TLR4 Monoclonal Antibody Research Articles

Mechanical Compression by Simulating Orthodontic Tooth Movement in an In Vitro Model Modulates Phosphorylation of AKT and MAPKs via TLR4 in Human Periodontal Ligament Cells.

Mechanical compression simulating orthodontic tooth movement in in vitro models induces pro-inflammatory cytokine expression in periodontal ligament (PDL) cells. Our previous work shows that TLR4 is involved in this process. Here, primary PDL cells are isolated and characterized to better understand the cell signaling downstream of key molecules involved in the process of sterile inflammation via TLR4. The TLR4 monoclonal blocking antibody significantly reverses the upregulation of phospho-AKT, caused by compressive force, to levels comparable to controls by inhibition of TLR4. Phospho-ERK and phospho-p38 are also modulated in the short term via TLR4. Additionally, moderate compressive forces of 2 g/cm2, a gold standard for static compressive mechanical stimulation, are not able to induce translocation of Nf-kB and phospho-ERK into the nucleus. Accordingly, we demonstrated for the first time that TLR4 is also one of the triggers for signal transduction under compressive force. The TLR4, one of the pattern recognition receptors, is involved through its specific molecular structures on damaged cells during mechanical stress. Our findings provide the basis for further research on TLR4 in the modulation of sterile inflammation during orthodontic therapy and periodontal remodeling.

Role of Toll-like receptor 4 signaling pathway in sevoflurane anesthesia-induced cognitive dysfunction in aged rats

Objective To evaluate the role of Toll-like receptor 4(TLR4)signaling pathway in sevoflurane anesthesia-induced cognitive dysfunction in aged rats. Methods Twenty-seven SPF healthy male Sprague-Dawley rats, aged 20 months, weighing 550-750 g, were divided into 3 groups(n=9 each)using a random number table: control group(C group), sevoflurane anesthesia group(S group)and TLR4 antagonist plus sevoflurane anesthesia group(TS group). TLR4 monoclonal antibody 30 μl was injected into the lateral cerebral ventricle in group TS, and the equal volume of serum containing no antibody was injected into the lateral cerebral ventricle in C and S groups.At 10 min after completion of injection, S and TS groups inhaled the mixture of 4% sevoflurane and 30% oxygen for 6 h, and group C only inhaled the mixture of air and oxygen.Morris water maze test was performed at 24 h after the end of sevoflurane anesthesia.The animals were sacrificed after completion of Morris water maze test, brains were removed and hippocampi were isolated for determination of nerve cell apoptosis(using TUNEL)and expression of activated caspase-3(using immunofluorescent staining). Nerve cell apoptosis rate was calculated.The expression of high-mobility group box 1 protein(HMGB1)mRNA in hippocampi was measured by Northern blot assay at 6 h after the end of sevoflurane anesthesia.The expression of amyloid precursor protein(APP)and amyloid beta protein(Aβ)in hippocampi was assessed by Western blot at 24 h after the end of sevoflurane anesthesia. Results Compared with C group, the escape latency was significantly prolonged, nerve cell apoptosis rate was increased, the expression of activated caspase-3, HMGB1 mRNA, APP and Aβ was up-regulated in group S, and nerve cell apoptosis rate was increased, the expression of activated caspase-3, HMGB1 mRNA, APP and Aβ was up-regulated(P 0.05). Compared with S group, the escape latency was significantly shortened, nerve cell apoptosis rate was decreased, and the expression of activated caspase-3, HMGB1 mRNA, APP and Aβ was down-regulated in group TS(P<0.05). Conclusion Activation of TLR4 signaling pathway is involved in the mechanism of sevoflurane anesthesia-induced cognitive dysfunction in aged rats. Key words: Toll-like receptor 4; Anesthetics, inhalation; Cognition disorders; Aged

Effects of probiotics on Toll‑like receptor expression in ulcerative colitis rats induced by 2,4,6‑trinitro‑benzene sulfonic acid.

