To investigate the effects of cigarette smoking in different manners on acute lung injury in rats.The commercially available cigarettes with tar of 1,5, 11 mg were smoked in Canada depth smoking (health canada method, HCM) manner, and those with tar of 11 mg were also smoked in international standard (ISO) smoking manner. Rats were fixed and exposed to mainstream in a manner of nose-mouth exposure. After 28 days, the bronchoalveolar lavage fluids from left lung were collected for counting and classification of inflammatory cells and determination of pro-inflammatory cytokines IL-1β and TNF-α. The right lungs were subjected to histological examination and determination of myeloperoxidase (MPO) and superoxide dismutase (SOD) activities and glutathione, reactive oxygen species (ROS) and malondialdehyde (MDA) levels.In both HCM and ISO manners, the degree of lung injury was closely related to the tar content of cigarettes, and significant decrease in the body weight of rats was observed after smoking for one week. In a HCM manner, smoking with cigarette of 11 mg tar resulted in robust infiltration of macrophages, lymphocytes and neutrophils into lungs, significant increase in IL-1β and TNF-α levels and MPO activities, and significant decrease in GSH levels and SOD activities and increase in ROS and MDA levels (all<0.05). Smoking with cigarette of 5 mg tar led to moderate increase in IL-1β and TNF-α levels, and MPO activities (all<0.05), and moderate decrease in GSH levels and SOD activities and increase of ROS and MDA levels (all<0.05). However, smoking with cigarette of 1 mg tar affected neither inflammatory cell infiltration nor IL-1β and TNF-α levels.Cigarette smoking in nose-mouth exposure manner can induce acute lung injury in rats; and the degree of lung injury is closely related to the content of tar and other hazards in cigarettes.
Acute Lung Injury ; etiology ; pathology ; physiopathology ; Animals ; Bronchoalveolar Lavage Fluid ; chemistry ; cytology ; Chemotaxis, Leukocyte ; drug effects ; Glutathione ; analysis ; drug effects ; Interleukin-1beta ; analysis ; drug effects ; Lung ; chemistry ; pathology ; Lymphocytes ; drug effects ; pathology ; Macrophages ; drug effects ; pathology ; Male ; Malondialdehyde ; analysis ; Neutrophil Infiltration ; drug effects ; Neutrophils ; drug effects ; pathology ; Peroxidase ; analysis ; drug effects ; Rats ; Reactive Oxygen Species ; analysis ; Smoking ; adverse effects ; Superoxide Dismutase ; analysis ; drug effects ; Tobacco Products ; adverse effects ; classification ; Tumor Necrosis Factor-alpha ; analysis ; drug effects ; Weight Loss ; drug effects

Neutrophils play a key role in innate immunity, and the identification of new stimuli that stimulate neutrophil activity is a very important issue. In this study, we identified three novel peptides by screening a synthetic hexapeptide combinatorial library. The identified peptides GMMWAI, MMHWAM, and MMHWFM caused an increase in intracellular Ca2+ in a concentration-dependent manner via phospholipase C activity in human neutrophils. The three peptides acted specifically on neutrophils and monocytes and not on other non-leukocytic cells. As a physiological characteristic of the peptides, we observed that the three peptides induced chemotactic migration of neutrophils as well as stimulated superoxide anion production. Studying receptor specificity, we observed that two of the peptides (GMMWAI and MMHWFM) acted on formyl peptide receptor (FPR)1 while the other peptide (MMHWAM) acted on FPR2. Since the three novel peptides were specific agonists for FPR1 or FPR2, they might be useful tools to study FPR1- or FPR2-mediated immune response and signaling.
Animals ; Calcium/metabolism ; Cell Line ; Cells, Cultured ; Chemotaxis, Leukocyte/drug effects ; Humans ; Mice ; NIH 3T3 Cells ; Neutrophils/*cytology/*drug effects ; PC12 Cells ; Peptides/*pharmacology ; Rats ; Receptors, Formyl Peptide/agonists

