Inflammatory M1-like macrophages polarized by NK-4 undergo enhanced phenotypic switching to an anti-inflammatory M2-like phenotype upon co-culture with apoptotic cells.

BACKGROUND: NK-4 has been used to promote wound healing since the early-1950s; however, the mechanism of action of NK-4 is unknown. In this study, we examined whether NK-4 exerts a regulatory effect on macrophages, which play multiple roles during wound healing from the initial inflammatory phase until the tissue regeneration phase. RESULTS: NK-4 treatment of THP-1 macrophages induced morphological features characteristic of classically-activated M1 macrophages, an inflammatory cytokine profile, and increased expression of the M1 macrophage-associated molecules CD38 and CD86. Interestingly, NK-4 augmented TNF-α production by THP-1 macrophages in combination with LPS, Pam3CSK4, or poly(I:C). Furthermore, NK-4 treatment enhanced THP-1 macrophage phagocytosis of latex beads. These results indicate that NK-4 drives macrophage polarization toward an inflammatory M1-like phenotype with increased phagocytic activity. Efferocytosis is a crucial event for resolution of the inflammatory phase in wound healing. NK-4-treated THP-1 macrophages co-cultured with apoptotic Jurkat E6.1 (Apo-J) cells switched from an M1-like phenotype to an M2-like phenotype, as seen in the inverted ratio of TNF-α to IL-10 produced in response to LPS. We identified two separate mechanisms that are involved in this phenotypic switch. First, recognition of phosphatidylserine molecules on Apo-J cells by THP-1 macrophages downregulates TNF-α production. Second, phagocytosis of Apo-J cells by THP-1 macrophages and activation of PI3K/Akt signaling pathway upregulates IL-10 production. CONCLUSION: It is postulated that the phenotypic switch from a proinflammatory M1-like phenotype to an anti-inflammatory M2-like phenotype is dysregulated due to impaired efferocytosis of apoptotic neutrophils at the wound site. Our results demonstrate that NK-4 improves phagocytosis of apoptotic cells, suggesting its potential as a therapeutic strategy to resolve sustained inflammation in chronic wounds.

Tryptanthrin inhibits Th2 development, and IgE-mediated degranulation and IL-4 production by rat basophilic leukemia RBL-2H3 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Tryptanthrin is a compound isolated from Polygonum tinctorium, which is a known folk medicine with various biological activities. AIM OF THE STUDY: Allergic diseases are initiated by the development of allergen-specific T helper type 2 (Th2) cells and amplified by the degranulation of and cytokine release from basophils and mast cells during an effector phase. We found that Tryptanthrin could down-regulate IL-4 production by Th2 cells, while IFN-γ production by Th1 cells was not affected. Since IL-4 produced by basophils and effector Th2 cells has been shown to play important roles in the development and amplification of Th2-dominated allergic responses, we examined the effects of Tryptanthrin on the initiation and effector phase responses of Type I allergy in vitro. MATERIALS AND METHODS: To determine the mechanisms of Tryptanthrin-induced down-regulation of IL-4 production, the expression of Th2-specific transcription factors, c-Maf and GATA-3, was analyzed by RT-PCR. The effects of Tryptanthrin on Th cell differentiation were evaluated using CD4(+) T cells purified from spleen cells of Sugi basic protein (SBP)-immunized BALB/c mice. In primary cultures, cells were stimulated with SBP and antigen-presenting cells under neutral or Th2-skewing conditions in the presence or absence of Tryptanthrin. Cytokines produced by differentiated Th cells in secondary cultures were analyzed by ELISA. The effects of Tryptanthrin on IgE-mediated degranulation and IL-4 production were determined using rat basophilic leukemia (RBL-2H3) cells. Phosphorylation of ERK1/2 and Akt in Tryptanthrin-treated RBL-2H3 cells was analyzed to determine the mechanism of Tryptanthrin actions. RESULTS: Tryptanthrin suppressed c-Maf mRNA expression in Th2 clone cells, and even under Th2-skewing conditions, Tryptanthrin inhibited differentiation toward the Th2 phenotype, which is an essential event for the initiation phase of allergic diseases. Tryptanthrin also inhibited the IgE-mediated degranulation of and IL-4 production by RBL-2H3 cells, probably due to inhibiting IgE-mediated signaling pathways, including the phosphorylation of ERK1/2 and Akt. CONCLUSION: These findings suggest that Tryptanthrin effectively inhibits the effector and exacerbation responses, as well as the initiator responses, of Type I allergy. Thus, Tryptanthrin may have beneficial effects for immediate-type allergic responses.