The present study aimed to investigate the regulatory effect of probiotics on the expression of Toll-like receptors (TLRs) in an ulcerative colitis (UC) rat model, and to determine the role of probiotics in the underlying mechanisms through which UC develops and progresses in rat models. Rats were randomly allocated to one of the four following groups: i) The healthy control, ii) the model, iii) the Golden bifid treatment group, and iv) the TLR4 monoclonal antibody (TLR4mAb) intervention group. The UC rat model was established using 2,4,6-trinitro-benzene sulfonic acid. The general status and histological changes of rats were scored using the disease activity index and the histopathological scoring method, respectively. In these rats, the expression of TLR4 and TLR2 was measured using reverse transcription-quantitative polymerase chain reaction. The expression of TLR4 and TLR2 in the model group was significantly higher than that in the healthy control group. However, when compared with the model rats, those that received either Golden bifid treatment or TLR4mAb intervention exhibited significantly decreased mRNA expression levels of TLR4 and TLR2 (P<0.05). The development of UC is characterized by an abnormal immune response in the intestines. Probiotics alleviated inflammatory reactions in rats with UC. The underlying mechanism of UC may be associated with the expression of TLRs and the subsequent release of inflammatory cytokines.

Monoclonal antibody against Toll-like receptor4 attenuates ventilator-induced lung injury in rats by inhibiting MyD88- and NF-κB-dependent signaling.

The mechanisms through which mechanical ventilation causes non-infectious inflammatory diseases and lung injury are poorly understood. Animals models of this type of injury suggest that it involves signaling mediated by Toll‑like receptor (TLR)4and9. In this study, in order to gain further insight into the involvement of TLR4 in this type of injury, we performed invivo and invitro experiments to determine the mechanisms through which TLR4 triggers inflammation. We also examined whether the use of TLR4 monoclonal antibody(mAb) can alleviate this type of injury. For this purpose, rats were tracheotomized and administered intratracheal injections of anti‑TLR4 mAb or saline, and then ventilated for 4 h at a high tidal volume(HTV) of 40 ml/ kg or allowed to breathe spontaneously for the same period of time (controls). Alveolar macrophages(AMs) were isolated from the bronchoalveolar lavage fluid(BALF) of the rats and stimulated for 16h with tumor necrosis factor(TNF)‑α in the presence or absence of anti‑TLR4 mAb. Lung injury was assessed by examining lung histopathology, lung wet/dry weight ratio, BALF total protein and cytokine levels in BALF and plasma. The mRNA and protein expression levels of TLR4, TLR9, myeloid differentiation factor88(Myd88) and nuclear factor(NF)‑κB were measured in cultured macrophages. Compared to the controls (spontaneous breathing), the ventilated rats exhibited greater pulmonary permeability, more severe inflammatory cell infiltration/lung edema, and higher levels of interleukin(IL)‑1β, IL‑6 and TNF‑α in BALF and plasma. The AMs from the ventilated rats expressed higher mRNA and protein levels of TLR4, TLR9, Myd88 and NF‑κB compared with the macrophages from the spontaneously breathing rats. The ventilated rats pre‑treated with anti‑TLR4 mAb exhibited markedly attenuated signs of ventilation‑induced injury, such as less lung inflammation and pulmonary edema, fewer cells in BALF, and lower levels of ILs and TNF‑α in BALF and plasma. Similarly, the TNF‑α‑dependent increases in the mRNA and protein expression of TLR4, Myd88 and NF‑κB in AMs were attenuated when TNF‑α was co‑administered with anti‑TLR4 mAb than when TNF-α was administered alone. Co‑administering anti-TLR4 mAb also reduced the TNF‑α‑dependent secretion of ILs. On the whole, our data demonstrate that TLR4 contributes significantly to ventilation‑induced lung injury by activating the Myd88/NF‑κB pathway, and pre‑treating rats with anti‑TLR4 mAb partially protects them against this type of injury by inhibiting Myd88/NF-κB signaling.

Platelets Toll-like receptor-4 in Crohns disease.

