B-1 cells, which constitute a predominant lymphocyte subset in serosal cavities and produce most of natural antibodies, are subdivided into the CD5+ B-1a and CD5- B-1b cell subpopulations, but the differential roles of B-1a and B-1b cells are not well understood. We report that B-1a cells preferentially migrate out of the peritoneal cavity and upregulate the expression of CXCR4 with heightened sensitivity to CXCL12 and CXCL13 upon LPS treatment compared to B-1b and B-2 cells. Whereas B-1a cells were slightly more abundant than B-1b and B-2 cells in the homeostatic condition, the number of B-1a cells preferentially decreased 48 hr after LPS treatment. The decrease in the peritoneal B-1a cell number was accompanied with increased migration of B-1a cells toward CXCL-12 and CXCL-13 in in vitro transmigration assay using peritoneal B cells from LPS treated mice. The expression level of CXCR4, but not of CXCR5, was also more prominently increased in B-1a cells upon LPS stimulation. LPS-stimulated B-1a cells did not accumulate in omental milky spots in contrast to B-2 cells. These results suggest that B-1a cells actively migrate out of the peritoneal cavity through the regulation of the migratory responsiveness to chemokines and actively participate in systemic immune responses.
Adjuvants, Immunologic/pharmacology ; Animals ; B-Lymphocytes/cytology/*drug effects/immunology ; Cell Movement ; Cells, Cultured ; Chemokine CXCL12/metabolism/*pharmacology ; Chemokine CXCL13/metabolism/pharmacology ; Lipopolysaccharides/*pharmacology ; Mice ; Mice, Inbred C57BL ; Peritoneal Cavity/cytology ; Receptors, CXCR4/*metabolism ; Up-Regulation

OBJECTIVETo investigate the effects of peritoneal dialysis solution (PDS) on apoptosis and intracellular free calcium([Ca(2+)]i), cell surface ICAM-1 expression of rat peritoneal mesothelial cells (RPMCs).

METHODSThe RPMCs apoptosis rate were determined by flow cytometry. [Ca(2+)]i in the cells were monitered the fluorescence at 528 nm by confocus laser microscopy. Cell surface ICAM-1 expression were detected by flow cytometry.

RESULTAfter PDS treatment for 1 h, the RPMCs apoptosis rate were increased. Such increase was more manifest with higher glucose concentration in PDS and longer treatment time of the cells. At the same times, after 3 hours, ICAM-1 expressions of the PDS containing glucose and mannitol are all increased. With the increase of glucose concentrations, the descend of [Ca(2+)]i levels were aggravated.

CONCLUSIONPDS containing high- concentration glucose can induce significant apoptosis of RPMCs in vitro. This may be related with the enhanced level of ICAM-1 expressions and the decreased level of [Ca(2+)]i. Which may due to the occurrence of peritoneal fibrosis and ultrafiltrate failure in patients suffering long term peritoneal dialysis.

Animals ; Apoptosis ; drug effects ; Calcium ; metabolism ; Dialysis Solutions ; pharmacology ; Dose-Response Relationship, Drug ; Epithelial Cells ; cytology ; drug effects ; metabolism ; Gene Expression Regulation ; drug effects ; Glucose ; pharmacology ; Intercellular Adhesion Molecule-1 ; metabolism ; Intracellular Space ; drug effects ; metabolism ; Male ; Peritoneal Cavity ; cytology ; Peritoneal Dialysis ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the role of nicotinamide-adenine dinucleotide phosphate (NADPH) oxidasedependent formation of reactive oxygen species (ROS) in the transforming growth factor β1 (TGF-β1)-induced epithelial-mesenchymal transition (EMT) in rat peritoneal mesothelial cells (RPMCs), and the effect of Astragalus injection (AGI) intervention.

METHODSPrimary RPMCs were cultured to the second generation in vitro. After synchronization for 24 h, the cells were randomly assigned to the following groups: control (Group A), AGI (2 g/mL; Group B), TGF-β1 (10 ng/mL; Group C), TGF-β1 (10 ng/mL) + AGI (2 g/mL; Group D; pretreated for 1 h with AGI before TGF-β1 stimulation). Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis were employed to evaluate the mRNA and protein expression of the NADPH oxidase subunit p67phox, α-smooth muscle actin (α-SMA) and E-cadherin. The dichlorofluorescein-sensitive cellular ROS levels were measured by a fluorometric assay and confocal microscopy.