Trehalose prevents adipocyte hypertrophy and mitigates insulin resistance.

Trehalose has been shown to evoke lower insulin secretion than glucose in oral saccharide tolerance tests in humans. Given this hypoinsulinemic effect of trehalose, we hypothesized that trehalose suppresses adipocyte hypertrophy by reducing storage of triglyceride and mitigates insulin resistance in mice fed a high-fat diet (HFD). Mice were fed an HFD and given drinking water containing 2.5% saccharide (glucose [Glc], trehalose [Tre], maltose [Mal], high-fructose corn syrup, or fructose [Fru]) ad libitum. After 7 weeks of HFD and saccharide intake, fasting serum insulin levels in the Tre/HFD group were significantly lower than in the Mal/HFD and Glc/HFD groups (P < .05). Furthermore, the Tre/HFD group showed a significantly suppressed elevation of homeostasis model assessment-insulin resistance compared with the Mal/HFD group (P < .05) and showed a trend toward lower homeostasis model assessment-insulin resistance than the Glc/HFD group. After 8 weeks of feeding, mesenteric adipocyte size in the Tre/HFD group showed significantly less hypertrophy than the Glc/HFD, Mal/HFD, high-fructose corn syrup/HFD, or Fru/HFD group. Analysis of gene expression in mesenteric adipocytes showed that no statistically significant difference in the expression of monocyte chemoattractant protein-1 (MCP-1) messenger RNA (mRNA) was observed between the Tre/HFD group and the distilled water/standard diet group, whereas a significant increase in the MCP-1 mRNA expression was observed in the Glc/HFD, Mal/HFD, Fru/HFD, and distilled water/HFD groups. Thus, our data indicate that trehalose prevents adipocyte hypertrophy and mitigates insulin resistance in HFD-fed mice by reducing insulin secretion and down-regulating mRNA expression of MCP-1. These findings further suggest that trehalose is a functional saccharide that mitigates insulin resistance.

Inhibitory effects of 2-amino-3H-phenoxazin-3-one on the melanogenesis of murine B16 melanoma cell line.

Hyperpigmentations are a serious concern addressed by both the medical community and the cosmetic industry through the development of agents that block melanin biosynthesis. In this study, we found that 2-amino-3H-phenoxazin-3-one (APO), isolated from extracts of the edible mushroom Agaricus bisporus Imbach, exhibited potent inhibitory effects on melanogenesis in B16 cells, a murine melanoma cell line. APO inhibited melanin biosynthesis at 1,000 times lower concentrations (IC(50)=1.31+/-0.08 microM) than kojic acid (IC(50)=1.31+/-0.13 mM), without causing cellular toxicity. APO did not directly inhibit the enzyme activity of tyrosinase, the rate-limiting melanogenic enzyme. Further study showed that APO inhibited the protein expression of tyrosinase and microphthalmia-associated transcription factor (MITF), a melanogenic transcription factor that regulates the expression of tyrosinase. These results suggest that APO is a promising depigmenting agent with both therapeutic and cosmetic value in preventing melanogenesis.

Propolis prevents diet-induced hyperlipidemia and mitigates weight gain in diet-induced obesity in mice.