Platelets are activated in Crohn's disease (CD) and interplay with leukocytes. Engagement of Toll-like receptor-4 (TLR-4), which is expressed in human platelets, may be involved in crosstalks between platelets and leukocytes leading to their mutual activation for host defense. Human neutrophil peptides (HNPs), lipoprotein binding peptides, and sCD14 were determined by enzyme-linked immunosorbent assays in 42 patients with active CD, in 43 patients with CD in remission, and in 30 healthy individuals. Neutrophil-platelet aggregates and binding of the TLR-4 monoclonal antibody to platelets were determined by flow cytometry. Levels of HNPs were higher in patients with CD than in controls (P = 0.0003 vs. active CD and P = 0.01 vs. CD in remission). Likewise, neutrophils with adhering platelets were higher in patients with active CD than in controls (P = 0.004). Binding of the TLR-4 antibody in patients with active CD was similar to that in controls, while patients in remission had significantly higher binding capacities (P = 0.59 and P = 0.003). Incubation of plasma from patients with active disease or patients in remission with platelets from healthy controls confirmed lower binding of the TLR-4 antibody in the presence of plasma from active diseased patients compared to controls (P = 0.039), possibly due to high levels of lipopolysaccharides, as suggested by high levels of sCD14 and lipoprotein binding protein. Our study indicates involvement of platelet TLR-4 in enhancing the secretion of antimicrobial peptides from neutrophils. While platelet aggregation can be due to a variety of mechanisms in inflammatory disease, the mutual activation of platelets and neutrophils may augment host defense.

The effect of lysophosphatidic acid on Toll-like receptor 4 expression and the nuclear factor-κB signaling pathway in THP-1 cells.

Toll-like receptors (TLRs) are major receptors that mediate the innate immune and inflammatory responses, of which TLR4 has been found most closely related to human atherosclerosis. After ligands are polymerized and activated by TLR, the mitogen-activated protein kinase and nuclear factor-κB (NF-κB) pathways are activated, leading to promotion of NF-κB-regulated transcription of inflammatory factors, thus playing a role in the physiological and pathological processes in atherosclerosis. Oxidized lipoproteins or their components, oxidized lipids, have been confirmed as endogenous TLR receptors. Lysophosphatidic acid (LPA) is an active component of low-density lipoprotein that induces vascular endothelial lesions. However, the mechanism of the TLR4/NF-κB signaling system involved in LPA-induced atherosclerosis has not been fully elucidated. In this study, we investigated the effects of LPA on TLR4 expression, nuclear translocation of NF-κB p65 subunit, and changes in the cytokine tumor necrosis factor α (TNF-α) in human THP-1 cells. LPA upregulated expression of the TLR4 mRNA and protein in THP-1 cells in a dose- and time-dependent manner, induced NF-κB p65 activation synchronously in THP-1 cells, and increased TNF-α secretion. After TLR4 was blocked using TLR4 monoclonal antibody, NF-κB p65 expression and TNF-α secretion were inhibited significantly. These data suggest that LPA can significantly upregulate TLR4 expression and promote NF-κB activation and proinflammatory cytokine secretion in THP-1 cells; it is possible that the TLR4/NF-κB signaling pathway mediates the atherogenic effect of LPA.

Obese Patient-Derived Sera have Proinflammatory Effect

Aim: Chronic inflammation of adipose tissues plays a key role in obesity-induced metabolic disorders. We examined effects of sera from obese patients with or without different compositions of metabolic disorders on the activation of Toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB) signaling pathway in the human monocytic leukemia cell line (THP-1), which have never been reported. Method: With or without pretreatment of TLR4 monoclonal antibody, THP-1 cells were incubated for 48h by sera from 45 obese patients with or without metabolic disorders and 15 controls. The level of TLR4 in THP-1 cells, intracellular level of phosphorylated NF-κB p65, IL-1β and TNF-α levels in cell culture supernatants were measured. Results: Compared with controls, the expression level of TLR4, NF-κB p65, IL-1β and TNF-α were significantly increased (p < 0.05) after cells were incubated with sera from obese patients. The more compositions of metabolic disorders, the higher increase of these factors. Pretreatment with TLR4 monoclonal antibody suppressed the degree of increase in these factors (p < 0.05). Conclusion: Sera from obese patients could induce the activation of TLR4/NF-κB signaling pathway in THP-1 monocytes by different degrees. The TLR4/NF-κB signaling pathway plays an important role in the proinflammatory effect of obese patient-derived sera.

