In vivo dynamics of murine tumor necrosis factor-alpha gene expression. Kinetics of dexamethasone-induced suppression

Tumor necrosis factor-alpha (TNF) has been implicated as an important, proximal mediator of many of the pathophysiologic effects observed during septic shock. In vitro studies have demonstrated that the glucocorticoid dexamethasone (Dex) will suppress the production of TNF; yet, clinical studies have shown that glucocorticoids are not protective in septic shock. In this paper we described the in vivo effects of lipopolysaccharide (LPS) on the kinetics of local and systemic TNF production, the time dependent expression of TNF mRNA, and the suppression of both TNF mRNA and bioactive protein using a defined treatment protocol of Dex. Peritoneal macrophages were elicited by CBA/J mice in the injection of complete Freunds adjuvant and the mice challenged with an intraperitoneal injection of LPS 2 weeks later. Kinetic studies showed that the peak of TNF production occurred 1 hour post LPS injection and reached a maximum of 775 units/ml within the ascites and 26 units/ml within the plasma. Northern blot analysis of mRNA extracted from peritoneal cells showed a peak of mRNA 30 minutes post LPS challenge. Dose-response studies disclosed that 10 micrograms of LPS/mouse produced maximal TNF within the ascites fluid, and half-maximal stimulation occurred at 70 ng LPS/mouse. Mice treated with Dex in vivo before LPS challenge showed a dramatic reduction in TNF production within both the ascites and plasma, and Northern blot analysis showed a corresponding reduction in the TNF specific mRNA. Further studies revealed that mice treated with 4 mg/kg of Dex intraperitoneally 4 hours before, or at the time of LPS challenge, had dramatic reductions in TNF levels within both the ascites and plasma. However, delaying the treatment only 20 minutes after LPS injection failed to significantly reduce TNF in either compartment. These data may provide a rationale why glucocorticoids are not clinically efficacious in the treatment of septic shock, since there is rapid upregulation of LPS-induced TNF gene expression. By the time patients develop clinical signs and symptoms of septic shock there are already preformed, circulating levels of TNF.     

Differential induction of brain heme oxygenase-1 and heat shock protein 70 mRNA in sepsis

We examined gene expression of heat shock protein 70 (HSP70) and heme oxygenase-1 (HO-1), which is the rate limiting enzyme in heme catabolism and is also known as heat shock protein 32 (HSP32), in the rat brain using a sepsis model induced by bacterial lipopolysaccharide (LPS). Intraperitoneal injection of LPS (10 mg/kg) to rats caused the elevation of body temperature and white blood cell (WBC) counts as well as marked elevation of serum interleukin-6 (IL-6) level, showing the typical pathological characteristics of sepsis. In this model, HO-1 mRNA increased at 6 h after LPS administration and continued to rise until 30 h. In contrast, HSP70 mRNA increased only between 3 h and 6 h after LPS administration, returning completely to the control level by 12 h. HO-1 mRNA was expressed predominantly in the cortex and the medulla oblongata, while HSP70 mRNA was expressed mainly in the striatum. HO-1 and HSP70 mRNA levels thus showed distinctive time courses and tissue distribution in the brain, suggesting that gene expression of these heat shock proteins (HSPs) is separately regulated.     

Differential induction of nitric oxide synthase in various organs of the mouse during endotoxaemia: role of TNF-alpha and IL-1-beta.

BALB/c mice injected intraperitoneally with bacterial lipopolysaccharide (LPS) developed lethal septic shock. This was accompanied by significantly elevated concentrations of nitrite and nitrate in the plasma and expression of high levels of nitric oxide (NO) synthase activity in the lungs, heart, spleen and peritoneal macrophages. Mice pretreated with anti-tumour necrosis factor-alpha (TNF-alpha) monoclonal antibody or anti-interleukin-1 beta (IL-1 beta) polyclonal antibody were protected, in a dose-dependent manner, from endotoxin-induced mortality. This effect was accompanied by a significant reduction in plasma levels of nitrite and nitrate. Antibody treatment also reduced the level of NO synthase activity in peritoneal macrophages, spleen and heart but had no effect on enzyme expression in the lung. These results demonstrate that TNF-alpha and IL-1 beta play an important role in the induction of NO following administration of LPS and in the development of endotoxin-induced shock. In addition, NO synthase activity is differentially expressed in various organs and this may not always require TNF-alpha and IL-1 beta.     

