OBJECTIVETo research the effects of recombinant human beta-defensin-3 (hBD-3) on expression of interleukin-17A (IL-17A) and interleukin-22 (IL-22) in BEAS-2B cell.

METHODSThe BEAS-2B cells were stimulated with different concentrations of hBD-3 for 6 hours and 24 hours, respectively. Toll-like receptor 2 (TLR2), IL-17A and IL-22 mRNA expression levels were determined by real-time PCR, and the expression levels of IL-17A and IL-22 protein were examined by enzyme linked immune-sorbent assay.

RESULTSTLR2 mRNA in BEAS-2B cells were significantly increased in a concentration-and time-dependent manner after stimulating by hBD-3 for 24 hours compared to 6 hours. The IL-17A has significantly increased in mRNA and protein levels stimulated 24 hours in a concentration of 100 ng/ml, however, IL-17A mRNA expression has increased while protein didn't change stimulated 6 hours in a concentration of 50 ng/ml. The IL-22 mRNA and protein expression reached peak levels after stimulating in a concentration of 50 ng/ml of hBD-3 while IL-22 expression declined in mRNA and protein levels as the concentration of hBD-3 increased.

CONCLUSIONSRecombinant hBD-3 can up-regulated the expression of TLR2, IL-17A and IL-22, lower concentration of hBD-3 mainly increased the expression of IL-22 while higher concentration of hBD-3 mainly increased the expression of IL-17A. These results show that different concentrations of hBD-3 maybe activate different transcription factors which was mediated by TLR2, initiating host immune response.

Cell Line ; Humans ; Interleukin-17 ; genetics ; metabolism ; Interleukins ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Toll-Like Receptor 2 ; genetics ; metabolism ; beta-Defensins ; genetics ; metabolism

OBJECTIVETo study the role of MEKK2 on the production of IL-2 in Jurkat cells stimulated by PHA/anti-CD28 antibody.

METHODSThe MEKK2 and JNK kinase activities were measured in both dominant negative MEKK2 Jurkat (dnMEKK2 Jurkat) cells and parental Jurkat cells. The AP(1) and IL-2 promotor activities were measured by luciferase activity assay. The IL-2 mRNA and protein were detected by RT-PCR and Western blot.

RESULTSAfter stimulation by PHA/anti-CD28, JNK was activated in parental Jurkat cells but not in dnMEKK2 Jurkat cells. The luciferase report gene activities of AP1 and IL-2 promotors were increased by 4- and 5-folds in parental cells whereas only by 1 fold in dnMEKK2 Jurkat cells. The level of IL-2 mRNA and IL-2 protein were increased in parental Jurkat cells but not in dnMEKK2 Jurkat cells.

CONCLUSIONMEKK2 plays an important role on the production of IL-2 in Jurkat cell stimulated with PHA/anti-CD28 antibody. It is a potential drug target for the treatment of GVHD and autoimmune disease.

Humans ; Interleukin-2 ; biosynthesis ; genetics ; Jurkat Cells ; MAP Kinase Kinase 4 ; metabolism ; MAP Kinase Kinase Kinase 2 ; metabolism ; physiology

Adult ; Cytokines ; metabolism ; Female ; Gene Expression ; Humans ; Interleukin-2 ; blood ; Interleukin-4 ; blood ; Male ; Semaphorins ; genetics ; metabolism ; T-Lymphocytes, Helper-Inducer ; metabolism ; Thrombocytopenia ; genetics ; metabolism