Obese Patient-Derived Sera Have Proinflammatory Effect

Aim: Chronic inflammation of adipose tissues plays a key role in obesity-induced metabolic disorders. We examined effects of sera from obese patients with or without different compositions of metabolic disorders on the activation of Toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB) signaling pathway in the human monocytic leukemia cell line (THP-1), which have never been reported. Methods: With or without pretreatment of TLR4 monoclonal antibody, THP-1 cells were incubated for 48h by sera from 45 obese patients with or without metabolic disorders and 15 controls. The level of TLR4 in THP-1 cells, intracellular level of phosphorylated NF-κB p65, IL-1β and TNF-α levels in cell culture supernatants were measured. Results: Compared with controls, the expression level of TLR4, NF-κB p65, IL-1β and TNF-α were significantly increased (p <0.05) after cells were incubated with sera from obese patients. The more compositions of metabolic disorders , the higher increase of these factors. Pretreatment with TLR4 monoclonal antibody suppressed the degree of increase in these factors (p <0.05). Conclusions: Sera from obese patient s could induce the activation of TLR4/NF-κB signaling pathway in THP-1 monocytes by different degrees. The TLR4/NF-κB signaling pathway plays an important role in the proinflammatory effect of obese patient-derived sera.

Toll-like receptor 4 monoclonal antibody attenuates lipopolysaccharide-induced acute lung injury in mice.

Toll-like receptor 4 (TLR4) has an important role in the recognition of lipopolysaccharide (LPS) and in the activation of the inflammatory cascade. In the present study, the effect of TLR4 monoclonal antibody (mAb) on LPS-induced acute lung injury (ALI) was investigated in mice. A total of 45 male BALB/c mice were randomly divided into three groups, namely, the control (group C), sepsis (group S) and pretreatment groups (group P). Mice in group P were intraperitoneally treated with TLR4 mAb 1 h prior to the intraperitoneal administration of LPS. Following treatment with LPS for increasing times periods in groups S and P, the mRNA expression level of TLR4 in the lung tissue and the expression of inflammatory factors in the serum were analyzed by quantitative polymerase chain reaction and enzyme-linked immunosorbent assays, respectively. The degree of pulmonary edema, expressed as (wet weight - dry weight)/wet weight, as well as the lung injury scores, observed using a light microscope, were also analyzed. The results demonstrated that intraperitoneal administration of LPS in mice increased the mRNA expression levels of TLR4, the secretion of inflammatory factors in the serum, the degree of pulmonary edema and the lung injury score in a time-dependent manner. However, pretreatment with TLR4 mAb effectively attenuated the increased mRNA expression of TLR4 and the overproduction of inflammatory factors to correct the pulmonary edema and the elevated lung injury score induced by LPS. Therefore, TLR4 plays a critical role in LPS-induced ALI, and the TLR4 mAb decreases the secretion of inflammatory factors and attenuates the degree of pulmonary edema, thereby protecting the lungs from LPS-induced ALI.

Effect of precondition with Toll-like receptor 4 monoclonal antibody on LPS-induced acute lung injury in mice

Objective To investigate the effect of precondition with Toll-like receptor 4 monoclonal antibody (TLR4mAb) on lipopolysaccharide (LPS) -induced acute lung injury in mice.Methods A total of 45 male BALB/c mice were randomly divided into 3 groups:the control group ( group C),the sepsis group (group S) and the pretreatment group (group P).Mice in the group P and group S were injected intraperitoneally with LPS ( 10 mg/kg) to produce acute lung injury models.Mice in the group P was injected intraperitoneally with TLR4mAb (5 μg/g) 1 h before the injection of LPS.Expression of TLR4mRNA in lung tissue,expression of TNF-α and IL-6 in serum,water content of lung,and the pathomorphologic changes of lung were detected after 6 h,12 h and 24 h.One-way ANOVA was used for inter-group comparison and two-way ANOVA was used for intra-group comparison.Results Compared to group C,water content significantly increased at 12 h and 24 h in group S and group P; compared to group S,water content significantly decreased in group P at 12 h and 24 h.Compared to group C,the expression of IL-6 and TNF-α significantly increased in group S and group P at 6 h,12 h and 24 h; compared to group S,the expression of IL-6 and TNF-α significantly decreased at 6 h,12 h and 24 h in group P.Compared to group C,the expression of TLR4 mRNA increased significantly in group S and group P at 6 h,12 h and 24 h; compared to group S,the expression of TLR4 mRNA decreased significantly in group P at6 h,12 h and 24 h.Compared to group S,pathological damage of the lung was improved significantly in group P.Conclusions Precondition with TLR4mAb can attenuate LPS-induced acute lung injury,suppress the expression of inflammatory factors.Regulation of TLR4 pathway may be a promising therapeutic strategy for ALI. Key words: Acute lung injury; Sepsis; Toll-like receptor; Lipopolysaccharide; Precondition; Acute respiratory distress syndrome; Inflammatory reaction; Inflammatory factor