Prophylaxis of lipopolysaccharide-induced shock by alpha-galactosylceramide

The NKT cell ligand alpha-galactosylceramide and its synthetic homologue KRN7000 stimulate rapid and copious secretion of IFN-gamma and TNF-alpha release, both of which are key mediators of LPS-induced shock. We showed that KRN7000, injected before or within 2 h after LPS challenge, was able to prevent endotoxic shock. KRN7000 induced survival when the mice were injected 6, 9, or 12 days before the first injection of LPS, and this protective effect was associated with reduction upon subsequent challenge in the levels of IFN-gamma, TNF-alpha, MCP-1, and an increase of IL-10. Further analysis showed that the animals treated with KRN7000 prior to LPS challenge had lower numbers of F4/80+, NKT, and NK cells and lower percentages of NKT cells that stained for intracytoplasmic IFN-gamma when compared with mice that were not treated with KRN7000. When MCP-1 was injected in KRN7000-treated mice, the lethal effect of LPS challenge was restored, and the numbers of F4/80+, NKT, and NK cells increased to levels similar to those in untreated mice following LPS challenge. Taken together, our data demonstrated that KRN7000, injected from 6 to 12 days before the first administration of LPS, prevented endotoxin shock by inhibiting IFN-gamma, TNF-alpha, and MCP-1 release.     

Differential expression of tumor necrosis factor and interleukin-6 by peritoneal macrophages in vivo and in culture.

To investigate the differences in cytokine regulation in vitro as compared to in vivo, we examined the synthesis of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) by peritoneal macrophages in response to lipopolysaccharide (LPS). Mice (CBA/J) were primed with an intraperitoneal injection of complete Freund's adjuvant and after 2 weeks, peritoneal cells were harvested for culture or mice were injected intraperitoneally with LPS for in vivo studies. In ascites fluid, TNF-alpha peaked 1 hour after LPS and returned to baseline levels by 4 hours. In contrast, TNF-alpha in the media reached maximum at 7 hours. Expression of TNF-alpha messenger (m)RNA in vivo was rapid but transient, as levels peaked at 15 minutes and returned to baseline 1 hour after LPS. In contrast, TNF-alpha mRNA in vitro became maximal at 1 hour, but remained elevated to 5 hours after LPS. In vivo, IL-6 in ascites fluid peaked at 2 hours, whereas in vitro, IL-6 continued increasing to 24 hours. In vivo, IL-6 mRNA reached maximum at 30 minutes, but fell below baseline by 1.5 hours after LPS. In contrast, IL-6 mRNA in vitro was sustained at maximal expression between 5 to 9 hours after LPS. These results demonstrate that both TNF-alpha and IL-6 synthesis is more rapid in vivo than in vitro. The rapid kinetics of cytokine expression in vivo must considered when designing strategies to inhibit cytokine action in vivo.     

Excretion of radioactivity in faeces and urine of rats injected with 3H,14C-lipopolysaccharide

The route of excretion of lipopolysaccharide (LPS) and its possible degradation in vivo was studied in rats using biosynthetically radiolabelled LPS from Salmonella abortus equi, carrying 3H activity exclusively in fatty acids and 14C activity in fatty acids and sugars. Following intravenous injection of the above LPS in AS2 rats with or without anaesthesia, excretion of radioactivity occurred mainly in the faeces and to smaller extent in urine. The rate of excretion was slow, a large part of the radioactivity being still present in the liver after 14 days. In faeces the percent recovery of 3H (18%) was lower than that of 14C (32%) suggesting loss of tritium activity and thereby of fatty acids from the excreted LPS. A similar loss of tritium was also found in the material remaining in the liver and spleen 14 days after LPS administration. In urine the material recovered during 14 days (about 7% of injected) was different from the original LPS, 70% of 14C activity being dialysable and practically all 3H activity being volatile. Similar results were also obtained following administration of the LPS intraperitoneally under anaesthesia. However, when the LPS was administered intraperitoneally without anaesthesia, in the majority of the animals, 90% of 14C and 54% of 3H was excreted in faeces within 3 days, suggesting that both route of administration and use of anaesthesia during injection influence the subsequent rate of excretion of LPS.     

