Chemotactic Activity In Conditioned Medium Research Articles

Amnion-derived serum amyloid A1 participates in sterile inflammation of fetal membranes at parturition.

Sterile inflammation of fetal membranes is an indispensable event of normal parturition. However, triggers of sterile inflammation are not fully resolved. Serum amyloid A1 (SAA1) is an acute phase protein produced primarily by the liver. Fetal membranes can also synthesize SAA1 but its functions are not well defined. Given the role of SAA1 in the acute phase response to inflammation, we postulated that SAA1 synthesized in the fetal membranes may be a trigger of local inflammation at parturition. The changes of SAA1 abundance in parturition were studied in the amnion of human fetal membranes. The role of SAA1 in chemokine expression and leukocyte chemotaxis was examined in cultured human amnion tissue explants as well as primary human amnion fibroblasts. The effects of SAA1 on monocytes, macrophages and dendritic cells were investigated in cells derived from a human leukemia monocytic cell line (THP-1). SAA1 synthesis increased significantly in human amnion at parturition. SAA1 evoked multiple chemotaxis pathways in human amnion fibroblasts along with upregulation of a series of chemokines via both toll-like receptor 4 (TLR4) and formyl peptide receptor 2 (FPR2). Moreover, SAA1-conditioned medium of cultured amnion fibroblasts was capable of chemoattracting virtually all types of mononuclear leukocytes, particularly monocytes and dendritic cells, which reconciled with the chemotactic activity of conditioned medium of cultured amnion tissue explants collected from spontaneous labor. Furthermore, SAA1 could induce the expression of genes associated with inflammation and extracellular matrix remodeling in monocytes, macrophages and dendritic cells derived from THP-1. SAA1 is a trigger of sterile inflammation of the fetal membranes at parturition.

Cyclic Stretch Augments Production of Neutrophil Chemokines and Matrix Metalloproteinases‐1 (MMP‐1) from Human Decidual Cells, and the Production was Reduced by Progesterone

The purpose of this study was to evaluate the impact of mechanical stretch caused by uterine contraction and progesterone (P₄) on decidual cells (DC), neutrophil chemokines, and MMP-1 expression. DC were cultured, and cyclic stretch was applied using a computer-operated cell stretch system. Interleukin (IL)-8, growth-regulated oncogene (GRO) α, matrix metalloproteinase (MMP)-1, and mRNA and/or protein expression/activity was measured using RT-PCR and ELISA. Neutrophil chemotactic activity in conditioned media was evaluated using migration assays. The effect of P₄ was also studied. Cyclic mechanical stretch increased IL-8, GROα, mRNA and protein, and MMP-1 production and activity level. Supernatant from stretched cells induced neutrophil chemotactic activity significantly. P₄ suppressed the effect of stretch. The current study demonstrates that cyclic mechanical stretch stimulates the production of neutrophil chemokines and MMP-1 from human decidual cells, and the production was reduced by progesterone. These findings suggest that decidual cells are responding to mechanical and endocrine signals and induce biochemical factors and thereby contribute to the regulation of human labor.

Cyclic Stretch Augments Production of Neutrophil Chemokines and Matrix Metalloproteinase‐1 in Human Uterine Smooth Muscle Cells

The aim of this study was to investigate the impact of uterine contraction on the immune environment within the uterus during parturition. Uterine smooth muscle cells (USMC) were isolated from uterine myometrial tissues and cultured. The effects of cyclic stretch on mRNA and/or protein expression of IL-8, Groα, and pro-MMP-1 by USMC were measured using RT-PCR and ELISA. Neutrophil chemotactic activity in conditioned media was evaluated using migration assays. To evaluate the effect of progesterone (P(4) ), USMC were pretreated with P(4) for 24hr. Cyclic stretch increased IL-8 and Groα mRNA and protein and pro-MMP-1 production significantly. Supernatants from stretched cells induced neutrophil chemotactic activity significantly; these effects were abrogated by anti-IL-8 or Groα neutralizing antibodies. Stretch effects were reduced by P(4) . These results suggest that uterine contraction may induce neutrophil infiltration and MMP-1 production, which may contribute to cervical ripening and rupture of membrane. The inhibitory effects of P(4) may explain the mechanism by which progestin prevents preterm labor.

NF-κB Decoy Oligonucleotides Suppress RANTES Expression and Monocyte Chemotactic Activity via NF-κB Inactivation in Stromal Cells of Ectopic Endometrium

The nuclear factor kappa B (NF-kappaB) pathway is a critical mediator of regulated on activation, normal T cell expressed and secreted (RANTES) gene regulation and therefore represents a potential target for therapy of endometriosis-associated symptoms. The objective of this study was to investigate the effect of NF-kappaB decoy oligonucleotides (ODNs) on NF-kappaB activation, RANTES expression, and monocyte chemotactic activity in ectopic endometrial stromal cells in vitro. A specific sandwich enzyme-linked immunosorbent assay (ELISA) was used to quantify RANTES expression in ectopic and normal endometrial stromal cells stimulated by interleukin (IL)-1beta. Four hours after transfection of NF-kappaB decoy ODNs, 10 ng/ml IL-1beta was added to induce the ectopic endometrial stromal cells to secrete RANTES. The NF-kappaB activation, RANTES expression, and monocyte chemotactic activity in ectopic endometrial stromal cells were respectively evaluated by electrophoretic mobility shift assay, ELISA, and Boyden chambers. IL-1beta induced significantly higher levels (P < 0.05) of RANTES expression in a time-dependent manner in ectopic endometrial stromal cells compared with IL-1beta-untreated ectopic and normal endometrial stromal cells. The RANTES accounts for the majority (68%) of the monocyte chemotactic activity in conditioned media of ectopic endometrial stromal cells. In vitro transfection of NF-kappaB decoy ODNs dramatically decreased (P < 0.05) the NF-kappaB activation, RANTES expression, and monocyte chemotactic activity in IL-1beta-induced ectopic endometrial stromal cells. NF-kappaB decoy ODNs may exert anti-inflammatory effects in ectopic endometrial stromal cells via the suppression of NF-kappaB activation, RANTES expression, and monocyte chemotactic activity.