Serum of obesity induce the activation of TLR4/NF-κB signaling pathway on THP-1 cell line

To investigate the activation of TLR4/NF-κB signaling pathway and the level of inflammation in volunteers with varying degrees of metabolic disorders and the effect of intervention by TLR4 monoclonal antibody. THP-1 cell line was cultured with 10% serum of volunteers with various degrees of metabolic disorders (each 10 cases) for 24, 48 h. TLR4 and NF-κB p65 phosphorylation protein were measured by Western blot. TLR4 mRNA was measured by RT-PCR. The expression of IL-1β and TNF-α were detected by ELISA. The TLR4 mRNA and protein, the level of NF-κB p65 phosphorylation protein in THP-1 cell line and IL-1β and TNF-α expression in culture supernatant in normal, simple obesity, obesity with hyperglycemia, obesity with hyperlipidemia, obesity with three metabolic disorders groups had statistical significance (P < 0.05). The TLR4 mRNA and protein, the level of NF-κB p65 phosphorylation protein and the IL-1β and TNF-α expression in obesity with three metabolic disorders group were higher than those in other group, and those were time-dependent (P < 0.05). TNF-α expression in normal, simple obesity, obesity with hyperglycemia, obesity with hyperlipidemia, obesity with three metabolic disorders group at 48 h were (222 ± 32), (246 ± 52), (322 ± 32), (322 ± 34) and(490 ± 83)ng/L, respectively, IL-1β (ng/L) were (94 ± 19), (133 ± 19), (174 ± 22), (180 ± 30), (279 ± 38) (P < 0.05). The activation of TLR4/NF-κB signaling pathway on THP-1 cell line which is cultured by serum with various degrees of metabolic disorders is different, with the increasing of severity of metabolic disorders, TLR4/NF-κB signaling pathway activation levels also incr.

TLR2McAb and TLR4McAb decrease inflammatory cytokines expression and increase counts of Lactobacilli and Bifidobacteria in dextran sulfate sodium-induced colitis in mice

Objective To evaluate the effect of TLR2McAb and TLR4McAb on intestinal flora of DSS-induced colitis in mice. Methods Fifty healthy male BALB/c mice (SPF level), were randomly assigned into five groups: the control group( group A), the UC model group( group B), TLR2McAb intervention group( group C), TLR4McAb intervention group( group D) and TLR2McAb + TLR4McAb intervention group(group E). Clinical symptoms were evaluated by the disease activity index(DAI), while tissue sam ples were evaluated by histological scoring(HS). The quantities of mRNA for IFN-γ, IL-4 and IL-17 were determined by real-time PCR. Meanwhile, fecal samples were obtained directly from the cecum for microbiological studies. Results After the treatment with TLR2McAb and TLR4McAb, DAI and HS were decreased significantly. Compared with group A, inflammatory cytokines such as IFN-γ, IL-4 and IL-17 in group B were higher. Compared with group B, expression of these three cytokines in group C to E was all markedly decreased. Group A showed a considerable predominance of Lactobacillus spp and Bifidobacterium spp,while the UC model group showed a conspicuous increase of Escherichia coli and decreases of Lactobacillus spp and Bifidobacterium spp. After treatment with TLR2McAb or/and TLR4McAb, Lactobacillus spp and Bifidobacterium spp increased to the normal level. But counts of E. Coli in the three intervention groups were not changed. Conclusion TLR2McAb and TLR4McAb suppressed the development of DSS-induced colitis and increase cecum counts of Lactobacilli and Bifidobacteria. Key words: Ulcerative colitis; TLR2 monoclonal antibody(TLR2McAb); TLR4 monoclonal antibody (TLR4McAb); Intestinal flora