Excretion of radioactivity in faeces and urine of rats injected with 3H,14C-lipopolysaccharide.

The route of excretion of lipopolysaccharide (LPS) and its possible degradation in vivo was studied in rats using biosynthetically radiolabelled LPS from Salmonella abortus equi, carrying 3H activity exclusively in fatty acids and 14C activity in fatty acids and sugars. Following intravenous injection of the above LPS in AS2 rats with or without anaesthesia, excretion of radioactivity occurred mainly in the faeces and to smaller extent in urine. The rate of excretion was slow, a large part of the radioactivity being still present in the liver after 14 days. In faeces the percent recovery of 3H (18%) was lower than that of 14C (32%) suggesting loss of tritium activity and thereby of fatty acids from the excreted LPS. A similar loss of tritium was also found in the material remaining in the liver and spleen 14 days after LPS administration. In urine the material recovered during 14 days (about 7% of injected) was different from the original LPS, 70% of 14C activity being dialysable and practically all 3H activity being volatile. Similar results were also obtained following administration of the LPS intraperitoneally under anaesthesia. However, when the LPS was administered intraperitoneally without anaesthesia, in the majority of the animals, 90% of 14C and 54% of 3H was excreted in faeces within 3 days, suggesting that both route of administration and use of anaesthesia during injection influence the subsequent rate of excretion of LPS.     

An early response to lipopolysaccharide is the elicitation of macrophages specialized for antigen degradation with negative regulatory effects on the induction of specific immune responses.

The ability of macrophages to catabolize antigens is relevant both as a means to process complex antigens before presentation to T cells and as a way to down-regulate immune responses by destroying the antigenicity of polypeptides. With these considerations in mind, we investigated the regulation of macrophage catabolic activity by lipopolysaccharide (LPS). Catabolic activity was quantitated by following the distribution and molecular form of 125I-labeled surface components of heat-killed Listeria monocytogenes after their uptake by macrophages. We compared the catabolic activity of macrophages from peritoneal exudates of mice injected intraperitoneally with saline or LPS and found that LPS-elicited macrophages displayed a greatly enhanced (threefold) rate of catabolism. This increase in catabolic activity peaked 3 days after LPS injection and slowly declined thereafter, approaching a base-line level after 3 weeks. The enhancement of catabolic activity was under Lps gene control. Macrophages that were elicited 3 days after intraperitoneal injection of LPS rapidly destroyed the antigenicity of bacterial antigens, expressed low levels of Ia molecules, and processed and presented antigen slowly when tested as antigen-presenting cells in vitro. We also showed that an injection of LPS before infection with L. monocytogenes resulted in diminished development of T-cell reactivity to this organism. These results suggest that LPS elicits a macrophage population specialized for antigen degradation functions, with negative regulatory effects on the induction of specific immune responses.     

Decrease in neutrophil migration induced by endotoxin and suppression of interleukin-1 production by macrophages in lactic dehydrogenase virus-infected mice.

Neutrophil (PMN) migration into the peritoneal cavity after intraperitoneal injection of lipopolysaccharide (LPS), chemotactic activity of PMN, interleukin-1 (IL-1) production by macrophages (M phi) and its ability to attract PMN in mice chronically infected with lactic dehydrogenase virus (LDV) were compared with those in uninfected control mice. PMN migration into the peritoneal cavity decreased in infected mice when LPS was injected intraperitoneally. PMN chemotactic activity did not show any difference following infection. To assess the mechanism of this decreased PMN migration, IL-1 production, which is responsible for PMN attraction, was studied in LDV-infected mice. IL-1 production by M phi derived from infected mice decreased and its ability to attract PMN was weak. IL-1 production by M phi from control and infected mice increased after treatment by indomethacin and LPS. PMN migration into the peritoneal cavity increased after treatment with indomethacin and LPS in both control and infected mice. However, the rate of increase of IL-1 production and PMN migration was greater in infected mice. These results suggest that the excess activation of cyclo-oxygenase-derived products (prostaglandins) in infected mice might be responsible for the suppression of IL-1 production by M phi, resulting in decreased PMN migration induced by endotoxin.     