We examined the hypolipidemic effect of propolis in a mouse obesity model induced by a high fat-diet. C57BL/6N mice were fed a high-fat diet ad libitum and given propolis extract intragastrically at 0 mg/kg (control), 5 mg/kg or 50 mg/kg twice daily for 10 d. Compared with mice in the control group, mice in the propolis extract-administrated groups displayed a reduction in all of the following parameters: body weight gain, weight of visceral adipose tissue, liver and serum triglycerides, cholesterol, and non-esterified fatty acids. Real-time polymerase chain reaction analysis of the liver showed down-regulation of mRNA expression associated with fatty acid biosynthesis, including fatty acid synthase, acetyl-CoA carboxylase alpha, and sterol regulatory element binding protein in the propolis-administrated mice. Subsequently, obese C57BL/6N mice that had been administered a high-fat diet were given propolis extract at 0 mg/kg (control), 2.5 mg/kg or 25 mg/kg for 4 weeks. The propolis extract treated mice showed a decrease in weight gain, a reduction of serum non-esterified fatty acids, and lipid accumulation in the liver. These results suggest that propolis extract prevented and mitigated high-fat diet-induced hyperlipidemia by down-regulating the expression of genes associated with lipid metabolism.

Lactosucrose inhibits body fat accumulation in rats by decreasing intestinal lipid absorption.

Lactosucrose (LS, 4(G)-beta-D-galactosylsucrose) is a non-digestible oligosaccharide, and the consumption of LS selectively increases the proportion of intestinal bifidobacteria. We examined in this study the hypolipidemic potential of LS. An oral triolein tolerance test on rats indicated that LS reduced the elevation of serum triglyceride (TG) and free fatty acids (FFA). Furthermore, LS inhibited the enzymatic digestion of triolein by pancreatic lipase in vitro. NMR spectroscopy showed that LS formed an intermolecular complex with triolein. The long-term consumption of a diet containing 5% LS for 8 weeks significantly decreased the weight of abdominal adipose tissue when compared with that of the control group. Thus, LS may reduce adipose tissue accumulation by inhibiting intestinal lipid absorption via a direct interaction with TG.

Anti-inflammatory and immunomodulatory properties of 2-amino-3H-phenoxazin-3-one.

Accumulating evidence suggests that nitric oxide (NO) and prostaglandin E(2) (PGE(2)) are involved in the pathogenesis of various chronic inflammatory diseases and cancer. During the course of a screening program to identify natural anti-inflammatory substances, we isolated the compound 2-amino-3H-phenoxazin-3-one (APO) from an extract of the edible brown mushroom Agaricus bisporus IMBACH. APO inhibited NO production by mouse peritoneal macrophages in response to the pro-inflammatory stimuli lipopolysaccharide (LPS) and interferon (IFN)-gamma (LPS/IFN-gamma) at low concentrations (IC(50)=1.5 microM) through reduced inducible NO synthase protein expression. PGE(2) production by LPS/IFN-gamma-stimulated macrophages was inhibited by APO at much lower concentrations (IC(50)=0.27 microM) than those required for the inhibition of NO production. Mechanistic analysis showed that APO inhibited both cyclooxygenase (COX)-1 and COX-2 enzyme activities with almost equal selectivity. Secretion of NO and the pro-inflammatory cytokine IL-6 by IFN-gamma-activated RAW264.7 cells, a murine macrophage-like cell line, was also dose-dependently reduced by APO. Furthermore, APO increased the secretion of the anti-inflammatory cytokine IL-4 by antigen-stimulated T cells and promoted the polarization of CD4(+) Th cells toward the anti-inflammatory Th2 phenotype at equimolar concentrations that inhibited NO production. Our results suggested that APO induced polarization toward the Th2 subset, at least in part through the down-regulation of IL-12 production. Thus, APO appears to have potent anti-inflammatory and immunoregulatory properties that may provide a promising therapeutic strategy for the treatment of T cell-mediated inflammatory autoimmune diseases as well as for bacteria-induced chronic-inflammatory diseases.

