuclear translocation pathways in mice T lymphocytes, and this result was further confirmed by the COX‐1/2 luciferase reporter assay. Intriguingly, administration of WA inhibited the extent of mice ear swelling and decreased pro‐inflammatory cytokines production in mice blood serum. Based on these evidences, WA influences the mice T lymphocytes function through targeted inhibiting COX‐2 expression via MAPKs and NF‐κB nuclear translocation signaling pathways, and this would make WA a strong candidate for further study as an anti‐inflammatory agent. J. Cell. Biochem. 109: 532–541, 2010. © 2009 Wiley‐Liss, Inc.     

Adipose stromal cell and sarpogrelate orchestrate the recovery of inflammation‐induced angiogenesis in aged hindlimb ischemic mice

Aging population displays a much higher risk of peripheral arterial disease (PAD) possibly due to the higher susceptibility, poor prognosis, and fewer therapeutic options. This study was designed to examine the impact of combined multipotent adipose‐derived stromal cells (mADSCs) and sarpogrelate treatment on aging hindlimb ischemia and the mechanism of action involved. mADSCs (1.0 × 10⁷) constitutively expressing enhanced green fluorescent protein (eGFP) or firefly luciferase (Fluc) reporter were engrafted into the hindlimb of aged Vegfr2‐luc transgenic or FVB/N mice subjected to unilateral femoral artery occlusion, followed by a further administration of sarpogrelate. Multimodality molecular imaging was employed to noninvasively evaluate mADSCs' survival and therapeutic efficacy against aging hindlimb ischemia. Aged Tg(Vegfr2‐luc) mice exhibited decreased inflammatory response, and downregulation of vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor‐2 (VEGFR2) compared with young ones following hindlimb ischemia induction, resulting in angiogenesis insufficiency and decompensation for ischemia recovery. Engrafted mADSCs augmented inflammation‐induced angiogenesis to yield pro‐angiogenic/anti‐apoptotic effects partly via the VEGF/VEGFR2/mTOR/STAT3 pathway. Nonetheless, mADSCs displayed limited survival and efficacy following transplantation. Sarpogrelate treatment with mADSCs further upregulated mammalian target of rapamycin (mTOR)/STAT3 signal and modulated pro‐/anti‐inflammatory markers including IL‐1β/TNF‐α/IFN‐γ and IL‐6/IL‐10, which ultimately facilitated mADSCs' survival and therapeutic benefit in vivo. Sarpogrelate prevented mADSCs from hypoxia/reoxygenation‐induced cell death via a mTOR/STAT3‐dependent pathway in vitro. This study demonstrated a role of in vivo kinetics of VEGFR2 as a biomarker to evaluate cell‐derived therapeutic angiogenesis in aging. mADSCs and sarpogrelate synergistically restored impaired angiogenesis and inflammation modulatory capacity in aged hindlimb ischemic mice, indicating its therapeutic promise for PAD in the elderly.     

In situ accessibility of murine macrophage gangliosides

Gangliosides are implicated in cell signal transduction. Priorto investigating this phenomenon in macrophages, the in situaccessibility of gangliosides to macromolecules was assessedfor peritoneal macrophages isolated from normal C3H/HeN andendotoxin-hyporesponsive C3H/HeJ mice. C3H/HeJ resident andthioglycolate-elicited macrophage gangiioside patterns are thesame as normal strains, and no strain differences in galactoseoxidase accessibility for resident or thioglycolate-elicitedmacrophage gangliosides were found. The only gangliosides accessibleto galadose oxidase in resident macrophages are G_M1a structures.In thioglycolate-elicited macrophages, an additional ganglieosideis accessible. For Escherichia coli-activated macrophages, whereganglioside distribution differs between strains, a differencein galactose oxidase-accessible gangliosides also exists. Escherichiacoli-activated C3H/HeN patterns show three triplets absent inC3H/HeJ patterns. There were no differences in ganglioside accessibilityto Vibrio cholenre sialidase between the thioglycolate-elicitedC3H/HeJ and C3H/HeN macrophages. However, despite differencesin sialidase-sensitive ganglioside content between E.coli-activatedmacrophages of these strains, sialidase accessibility for E.coli-activatedmacrophages was also similar. Sialidase-susceptible G_M3. wascryptic in either strain under all conditions examined. Theaccessibility of murine macrophage gangliosides to galactoseoxidase or sialidase was independent of their sialic acid speciesand chain length of the ceramide fatty acid. With the exceptionof G_M3, major murine macrophage gangliosides are accsdble insitu to macromolecules, especially to exogenous pathogenic bacterialsialidase which can alter macrophage cell surface characteristics.Altered macrophage ganglioside accessibility appears sometimesas a consequence, but not a cause, of C3H/HeJ endotoxin hyporesponsiveness. accessibility galactose oxide gangliosides macrophages sialidase     

Cellular mechanism of endotoxin unresponsiveness in C3H/HeJ mice.

