Plasma glutamate-modulated interaction of A2AR and mGluR5 on BMDCs aggravates traumatic brain injury-induced acute lung injury.

The bone marrow-derived cell (BMDC)-associated inflammatory response plays a key role in the development of acute lung injury (ALI). Activation of adenosine A2A receptor (A2AR) is generally considered to be antiinflammatory, inhibiting BMDC activities to protect against ALI. However, in the present study, we found that in a mouse model of neurogenic ALI induced by severe traumatic brain injury (TBI), BMDC A2AR exerted a proinflammatory effect, aggravating lung damage. This is in contrast to the antiinflammatory effect observed in the mouse oleic acid-induced ALI model (a nonneurogenic ALI model.) Moreover, the A2AR agonist CGS21680 aggravated, whereas the antagonist ZM241385 attenuated, the severe TBI-induced lung inflammatory damage in mice. Further investigation of white blood cells isolated from patients or mouse TBI models and of cultured human or mouse neutrophils demonstrated that elevated plasma glutamate after severe TBI induced interaction between A2AR and the metabotropic glutamate receptor 5 (mGluR5) to increase phospholipase C-protein kinase C signaling, which mediated the proinflammatory effect of A2AR. These results are in striking contrast to the well-known antiinflammatory and protective role of A2AR in nonneurogenic ALI and indicate different therapeutic strategies should be used for nonneurogenic and neurogenic ALI treatment when targeting A2AR.

Local glutamate level dictates adenosine A2A receptor regulation of neuroinflammation and traumatic brain injury.

During brain injury, extracellular adenosine and glutamate levels increase rapidly and dramatically. We hypothesized that local glutamate levels in the brain dictates the adenosine-adenosine A(2A) receptor (A(2A)R) effects on neuroinflammation and brain damage outcome. Here, we showed that, in the presence of low concentrations of glutamate, the A(2A)R agonist 3-[4-[2-[[6-amino-9-[(2R,3R,4S,5S)-5-(ethylcarbamoyl)-3,4-dihydroxy-oxolan-2-yl]purin-2-yl]amino]ethyl]phenyl]propanoic acid (CGS21680) inhibited lipopolysaccharide (LPS)-induced nitric oxide synthase (NOS) activity of cultured microglial cells, an effect that was dependent on the protein kinase A (PKA) pathway. However, in high concentrations of glutamate, CGS21680 increased LPS-induced NOS activity in a protein kinase C (PKC)-dependent manner. Thus, increasing the local level of glutamate redirects A(2A)R signaling from the PKA to the PKC pathway, resulting in a switch in A(2A)R effects from antiinflammatory to proinflammatory. In a cortical impact model of traumatic brain injury (TBI) in mice, brain water contents, behavioral deficits, and expression of tumor necrosis factor-alpha, interleukin-1 mRNAs, and inducible NOS were attenuated by administering CGS21680 at post-TBI time when brain glutamate levels were low, or by administering the A(2A)R antagonist ZM241385 [4-(2-{[5-amino-2-(2-furyl)[1,2,4]triazolo[1,5-a][1,3,5]triazin-7-yl]amino}ethyl)phenol] at post-TBI time when brain glutamate levels were elevated. Furthermore, pre-TBI treatment with the glutamate release inhibitor (S)-4C3HPG [(S)-4-carboxy-3-hydroxyphenylglycine] converted the debilitating effect of CGS21680 administered at post-TBI time with high glutamate level to a neuroprotective effect. This further indicates that the switch in the effect of A(2A)R activation in intact animals from antiinflammatory to proinflammatory is dependent on glutamate concentration. These findings identify a novel role for glutamate in modulation of neuroinflammation and brain injury via the adenosine-A(2A)R system.

Clinical relevance of IL-1beta promoter polymorphisms (-1470, -511, and -31) in patients with major trauma.