Effects of dietary lactosucrose (4G-beta-D-galactosylsucrose) on the IgE response in mice.

In this study, we examined the effects of dietary lactosucrose (LS, a non-digestible oligosaccharide) on the IgE response in mice immunized with ovalbumin (OVA)/alum. In addition to IgG1 and IgG2a responses, the anti-OVA IgE response in mice fed LS diets was dose-dependently suppressed, as compared with the control mice, while the serum total IgG levels were comparable. Moreover, dietary LS feeding inhibited antigen-specific IgE and IgG1 productions even after a second immunization. Regarding with cytokine production, when stimulated in vitro with OVA, splenocytes obtained from LS-fed mice produced a similar level of IFN-gamma, and lower levels of IL-4 and IL-5, as compared with the control mice. But IL-10 production by OVA-stimulated splenocytes was augmented in LS-fed mice, suggesting that IL-10 producing cells are responsible for the immunoregulatory effect of LS. Our findings indicate the further possibility that dietary LS supplementation can be used to prevent IgE-mediated allergic diseases.

Inhibition of chlamydia trachomatis growth by human interferon-alpha: mechanisms and synergistic effect with interferon-gamma and tumor necrosis factor-alpha.

We have evaluated the effect of natural human interferon (IFN)-alpha on the growth of chlamydia trachomatis in human epithelial cells in vitro and revealed that IFN-alpha has reduced both growth and infectivity of C. trachomatis. The effect of IFN-alpha was reversed by the addition of exogenous L-tryptophan and iron to the culture medium, suggesting that antichlamydial effect of IFN-alpha was caused by depletion of intracellular tryptophan and iron, both of which are essential for chlamydial growth. When IFN-alpha was combined with another antichlamydial cytokines, IFN-gamma and tumor necrosis factor (TNF)-alpha, the effect was synergistically enhanced. Therefore, IFN-alpha would act coordinately with other cytokines such as IFN-gamma and TNF-alpha, and play an important role in host defense against infection and in the establishment of persistent chlamydial infection of host, in which the organism remains viable, but in a culture-negative state.

Identification of a collagen production-promoting factor from an extract of royal jelly and its possible mechanism.

We have previously shown that royal jelly (RJ) promoted collagen production by skin fibroblasts in the presence of ascorbic acid-2-O-alpha-glucoside (AA-2G). In this study, we purified the honeybee RJ-derived collagen production-promoting factor (HBRJ-CPF) from an alkali-solubilized fraction of RJ by C18 reverse-phase column chromatography. The elution profile by the C18 column chromatography and the molecular mass of the purified HBRJ-CPF material coincided with those of 10-hydroxy-2-decenoic acid (10H2DA). We then examined the collagen production-promoting activities of several commercially available fatty acids contained in RJ. We found that 10H2DA and 10-hydroxydecanoic acid increased the collagen production in a dose-dependent manner. Furthermore, 10H2DA induced the fibroblast cell line, NHDF, to produce transforming growth factor-beta 1 (TGF-beta 1) which is an important factor for collagen production. As expected, the collagen production-promoting activity of 10H2DA was neutralized by the anti-TGF-beta 1 antibody. These result suggest that HBRJ-CPF identified as 10H2DA promoted the collagen production of AA-2G-treated fibroblasts by inducing TGF-beta 1 production.

Immuno-potentiating effects of the antler-shaped fruiting body of Ganoderma lucidum (Rokkaku-Reishi).