Dehydroxymethylepoxyquinomicin (DHMEQ) can suppress tumour necrosis factor-α production in lipopolysaccharide-injected mice, resulting in rescuing mice from death in vivo

Dehydroxymethylepoxyquinomicin (DHMEQ), a new nuclear factor (NF)-κB inhibitor, has several beneficial effects, including the suppression of tumour growth and anti-inflammatory effects. DHMEQ can also suppress the production of tumour necrosis factor (TNF)-α induced by lipopolysaccharide (LPS) in vitro. In the present study, we examine the effects of DHMEQ on TNF-α production in vivo and on the survival of mice injected with LPS. When DHMEQ was injected into mice 2 h before LPS injection, the survival of the LPS-injected mice was prolonged. When DHMEQ was injected twice (2 h before LPS injection and the day after LPS injection), all the mice were rescued. The injection of DHMEQ 1 h after LPS injection and the day after LPS injection also resulted in the rescue of all mice. The serum levels of TNF-α in the mice that received both LPS and DHMEQ were suppressed compared to the mice that received only LPS. These results suggest that DHMEQ can be utilized for the prevention and treatment of endotoxin shock.     

Effect of a traditional Chinese herbal medicine ren-shen-yang-rong-tang (Japanese name: ninjin-youei-to) on hematopoietic stem cells in mice

Cell dynamics after intraperitoneal (i.p.) and oral administration of a traditional herbal medicine, ren-shen-yang-rong-tang (Japanese name: ninjin-youei-to, NYT), were investigated. When NYT was injected i.p. into C3H/He mice, numbers of spleen and peritoneal cells significantly increased in a dose-dependent manner and showed high levels from 4 to 21 days. Two peaks in the total cell number were observed on days 7 and 14 in the peritoneal cavities and spleen of C3H/He mice administered NYT. A marked accumulation of PMN cells in the peritoneal cavity and spleen was detected at 7 days after injection. The numbers of macrophages and lymphocytes also increased by i.p. administration of NYT. The thymus cell number decreased transiently between 4 and 7 days and thereafter returned to the control level. No significant change in the cell number of lymph nodes was observed. Such cellular accumulation was also detected in C3H/HeJ mice, a nonresponder strain to bacterial endotoxin, and athymic nude mice. The activity of colony-forming units in the spleen (CFU-S) of C3H/He as well as C3H/HeJ mice was markedly augmented by i.p. administration of NYT. NYT induced significant CSF production as detectable by its activity in the sera. In addition, oral administration of NYT for 10 days induced a significant increment of peripheral leukocytes and spleen cells and enhanced CFU-S activity of bone marrow cells as induced by i.p. administration, indicating that NYT acts on hematopoietic stem cells capable of differentiating to lymphocytes, macrophages and PMN cells into the periphery.     

Modulation of macrophage Ia expression by lipopolysaccharide: stem cell requirements, accessory lymphocyte involvement, and IA-inducing factor production.

