Rab7 is involved in enhanced bacterial clearance by BLP-tolerized macrophages

AIM: To explore whether Rab GTPases are involved in enhanced bacterial clearance by bacterial lipoprotein (BLP)-tolerized macrophages (bone marrow-derived macrophages, BMDMs).METHODS: Real-time PCR was used to compare the mRNA levels of Rab5a, Rab5b, Rab7, Rab9, Rab9b, Rab11a, Rab11b, Rab12, Rab32 and Rab34 between the naive BMDMs and the BLP-tolerized BMDMs. The up-regulation of Rab7 was further confirmed in BLP-tolerized BMDMs infected with E. coli by real-time PCR and Western blot. Following down-regulating the expression of Rab7 using RNA interference (RNAi), the bacterial uptake and intracellular bacterial killing of BLP-tolerized BMDMs treated with E. coli were detected.RESULTS: The mRNA level of Rab7 was significantly increased in BLP-tolerized BMDMs, which was 1.4 folds as high as that in the naive BMDMs, and the mRNA and protein levels of Rab7 in BLP-tolerized BMDMs stimulated with bacteria were further confirmed to be increased. Down-regulating the expression of Rab7 had no effect on phagocytosis of BLP-tolerized BMDMs for bacteria, but the intracellular bacterial killing ability was significantly attenuated. CONCLUSION: BLP tolerance plays an important role during intracellular bacterial killing in macrophages through up-regulating the expression of Rab7.     

Effect of P2X7R gene silencing by RNA interference on proliferation and phagocytosis of murine macrophage cell line RAW264.7

AIM: To establish a cell line of stable silencing of P2X₇ receptor (P2X₇R) expression through short hairpin RNA (shRNA)-mediated interference in murine RAW264.7 macrophages, and to investigate the proliferation and apoptosis in the cell line. METHODS: Stable silencing of P2X₇R gene in the RAW264.7 cells was achieved by recombinant shRNA plasmid targeting murine P2X₇R gene via liposome mediated transfection, followed by G418 selection. The efficacy of plasmid transfection and P2X₇R silencing in G418 resistant cells was verified by immunofluorescent microscopy and real-time PCR, respectively. The proliferative activity was analyzed by CCK-8 assay and EdU cell proliferation assay. The cell cycle distribution and apoptosis were evaluated by flow cytometry. RESULTS: The expression of P2X₇R at mRNA and protein levels was down-regulated by 80% in sh P2X₇R group compared with negative control (NC) plasmid transfection. In addition, P2X₇R-silencing cells exhibited higher proliferative activity compared with NC and wild-type RAW264.7 cells (P<0.05). Compared with NC cells, P2X₇R silencing resulted in an increase in the phagocytosis of the cells (P<0.05). CONCLUSION: A cell line RAW264.7 of stable silencing of P2X₇R expression was successfully established. P2X₇R gene silencing stimulates the proliferation, and changes phagocytic function in murine RAW264.7 macrophages.     

Effects of airway epithelial cells on phenotype and phagocytosis of macrophages and roles of HIF-1α