TLR4 monoclonal antibody blockade suppresses dextran‐sulfate‐sodium‐induced colitis in mice

Ulcerative colitis (UC) refers to a kind of inflammatory bowel disease, of which the accurate pathogenesis is not yet well understood. Recently, the toll-like receptor 4 (TLR4) and the TLR4 signaling pathway have been proved as playing an important role in the pathogenesis of UC. The objective of this study was to evaluate the effect of TLR4 monoclonal antibody on dextran-sulfate-sodium-induced colitis in a mouse model. We evaluated the effects of the TLR4 monoclonal antibody (TLR4mAb) on the development of dextran-sulfate-sodium-(DSS)-induced colitis. Tissue samples were evaluated by the disease activity index and histopathological score. Meanwhile, the mucosal mRNA expression of cytokines, tumor necrosis factor-alpha, interferon-gamma and interleukin-1beta were analyzed by semiquantitative reverse transcription polymerase chain reaction. The mucosal protein P38-MAPK, c-jun and c-fos expressions of the TLR4-P38MAPK pathway were analyzed using Western blot. After the treatment with TLR4mAb against DSS-induced colitis, the bodyweight was significantly increased and both disease activity index and histopathological score were decreased significantly. Furthermore, the mucosal expression of messenger RNA of tumor necrosis factor-alpha, interferon-gamma and interleukin-1beta were observed to be 8-15-fold more than the baseline, whereas the mucosal expressions of P38MAPK and c-jun were found to be decreased. Blocking TLR4 by TLR4mAb can prevent the development of DSS-induced colitis through the TLR4-P38MAPK-c-jun pathway.

Labour increases the surface expression of two toll‐like receptors in the cord blood monocytes of healthy term newborns

Mode of delivery may influence the innate immune system in newborns. We investigated the effect of maternal labour on the expression of two Toll-like receptors, TLR2 and TLR4, in monocytes obtained from healthy full-term newborns. Monocytes were obtained from cord blood of 48 newborns that have been vaginally delivered (VD) and 14 newborns delivered by elective caesarean section (CS) without labour. Peripheral blood was also obtained from 17 healthy adults. Surface expression of TLR2 and TLR4 in the monocytes was measured by antihuman TLR2 or TLR4 monoclonal antibody and immunofluorescence flow cytometry. TLR2 and TLR4 mRNA levels were evaluated by real-time PCR. CS newborns had a significantly lower level of TLR2 and TLR4 surface expression on monocytes than VD newborns. No significant difference was found between the surface expression of VD newborns and healthy adults. TLR2 and TLR4 mRNA levels in monocytes did not vary among the three study groups. Labour may up-regulate TLR2 and TLR4 on the cord blood monocytes of newborns at the protein level. Since TLRs are an important part of the innate immune system, our findings suggest that labour may be immunologically beneficial to normal newborns.

Reishi Polysaccharides Induce Immunoglobulin Production through the TLR4/TLR2-mediated Induction of Transcription Factor Blimp-1