Recent reports have indicated that IL-1[beta] is excessively released into the circulation during sepsis, and the expression level is closely correlated with the clinical course. Polymorphisms in the promoter region of IL-1B have been shown to affect LPS-induced IL-1[beta] transcription in vitro and IL-1[beta] plasma levels in healthy adults and to confer susceptibility to inflammatory diseases. However, it is not clear whether they confer susceptibility to sepsis after major trauma. The aim of this study was to search for association of polymorphisms (-1470G/C, -511T/C, and -31C/T) in the IL-1B gene promoter with the susceptibility to sepsis in a cohort of 238 major trauma patients. Genotyping of each patient for the three single-nucleotide polymorphisms was performed by restriction fragment length polymorphism-polymerase chain reaction method. Multivariate logistic regression analysis showed that the -1470 and -31 polymorphisms were associated with IL-1[beta] production by peripheral leukocytes in response to ex vivo LPS stimulation in an allele dose-dependent manner. Moreover, trauma patients carrying the -1470G or -31T alleles were more likely to develop sepsis compared with those with the -1470C or -31C allele (P = 0.014 and P = 0.038, respectively). Of the eight possible haplotypes composed of the three loci, the GCT and CTC haplotypes were associated with significantly higher and lower IL-1[beta] secretion (P = 0.005 and P = 0.035, respectively). Moreover, the GCT haplotype imparted higher risk of sepsis after severe injury (P = 0.04; odds ratio, 1.131; 95% confidence interval, 1.013-1.678). GCT homozygote patients also showed higher multiple organ dysfunction scores than CTC homozygote patients (P = 0.048). These data suggest that the IL-1[beta] promoter polymorphisms -1470G/C, -511T/C, and -31C/T may be functional both in vitro and in vivo. It may be possible to use these polymorphisms as relevant risk estimates for sepsis in trauma patients.

Distributions of glucocorticoid receptor gene polymorphisms in a Chinese Han population and associations with outcome after major trauma.

AIM: To investigate in a Chinese population the occurrence of polymorphisms Bcl I, N363S and ER22/23EK in the glucocorticoid receptor and their association with outcome of trauma. METHODS: In all, 266 healthy volunteers and 95 victims of major trauma were recruited. The presence of glucocorticoid receptor polymorphisms (ER22/23EK, N363S and Bcl I) was sought by restriction fragment length polymorphism analysis. The injured group were monitored as to respiratory, renal, hepatic, cardiovascular, haematological and central nervous functions. The association was determined between polymorphisms and the development of multiple organ dysfunction syndrome and sepsis after trauma. RESULTS: Only the Bcl I polymorphism was identified. The frequency of its G allele was 23.5% among volunteers and 26.3% among casualties. There were no significant differences in MOD score or sepsis rate between participants classified according to genotype. CONCLUSIONS: Only the Bcl I polymorphism of the glucocorticoid receptor gene is common in the Chinese Han population; it may not influence the development of complications following major trauma.

[Burn-blast combined injury].

Burn-blast combined injury is caused by two injury factors--heat and blast, which inflict the body at the same time or in sequence. The incidence of the combined injury is high either in wartime or in peacetime, and the mortality is much higher than that of an injury due to either one injury factor. In order to elucidate the mechanism, characteristics of the injury and the treatment of the combined injury, lots of studies were carried out both at home and abroad. The paper presents the data of burn-blast injury from a part of experimental studies and some clinical experience in the past forty years. The paper may be useful to medical doctors who may treat burn-blast injury in future.

Expressions of scavenger receptor, CD14 and protective mechanisms of carboxymethyl-beta-1, 3-glucan in posttraumatic endotoxemia in mice.

BACKGROUND: Previous studies in our laboratory have demonstrated the downregulation of surface expression of scavenger receptor (SR) and upregulation of CD14 in the presence of endotoxemia, which directly correlates to the excessive inflammatory response in lung injuries. This study aims to analyze the dynamics of the expressions of SR and CD14 in traumatic endotoxemia, and to investigate the receptor mechanism of immunomodulator, carboxymethyl-beta-1, 3-glucan (CMG), on the protection of traumatic infections. METHODS: By using a sublethal fracture plus endotoxemia model, experimental mice were assigned to sham group (Sham), trauma group (T), traumatic endotoxemia group (TE), and traumatic endotoxemia plus CMG group (TE + C). Alveolar macrophages were isolated from each group. Expressions of SR and CD14 were examined at the cell and tissue levels by immunohistochemistry assay. The effects of CMG on the phagocytosis of alveolar macrophages, tissue injury, and mortality were also determined. RESULTS: Expressions of SR and CD14 in lungs and livers decreased and increased, respectively. Alteration of SR and CD14 levels was more evident in lungs than in livers in posttraumatic endotoxemia. CMG up-regulated the SR expression in lipopolysaccharide-stimulated alveolar macrophages, alleviated the tissue injury, reduced mice mortality, and increased the opsonin-independent phagocytosis of Staphylococcus aureus, which was inhibited by SR mono-antibody. CONCLUSION: Significant correlation was found between inflammatory responses and the imbalance between SR and CD14 in posttraumatic endotoxemia. The more dramatic changes in lungs might be related to the sequential preferred injury in uncontrolled inflammation. CMG could be a promising bioactive reagent in immunomodulating sepsis.