AIM: To investigate the effects of airway epithelial cells on the phenotype and phagocytosis of macrophages and the roles of hypoxia-inducible factor-1α (HIF-1α).METHODS: Human bronchial epithelial (HBE) cells treated with CoCl₂ (0, 100, 200, 400 and 800 μmol/L) or transfected with HIF-1α siRNA were co-cultured with the macrophages differentiated from human monocyte line THP-1 induced by phorbol 12-myristate 13-acetate (PMA). The mRNA expression of HIF-1α in the HBE cells was detected by RT-qPCR. The expression of macrophage surface markers and the phagocytosis rate of E.coli by macrophages were analyzed by flow cytometry.RESULTS: CoCl₂ upregulated the mRNA expression of HIF-1α in the HBE cells in a concentration-dependent manner and peaked at 8 h. HBE cells treated with CoCl₂ increased the fluorescence intensity ratio of CCL3, CD163, CD206 and CCL18 in co-cultured macrophages, and the strongest effect was seen in the macrophages co-cultured with HBE cells treated with CoCl₂ at 800 μmol/L. The fluorescence intensity ratio of CCL3 in co-cultured macrophages increased most obviously at 8 h and 12 h, while the fluorescence intensity ratio of CD163, CD206 and CCL18 increased more prominently in the macrophages co-cultured for 24 h. The stimulating effects of the HBE cells transfected with HIF-1α-Homo-488 siRNA on CCL3, CD163, CD206 and CCL18 in the macrophages were significantly attenuated. The phagocytosis rate of E.coli by macrophages co-cultured with HBE cells treated with different concentrations of CoCl₂ for 24 h initially increased (up to 60 min), and then it gradually decreased. Compared with normal HBE co-culture group, the phagocytosis rate in 400 and 800 μmol/L stimulation groups decreased at each time point, and that in 800 μmol/L stimulation group was the most.CONCLUSION: In hypoxia environment, airway epithe-lial cells initially transform macrophages predominantly to an M1-phenotype. However, the long-term hypoxia-stimulated airway epithelial cells inhibit the phagocytosis of macrophages and convert them to M2 superiority. HIF-1α may be an important mediator in these processes.     

Effects of Mcl-1 silencing on apoptosis of mouse peritoneal macrophages infected with different virulence of Mycobacterium tuberculosis

AIM: To investigate the effect of inhibiting Mcl-1 gene expression on apoptosis of mouse peritoneal macrophages infected with different virulence of Mycobacterium tuberculosis using a technique of RNA interference. METHODS: The BALB/c mice were infected with prepared bacterium of the virulence strains of Xinjiang, H37Rv, H37Ra and BCG. Mcl-1-shRNA was applied to the mouse model of infection, and the control groups were set up. On 1 d, 3 d, 5 d and 7 d, the mouse peritoneal macrophages were collected. The expression of Mcl-1 at mRNA and protein levels was determined by real-time PCR and Western blot. The apoptotic rate of peritoneal macrophages was analyzed by flow cytometry. RESULTS: The expression of Mcl-1 at mRNA and protein levels was up-regulated in the peritoneal macrophages from the mice infected with different virulence of Mycobacterium tuberculosis, and the cells from the mice infected with virulence strains of Xinjiang and H37Rv expressed higher level of Mcl-1 than the uninfected control cells (P<0.05). The expression of Mcl-1 at mRNA and protein levels was reduced by RNA interference as compared with control group (P<0.05). Inhibition of Mcl-1 expression induced apoptosis of peritoneal macrophages in the mice. CONCLUSION: The Mcl-1 expression at mRNA and protein levels in mouse peritoneal macrophages infected with different virulence of Mycobacterium tuberculosis was effectively suppressed by Mcl-1-shRNA, which can induce macrophage apoptosis.     

Phagocytosis of viable apoptotic cells inhibits the activation of T lymphocytes

AIM: To investigate whether viable apoptotic cells and phagocytosis of them affect the activation of T lymphocytes. METHODS: Ultraviolet irradiation was used to induce apoptotic cells in vitro and the model of phagocytosis of these cells was established. Cytokine TGF_β1 was detected by ELISA. The rate of apoptotic cells and phagocytosis of them were assessed by flow cytometry. Furthermore, flow cytometry was also employed to examine the expression of activation signs, such as CD69, CD25, CD71, of T lymphocytes under the intervention of apoptotic cells and macrophage which ingested apoptotic cells, to reflect whether the apoptotic cells and the phagocytosis of these cells could influence the activation of lymphocytes stimulated by Con A. RESULTS: Ingestion of apoptotic cells increased TGF_β1 secretion. Only the macrophages that had ingested apoptotic cells could suppress the activation of lymphocytes. The expression of the markers of lymphocytes activation such as CD69, CD25, CD71 had been restrained. These inhibition effects were abolished by monoclonal anti-TGF_β1 antibody. CONCLUSION: The macrophages that have ingested apoptotic cells inhibit expression of CD69, CD25 and CD71 of T lymphocytes stimulated by ConA. This effect is dependent on the increase in TGF_β1 secretion in local site.     