B cells from C3H/HeJ mice fail to respond to an endotoxin (LPS K235) which is mitogenic for normal mice including the closely related C3H/HeN strain. The cellular basis for this unresponsive state has been investigated. The C3H/HeJ mice have normal numbers of B cells, which are capable of normal responses to other B cell mitogens, such as polyinosinic acid (Poly I). Addition of normal macrophages or spleen cells fails to reconstitute the normal response. Furthermore, neither macrophages nor spleen cells from the C3H/HeJ strain suppress the normal C3H/HeN spleen cells. Finally, spleen cells enriched for B cells by the removal of macrophages or T cells demonstrate the same differences in responsiveness to LPS. These results indicate that LPS unresponsiveness is a defect of the B cell itself and not due to suppressor cells or the absence of helper cells. When LPS is added to Poly I-stimulated cultures, there is additional enhancement of the response of normal C3H/HeN spleen cells. However, LPS causes a dose-dependent suppression of the Poly I response of C3H/HeJ spleen cells. This suppression is dependent on the time of addition of LPS to the Poly I-stimulated cultures. These data are interpreted as indicating that the binding of LPS to the membrane of C3H/HeJ B cells results in their inactivation or suppression, and that this is the basis of LPS unresponsiveness in this mouse strain.     

Oral administration of poly-gamma-glutamate induces TLR4- and dendritic cell-dependent antitumor effect

Previously, we reported that the oral administration of high molecular mass poly-γ-glutamate (γ-PGA) induced antitumor immunity but the mechanism underlying this antitumor activity was not understood. In the present study, we found that application of high molecular mass γ-PGA induced secretion of tumor necrosis factor (TNF)-α from the bone-marrow-derived macrophages of wild type (C57BL/6 and C3H/HeN) and Toll-like receptor 2 knockout (TLR2^−/−) mice, but not those of myeloid differentiation factor 88 knockout (MyD88^−/−) and TLR4-defective mice (C3H/HeJ). Production of interferon (IFN)-γ-inducible protein 10 (IP-10) in response to treatment with γ-PGA was almost abolished in C3H/HeJ mice. In contrast to LPS, γ-PGA induced productions of TNF-α and IP-10 could not be blocked by polymyxin B. Furthermore, γ-PGA-induced interleukin-12 production was also impaired in immature dendritic cells (iDCs) from MyD88^−/− and C3H/HeJ mice. Downregulation of MyD88 and TLR4 expression using small interfering RNA (siRNA) significantly inhibited γ-PGA-induced TNF-α secretion from the RAW264.7 cells. γ-PGA-mediated intracellular signaling was markedly inhibited in C3H/HeJ cells. The antitumor effect of γ-PGA was completely abrogated in C3H/HeJ mice compared with control mice (C3H/HeN) but significant antitumor effect was generated by the intratumoral administration of C3H/HeN mice-derived iDCs followed by 2,000 kDa γ-PGA in C3H/HeJ. These findings strongly suggest that the antitumor activity of γ-PGA is mediated by TLR4. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Anti‐inflammatory and anti‐endotoxin properties of peptides derived from the carboxy‐terminal region of a defensin from the tick Ornithodoros savignyi

Antimicrobial peptides are small cationic peptides that possess a large spectrum of bioactivities, including antimicrobial, anti‐inflammatory and antioxidant activities. Several antimicrobial peptides are known to inhibit lipopolysaccharide (LPS)‐induced inflammation in vitro and to protect animals from sepsis. In this study, the cellular anti‐inflammatory and anti‐endotoxin activities of Os and Os‐C, peptides derived from the carboxy‐terminal of a tick defensin, were investigated. Both Os and Os‐C were found to bind LPS in vitro, albeit to a lesser extent than polymyxin B and melittin, known endotoxin‐binding peptides. Binding to LPS was found to reduce the bactericidal activity of Os and Os‐C against Escherichia coli confirming the affinity of both peptides for LPS. At a concentration of 25 µM, the nitric oxide (NO) scavenging activity of Os was higher than glutathione, a known NO scavenger. In contrast, Os‐C showed no scavenging activity. Os and Os‐C inhibited LPS/IFN‐γ induced NO and TNF‐α production in RAW 264.7 cells in a concentration‐dependent manner, with no cellular toxicity even at a concentration of 100 µM. Although inhibition of NO and TNF‐α secretion was more pronounced for melittin and polymyxin B, significant cytotoxicity was observed at concentrations of 1.56 µM and 25 µM for melittin and polymyxin B, respectively. In addition, Os, Os‐C and glutathione protected RAW 264.7 cells from oxidative damage at concentrations as low as 25 µM. This study identified that besides previously reported antibacterial activity of Os and Os‐C, both peptides have in addition anti‐inflammatory and anti‐endotoxin properties. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Lack of responsiveness of C3H/HeJ macrophages to lipopolysaccharide: the cellular basis of LPS-stimulated metabolism.

The rate of glucose utilization has been used as a measure of LPS-induced activation of cultures of C3H/HeN and C3H/HeJ spleen cells, peritoneal cells, and purified peritoneal adherent cells. Peritoneal cells utilized 40 to 60 times more glucose than did spleen cells and purified adherent monolayers were more active than mixed peritoneal cells, suggesting that only macrophage metabolism was being measured. The cell preparations for C3H/HeJ mice were not activated by Escherichia coli K235 LPS prepared by extensive phenol extraction, whereas C3H/HeN cells were activated by the LPS. Cells from both strains were activated by a commercially obtained E. coli 0111:B4 LPS and butanol-extracted K235 LPS. The addition of 10% C3H/HeN spleen cells to C3H/HeJ peritoneal cells resulted in a marked enhancement of glucose utilization. These findings suggest that LPS-induced enhancement of macrophage metabolism occurs both by direct action of LPS on macrophages as well as indirectly through activated lymphocytes.