The polysaccharides of Ganoderma lucidum (Reishi) possess immunomodulation activities; however, their mode of molecular action in regulating each cellular subset in the immune system is still not clear. Here, we investigate the function of the main polysaccharide fraction of Reishi (Reishi-F3) in B lymphocyte activation/differentiation. We find that Reishi-F3 causes mouse splenic B cell activation and differentiation to IgM-secreting plasma cells, and the process depends on Reishi-F3-mediated induction of Blimp-1, a master regulator capable of triggering the changes of a cascade of gene expression during plasmacytic differentiation. In human peripheral B lymphocytes, although Reishi-F3 fails to induce their activation, it is able to enhance antibody secretion, which is associated with Blimp-1 mRNA induction. The function of Reishi-F3 depends on the Toll-like receptors TLR4/TLR2 as neutralizing antibodies against TLR4/TLR2 block Reishi-F3-mediated induction of Blimp-1 mRNA and Ig secretion. We have shown that interaction of Reishi-F3 with TLR4/TLR2 followed by signaling through p38 MAPK is involved in the induction of Blimp-1 mRNA, whereas signaling through ERK, p38 MAPK, JNK, and IKK complex is involved in Reishi-F3-mediated Ig secretion. Furthermore, the differential mechanism of Reishi-F3 in mouse and human B cell activation is probably due to the presence of Blimp-1 regulatory site in human CD86 promoter. These results establish the signaling and molecular mechanisms of Reishi-F3 on promoting antibody secretion.

Immune receptors for polysaccharides from Ganoderma lucidum

This study was designed to identify and characterize the immune receptors for polysaccharides from Ganoderma lucidum, a Chinese medicinal fungus that exhibits anti-tumor activities via enhancing host immunity. We herein demonstrate that G. lucidum polysaccharides (GLPS) activated BALB/c mouse B cells and macrophages, but not T cells, in vitro. However, GLPS was unable to activate splenic B cells from C3H/HeJ mice that have a mutated TLR4 molecule (incapable of signal transduction) in proliferation assays. Rat anti-mouse TLR4 monoclonal antibody (Ab) inhibited the proliferation of BALB/c mouse B cells under GLPS stimulation. Combination of Abs against mouse TLR4 and immunoglobulin (Ig) achieved almost complete inhibition of GLPS-induced B cell proliferation, implying that both membrane Ig and TLR4 are required for GLPS-mediated B cell activation. In addition, GLPS significantly inhibited the binding of mouse peritoneal macrophages with polysaccharides from Astragalus membranaceus, which is known to bind directly with TLR4 on macrophage surface. Moreover, GLPS induced IL-1β production by peritoneal macrophages from BALB/c, but not C3H/HeJ, mice, suggesting that TLR4 is also involved in GLPS-mediated macrophage activation. We Further identified a unique 31 kDa serum protein and two intracellular proteins (ribosomal protein S7 and a transcriptional coactivator) capable of binding with GLPS in co-precipitation experiments. Our results may have important implications for our understanding on the molecular mechanisms of immunopotentiating polysaccharides from traditional Chinese medicine.

Micrococcus luteus teichuronic acids activate human and murine monocytic cells in a CD14- and toll-like receptor 4-dependent manner.

Teichuronic acid (TUA), a component of the cell walls of the gram-positive organism Micrococcus luteus (formerly Micrococcus lysodeikticus), induced inflammatory cytokines in C3H/HeN mice but not in lipopolysaccharide (LPS)-resistant C3H/HeJ mice that have a defect in the Toll-like receptor 4 (TLR4) gene, both in vivo and in vitro, similarly to LPS (T. Monodane, Y. Kawabata, S. Yang, S. Hase, and H. Takada, J. Med. Microbiol. 50:4-12, 2001). In this study, we found that purified TUA (p-TUA) induced tumor necrosis factor alpha (TNF-alpha) in murine monocytic J774.1 cells but not in mutant LR-9 cells expressing membrane CD14 at a lower level than the parent J774.1 cells. The TNF-alpha-inducing activity of p-TUA in J774.1 cells was completely inhibited by anti-mouse CD14 monoclonal antibody (MAb). p-TUA also induced interleukin-8 (IL-8) in human monocytic THP-1 cells differentiated to macrophage-like cells expressing CD14. Anti-human CD14 MAb, anti-human TLR4 MAb, and synthetic lipid A precursor IV(A), an LPS antagonist, almost completely inhibited the IL-8-inducing ability of p-TUA, as well as LPS, in the differentiated THP-1 cells. Reduced p-TUA did not exhibit any activities in J774.1 or THP-1 cells. These findings strongly suggested that M. luteus TUA activates murine and human monocytic cells in a CD14- and TLR4-dependent manner, similar to LPS.