Preliminary mechanism of Mcl-1 silencing in regulating apoptosis of mouse peritoneal macrophages infected with MTB

AIM: To investigate the mechanism of myeloid cell leukemia-1 (Mcl-1) silencing in regulating the apoptosis of mouse peritoneal macrophages infected with Mycobacterium tuberculosis (MTB) by observing the changes of Bcl-2 and Bax expression. METHODS: The suspensions of MTB strains with different virulence, BCG, H37Ra, H37Rv and XJ-MTB, were prepared to infect BALB/c mice. The transfection of Mcl-1-shRNA plasmid was used to establish a mouse model, and a corresponding control group at the same time was set up. The mice were executed and their peritoneal macrophages were collected 1 d, 3 d, 5 d and 7 d after the treatment. The apoptosis of the macrophages treated with diffe-rent virulence of MTB strains at different time points was analyzed by flow cytometry. The expression of Bcl-2 and Bax at mRNA and protein levels was determined by real-time PCR and Western blot. RESULTS: The apoptotic rate of mouse peritoneal macrophages increased to some extent after transfection with Mcl-1-shRNA plasmid compared with control group. The order of apoptotic rates was BCG > H37Ra > H37Rv≈XJ-MTB (P<0.05). The expression of Bcl-2 at mRNA and protein levels was significantly decreased, while the expression of Bax at mRNA and protein levels was significantly increased. The changes in BCG infection group were the most significant, and the negative correlation between the Bcl-2/Bax ratio at mRNA level and the virulence of the MTB strains was observed (P<0.05). CONCLUSION: Inhibition of Mcl-1 expression significantly promotes the apoptosis of peritoneal macrophages in mice infected with different virulence of MTB strains. The regulatory mechanism may be closely related to the protein expression of Bcl-2 and Bax and the virulence of MTB strains.     

CCK-8 up-regulats signal pathway of LPS-induced HO-1 expression in rat lungs

AIM: To study the signal pathway involved in up-regulation of LPS-induced HO-1 expression by CCK-8. METHODS: Forty-two SD rats were divided into 7 groups (six rats each) randomly as follows: control group, LPS group, LPS+SP600125 (JNK-specific inhibitor) group, CCK-8+LPS group, CCK-8+LPS+SP600125 group, CCK-8 group and CCK-8 +SP600125 group. Lungs from the rats in these 7 groups were excised 6 h after the agents were administered. HO-1 mRNA expression was examined by RT-PCR. The protein expression of HO-1 was detected by Western blotting and immunofluorescence flow cytometry (FCM). RESULTS: There were significant positive expression of HO-1 mRNA in LPS group compared to control group. CCK-8 enhanced LPS-induced HO-1 mRNA expression and CCK-8 alone induced HO-1 mRNA expression as well. The mRNA expressions of HO-1 in LPS group, CCK-8+LPS group and CCK-8 group were 3.01 (P<0.01), 5.88 (P<0.01) and 3.45 (P<0.01) times as many as that in control group, respectively. SP600125 inhibited the mRNA expression of HO-1 induced by CCK-8 and (or) LPS. The change of HO-1 protein expression was in accordance with that of HO-1 mRNA expression by Western blotting and immunofluorescence FCM. CONCLUSION: These results suggest that JNK/c-Jun signal pathway plays an important role in the up-regulation of LPS-induced HO-1 expression by CCK-8.     