Vascular endothelial growth factor and its receptor expression during the process of fracture healing.

OBJECTIVE: To study the expression regularity of vascular endothelial growth factor (VEGF) during the process of fracture healing, and the type of VEGF receptor expressed in the vascular endothelial cells of the fracture site. METHODS: The fracture model was made in the middle part of left radius in 35 rabbits. The specimens from the fracture site were harvested at 8, 24, 72 hours and 1, 3, 5, 8 weeks, and then fixed, decalcified, and sectioned frozenly to detect the expression of VEGF and its receptor at the fracture site by in situ hybridization and immunochemical assays. RESULTS: VEGF mRNA and VEGF expression was detected in many kinds of cells at the fracture site during 8 hours to 8 weeks after fracture. Flt1 receptor of VEGF was found in the vascular endothelial cells at the fracture site during 8 hours to 8 weeks after fracture, and strong expression of flk1 receptor was detected from 3 days to 3 weeks after fracture. CONCLUSIONS: The expression of VEGF and flt1 receptor appears during the whole course of fracture healing, especially from 1 to 3 weeks. Flk1 receptor is highly expressed in a definite period after fracture. VEGF is proved to be involved in the vascular reconstruction and fracture healing.

[Different effects of adenosine A2A receptors in the models of traumatic brain injury and peripheral tissue injury].

Recently, activation of the adenosine A2A receptors has been shown to exert protection against peripheral tissue injuries but aggravation in the central nervous system (CNS) injuries. To explore the different effects of adenosine A2A receptors and try to perform some new treatment strategies for peripheral tissue and CNS traumas, we constructed the mouse models of skin trauma, skin combined radiation-impaired wound and traumatic brain injury (TBI), respectively. Wild type mice and A2A receptor gene knockout mice were both used in the experiments. In skin trauma and combined radiation-impaired wound models, the time of wound healing was observed, while in TBI model, neurological deficit scores, water content in injured brain and glutamate concentration in cerebral spinal fluid (CSF) were detected at 24 h after TBI. The results showed that in skin trauma and combined radiation-impaired wound models, CGS21680 (an agonist of the A2A receptors) promoted while A2A receptor gene knockout delayed the course of skin wound healing. On the contrary, in TBI model, A2A receptor gene knockout, not CGS21680, showed a protective role by inhibition of glutamate release. These data further indicate that promoting glutamate release may account for the different effects of A2A receptor activation in CNS injury and peripheral tissue injury models. These findings may provide some experimental evidence and a new strategy for clinical treatment of peripheral tissue damages by agonists of A2A receptors, while treatment of CNS injuries by antagonists of A2A receptors.

Identification of interleukin-6 promoter polymorphisms in the Chinese Han population and their functional significance.

OBJECTIVE: Interleukin-6 (IL-6) is a pivotal cytokine in both innate and adaptive immunity. Several polymorphisms in the IL-6 promoter have been reported in Western populations. However, little is known about their occurrence in the Chinese population. The functionality of IL-6 promoter polymorphisms remains controversial. DESIGN: Genetic, functional, and association studies. SETTING: National key laboratory of trauma and departments of traumatic surgery in two teaching hospitals. SUBJECTS: A total of 348 healthy blood donors and 105 patients with major trauma. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Identification of polymorphisms in the IL-6 promoter was performed using sequencing and restriction fragment length polymorphism methods. Their functionality was assessed by observation of transcription activity, IL-6 production, and their clinical relevance in 105 patients with major trauma. Only one variant (C-572G) was identified in IL-6 promoter in Chinese Han population. This polymorphism was associated with IL-6 production by peripheral leukocytes in response to ex vivo lipopolysaccharide stimulation in an allele-dose-dependent effect. The C-->G variation at position -572 could reduce transcriptional activity of the IL-6 promoter as shown in both U-937 and K562 cell lines. Moreover, the -572 polymorphism was associated with lower risk of sepsis in major trauma patients. CONCLUSIONS: The -572 polymorphism, a unique variation in the IL-6 promoter in the Chinese Han population, may be used as a relevant risk estimate for sepsis in trauma patients.