Regulation of P2X7 receptor expression is of interest because activation of this receptor by extracellular ATP triggers a wide variety of cell functions in leukocytes. However, its expression and modulation in human peripheral blood mononuclear cells (PBMC) and monocytes remain unclear. RT-PCR was used to detect the constitutive level of P2X7 receptor and the levels upon stimulation with bacteria, bacterial product, mitogen and various cytokines in human PBMC and monocytes. P2X7 receptor mRNA was detected in PBMC and monocytes. P2X7 receptor expression in PBMC was up-regulated by interleukin-2, -4, -6 (IL-2, IL-4, IL-6) tumour necrosis factor-alpha (TNF-alpha), lipopolysaccharide (LPS) and heat-inactivated Staphylococcus aureus Cowan strain I (SAC). However, interferon-gamma (IFN-gamma), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF) and phytohemagglutinin-M (PHA-M) had little effect on the expression of P2X7 receptor. Furthermore, LPS and M-CSF could up-regulate P2X7 receptor expression in monocytes, while IFN-gamma, TNF-alpha and GM-CSF had weak effects, but pretreatment with these inducers could not further enhance LPS-stimulated P2X7 receptor expression in monocytes. The results obtained demonstrate that inflammatory stimuli drive P2X7 expression, thus supporting the hypothesis that P2X7 receptor may play a role in the inflammatory responses against bacteria infection, which need further verification.
Humans ; Interleukin-2 ; physiology ; Interleukin-4 ; physiology ; Leukocytes, Mononuclear ; drug effects ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Receptors, Purinergic P2X7 ; genetics ; metabolism ; Tumor Necrosis Factor-alpha ; physiology

This paper aims to explore the effects of chicken interferon-γ (ChIFN-γ) and interleukin-2 (ChIL-2) on type 1 helper (Th1) T lymphocyte differentiation. To be specific, ChIFN-γ and ChIL-2 were first expressed in Escherichia coli competent cells and then purified by Ni-NTA affinity chromatography. Different concentration of ChIFN-γ and ChIL-2 were employed to stimulate the lymphocytes in chicken peripheral blood which had been activated by concanavalin A (Con A), and the mRNA levels of cytokines related to Th1 cell differentiation were detected by real-time quantitative PCR (RT-qPCR). The results showed that both ChIFN-γ and ChIL-2 can significantly up-regulate mRNA levels of cytokines related to Th1 cell differentiation and the optimal concentration was 12.5 μg/mL and 25.0 μg/mL, respectively. In addition, specific-pathogen-free (SPF) chickens were immunized with ChIL-2 or ChIFN-γ together with H9N2 vaccine, or H9N2 vaccine alone by oral administration or intramuscular injection, respectively. The mRNA levels of cytokines related to Th1 cell differentiation were detected after immunization. The results showed that ChIFN-γ and ChIL-2 significantly up-regulated the mRNA levels of cytokines related to Th1 cell differentiation induced by H9N2 vaccine compared with H9N2 vaccine alone, and that the intramuscular injection was better than oral administration. In this study, we verified that ChIFN-γ and ChIL-2 can significantly enhance mRNA levels of cytokines related to Th1 cell differentiation induced by ConA or H9N2 vaccine in vitro and in vivo. The results of this study can lay a theoretical basis for using ChIFN-γ and ChIL-2 as vaccine adjuvants.
Animals ; Cell Differentiation ; Chickens ; Concanavalin A ; Cytokines/genetics* ; Influenza A Virus, H9N2 Subtype/genetics* ; Interferon-gamma/metabolism* ; Interleukin-2/genetics* ; RNA, Messenger