The immuno-potentiating effects of the antler-shaped fruiting body of Ganoderma lucidum (Rokkaku-Reishi, RR), which has been used as a traditional supplement for human health, were investigated in mice. BALB/c mice were administered orally with RR for 3 days at a dose of 50 mg/kg or 500 mg/kg, and interferon-gamma (IFN-gamma) production by splenocytes in response to lipopolysaccharide (LPS) was examined on day 4. The oral administration of 500 mg/kg of RR resulted in a significant increase (p<0.05) in IFN-gamma production. Stimulation of splenic adherent cells from these mice with LPS also resulted in a significant increase (p<0.05) in interleukin-12 (IL-12) production compared with that from the control mice, suggesting that splenic macrophages were activated by RR administration. Furthermore, 500 mg/kg of RR administered for 14 days resulted in a significant increase (p<0.05) in IFN-gamma production by splenocytes in response to both LPS and concanavalin A (Con A). These results suggest that not only splenic macrophages but also T cells were activated by the long-term treatment with RR in vivo. On the other hand, the production of interleukin-4 (IL-4), which is known as an allergic disease-related cytokine, was not affected by the long-term treatment with RR. Our results suggest that the oral administration of RR resulted in Th1-associated immuno-potentiating activities in vivo.

Characterization of royal jelly proteins in both Africanized and European honeybees (Apis mellifera) by two-dimensional gel electrophoresis.

In this study, the proteins contained in royal jelly (RJ) produced by Africanized honeybees and European honeybees (Apis mellifera) haven been analyzed in detail and compared using two-dimensional gel electrophoresis, and the N-terminal amino acid sequence of each spot has been determined. Most spots were assigned to major royal jelly proteins (MRJPs). Remarkable differences were found in the heterogeneity of the MRJPs, in particular MRJP3, in terms of molecular weights and isoelectric points between the two species of RJ. Furthermore, during the determination of the N-terminal amino acid sequence of each spot, for the first time, MRJP4 protein has been identified, the existence of which had been only implied by cloning of its cDNA sequence. The presence of heterogeneous bands of glucose oxidase was also identified. Thus, the results suggest that two-dimensional gel electrophoresis provides a suitable method for the qualitative analysis of the proteins contained in RJ derived from different honeybee species.

Royal jelly inhibits the production of proinflammatory cytokines by activated macrophages.

In this study, we have examined the anti-inflammatory actions of royal jelly (RJ) at a cytokine level. When supernatants of RJ suspensions were added to a culture of mouse peritoneal macrophages stimulated with lipopolysaccharide and IFN-gamma, the production of proinflammatory cytokines, such as TNF-alpha, IL-6, and IL-1, was efficiently inhibited in a dose-dependent manner without having cytotoxic effects on macrophages. This suggests that RJ contains factor(s) responsible for the suppression of proinflammatory cytokine secretion. We named the factor for honeybees RJ-derived anti-inflammatory factor (HBRJ-AIF), and further investigated the molecular aspects of it. Size fractionation study showed that HBRJ-AIF is composed of substances of low (< 5 kDa) and high (> 30 kDa) molecular weights, with the former being a major component. Chromatographic analysis showed that MRJP3 is one candidate for the HBRJ-AIF with high molecular weights. Thus, our results suggest that RJ has anti-inflammatory actions through inhibiting proinflammatory cytokine production by activated macrophages.

Royal Jelly prolongs the life span of C3H/HeJ mice: correlation with reduced DNA damage.

In this study, we investigate the effect of dietary Royal Jelly (RJ) on tissue DNA oxidative damage and on the life span of C3H/HeJ mice. In C3H/HeJ mice that were fed a dietary supplement of RJ for 16 weeks, the levels of 8-hydroxy-2-deoxyguanosine (8-OHdG), a marker of oxidative stress, were significantly reduced in kidney DNA and serum. Secondly, we determined the effect of dietary RJ on the life span in C3H/HeJ mice. The 50% mice survivals of intermediate- (about 6 mg/kg weight) and high-dose groups (about 60 mg/kg weight) were reached at significantly longer times than that of the control group according to the generalized Wilcoxon test (p<0.05). The average survival times were 88 weeks for the control group vs. 79 weeks for the low-dose group (about 0.6 mg/kg weight), 112 weeks for the intermediate-dose group and 110 weeks for the high-dose group, respectively, showing that RJ extended the average survival time by about 25% compared to the control group. However, RJ did not extend the total life span. These results indicated that dietary RJ increased the average life span of C3H/HeJ mice, possibly through the mechanism of reduced oxidative damage.