Regulation of vascular endothelial growth factor on the expression of fracture healing-related factors.

OBJECTIVE: To study the effect of vascular endothelial growth factor (VEGF)and anti-VEGF on the expression of fracture healing-related factors and observe pathological changes at fractured sites. METHODS: Fracture models were established in 105 New Zealand white rabbits and they were randomly divided into control group, VEGF group and anti-VEGF group. The relevant factors expression at fractured sites was assayed and pathological changes were observed in decalcified samples at 8, 24, 72 hours and 1,3,5,8 weeks after fracture. RESULTS: After application of VEGF, the expression of BMP appeared earlier and expression time lasted longer. On the contrary, anti-VEGF completely inhibited the expression of BMP. The fractured sites were filled with fibrous callus, cartilaginous callus and bony callus at the 3rd week and woven bone was constructed at the 5th week. Fracture healing was accomplished at the 8th week in VEGF group. In anti-VEGF polyclonal antibody group, cellular necrosis increased at early period. Continuous focal necrosis was seen in the fractured sites from the 1st week to 5th week. Vascularization reduced obviously at the 3rd week. CONCLUSIONS: Fracture healing is a result of mutual regulation and coordination among many factors. VEGF may be an important factor in fracture healing.

[Establishment of a selective inactivation adenosine A2A receptors mice model].

OBJECTIVE: To establish a mice model with selective inactivation adenosine A2A receptors (A2ARs) in peripheral white blood cells (PWBC). METHODS: A2ARs were selectively inactivated in PWBCs by transplanting bone marrow cells (BMCs) from A2AR knockout (KO) mice into their wild type (WT) littermates after a single total body irradiation of 9.5 Gy or fractionated total body irradiation of 6.2 Gy x 2. The efficiency of reconstitution of bone marrow-derived cells in chimeric mice was assessed. RESULTS: PCR band patterns changed from the recipient pattern (one band of 330 bp) to the donor (two bands of 300 and 330 bp) pattern. Immunohistochemistry analysis showed that 10.21% of cells were A2AR+ in PWBCs in KO--> WT mice, whereas 96.72% of cells were A2AR+ in WT mice. The survival rates of mice irradiated with 6.2 Gy x 2 and transplanted with more than 6 x 10(6) BMCs were about 91%. CONCLUSION: A murine model of selective inactivation adenosine A2A receptors in PWBCs was established successfully.

Functional significance of the TLR4/11367 polymorphism identified in Chinese Han population.

Toll-like receptor 4 (TLR4) is the central signaling receptor for lipopolysaccharide (LPS) in mammals. This study was designed to investigate the functional significance of the G11367C polymorphism, which is a novel variant we identified in the 3' untranslated region of TLR4 gene in Chinese Han population. Three hundred seventy healthy volunteers were selected. The TLR4/11367 polymorphism was genotyped using single-tube bidirectional allele-specific amplification method. The TLR4 protein expression on peripheral leukocytes and plasma tumor necrosis factor alpha levels were determined by means of flow cytometry and enzyme-linked immunosorbent assay. The post-transcriptional effect of the 11367 polymorphism was evaluated by means of reporter gene assay and real-time quantitative polymerase chain reaction. The G11367C polymorphism is a common allele in Chinese Han population, with minor allele frequency of 14.7%. In response to ex vivo LPS stimulation, the TLR4 expression on the surface of peripheral leukocytes and the plasma tumor necrosis factor alpha levels were significantly lower in carriers of 11367C variant allele than in carriers of 11367G allele. This association was allele dose dependent. We also found that the activity and the mRNA expression of luciferase was significantly smaller in human embryonic kidney 293 cells transfected with construct containing 11367C allele than in those transfected with construct containing 11367G allele. Together, these results suggest that the TLR4/11367 polymorphism may be a functional single nucleotide polymorphism, which could attenuate the LPS-induced transmembrane signaling through the alteration of post-transcriptional regulation of 3' untranslated region and target gene expression.

[Relationship of myeloid differentiation-2 gene promoter polymorphisms with susceptivity of complications after severe trauma in Chinese Han population].