Expression of BiP in rat splenic macrophages with mild heat stress

AIM: To investigate the role of mild heat stress towards the immunological function of splenic macrophages and significance of BiP protein expression.METHODS: Primary cultured splenic macrophages were prepared and placed in 41 ℃ thermostat for thermal stress and restored to 37 ℃ after an hour. Heat stressed macrophages were separated into groups at time points of 0 min, 30 min, 60 min, 120 min and 180 min, and their BiP mRNA and BiP protein expressions were detected. At the same time, the phagocytic function, cytotoxicity and chemotaxis of the macrophages were detected.RESULTS: After heat stress, the expressions of BiP mRNA and BiP protein in splenic macrophages remarkably increased, and reached to peak after 30-60 min, still remained higher than control group after 120 min and restored to normal level after 180 min. At the same time, mild heat stress enhanced the phagocytotic, cytotoxicity and chemotactic activities of splenic macrophages, and reached to peak after 60 min then gradually decreased.CONCLUSION: The expression of BiP protein is enhanced after mild heat stress, synchronous changes happen both in BiP protein expression and cellular function. There is close relation between BiP protein expression and increased functions of splenic macrophages induced by mild heat stress.     

Effect of interferon-inducible protein p204 on proliferation of vascular smooth muscle cells in rats

AIM: To study the effect of interferon-inducible protein p204 on the proliferation of vascular smooth muscle cells (VSMCs) in rats. METHODS: Cultured VSMCs were treated with interferon alpha (IFN-α) and p204 gene (Ifi204) small interfering RNA (siRNA) in vitro instantaneously. The cell vitality was detected by MTT me-thod,and the cell cycle was analyzed by flow cytometry. The expression of mRNA and proteins was determined by real-time qRT-PCR and Western blotting,respectively.RESULTS: IFN-α induced the increase in the expression of p204 at mRNA and protein levels, reduced the cell vitality, inhibited the cell cycle of G₁/S transition, and down-regulated the expression of Ras protein in VSMCs. Meanwhile, the phosphorylation levels of Raf and ERK were decreased. Transfection of Ifi204 siRNA restrained the expression of p204, increased the cell vitality and promoted the cell cycle of G₁/S transition in VSMCs. The up-regulation of Ras protein expression and the increased phosphorylation levels of Raf and ERK were also observed.CONCLUSION: The expression of p204 restrains the proliferation of VSMCs in rats by inhibiting the activation of Ras/Raf/MEK/ERK signal pathway.     

Adipose-derived stem cells mediate immunosuppression of Th17 in multiple sclerosis

AIM: To investigate how human adipose-derived stem cells (hASCs) regulates the differentiation of Th17 cells in multiple sclerosis. METHODS: hASCs were isolated from the adipose tissues. Magnetic-activated cell sorting (MACS) kit was used to isolate CD4⁺ T cells from peripheral blood mononuclear cells (PBMCs) which were isolated by density gradient centrifugation. The percentage of CD4⁺ T cells was detected by flow cytometry. The activated CD4⁺ T cells were co-cultured with hASCs for about 4 d at different ratios of hASCs to CD4⁺ T cells (1:4 and 1:10) in a Th17 polarised condition. Another group adding anti-leukemia inhibitory factor (LIF) antibody was set up. Th17 cell proportion of the CD4⁺ T cells was determined by flow cytometry. The level of LIF in the supernatant of co-cultured system was measured by ELISA. The mRNA expression of retinoid-related orphan receptor γt (RORγt), interleukin-6 receptor (IL-6R), interleukin-23 receptor (IL-23R), LIF and leukemia inhibitory factor receptor (LIFR) was detected by real-time PCR. RESULTS: The result of flow cytometry suggested there were mainly hASCs, and the percentage of CD4⁺ T cells in the PBMCs were above 90% after MACS. The Th17 cell proportion decreased in 1:4 and 1:10 co-cultured groups in a dose-dependent manner. The mRNA expression of IL-6R, IL-23R and RORγt was downregulated and the expression of LIFR and LIF was up-regulated. When the anti-LIF was added into the co-cultured system, the ratio of Th17 cells increased and reached to the control level. The protein level of LIF obviously increased after co-cultured. After anti-LIF added, the mRNA expression of RORγt and IL-6R was up-regulated. CONCLUSION: hASCs inhibits the differentiation of Th17 cells from multiple sclerosis patients through the competitive inhibition of LIF/IL-6 by secreting LIF.     