Oral administration of royal jelly inhibits the development of atopic dermatitis-like skin lesions in NC/Nga mice.

We have shown previously that in addition to IL-4, IL-5 and IL-10, antigen-specific interferon-gamma (IFN-gamma) production by spleen cells from ovalbumin (OVA)/Alum-immunized mice is inhibited by the administration of royal jelly (RJ). Since it has been shown that both Th1 and Th2 cytokines play pathogenic roles in the generation of atopic dermatitis (AD), we have examined whether RJ suppresses the development of AD-like skin lesions in NC/Nga mice induced by repeated application of picryl chloride (PiCl) under specific pathogen-free (SPF) conditions. Oral administration of RJ to the PiCl-treated NC/Nga mice inhibited the development of AD-like skin lesions in these mice as exemplified by the significant decrease in the total skin severity scores and the decrease in hypertrophy, hyperkeratosis, and infiltration of the epidermis and corium by inflammatory cells. IFN-gamma production by spleen cells from PiCl-treated NC/Nga mice in response to TNP-KLH was partially but significantly inhibited by the oral administration of RJ, while IFN-gamma production by Con A-stimulated spleen cells was not affected. Since inducible nitric oxide (NO) synthase (iNOS)-derived NO has been suggested as an important immunoregulatory mediator in inflammatory autoimmune diseases, we have also examined the expression of iNOS in the dorsal skin lesions of PiCl-treated NC/Nga mice. Interestingly, the expression of iNOS was significantly increased in the skin lesions of RJ-administered mice compared with those of control PBS-administered mice. Thus, our results suggest that RJ suppresses the development of AD-like skin lesions in PiCl-treated NC/Nga mice, possibly by a combination of down-regulating TNP-specific IFN-gamma production and up-regulating iNOS expression.

Major royal jelly protein 3 modulates immune responses in vitro and in vivo.

We have recently shown that royal jelly has potent antiallergic properties in a mouse model of immediate hypersensitivity. However, it is still unclear which components of royal jelly exhibit antiallergic activity. In this study, we have screened for antiallergic factors in royal jelly based on inhibition of IL-4 production by anti-CD3 stimulated spleen cells derived from OVA/alum-immunized mice. Using a series of column chromatographies, we purified a 70 kDa glycoprotein, major royal jelly protein 3 (MRJP3), that suppresses IL-4 production. In in vitro experiments, MRJP3 suppressed the production of not only IL-4 but also that of IL-2 and IFN-gamma by T cells concomitant with inhibition of proliferation. The MRJP3-mediated suppression of IL-4 production was also evident when lymph node cells from OVA/alum-immunized mice were stimulated with OVA plus antigen presenting cells. We next examined the purified suppressive factor on OVA/alum-induced allergic responses in mice. Interestingly, in spite of the antigenicity of MRJP3 itself as an extraneous foreign protein, intraperitoneal administration of MRJP3 inhibited serum anti-OVA IgE and IgG1 levels in immunized mice. In addition, heat-treated soluble MRJP3 treatment reduced its antigenicity while maintaining its inhibitory effects on antibody responses to OVA. These results indicate that MRJP3 can exhibit potent immunoregulatory effects in vitro and in vivo. Furthermore, considering the intriguing immunomodulatory effects of MRJP3, it may be of clinical significance to design MRJP3-derived antiallergic peptides by identifying the associated polypeptide regions.

Promoting effect of kaempferol on the differentiation and mineralization of murine pre-osteoblastic cell line MC3T3-E1.