RESULTSTGF-β1 significantly induced NADPH oxidase subunit p67phox mRNA and protein expression in RPMCs, as well as inducing the production of intracellular ROS. AGI inhibited this TGF-β1-induced up-regulation by 39.3% and 47.8%, respectively (P<0.05), as well as inhibiting the TGF-β1-induced ROS generation by 56.3% (P<0.05). TGF-β1 also induced α-SMA mRNA and protein expression, and down-regulated E-cadherin mRNA and protein expression (P<0.05). This effect was suppressed by AGI (P<0.05).

CONCLUSIONSNADPH oxidase-dependent formation of ROS may mediate the TGF-β1-dependent EMT in RPMCs. AGI could inhibit this process, providing a theoretical basis for AGI in the prevention of peritoneal fibrosis.

Animals ; Base Sequence ; DNA Primers ; Epithelial-Mesenchymal Transition ; physiology ; Epithelium ; NADPH Oxidases ; metabolism ; Peritoneal Cavity ; cytology ; Polymerase Chain Reaction ; Rats ; Reactive Oxygen Species ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Transforming Growth Factor beta1 ; physiology

Atrial natriuretic peptide (ANP), a 28 amino acid basic polypeptide, is known to induce histamine release from human and rat mast cells in vitro and cause a wheel formation in rat skin. However, cellular events associated with histamine release are not clearly understood. In this study, we have examined the calcium flux and cGMP formation associated with histamine release in the ANP-treated mast cells. ANP, in vitro, induced mast cell degranulation and histamine release in a dose-dependent manner. ANP also induced an enhanced calcium uptake into cells and increased the cellular level of cGMP in mast cells. A high level of calcium in the media caused an inhibition of ANP-dependent histamine release but enhanced the level of intracellular cGMP of mast cells. ANP inducing a dose-dependent increase in vascular permeability of rat skin was confirmed by the extravasation of the circulating Evans blue. The results indicate ANP induced the histamine release and an increase in vascular permeability through mast cell degranulation in cGMP-independent and calcium uptake-dependent manner.
Animal ; Atrial Natriuretic Factor/*pharmacology ; Biological Transport ; Calcium/*metabolism ; Capillary Permeability ; Cell Degranulation ; Cyclic GMP/*metabolism ; Dose-Response Relationship, Drug ; *Histamine Release ; Mast Cells/*drug effects ; Peritoneal Cavity/cytology ; Rats

Pannexin-1 (Panx1) forms nonselective large channel in cell plasma membrane and has been shown to be associated with NLRP3 inflammasome activation, ATP release and phagocytes recruitment. In the current study, by manipulation of Panx1 expression in human myeloid cells and application of Panx1 deficient mice, we failed to find a correlation between Panx1 and NLRP3 inflammasome activation, although an interaction between these two proteins was evident. However, in thioglycollate induced peritonitis, Panx1 deficient mice showed much more phagocytes infiltration. Further analyses showed that mice deficient for Panx1 exhibited enlarged F4/80(low)Gr1(-)Ly6C(-)cell population in the peritonea. Our study thus reveals an important role for Panx1 in regulation of peritoneal cell population and peritonitis development.
Animals ; Carrier Proteins ; metabolism ; Cell Line ; Connexins ; antagonists & inhibitors ; deficiency ; genetics ; metabolism ; HEK293 Cells ; Humans ; Inflammasomes ; metabolism ; Macrophages ; cytology ; metabolism ; Mice ; Mice, Inbred C57BL ; NLR Family, Pyrin Domain-Containing 3 Protein ; Nerve Tissue Proteins ; antagonists & inhibitors ; deficiency ; genetics ; metabolism ; Peritoneal Cavity ; cytology ; Peritonitis ; chemically induced ; metabolism ; pathology ; RNA Interference ; RNA, Small Interfering ; metabolism ; Thioglycolates ; toxicity

OBJECTIVETo study the effect of ligustrazine nanoparticles nano spray (LNNS) on transforming growth factor β (TGF-β)/Smad signal protein of rat peritoneal mesothelial cells (RPMC) induced by tumor necrosis factor α (TNF-α), and the anti-adhesion mechanism of LNNS in the abdominal cavity.