Role of Api6 in lung inflammation induced by high fat/cholesterol food in C57BL/6J mice

AIM：To investigate the function of apoptosis inhibitor 6 (Api6) in lung inflammation induced by high-fat high-cholesterol diet (HFD/HCD) in male C57BL/6J mice. METHODS：Male C57BL/6J mice (6~8 weeks old) were randomly divided into 2 groups and treated with regular diet and HFD/HCD, respectively. After 16 weeks of feeding, the lung tissues were collected and the pulmonary inflammatory status was determined by immunohistochemistry and ELISA. The mRNA and protein expression levels of Api6 were determined by real-time PCR and Western blotting. The apoptotic rate of bronchioalveolar lavage cells was examined by flow cytometry. RAW264.7 cells were cultured in vitro and the apoptosis induced by oxidized low-density lipoprotein (oxLDL) was detected by flow cytometry. RESULTS：Accumulation of macrophages and increases in both tumor necrosis factor α and monocyte chemoattractant protein 1 were observed in the lung tissues of 16-week HFD/HCD-fed C57BL/6J mice. Compared with the regular diet-fed mice, the expression of Api6 at mRNA and protein levels in the lung tissues was highly increased in the HFD/HCD-fed mice (P<0.01). Meanwhile, the apoptotic rate of bronchioalveolar lavage macrophages from the HFD/HCD-fed mice was highly inhibited (P<0.01). In vitro, 500 μg/L recombinant Api6 significantly inhibited the apoptosis of RAW264.7 cells induced by oxLDL (P<0.05). CONCLUSION：HFD/HCD feeding results in the accumulation of macrophages in the lung of C57BL/6J mice, which may partly due to the increased expression of Api6 and its anti-apoptotic role in macrophages.     

SUMO-specific protease 3 aggravates CaPO4-induced mouse abdominal aortic aneurysm formation by promoting macrophage polarization

AIMTo investigate the role of SUMO-specific protease 3 (SENP3) in macrophage polarization and calcium phosphate (CaPO₄)-induced abdominal aortic aneurysm (AAA) formation in mice. METHODS（1） Bone marrow-derived monocytes (BMDMs) in Senp3^flox/flox (wild-type, WT) mice and Senp3^flox/flox; Lyz2-Cre (monocyte-specific SENP3 knockout, i.e. conditioned knockout, cKO) mice were isolated and induced for M1 and M2 polarization. The mRNA and protein expression level of SENP3 were detected by RT-qPCR, Western blot and immunocytofluorescence, and the differential distribution of M1/M2 BMDMs from WT and cKO mice was analyzed. （2） CaPO₄ was administrated to induce AAA model in 8~12-week-old male WT and cKO mice. The AAA incidence, survival rate and maximal aortic diameter were analyzed between the 2 groups. Aortic aneurysm tissues were collected for pathological analysis, and the expression levels of SENP3, interleukin-1β (IL-1β), tumor necrosis factor-α (TNFα), IL-6 and matrix metalloproteinases-9 (MMP-9) were measured by RT-qPCR and Western blot. Dihydroethidium staining in situ in frozen sections was used to analyze the production of reactive oxygen species (ROS). （3） To explore the potential mechanisms, Western blot and co-immunoprecipitation were used to verify the de-SUMO modification of mitogen-activated protein kinase kinase 7 (MKK7) induced by SENP3. Besides, BMDMs were transfected with Flag-MKK7 wild type (Flag-MKK7 WT) and SUMO-modified site K18 mutant (Flag-MKK7 K18R mutant), and then M1 polarization of the cells was induced. The protein levels of p-JNK and MMP-9 in the 2 groups were determined by Western blot. RESULTS(1) SENP3 expression was up-regulated in M1 polarized macrophages (P<0.01), but was down-regulated in M2 polarized macrophages (P<0.01). The expression of SENP3 was decreased during the transformation of M1 to M2 in the macrophages (P<0.01), but was significantly up-regulated during the opposite process (P<0.01). Besides, more M1 macrophages and less M2 macrophages after induction were observed in the BMDMs from cKO mice than those from WT mice. (2) SENP3 expression was up-regulated in AAA tissues (P<0.05). The AAA incidence of cKO mice was significantly reduced after CaPO₄ induction (P<0.01), the survival rate was significantly improved (P<0.05), and maximal aortic diameter was significantly reduced in cKO group (P<0.01). The levels of IL-1β, IL-6 and TNFα, and the production of ROS were significantly down-regulated (P<0.01), meanwhile MMP-9 expression was also down-regulated in cKO mice (P<0.05). (3) the SUMO2/3 modification of MKK7 was reduced during M1 polarization, and MKK7 interaction with SENP3 was enhanced. Significantly up-regulated protein level of p-JNK and MMP-9 were verified in the M1 macrophages transfected with Flag-MKK7 K18R mutant (P<0.05). CONCLUSION SENP3 activates the MAPK/JNK pathway via de-SUMOylation of MKK7, regulates the M1/M2 polarization of macrophages and promotes the protein level of MMP-9, thus aggravating AAA formation.     