Secondary lymphoid tissue chemokine (SLC), which is expressed in T cell zones of secondary lymphoid organs, including the spleen and lymph nodes, strongly recruits both T lymphocytes and mature dendritic cells. As appropriate interaction of tumor-specific T cells and mature dendritic cells, equipped with tumor antigens, is a prerequisite for effective T cell immunity against established tumors, we mobilized lymphocytes and dendritic cells to tumor sites by intratumoral injection of secondary lymphoid tissue chemokine-Fc (SLC-Fc) fusion protein using the B16F10 murine melanoma model. Activation of dendritic cells, another prerequisite for the effective activation of naive tumor-specific T cells, was achieved by the addition of immunostimulatory cytosine-phosphorothioate-guanine oligodeoxynucleotide (CpG-ODN) into the tumor site. Intratumoral administration of SLC-Fc or CpG-ODN revealed antitumor effects against B16F10 murine melanoma grown in the subcutaneous space. Co-treatment of SLC-Fc and CpG-ODN displayed synergistic effects in reducing the tumor size. The synergistic antitumor effect in co-treatment group was correlated with the synergistic/additive increase in the infiltration of CD4+ T cells and CD11c+ dendritic cells in the tumor mass compared to the single treatment groups. These results suggest that the combined use of chemokines and adjuvant molecules may be a possible strategy in clinical tumor immunotherapy.
Animals ; Antigens, CD11c/immunology ; CD4-Positive T-Lymphocytes/immunology ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Chemokine CCL21/*administration & dosage/pharmacology ; Chemotaxis, Leukocyte ; Dendritic Cells/immunology/metabolism ; Immunotherapy ; Injections, Intralesional ; Melanoma, Experimental/*immunology/*therapy ; Mice ; Mice, Inbred C57BL ; Oligodeoxyribonucleotides/*administration & dosage/pharmacology ; T-Lymphocytes/immunology/metabolism

OBJECTIVETo construct the murine CCL21 eukaryotic expression plasmid, and to investigate the chemotactic function of its products.

METHODSMurine CCL21 cDNA was amplified by RT-PCR from murine total RNA, and was inserted into eukaryotic expression plasmid pcDNA3.1 after confirmation of sequencing. The recombinant CCL21 plasmid was transferred into mouse forestomach carcinoma (MFC) cells and the chemotactic function of expressed products was detected by chemotaxis assay.

RESULTGene sequencing, gel electrophoresis of PCR products and restrictive digestion proved the successful construction of CCL21, and its expression was confirmed by Western Blot. The transfected tumor cells had a significant chemotactic function to DC.

CONCLUSIONThe recombinant murine CCL21 eukaryotic expression plasmid has been successfully constructed, and its expression products in tumor cells have a marked chemotactic function to DC.

Animals ; Base Sequence ; Chemokine CCL21 ; biosynthesis ; genetics ; Chemotaxis, Leukocyte ; Cloning, Molecular ; DNA, Complementary ; genetics ; Dendritic Cells ; drug effects ; immunology ; Genetic Vectors ; genetics ; Mice ; Mice, Inbred C57BL ; Molecular Sequence Data ; Plasmids ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Stomach Neoplasms ; metabolism ; pathology ; Transfection ; Tumor Cells, Cultured

Serum amyloid A (SAA) has been regarded as an important mediator of inflammatory responses. The effect of several formyl peptide receptor-like 1 (FPRL1) ligands on the production of IL-8 by SAA was investigated in human neutrophils. Among the ligands tested, LL-37 was found to specifically inhibit SAA-induced IL-8 production in transcriptional and post-transcriptional levels. Since SAA stimulated IL-8 production via ERK and p38 MAPK in human neutrophils, we tested the effect of LL-37 on SAA induction for these two MAPKs. LL-37 caused a dramatic inhibition of ERK and p38 MAPK activity, which is induced by SAA. LL-37 was also found to inhibit SAA-stimulated neutrophil chemotactic migration. Further, the LL-37-induced inhibitory effect was mediated by FPRL1. Our findings indicate that LL-37 is expected to be useful in the inhibition of SAA signaling and for the development of drugs against SAA-related inflammatory diseases.
Animals ; Antimicrobial Cationic Peptides/*pharmacology ; Cell Line, Tumor ; Cell Movement ; Chemotaxis, Leukocyte ; Humans ; Interleukin-8/*biosynthesis ; MAP Kinase Kinase Kinases/metabolism ; Neutrophils/drug effects/*immunology ; Proto-Oncogene Proteins/metabolism ; Rats ; Receptors, Formyl Peptide/metabolism ; Receptors, Lipoxin/metabolism ; Serum Amyloid A Protein/*antagonists & inhibitors ; Signal Transduction ; Transcription, Genetic