METHODSThe primary culture and subculture of rat peritoneal mesothelial cells (RPMC) was processed by trypsin digestion method in vitro. The third generation was identifified for experiment and divided into 5 groups: a blank group: RPMC without treatment; a control group: RPMC stimulated with TNF-α; RPMC treated by a low-dosage LNNS group (2.5 mg/L); RPMC treated by a medium-dosage LNNS group (5 mg/L); and RPMC treated by a high-dosage LNNS group (10 mg/L). Reverse transcription-polymerase chain reaction was applied to test the expression of fifibronectin, collagen I (COL-I), TGF-β mRNA, and Western blot method to test the Smad protein 7 expression of RPMC.

RESULTSCompared with the blank group, a signifificant elevation in fifibronectin (FN), COL-I and TGF-β mRNA expression of RPMC were observed in the control group (P<0.05). Compared with the control group, LNNS suppressed the expressions of FN, COL-I and TGF-β mRNA in a concentrationdependent manner (P<0.05). The expression of Smad7 protein of RPMC was down-regulated by TNF-α stimulation, and up-regulated with the increase of LNNS dose (P<0.05).

CONCLUSIONSTNF-α may induce changes in RPMC's viability, leading to peritoneal injury. LNNS could reverse the induction of fifibrosis related cytokine FN, COL-I and TGF-β, up-regulating the expression of Smad7 by TNF-α in RPMC, thus attenuate peritoneal injury by repairing mesothelial cells.

Animals ; Collagen Type I ; genetics ; metabolism ; Epithelium ; drug effects ; metabolism ; Fibronectins ; metabolism ; Male ; Nanoparticles ; chemistry ; ultrastructure ; Particle Size ; Peritoneal Cavity ; cytology ; Pyrazines ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Rats, Sprague-Dawley ; Signal Transduction ; drug effects ; Smad Proteins ; metabolism ; Transforming Growth Factor beta ; genetics ; metabolism ; Tumor Necrosis Factor-alpha ; pharmacology

OBJECTIVETo investigate the impact of human peritoneal mesothelial cells (HPMC) on angiogenesis factor expression and secretion of ovarian carcinoma cell line SKOV3.

METHODSThe conditioned medium with HPMC was tested by ELISA for tumor necrosis factor-alpha (TNF-alpha) and interleukin 10 (IL-1beta). Millicell was used to co-culture HPMC and ovarian carcinoma cell line SKOV3 in the presence or absence of neutralizing antibody against TNF-alpha or IL-1beta. RT-PCR was used to detect vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) gene expression in SKOV3 cells. VEGF and bFGF protein levels in the SKOV3 conditioned medium were assessed by ELISA.

RESULTSConditioned medium with HPMC contained both TNF-alpha and IL-1beta. SKOV3 co-cultured with HPMC expressed higher levels of VEGF and bFGF mRNA and secreted at increased levels of both VEGF and bFGF, in comparison with those in SKOV3 cells cultured alone (P < 0.01). Addition of neutralizing antibody against TNF-alpha or IL-1beta during co-cultures resulted in decrease in mRNA expression and secretion of VEGF and bFGF in SKOV3 cells. When both antibodies were administered during co-culture, additive decrease was observed.

CONCLUSIONHPMC can act in a paracrine fashion to stimulate ovarian tumor cells to produce and secret at increased levels of VEGF and bFGF through TNF-alpha and IL-1beta, and contribute to angiogenesis and peritoneal metastasis of ovarian cancer.

Antibodies ; pharmacology ; Cell Line, Tumor ; Cells, Cultured ; Coculture Techniques ; Culture Media, Conditioned ; metabolism ; Cystadenocarcinoma, Serous ; genetics ; metabolism ; pathology ; Enzyme-Linked Immunosorbent Assay ; Epithelial Cells ; secretion ; Female ; Fibroblast Growth Factor 2 ; genetics ; secretion ; Gene Expression ; drug effects ; Humans ; Interleukin-1beta ; immunology ; secretion ; Ovarian Neoplasms ; genetics ; metabolism ; pathology ; Peritoneal Cavity ; cytology ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Tumor Necrosis Factor-alpha ; immunology ; secretion ; Vascular Endothelial Growth Factor A ; genetics ; secretion