Effects of TGF-β1 and signal protein Smad3 on rat cardiac myocyte hypertrophy

AIM: To investigate the effects of TGF-β1 and signal protein Smad3 on rat cardiac myocyte hypertrophy. METHODS: The total protein was analyzed by flow cytometry and the ANF mRNA expression was measured by RT-PCR to judge the hypertrophy of cultured neonatal cardiac myocytes. Smad3 mRNA expression in cardiac myocytes was measured by RT-PCR, and the protein expression of Smad3 was analyzed by Western blotting. RESULTS: TGF-β1 significantly increased the total protein in cardiac myocytes and promoted ANF mRNA expression, compared with control group. In cultured neonatal myocytes, AS-ODN of Smad3 inhibited myocyte hypertrophy induced by TGF-β1. Smad3 mRNA and protein expression increased at 15 min after incubated with TGF-β1, reached the peak at 1 h, and declined at 4 h. CONCLUSION: TGF-β1 and signal protein Smad3 may participate in the progress of rat cardiac myocyte hypertrophy.     

Effect of MIF antisense oligonucleotides on expression of MIF in macrophages

AIM: To investigate the effect of antisense oligonucleotides on expression of macrophage migration inhibitory factor (MIF) in macrophages. METHODS: MIF phosphorothioate oligonucleotides was designed and synthesized. The phosphorothioate antisense, sense and missense oligonucleotides of mouse MIF was transfected into macrophages, separately. After that, macrophages were incubated with LPS. Cell culture medium was collected for MIF protein detection by EIA. Cellular RNA was extracted and the expression of MIF mRNA was examined by RT-PCR analysis. RESULTS: LPS stimulation resulted in a specific time-dependent expression of MIF derived from macrophages. MIF mRNA and MIF protein level increased at 6 h and reached a plateau at 9-12 h after LPS stimulation. The macrophages treated with antisense oligonucleotides showed a significant decrease in MIF mRNA and MIF protein after LPS stimulation than those with LPS stimulation only and LPS plus sense or missense oligonucleotides (P<0.05). CONCLUSION: Antisense oligonucleotides of MIF inhibit the expression of MIF mRNA and MIF protein in macrophages treated with LPS.     