A number of agents have been reported to influence osteoblastic differentiation and to prevent and treat bone loss. We found that kaempferol, a flavonoid identified in extracts of the medicinal plant, Polygonum tinctorium. Lour, had stimulatory effects on the differentiation and mineralization of the murine pre-osteoblastic cell line, MC3T3-E1. After enhancing the alkaline phosphatase activity, significant augmentation of calcification by kaempferol was observed between concentrations of 10 and 20 microM, without any marked effect on cell proliferation. When kaempferol was combined with ipriflavone, which is clinically applied to treat bone loss, calcification was synergistically augmented, suggesting that these two flavonoids may have different mechanisms of action. These results suggest that kaempferol may be a promising agent for the prevention or treatment of bone loss, especially when combined with ipriflavone.

Tryptanthrin inhibits interferon-gamma production by Peyer's patch lymphocytes derived from mice that had been orally administered staphylococcal enterotoxin.

Tryptanthrin, a biologically active compound found in the medicinal plant Polygonum tinctorium, reportedly has several biological activities. We investigated the effects of tryptanthrin on cytokine production by lymphocytes in response to staphylococcal enterotoxin B (SEB), which causes a variety of disorders in humans based on its induction of large amounts of immunostimulatory cytokines. Tryptanthrin dose-dependently inhibited interferon-gamma (IFN-gamma) and interleukin-2 production by mouse spleen cells and Peyer's patch (PP) lymphocytes in vitro. The efficacy of tryptanthrin was further studied in a mouse model in vivo. Tryptanthrin was administered orally 2 h after an oral challenge with SEB. Nineteen hours after SEB administration, PP lymphocytes were prepared, and IFN-gamma production by PP lymphocytes was examined. The production of IFN-gamma increased after SEB administration, and the elevated IFN-gamma production was significantly inhibited by tryptanthrin treatment. These results suggest that tryptanthrin may be effective in the treatment of disorders of the intestines, such as food poisoning, that are associated with activated lymphocytes.

Measurements of endothelial cell-to-cell and cell-to-substrate gaps and micromechanical properties of endothelial cells during monocyte adhesion.

The interaction between monocytes and endothelial cells is considered to play a major role in the early stage of atherosclerosis, and the involved endothelial cell micromechanics may provide us with important aspects of atherogenesis. In the present study, we evaluated (i) the endothelial cell-to-cell and cell-to-substrate gaps with the electric cell-substrate impedance sensing system, which can detect the nanometer order changes of cell-to-cell and cell-to-substrate distances separately, and (ii) the endothelial cell micromechanical properties with an atomic force microscope after application of monocytes to endothelial cells. Application of monocytic THP-1 cells to IL-1beta-stimulated human umbilical vein endothelial cells immediately decreased the electrical resistance of the endothelial cell-to-substrate (increase of the cell-to-substrate gap), whereas the endothelial cell-to-cell resistance (cell-to-cell gap) did not change. The elastic modulus of the endothelial cells decreased after 2-h monocyte application, indicating an increase of endothelial cell deformability. In conclusion, the interaction of the monocytes to the endothelial cells reduced the adhesiveness to the substrate and increased the deformability of endothelial cells. These changes in the adhesiveness and the deformability may facilitate migration of monocytes, a key process of atherogenesis in the later stage.

Lumin, a cyanine dye, enhances interleukin 12-dependent interferon gamma production by lipopolysaccharide-stimulated mouse splenocytes.

Lumin was orally administered to mice daily for 3 d, and on the day following the final administration, mice were sacrificed and splenocytes were stimulated with lipopolysaccharide (LPS). Splenocytes obtained from lumin-treated mice showed enhanced production of interferon gamma (IFN-gamma) and increased percentages of CD3+ cells. Although T cells are considered to be the source of IFN-gamma, it is unlikely that LPS directly stimulates T cells. Next we performed neutralization experiments using a monoclonal antibody (mAb) against interleukin (IL-)12 because this cytokine, which is produced by macrophages, has the direct ability to induce IFN-gamma production and the proliferation of activated T cells. This antibody inhibited IFN-gamma production by splenocytes. We thus show that orally administered lumin enhances IFN-gamma production by splenocytes when the latter are stimulated with LPS, a phenomenon that was observed in correlation with activation of T cells by IL-12, that is produced by macrophages.