To study the effect of anemoside B4 on rats with chronic obstructive pulmonary disease (COPD).Seventy-two SD male rats were randomly divided into blank group and model group.The method of exposure to cigarette smoke and combined with lipopolysaccharide (LPS) was used to replicate the rat model of COPD.After the model was maintained for 5 weeks,the rats were randomly divided into model group,dexamethasone group (0.81 mg·kg~(-1)) and anemoside B4 low,medium and high (2,4,8 mg·kg~(-1)) dose groups,a group of 12 animals were administered,and then the administration was started.The administration was maintained until the28th day,and the pulmonary function parameters of rats were measured by an animal pulmonary function instrument.After testing the rat lung function parameters,immediately draw rat alveolar lavage fluid (BALF),and use high-throughput protein chip technology to determined the expression levels of inflammatory cytokines in rat BALF.HE staining was used to observe the general pathological changes of rat lung and tracheal tissue.Masson staining was used to observe the collagen deposition in rat lung tissue.Real-time q PCR method was used to determine the mRNA expression level of related genes in rat lung tissue.Western blot method was used to determine the expression levels of related proteins in rat lung tissues.According to the findings,compared with the model group,the dexamethasone group and the anemoside B4 drug groups had different degrees of increase in the lung function parameters of rats (P<0.01,P<0.05),improved the expression level of inflammatory cytokines in the BALF of rats to varying degrees (P<0.01,P<0.05),and improved the pathological structure of rat lung tissue to varying degrees.Relative mRNA expressions of matrix metalloproteinase 2 (MMP-2),matrix metalloproteinase 12 (MMP-12),matrix metalloproteinase inhibitor 1 (TIMP-1),interleukin-6 (IL-6),and transforming growth factor-β1 (TGF-β1) were significantly reduced (P<0.01);whereas relative mRNA expressions of matrix metalloproteinase 9(MMP-9) and matrix metalloproteinase inhibitor 2 (TIMP-2) were increased significantly (P<0.01).The mRNA and protein expression levels of T-box transcription factor (T-bet),interleukin-12 (IL-12) and signal transducer and activator of transcription 4(STAT4) reduced to varying degrees (P<0.01,P<0.05).The mRNA of transcription factor GATA3 (binding protein-3),interleukin-4 (IL-4) and signal transducer and activator of transcription 6 (STAT6) in rat lung tissues and the protein expression levels of IL-4 and STAT6 were increased to varying degrees (P<0.01,P<0.05).In conclusion,anemoside B4 has a certain protective effect on COPD rats caused by cigarette smoke exposure and combined with LPS.The mechanism of action may be related to the regulation of IL-12/STAT4 and IL-4/STAT6 signaling pathways.
Animals ; Interleukin-12 ; Interleukin-4 ; Lung/metabolism* ; Male ; Matrix Metalloproteinase 2 ; Pulmonary Disease, Chronic Obstructive/genetics* ; Rats ; STAT4 Transcription Factor/metabolism* ; STAT6 Transcription Factor/metabolism* ; Saponins