Serum amyloid A (SAA) has been regarded as an important mediator of inflammatory responses. The effect of several formyl peptide receptor-like 1 (FPRL1) ligands on the production of IL-8 by SAA was investigated in human neutrophils. Among the ligands tested, LL-37 was found to specifically inhibit SAA-induced IL-8 production in transcriptional and post-transcriptional levels. Since SAA stimulated IL-8 production via ERK and p38 MAPK in human neutrophils, we tested the effect of LL-37 on SAA induction for these two MAPKs. LL-37 caused a dramatic inhibition of ERK and p38 MAPK activity, which is induced by SAA. LL-37 was also found to inhibit SAA-stimulated neutrophil chemotactic migration. Further, the LL-37-induced inhibitory effect was mediated by FPRL1. Our findings indicate that LL-37 is expected to be useful in the inhibition of SAA signaling and for the development of drugs against SAA-related inflammatory diseases.
Animals ; Antimicrobial Cationic Peptides/*pharmacology ; Cell Line, Tumor ; Cell Movement ; Chemotaxis, Leukocyte ; Humans ; Interleukin-8/*biosynthesis ; MAP Kinase Kinase Kinases/metabolism ; Neutrophils/drug effects/*immunology ; Proto-Oncogene Proteins/metabolism ; Rats ; Receptors, Formyl Peptide/metabolism ; Receptors, Lipoxin/metabolism ; Serum Amyloid A Protein/*antagonists & inhibitors ; Signal Transduction ; Transcription, Genetic

In this study, we observed that lysophosphatidylglycerol (LPG) completely inhibited a formyl peptide receptor like-1 (FPRL1) agonist (MMK-1)-stimulated chemotactic migration in human phagocytes, such as neutrophils and monocytes. LPG also dramatically inhibited IL-1beta production by another FPRL1 agonist serum amyloid A (SAA) in human phagocytes. However, LPG itself induced intracellular calcium increase and superoxide anion production in human phagocytes. Keeping in mind that phagocytes migration and IL-1beta production by FPRL1 are important for the induction of inflammatory response, our data suggest that LPG can be regarded as a useful material for the modulation of inflammatory response induced by FPRL1 activation.
Chemotaxis, Leukocyte/*drug effects ; Humans ; Interleukin-1beta/*biosynthesis ; Lysophospholipids/*pharmacology ; Monocytes/drug effects/immunology/metabolism/physiology ; Neutrophils/drug effects/immunology/metabolism/physiology ; Peptides/metabolism/pharmacology ; *Phagocytes/drug effects/immunology/metabolism/physiology ; Receptors, Formyl Peptide/*metabolism ; Receptors, Lipoxin/*metabolism ; Serum Amyloid A Protein/metabolism/pharmacology

9-cis-retinoic acid (9CRA) plays an important role in the immune response; this includes cytokine production and cell migration. We have previously demonstrated that 9CRA increases expression of chemokine receptors CCR1 and CCR2 in human monocytes. To better understand how 9CRA induces CCR1 and CCR2 expression, we examined the contribution of signaling proteins in human monocytic THP-1 cells. The mRNA and surface protein up-regulation of CCR1 and CCR2 in 9CRA-stimulated cells were weakly blocked by the pretreatment of SB202190, a p38 MAPK inhibitor, and PD98059, an upstream ERK inhibitor. Activation of p38 MAPK and ERK1/2 was induced in both a time and dose-dependent manner after 9CRA stimulation. Both p38 MAPK and ERK1/2 phosphorylation peaked at 2 h after a 100 nM 9CRA treatment. 9CRA increased calcium influx and chemotactic activity in response to CCR1-dependent chemokines, Lkn-1/CCL15, MIP-1alpha/CCL3, and RANTES/CCL5, and the CCR2-specific chemokine, MCP-1/CCL2. Both SB202190 and PD98059 pretreatment diminished the increased calcium mobilization and chemotactic ability due to 9CRA. SB202190 inhibited the expression and functional activities of CCR1 and CCR2 more effectively than did PD98059. Therefore, our results demonstrate that 9CRA transduces the signal through p38 MAPK and ERK1/2 for CCR1 and CCR2 up-regulation, and may regulate the pro-inflammatory process through the p38 MAPK and ERK-dependent signaling pathways.
Calcium Signaling/drug effects ; Cell Line ; Chemokines/pharmacology ; Chemotaxis, Leukocyte/drug effects ; Enzyme Activation/drug effects ; Extracellular Signal-Regulated MAP Kinases/*metabolism ; Flavonoids/pharmacology ; Gene Expression Regulation/*drug effects ; Humans ; Imidazoles/pharmacology ; Mitogen-Activated Protein Kinase 1/metabolism ; Mitogen-Activated Protein Kinase 3/metabolism ; Monocytes/drug effects/*enzymology ; Pyridines/pharmacology ; RNA, Messenger/genetics/metabolism ; Receptors, CCR1 ; Receptors, CCR2 ; Receptors, Chemokine/*genetics/metabolism ; Tretinoin/*pharmacology ; p38 Mitogen-Activated Protein Kinases/*metabolism