The mechanism of induction of murine macrophage Ia expression by lipopolysaccharide (LPS) was studied. Intraperitoneal injection of 1 microgram of LPS resulted in a 3- to 10-fold increase in the number of IA-positive peritoneal macrophages (flow cytometry and immunofluorescence and a 6-to 16-fold increase by radioimmunoassay. The isolated lipid A moiety of LPS was a potent inducer of macrophage Ia expression. Ia induction required a functional myelopoietic system as indicated by the finding that the response to LPS was eliminated in irradiated (900 rads) mice and reinstated by reconstitution with bone marrow cells. Comparison of LPS-induced Ia expression in normal and LPS-primed mice revealed a faster secondary response to LPS. The memory response could be adoptively transferred to normal mice with nonadherent spleen cells prepared 60 days after LPS injection. Spleen cells prepared 5 days after LPS injection caused Ia induction in LPS-nonresponder mice; such induction was not observed in irradiated (900 rads) recipients. The cell responsible for this phenomenon was identified as a Thy-1+, immunoglobulin-negative nonadherent cell. The biosynthesis and expression of Ia were not increased by direct exposure of macrophages to LPS in vitro. Small amounts of LPS inhibited Ia induction by gamma interferon. LPS showed positive regulatory effects on Ia expression by delaying the loss of Ia expression on cultured macrophages and by stimulating the production of Ia-inducing factors. Supernatants from cultured spleen cells stimulated with LPS in vitro contained antiviral and Ia-inducing activity that was acid labile, indicating that the active factor is gamma interferon. We conclude that induction of Ia expression by LPS in vivo is a bone-marrow-dependent, radiation-sensitive process which involves the stimulation of a gamma interferon-producing accessory lymphocyte and a delay in Ia turnover.     

Role of invariant NKT cells in lipopolysaccharide-induced lethal shock during encephalomyocarditis virus infection

Viral infections can give rise to secondary bacterial infections. In the present study, we examined the role of invariant natural killer T (iNKT) cells in lipopolysaccharide (LPS)-induced lethal shock during encephalomyocarditis virus (EMCV) infection. Wild-type (WT) mice and Jα18 gene knockout (Jα18 KO) mice were inoculated with EMCV, 5 days prior to challenging with LPS. The survival rate of Jα18 KO mice subjected to EMCV and LPS was significantly higher than that of WT mice. TNF-α and nitric oxide (NO) production were increased in WT mice, than that in Jα18 KO mice, after the administration of EMCV and LPS. EMCV infection increased the number of iNKT cells and IFN-γ production by iNKT cells in WT mice. Moreover, EMCV infection enhanced the expression of Toll-like receptor 4 (TLR4) in the lung and spleen. IFN-γ also increased the expression of TLR4 in splenocytes. These findings indicated that EMCV infection activated iNKT cells, and IFN-γ secreted from the iNKT cells up-regulated the expression of TLR4 in various tissues. As a result, EMCV-infected mice were susceptible to LPS and easily developed the lethal shock. In conclusion, iNKT cells were involved in the development of LPS-induced lethal shock during EMCV infection.     

脂多糖刺激干扰素调节因子3促进小鼠腹腔巨噬细胞白介素-17表达

目的：探讨干扰素调节因子3（IRF3）对脂多糖（LPS）刺激小鼠腹腔巨噬细胞促进白细胞介素-17（IL-17） 表达的初步作用机制。方法：小鼠腹腔注射3%巯基乙酸肉汤,3 d 后提取C57BL/6 野生和IRF3 基因敲除小鼠的 腹腔巨噬细胞,培养过夜后添加LPS。收集细胞培养上清液,酶联免疫吸附试验检测细胞因子IL-17 和白细胞介素-6 （IL-6）的表达;提取细胞蛋白质,免疫印迹检测细胞核因子κB抑制蛋白（IκB）α、IRF3、磷酸化信号转导子和 转录激活子3（STAT3）的蛋白水平。结果：LPS 刺激小鼠腹腔巨噬细胞后,IRF3 可显著地促进IL-17 的产生,同 时伴随着IL-6 的产生、IκBα 蛋白的降解及STAT3 的磷酸化。结论：IRF3 可能通过促进IL-6 的产生,间接地激活 STAT3 的磷酸化,进而促进IL-17 的产生。     

Increase of heat-shock protein and induction of gamma/delta T cells in peritoneal exudate of mice after injection of live Fusobacterium nucleatum.