Objective: To investigate the role and mechanism of tumor-derived mesenchymal stem cells in regulating the M2 polarization of macrophages within gastric cancer microenvironment. Methods: Gastric cancer tissues and the adjacent non-cancerous tissues were collected from patients underwent gastric cancer resection in the First People's Hospital of Lianyungang during 2018. In our study, THP-1-differentiated macrophages were co-cultured with gastric cancer-derived mesenchymal stem cells (GC-MSCs). Then, the M2 subtype-related gene, the markers expressed on cell surface and the cytokine profile were analyzed by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), flow cytometry and Luminex liquid chip, respectively. The key cytokines mediating the inducing effect of GC-MSCs on macrophage polarization into the M2 subtype were detected and screened by Luminex liquid chip, which were further confirmed by the neutralizing antibody test. The expressions of macrophage proteins involved in M2 polarization-related signaling pathways under the different co-culture conditions of GC-MSCs were detected by western blot. Results: In Mac+ GC-MSC-culture medium (CM) group, the expression levels of Ym-1 and Fizz-1 (1.53±0.32 and 13.22±1.05, respectively), which are markers for M2 subtype, were both significantly higher than those of Mac group (1.00±0.05 and 1.21±0.38, respectively, P<0.05). The level of iNOS in Mac+ GC-MSC-CM group (0.60±0.41) was significantly lower than that of Mac group (1.06±0.38, P=0.023). In Mac+ GC-MSC-Transwell (TW) group, the expression levels of Ym-1 and Fizz-1 (1.47±0.09 and 13.16±2.77, respectively) were both significantly higher than those of Mac group (1.00±0.05 and 1.21±0.38, respectively, P<0.05). The level of iNOS in Mac+ GC-MSC-CM group (0.56±0.03) was significantly lower than that of Mac group (1.06±0.38, P=0.026). The ratios of CD163(+) /CD204(+) cells in Mac+ GC-MSC-CM and Mac+ GC-MSC-TW groups (3.80% and 4.40%, respectively) were both remarkably higher than that of Mac group (0.60%, P<0.05). The expression levels of IL-10, IL-6, MCP-1 and VEGF in Mac+ GC-MSC-CM group were (592.60±87.52), (1 346.80±64.70), (11 256.00±29.03) and (1 463.90±66.67) pg/ml, respectively, which were significantly higher than those of Mac group [(41.03±2.59), (17.35±1.79), (5 213.30±523.71) and (267.12±12.06) pg/ml, respectively, P<0.05]. The levels of TNF-α, IP-10, RANTES and MIP-1α were (95.57±9.34), (410.48±40.68), (6 967.30±1.29) and (1 538.70±283.04) pg/ml, which were significantly lower than those of Mac group [(138.01±24.31, (1 298.60±310.50), (14 631.00±4.21) and (6 633.20±1.47) pg/ml, respectively, P<0.05]. The levels of IL-6 and IL-8 in GC-MSCs [(11 185.02±2.82) and (12 718.03±370.17) pg/ml, respectively] were both strikingly higher than those of MSCs from adjacent non-cancerous gastric cancer tissues [(270.71±59.38) and (106.04±32.84) pg/ml, repectively, P<0.05]. The ratios of CD86(+) cells in Mac+ IL-6-blocked-GC-MSC-CM and Mac+ IL-8-blocked-GC-MSC-CM groups (28.80% and 31.40%, respectively) were both higher than that of Mac+ GC-MSC-CM group (24.70%). Compared to Mac+ GC-MSC-CM group (13.70%), the ratios of CD204(+) cells in Mac+ IL-6-blocked-GC-MSC-CM and Mac+ IL-8-blocked-GC-MSC-CM groups (9.90% and 8.70%, separately) were reduced. The expression levels of p-JAK2 and p-STAT3, which are proteins of macrophage M2 polarization-related signaling pathway, in Mac+ GC-MSC-CM group (0.86±0.01 and 1.08±0.01, respectively) were significantly higher than those of Mac group (0.50±0.01 and 0.82±0.01, respectively, P<0.05). The expression levels of p-JAK2 in Mac+ IL-6-blocked-GC-MSC-CM group (0.47±0.02) were significantly lower those that of Mac+ GC-MSC-CM group (0.86±0.01, P<0.05). The expression levels of p-JAK2 and p-STAT3 in Mac+ IL-8-blocked-GC-MSC-CM group (0.50±0.01 and 0.85±0.01, respectively) were both significantly lower than those of Mac+ GC-MSC-CM group (0.86±0.01 and 1.08±0.01, P<0.05). The expression levels of p-JAK2 and p-STAT3 in Mac+ IL-6/IL-8-blocked-GC-MSC-CM group (0.37±0.01 and 0.65±0.01, respectively) were both significantly lower than those of Mac+ GC-MSC-CM group (0.86±0.01 and 1.08±0.01, P<0.05). Conclusion: GC-MSCs promote the activation of JAK2/STAT3 signaling pathway in macrophages via high secretions of IL-6 and IL-8, which subsequently induce the macrophage polarization into a pro-tumor M2 subtype within gastric cancer microenvironment.
Humans ; Interleukin-6/genetics* ; Interleukin-8/pharmacology* ; Janus Kinase 2/metabolism* ; Macrophages/metabolism* ; Mesenchymal Stem Cells ; STAT3 Transcription Factor/metabolism* ; Signal Transduction ; Stomach Neoplasms/pathology* ; Tumor Microenvironment

OBJECTIVETo investigate the effects of interleukin 7 (IL-7) on B7 molecules expression and immunogenicity of acute leukemia (AL) cells.

METHODSThe B7 molecules expression on fresh acute leukemia cells and on the IL-7 exposed leukemia cells was detected by FACScan cytometer. B7-1 and B7-2 mRNA in IL-7 treated HL-60 cells were detected by reverse transcription-PCR (RT-PCR). The stimulation of proliferation of allogeneic peripheral blood mononuclear cells (PBMNC) by IL-7 treated leukemia cells was detected by MTT method. The level of interferon-gamma (IFN-gamma) secreted by the stimulated PBMNC was determined using enzyme-linked immunosorbent assays (ELISA). The blocking experiments were performed using monoclonal antibodies against B7-1, B7-2 and W6/32.