OBJECTIVE: To investigate the polymorphisms of myeloid differentiation-2 (MD-2) gene promoters, and to explore whether such polymorphisms are associated with the susceptibility to multiple organ dysfunction syndrome (MODS) and sepsis in Chinese Han population. METHODS: Using polymerase chain reaction-restriction fragment length polymorphism method, the authors detected the single nucleotide polymorphisms of the promoter region of MD-2 gene at position - 1625C/G in 105 severe trauma patients (42 with sepsis). The organ function was scored. RESULTS: The frequency of CC genotype in MD-2 gene promoter region at position - 1625 was 0.5 (21/42) in septic patients and 0.7 (44/63) in non-septic patients. The frequency of CG genotype was 0.38 (16/42) in septic patients and 0.27 (17/63) in non-septic patients. The frequency of GG genotype was 0.12 (5/42) in septic patients and 0.03 (2/63) in non-septic patients. The MODS scores in trauma patients carrying G allele at position - 1625 were significantly higher than those carrying C allele (P<0.001 for dominant effect, and P>0.05 for recessive effect). Moreover, trauma patients carrying G allele appeared to have higher risk of sepsis comparing to those carrying C allele (OR 0.477, 95% CI 0.266-0.855, P<0.05). Sepsis morbidity was significantly different between subjects with C and G alleles (P<0.05 for dominant effect, P>0.05 for recessive effect). CONCLUSIONS: The polymorphisms of the promoter region of MD-2 gene at position - 1625 C/G is correlated with MODS and sepsis after severe trauma in Chinese Han population. The people with - 1625 G allele in the promoter region of MD-2 gene may be a risk factor of severe complications.

Comparative observation of protective effects of earplug and barrel on auditory organs of guinea pigs exposed to experimental blast underpressure.

OBJECTIVE: To explore the protective effects of earplug and barrel on auditory organs of guinea pigs exposed to experimental blast underpressure (BUP). METHODS: The hearing thresholds of the guinea pigs were assessed with auditory brainstem responses (ABR). The traumatic levels of tympanic membrane and ossicular chain were observed under stereo-microscope. The rate of outer hair cells (OHCs) loss was analyzed using a light microscope. The changes of guinea pigs protected with barrel and earplug were compared with those of the control group without any protection. RESULTS: An important ABR threshold shift of the guinea pigs without any protection was detected from 8h to 14d after being exposed to BUP with a peak ranging from -64.5 kPa to -69.3 kPa ( P<0.01). The rate of perforation of tympanic membrane reached 87.5% and that of total OHCs loss was 19.46% +/- 5.38% at 14d after exposure. The guinea pigs protected with barrel and earplug had lower ABR threshold and total OHCs loss rate compared with the animals without any protection (P<0.01). All of the tympanic membrane and ossicular chain of the protected animals maintained their integrities. Meanwhile, the guinea pigs protected with the barrel had lower ABR threshold and total OHCs loss rate than those with earplug (P<0.01). CONCLUSIONS: The earplug and barrel have protective effects against BUP-induced trauma on auditory organs of the guinea pigs and the protective effects of barrel are better than those of earplug.

[The relationship of the expression of principal pattern recognition receptor with the pulmonary injury in abdominal infection-induced sepsis].

OBJECTIVE: In order to investigate the evidence of the synergistic effects of bacterial components, to observe the relationship of the expression of lipopolysaccharide (LPS) receptors [CD14, Toll-like receptor 4 (TLR4), scavenger receptor (SR)], lipoprotein receptor (TLR2) and bacterial DNA receptor (TLR9) with pulmonary injury in abdominal infection-induced sepsis. METHODS: 30 mice were used and randomly divided into cecal ligation puncture (CLP) (n = 15) and sham (n = 15) groups. The animals were respectively sacrificed 8, 12 and 24 (each point n = 5) hours following CLP and sham CLP. The lungs were removed and immediately stored in liquid nitrogen for TLRs mRNA, tumor necrosis factor (TNF) alpha and myeloperoxidase (MPO) assay. To detect the expression of CD14, TLR4, SR, TLR2 and TLR9 mRNA by reverse-transcription polymerase chain reaction, to detect the TNF-alpha content of the lung tissue by enzyme-labeled immunosorbent assay, and to assay the MPO activity of the lung tissue spectrophotometer. RESULTS: It was found that the expression of receptors for LPS, BLP and bacterial DNA in pulmonary tissues was markedly changed in CLP-induced sepsis, showing upregulation of CD14 mRNA (1.143 +/- 0.139, t = 0.022, P < 0.05), TLR2 mRNA (0.418 +/- 0.102, t = 0.021, P < 0.05), TLR4 mRNA (0.595 +/- 0.052, t = 0.0001, P < 0.01) and TLR9 mRNA (0.743 +/- 0.178, t = 0.0023, P < 0.01) at different degrees (P < 0.05 or P < 0.01) after postinjury 8 h, among which the expression of TLR9 mRNA kept increasing. The expression of SR mRNA (8 h: 0.659 +/- 0.159; 12 h: 0.429 +/- 0.061; 24 h: 0.300 +/- 0.045; t = 0.029, P < 0.05; t = 0.001, P < 0.01; t = 0.003, P < 0.01) showed continuous down-regulation. CONCLUSION: There was a marked correlation between the changes of pattern-recognition receptor expression and the increases of MPO and TNF-alpha levels in pulmonary tissues.