Fusobacterium nucleatum and Actinobacillus actinomycetemcomitans are Gram-negative rod periodontal pathogens. The peritoneal cavity of Institute of Cancer Research (ICR) mice was used as the local infection model. In vivo production of heat-shock proteins (hsp) was studied by injection of 1/10 minimum lethal dose (MLD) of each live bacteria into mice. Heat-shock proteins 70 and 60 were examined in the extract of peritoneal exudate cells (PEC) from mice injected intraperitoneally with either F. nucleatum or A. actinomycetemcomitans by using sodium dodecylsulphate-polyacrylamide gel electrophoresis and immunoblotting analysis. Although hsp are present in PEC without injection of the bacteria, both hsp increased and reached a peak on day 3 after F. nucleatum injection but not after A. actinomycetemcomitans. Kinetic study of gamma/delta cells in PEC after injection of bacteria showed that the increase of gamma/delta T cells was observed only in the PEC from mice injected with F. nucleatum but not A. actinomycetemcomitans. The gamma/delta T cells in PEC were either CD3+ and CD4+ or CD3+ and CD8+. The differential cell count of PEC suggested that gamma/delta T-cell induction is related to the expansion of the macrophage population. The phagocytic and chemiluminescence responses of macrophages against the same bacteria were compared after intensive immunization with live F. nucleatum and A. actinomycetemcomitans. Elevations of chemiluminescence response and phagocytic function by immunization were observed in the macrophages of mice immunized with F. nucleatum. These results suggest the sequential appearance of hsp, gamma/delta T cells and macrophage activation after fusobacterial infection.     

Lactoferrin protects against development of hepatitis caused by sensitization of Kupffer cells by lipopolysaccharide.

BALB/c mice were intravenously injected with lipopolysaccharide (LPS) (0.05 microg/g of body weight) 7 days after being primed with zymosan. Recombinant human lactoferrin (250 microg/g of body weight), intravenously administered 1 day before the injection of LPS, significantly lessened the severity of hepatitis, as assessed by levels of serum alanine transaminase compared to those seen when casein was administered. The transient rise of serum tumor necrosis factor alpha (TNF-alpha) after LPS treatment was also significantly lowered by the intravenous administration of lactoferrin, suggesting that the effect of lactoferrin was due to the suppression of TNF-alpha production. The following results indicate that the sites of action of lactoferrin for the suppression of the development of this type of hepatitis are Kupffer cells. Gadolinium chloride, a substance known to eliminate Kupffer cells, administered 1 day before LPS, inhibited the transient rise of TNF-alpha and protected against the development of hepatitis. Kupffer cells isolated from mice intraperitoneally injected with recombinant human lactoferrin became refractory to LPS. The specific interaction of recombinant human lactoferrin with the Kupffer cells was shown by a binding assay, which revealed two types of binding sites on mouse Kupffer cells. Of the two dissociation constants determined in this way, the lower dissociation constant, 0.47 x 10(-6) M, was within the range of the 50% effective doses for the suppression of TNF-alpha production. These results suggest that recombinant human lactoferrin administered to mice suppresses the production of TNF-alpha by Kupffer cells by directly associating with the binding sites on these cells.     

脂多糖对蜕膜NK细胞表达NKG2D水平的促进作用

为探讨脂多糖(LPS)及其受体TLR4相互作用并影响蜕膜NK细胞的可能机制,在孕E6.5给BALB/c×C57BL/6孕鼠腹腔注射LPS,而E10.5采用流式细胞术检测小鼠蜕膜NKG2D+TGF-β-NK细胞构成比,计算小鼠胚胎吸收率。此外,采用磁珠亲和细胞分选术纯化E10.5小鼠蜕膜NK细胞并培养,用LPS刺激所培养的细胞,并观察刺激后NK细胞表达NKG2D水平的变化。研究发现,在体内和体外实验中LPS刺激均可显著增高BALB/c×C57BL/6孕鼠蜕膜NK细胞NKG2D的表达水平,其中腹腔注射可显著增高胚胎吸收率。在体外细胞培养实验中,预先在培养基中加入抗TLR4抗体,则LPS刺激后细胞NKG2D表达水平无显著升高。这些结果提示,LPS与TLR4相互作用可增强小鼠蜕膜NK细胞NKG2D的表达,而NKG2D的过高表达不利于同种异基因妊娠成功。     