AIMTo study the inhibitory effect of ginkgolide B (BN52021) on the PAF induced changes of chemotaxis of murine peritoneal macrophages and the related polymerization of F-actin.

METHODSChemotaxis assays were performed using a modified 48-well Boyden chamber. Actin polymerization of murine peritoneal macrophages was analyzed by flow cytometry using a specific fluorescent stain.

RESULTSPeritoneal macrophages significantly migrated toward platelet-activating factor (PAF) through a micropore filter; however, in the presence of PAF receptor antagonist BN52021 (0.01 nmol x L(-1) -0.1 micromol x L(-1)), the migration was significantly inhibited. Moreover, BN52021 inhibited the actin polymerization of murine peritoneal macrophages induced by PAF in the presence of Ca2+, but not in Ca2+ -free medium.

CONCLUSIONThe results suggested that preventing polymerization of F-actin may be a pathway by BN52021 to inhibit the chemotaxis of macrophages, and this effect seems to be Ca2+ dependent. The data further indicated that inhibition of PAF induced macrophage chemotaxis is an important mechanism underlying the anti-inflammatory action of BN52021.

Actins ; metabolism ; Animals ; Chemotaxis, Leukocyte ; drug effects ; Diterpenes ; isolation & purification ; pharmacology ; Ginkgo biloba ; chemistry ; Ginkgolides ; Lactones ; isolation & purification ; pharmacology ; Macrophages, Peritoneal ; metabolism ; physiology ; Mice ; Mice, Inbred C57BL ; Plants, Medicinal ; chemistry ; Platelet Activating Factor ; antagonists & inhibitors

OBJECTIVETo explore the regularity of recipe composition by observing inhibitory effects of disassembled compositions of Mahuang decoction (MHD) on chemotaxis and leukotriene production from neutrophils in rats.

METHODNeutrophil aggregation was induced by intraperitoneal injection of glycogen in rats. Intraperitoneal lavage fluid (PLF) was collected and neu-trophils were removed. Neutrophils were stimulated by calciumionophore A23187 in vitro to produce leukotriene B4. The concentrations of leukotriene B4 was measured by reversed-phase high performance liquid chromatography(HPLC), chemotatic chamber assay was used to investigate the regulative role of MHD on chenmotaxis of the neutrophils in response to LPS stimulation.

RESULTDisassembled compositions of MHD could inhibite chemotaxis and leukotriene production from neutrophils in rats. Inhibitory effects of MHD on mast cells were different.

CONCLUSIONMHD has significantly inhibitory effects on chemotaxis and leukotriene production from neutrophils in rats. The original formula (MHD) works best. These results have confirmed the rationality and scientific level of MHD.

Animals ; Chemotaxis, Leukocyte ; drug effects ; Drug Combinations ; Drugs, Chinese Herbal ; administration & dosage ; isolation & purification ; pharmacology ; Female ; Leukotriene B4 ; biosynthesis ; Male ; Neutrophils ; drug effects ; secretion ; Plants, Medicinal ; chemistry ; Rats ; Rats, Sprague-Dawley