Effects of haplotypes in the interleukin 1beta promoter on lipopolysaccharide-induced interleukin 1beta expression.

Previous studies have indicated that there are 3 common haplotypes composed of the -1470, -511, and -31 loci in the interleukin 1beta (IL-1beta) promoter in the Chinese population. The purpose of this study was to investigate the relationship between these haplotypes and lipopolysaccharide (LPS)-stimulated IL-1beta expression by whole blood leukocytes in vitro and to evaluate the effects of these haplotypes on IL-1beta gene transcription. Genomic DNAs were obtained from 105 healthy subjects. The genotypes at the 3 sites of the IL-1beta promoter were determined by restriction fragment length polymorphism analysis. Haplotype frequency was evaluated by using the Arlequin software. Plasma IL-1beta level was measured by enzyme-linked immunosorbent assay. The transcriptional activity of the haplotypes was determined by in vitro reporter gene. The results indicated that after the exposure to LPS, whole blood leukocytes from subjects with the homozygous haplotype -1470G, -511C, and -31T (G-C-T) produced more IL-1beta in vitro than those from subjects with haplotype -1470C, -511T, and -31C (C-T-C) and that the transcriptional activity of the haplotype G-C-T was also higher than that of the haplotype C-T-C. It is suggested that the haplotypes of the IL-1beta promoter influence the expression and transcriptional activity of the IL-1beta gene and that the upregulation of IL-1beta gene expression after LPS exposure in subjects with haplotype G-C-T may be due to an increased transcriptional activity of the haplotype.

Silencing effect of recombinant plasmids PPARgamma-pSUPER-EGFP on the expression of peroxisome proliferator-activated receptor gamma.

OBJECTIVE: To investigate the silencing effect of gene encoding peroxisome proliferator-activated receptor gamma (PPARgamma) on the expression of tumor necrosis factor alpha (TNFalpha) by constructing vectors for RNA interference in RAW264.7 cells. METHODS: The pSUPER-EGFP vectors were used to transcribe functional small interfering RNA (siRNA). Four pairs of oligonucleotides (64 nt) targeting PPARgamma gene were inserted into the downstream of the H1 promotor, with their veracity confirmed by double digestion and sequencing. Western blotting and immunofluorescence assay were used to examine the silencing effect of PPARgamma gene in RAW264.7 cells. Meanwhile, the TNFalphalevel was determined by Sandwich ELISA. RESULTS: Compared with other recombinant pSUPER-EGFP vectors (R-pSUPER.EGFP), R-pSUPER.EGFP2 induced the best silencing effect on the expression of PPARgamma in RAW264.7 cells, which played an obvious inhibitory role in down-regulating the TNFalphaexpression after the curcumin and lipopolysaccharide (LPS) stimulation. CONCLUSIONS: PPARgamma-pSUPER-EGFP inducing a silencing effect on the expression of PPARgamma can efficiently play a negative role in controlling the inflammatory responses of RAW264.7 cells.

[The synergistic effects of lipopolysaccharide, bacterial lipoprotein and bacterial DNA on mouse alveolar macrophage activation].