Gene expression of heme oxygenase-1 during glial activation by lipopolysaccharide

The effect of bacterial lipopolysaccharide (LPS) on the expression of mRNA encoding heme oxygenase-1 (HO-1), which is the rate limiting enzyme in heme catabolism and is also known as heat shock protein 32 (HSP32), was examined in primarily cultured rat glial cells. Treatment of cells with LPS elicited an increase in HO-1 mRNA, accompanying down regulation of delta-aminolevulinate synthase, in a dose-dependent fashion. HO-1 mRNA increased markedly at 12 h after LPS treatment (10 microg/ml) and reached a maximum at 24 h. In contrast, HSP70, a major heat shock protein, slightly increased only at 6 h after LPS treatment and returned to the control level by 12 h. These results suggest that HSPs are induced under separate regulation during glial activation by LPS through oxidative stress, a part of which is likely mediated by intracellular free heme.     

Activation of murine peritoneal macrophages by intraperitoneal administration of a traditional Chinese herbal medicine, xiao-chai-hu-tang (Japanese name: shosaiko-to)

Macrophage activation by a traditional Chinese herbal medicine, xiao-chai-hu-tang (Japanese name: shosaiko-to), was investigated. Intraperitoneal (i.p.) administration of shosaiko-to into (BALB/c x DBA/2)F1 mice resulted in marked activation of macrophages with respect to phagocytic and lysosomal enzyme activities (acid phosphatase and N-acetyl-beta-D-glucosaminidase) compared with the control. The maximal responses were induced by an i.p. injection of 3 mg shosaiko-to 4 days previously. Enhanced activities induced by shosaiko-to were also seen in C3H/HeJ mice, which is a non-responder strain to bacterial lipopolysaccharide (LPS). Significant macrophage accumulation in the peritoneal cavity and increased lysosomal enzyme activities were observed in mice injected with shosaiko-to. Shosaiko-to exhibited significant cytostasis-inducing activity. In addition, the administration of shosaiko-to led to a moderate expression of Ia antigen on the surface of peritoneal macrophages. These results suggest that shosaiko-to is a potent macrophage activator.     

Role of colony-stimulating activity in antitumor activity of Lactobacillus casei in mice

Wide-ranging differences were observed between the antitumor activities of 23 lactobacilli (13 species; 23 strains) and their capacities to elevate the level of serum colony-stimulating activity (CSA) by intraperitoneal administration in mice, and a good correlation existed between the two activities. The mechanism of enhanced production of CSA by administration of Lactobacillus casei YIT 9018 (LC 9018), one of the bacteria that had the strongest activities, and the role of CSA in antitumor activity of LC 9018 were studied. Colony-stimulating activity in the washing fluid from the peritoneal cavity of mice that had been administered LC 9018 intraperitoneally was elevated at 3 to 24 h after the injection, and CSA was also detected at elevated levels in the serum of the mice 6 to 12 h after injection. The cells responsible for the production of CSA after stimulation with LC 9018 seem to be the resident macrophages at the site of administration, because the resident macrophages of mice lavaged 1 h after an intraperitoneal administration of LC 9018 released CSA when they were cultured in vitro. Moreover, resident peritoneal macrophages of normal mice cultured with LC 9018 in vitro also produced CSA. Similar results were obtained with athymic nude mice, and the CSA-inducing activity of LC 9018 was diminished in the mice pretreated with carrageenan, which is selectively toxic to mature macrophages. Bone marrow cells matured to macrophages and polymorphonuclear cells by culture with the CSA induced by LC 9018 for 7 days. These matured macrophages showed strong antitumor activity both in vivo and in vitro. These results suggest that CSA plays important roles in the antitumor activity of LC 9018: it enhances not only the multiplication of committed precursor cells for macrophages and polymorphonuclear cells, but also the functional maturation of the precursor cells for macrophages which serve as potent effectors for tumor cells.