Effects of resveratrol on proliferation of ARPE-19 cells

AIM:To investigate the effects of resveratrol (Res) on the proliferation of ARPE-19 cells and to explore the possible mechanisms. METHODS:After ARPE-19 cells were treated with Res at concentrations of 0, 50, 100, 150, 200 and 300 μmol/L for 24 h, 48 h and 72 h, the effects of Res on the proliferation of the cells were tested by CCK-8 assay. The ARPE-19 cells were treated with Res at concentrations of 0, 100, 150 and 200 μmol/L for 48 h. The effects of Res on the cell cycle and apoptosis were detected by flow cytometry with Annexin V-FITC/PI staining. The protein expression of proliferating cell nuclear antigen (PCNA) was detected by immunofluorescent assay. The mRNA expression of PCNA, P21 and P27 was determined by real-time PCR. RESULTS: The results of CCK-8 assay showed that Res inhibited the proliferation of ARPE-19 cells in a time- and dose-dependent manner. The treatment with Res for 48 h resulted in an arrest of cell cycle at S phase without increasing cell apoptosis. Res inhibited the protein expression of PCNA in ARPE-19 cells. The results of real-time PCR showed that Res increased the mRNA expression of P21 and P27, and decreased the mRNA expression of PCNA. CONCLUSION: Res inhibits the proliferation of ARPE-19 cells and induces the cell cycle arrest at S phase. The mechanism may be related to up-regulation of P21 and P27, and down-regulation of PCNA.     

HCMV-infected human THP-1 cells induce expression of HLA-G and its receptors

AIM: To investigate the differential expression of human leukocyte antigen-G (HLA-G) isoforms and its receptors in human monocyte line THP-1 after human cytomegalovirus (HCMV) infection for exploring the role of HLA-G in HCMV escaping the immune response of the organism.METHODS: THP-1 cells were infected with HCMV Towne strain. The expression of HLA-G isoforms at mRNA and protein levels was determined by RT-PCR and Western blot, respectively. The surface expression of HLA-G and its receptors ILT2/ILT4 and the cell viability were analyzed by flow cytometry. The levels of soluble HLA-G (sHLA-G) and IL-10 were measured by ELISA.RESULTS: After infection of the THP-1 cells with HCMV, no obvious apoptosis in the cells was observed, and the viability of the cells was high. A significant up-regulation of HLA-G1, -G3, -G4 and -G5 at mRNA expression level 1 d after infection was found, while the protein expression of HLA-G1 and HLA-G5 isoforms was mainly detected. The expression of HLA-G/ILT2/ILT4 was evidently up-regulated 1 d after infection. The level of sHLA-G was significantly increased 1 d after infection as compared with control group (P<0.01). The expression of IL-10 was obviously up-regulated 1 d post-infection as compared with control group.CONCLUSION: The differential expression of HLA-G isoforms and secretion of the receptors ILT2/ILT4 and IL-10 in the THP-1 cells are induced after HCMV infection. This study provides experimental evidence for evaluating the immune mechanism of HCMV infection.     

Role of homocysteine in tissue factor expression in human vascular smooth muscle cells

AIM: To investigate the expression of tissue factor (TF) induced by homocysteine (Hcy) and the effect of Hcy on activation of nuclear factor-κB (NF-κB) in human vascular smooth muscle cells (VSMCs).METHODS: Human umbilical artery VSMCs were cultured by tissue explanting method,and were incubated with different dosage of Hcy/ PDTC (NF-κB inhibitor) in different time.RT-PCR was used to measure the expression of TF mRNA,and flow cytometry was used to detect the expression of TF on the surface of VSMCs.Western blotting was performed to measure the NF-κB protein level in the nuclear,flow cytometry was used to examine the expression of iNOS and the expression of TF on the surface of VSMCs.RESULTS: Hcy induced VSMCs TF mRNA expression significantly after the VSMCs were incubated with Hcy at concentrations of 10,100,500 μmol/L,respectively.There was low expression level of TF protein on the surface of the control VSMCs and Hcy also induced VSMCs TF protein expressionn on the cell surface at different concentrations.Additionally,Hcy rapidly induced the activation of NF-κB and inhibited this effect significantly by PDTC.Hcy alone did not induce the expression of iNOS in VSMCs.CONCLUSION: Hcy induces human VSMCs expression of TF in mRNA and protein.These effects were partly mediated by NF-κB.These results suggest that Hcy may play an important role in atherosclerosis and thrombosis.