OBJECTIVE: To investigate the synergistic effects of lipopolysaccharide (LPS), bacterial lipoprotein (BLP), and bacterial DNA on the expression of pattern recognition receptors (PRRs) on the cell surface of mouse alveolar macrophages and cellular activation at the level of receptor and its possible mechanism. METHODS: Mouse alveolar macrophages were isolated, cultivated and randomly divided into 7 groups: control group, LPS group, CpG oligonucleoetide (CpG-ODN) group, BLP group, LPS + BLP group, LPS + CpG-ODN group, and LPS + BLP + CpG-ODN group. Six hours later the supernatants were collected to detect the level of tumor growth factor alpha (TNFalpha) by ELISA. RT-PCR was used to detect the expression of the main PRRs: CD14, SR, TLR2, TLR4, and TLR9. RESULTS: The TNFalpha levels in the supernatant were 234 pg/ml +/- 30 pg/ml in the LPS group, 274 pg/ml +/- 30 pg/ml in the BLP group, and 308 pg/ml +/- 28 pg/ml in the CpG-ODN group, all significantly higher than that in the control group (92 pg/ml +/- 27 pg/ml, P < 0.01 or P < 0.01). The TNFalpha levels in the supernatant were 483 pg/ml +/- 31 pg/ml in the LPS + BLP group, and 511 pg/ml +/- 46 pg/ml in the LPS + CpG-ODN group, both significantly higher than those of the groups of the 3 factor alone (all P < 0.05). And the TNFalpha levels in the supernatant was 665 pg/ml +/- 24 pg/ml in the LPS + BLP + CpG-ODN group, significantly higher than those of the LPS + ODN group and LPS + BLP group (both P < 0.05). LPS, BL, and CpG-ODN alone, combinations of any 2 of them, and the combination of the three all up-regulated the expression of CD14 mRNA more and more strongly in sequence. LPS, BLP, and CpG-ODN alone all up-regulated the expression of SR mRNA (all P < 0.01), however, the combinations of any 2 factors or of the 3 factors failed to further up-regulate the expression of SR. LPS and BLP up-regulated the expression of TLR2 mRNA (both P < 0.05), LPS combined with BLP showed a stronger up-regulation of TLR2 mRNA (P < 0.05) than those by LPS and BLP alone. CPG-ODN alone failed to up-regulate the expression of TLR2 mRNA (P > 0.05) but significantly increased the up-regulation by LPS (P < 0.05). In comparison with the combinations of any 2 factors, LPS and BLP with CPG-ODN together up-regulated the expression of TLR2 mRNA more strongly (all P < 0.05). LPS, BLP, and CpG-ODN alone did not significantly up-regulate the expression of TLR4 mRNA (P > 0.05), LPS + BLP significantly regulated the expression of TLR4 mRNA than the groups of any factor alone (all P < 0.05). LPS + BLP + CpG-ODN further up-regulated the expression of TLR4 mRNA. LPS and CpG-ODN, especially LPS + CpG-ODN significantly up-regulated the expression of TLR9 mRNA (all P < 0.05). BLP failed to up-regulate the expression of TLR9 mRNA (P > 0.05) and did not coordinate the upregulation by LPS, however, in comparison with any combinations of the 3 factors, the combination of LPS, BLP, and ODN up-regulated the expression of TLR9 mRNA the most strongly (all P < 0.05). CONCLUSION: Bacterial LPS, BLP and bacterial DNA not only up-regulate the expression of PRRs of each other, but also synergistically increase the each other's effects on the cell surface of mouse alveolar macrophages.

[The effects of TNF alpha and IFN gamma on the expression of pattern recognition receptors on the surface of mouse alveolar macrophages].

OBJECTIVE: To investigate the effects of tumor necrosis factor alpha (TNF alpha) and interferon gamma (IFN gamma) on the expression of pattern recognition receptors (PRRs) on the surface of mouse alveolar macrophages. METHODS: Alveolar macrophages from mouse were cultured in DMEM supplemented with 10% (V/V) endotoxin-free calf serum. After the alveolar macrophages were stimulated with TNF alpha and IFN gamma (concentration, 20 ng/ml) for 3 h, 6 h and 12 h, the expression of PRRs, including cluster of differentiation 14 (CD14), scavenger receptor (SR), toll-like receptor 4 (TLR4), TLR2 and TLR9 mRNA and proteins were examined by RT-PCR and immunohistochemistry. RESULTS: The expressions of CD14, TLR2 and TLR9 receptors, which were related with cellular activation, were up-regulated by the stimulation of TNF alpha and IFN gamma (P < 0.05), while SR, which was related with cellular defense action, was down-regulated (P < 0.05). Although the expression of TLR4 was up-regulated, there was no statistical significance (P > 0.05). CONCLUSIONS: The cytokines such as TNF alpha and IFN gamma could also produce feedback regulation on the expression of PRRs at the levels of genes and proteins. Such regulation on the PRRs expression would be significant for further amplification of inflammation cascade and eventually leading to uncontrolled inflammation.