RESULTSB7-1 was weakly expressed in three, whereas B7-2 did in only one of eleven AL patients. IL-7 significantly enhanced B7 molecules expression on AL cells in a time-dependent manner. Furthermore, IL-7 could induce higher expression of B7-1 and B7-2 mRNAs on HL-60 cells. IL-7 treated leukemia cells could stimulate PBMNC proliferation and promote their IFN-gamma production. Anti-B7-1 and anti W6/32 but not anti-B7-2 monoclonal antibodies significantly inhibited the stimulated PBMNC proliferation and IFN-gamma secretion.

CONCLUSIONFresh AL cells express low level of B7-1 and B7-2 molecules. IL-7 enhances the B7 molecules expression on AL cells. The IL-7-treated leukemia cells can significantly stimulate the proliferation of allogeneic PBMNC and induce their IFN gamma secretion.

B7-1 Antigen ; genetics ; metabolism ; B7-2 Antigen ; genetics ; metabolism ; Humans ; Interleukin-7 ; pharmacology ; Leukemia ; immunology ; metabolism ; RNA, Messenger ; genetics ; Tumor Cells, Cultured ; Up-Regulation ; drug effects

RT-PCR was used to clone DNA fragment of the extracellular domain of 4-1BBL from human THP-1 cells (human monocyte), and the expression vector pAYZ4-1BBL was constructed by cloning the extracellular domain of 4-1BBL into the expression vector pAYZ. The extracellular domain of 4-1BBL was expressed in E. coli 16C9 and purified by affinity chromatography. SDS-PAGE and Western blot analysis showed that the relativae molecular weight of soluble 4-1BBL is 22kD which was consistent with the theoretically predicted value. So far as we know, it is the first time that the soluble expression of 4-1BBL in E. coli was achieved 4-1BBL induced a significant release of IL-2 in stimulated Jurkat cells after 48h incubation, especially in the presence of tumor cell. At the same time the apoptosis level of Jurkat cell reduce more than 50%. In conclusion, 4-1BBL may be useful in cancer immunotherapy.
4-1BB Ligand ; biosynthesis ; genetics ; Apoptosis ; genetics ; Cell Line ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Extracellular Space ; metabolism ; Humans ; Interleukin-2 ; biosynthesis ; Jurkat Cells ; Recombinant Proteins ; biosynthesis ; genetics

To construct a recombinant adenovirus co-expressing the E2 protein of classical swine fever virus (CSFV) and the porcine interleukin 2 (pIL-2), the CSFV E2 gene and pIL-2 gene were amplified respectively from the plasmids pMD19-T-E2 and pMD19-T-pIL-2 by PCR. E2-pIL-2 fusion gene was obtained by using 5 consecutive glycine codons as a linker and cloned into the adenoviral shuttle plasmid AdTrack. The AdTrack-E2-pIL-2 was linearized and transformed into E. coli BJ5183 with the backbone plasmid AdEasy1. The resultant recombinant plasmid AdEasy-E2-pIL-2 was transfected into the 293 cells where the recombinant adenovirus rAd-E2-pIL-2 was produced. The immunogenicity of rAd-E2-pIL-2 was evaluated in rabbits. The results of RT-PCR and Western-blotting showed that rAd-E2-pIL-2 could carry and express E2 and pIL-2 proteins. The titer of the rAd-E2-pIL-2 was 10(8.12) PFU/mL. After immunized with rAd-E2pIL-2, The injected rabbits developed a high level of CSFV specific antibodies. Regular fever was not detected in the rAd-E2-pIL-2-immunized rabbits upon challenge with CSFV C stain, and specific lymphoproliferative responses to the CSFV was detected in the lymphocytes from the immunized rabbits. In conclusion, rAd-E2-pIL-2 was constructed successfully and it could be an attractive vaccine candidate against CSFV.
Adenoviridae ; genetics ; metabolism ; Animals ; Cell Line ; Classical swine fever virus ; genetics ; Humans ; Interleukin-2 ; genetics ; immunology ; Rabbits ; Swine ; Viral Proteins ; genetics ; immunology ; metabolism ; Viral Vaccines ; immunology