Ectopic, autologous eutopic and normal endometrial stromal cells have altered expression and chemotactic activity of RANTES

Objective To evaluate if the expression and chemotactic activity of RANTES are different in IL-1β treated autologous eutopic endometrial stromal cells compared to ectopic and normal endometrium. Study design Conditioned media from IL-1β-treated ectopic, autologous eutopic and normal endometrial stromal cells were analyzed with a specific sandwich ELISA to quantify RANTES. The monocyte chemotactic activity of RANTES was assayed in a Boyden Chamber. Results RANTES expression in IL-1β-treated autologous eutopic and normal endometrial stromal cells was significantly lower than ectopic endometrium. Autologous eutopic endometrial stromal cells showed a significant increase in RANTES expression compared to normal endometrium after IL-1β stimulation for 60 h. The monocyte chemotactic activities of these conditioned media were highly correlated with the immunoreactive RANTES concentration. We observed significantly increased monocyte chemotactic activity in conditioned media of ectopic stromal cells compared to autologous eutopic and normal endometrium. The different chemotactic activity of RANTES between the autologous eutopic and normal endometrial stromal cells was also statistically significant. RANTES accounts for the majority (62%) of the monocyte chemotactic activity in ectopic endometrial stromal cells conditioned media and 55% of that activity in autologous eutopic endometrium. Conclusions Although the eutopic endometric of women with and without endometriosis are histologically similar, our findings confirm that different expression and chemotactic activity of RANTES exist between autologous eutopic and normal endometrium. The altered expression of RANTES and monocyte chemotactic activity observed in ectopic, autologous eutopic and normal endometrium suggest the autologous eutopic endometrium may contribute to the pathogenesis of endometriosis.

Soluble Interleukin-6 Receptor α Inhibits the Cytokine-Induced Fractalkine/CX3CL1 Expression in Human Vascular Endothelial Cells in Culture

Soluble form of IL-6 receptor α (sIL-6R) is known to serve as an agonist, without exogenous IL-6, on endothelial cells which do not express IL-6R but have only IL-6 receptor β chain, gp130. We investigated the effect of sIL-6R on fractalkine expression in human umbilical vein endothelial cells (HUVECs) in culture. sIL-6R markedly inhibited HUVEC fractalkine/CX3CL1 expression induced by interleukin (IL)-1α, tumor necrosis factor (TNF)-α, or interferon (IFN)-γ. IL-1α-induced fractalkine expression was inhibited by sIL-6R in time- and concentration-dependent manners. The experiment using actinomycin D indicated that sIL-6R lowered the stability of fractalkine mRNA. The inhibitory effect of sIL-6R was reversed by anti-gp130 neutralizing antibody. sIL-6R inhibited adhesion of mononuclear cells (MNCs) to HUVEC monolayers stimulated with IFN-γ, but it did not inhibit the adhesion to monolayers stimulated with IL-1α. MNC chemotactic activity of conditioned medium of HUVEC stimulated with IL-1α or IFN-γ was inhibited by co-treatment with sIL-6R. sIL-6R may play a regulatory role in immune responses by modulating the interaction between leukocytes and the vascular endothelium.

Cell-Associated Human Retinal Pigment Epithelium Interleukin-8 and Monocyte Chemotactic Protein-1: Immunochemical and In-situ Hybridization Analyses

Human retinal pigment epithelial (RPE) cells secrete chemokines, interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) in response to pro-inflammatory cytokines. In this study we (1) examined the efficiency of human RPE IL-8 and MCP-1 secretion, (2) determined the amount of neutrophil and monocyte chemotactic activity in human RPE cell conditioned media and cell extracts that is attributable to IL-8 and MCP-1, respectively, and (3) assessed the sensitivity of immunohistochemistry and in situ hybridization for detecting chemokine production by cytokine-stimulated human RPE cells. Conditioned media and extracts from human RPE cells stimulated with various physiologic concentrations of interleukin-1 beta (IL-1β) (0.2–20 ng ml−1), tumor necrosis factor (TNF-α) (0.2–20 ng ml−1) or interferon-gamma (IFN-γ) (10–1000 U ml−1) were examined to compare secreted and cell associated levels of IL-8 and MCP-1 at various time points up to 24 hr.ELISA demonstrated that IL-8 and MCP-1 are both efficiently secreted by pro-inflammatory cytokine treated human RPE cells. Substantial dose- and time-dependent RPE secretion of IL-8 was observed following stimulation with IL-1β or TNF-α, but cell associated IL-8 was detectable only after high dose (20 ng ml−1) IL-1β stimulation and comprised less than 1% of the total IL-8 induced. Dose- and time-dependent RPE cell MCP-1 secretion was also observed following IL-1β>TNF-α>IFN-γ stimulation, with an average of 4% of the total MCP-1 retained within RPE. Bioassays demonstrated neutrophil and monocyte chemotactic activity in conditioned media from stimulated RPE cells, but not in human RPE cell extracts. Inhibition of conditioned media-induced chemotaxis by specific anti-IL-8 or anti-MCP-1 antibodies demonstrated that IL-8 and MCP-1 were responsible for the majority of HRPE-derived neutrophil (>60%) and monocyte (53–57%) chemotactic activity, respectively.Using in situ hybridization IL-8 mRNA was readily detected within IL-1β>TNF-α stimulated RPE cells and MCP-1 mRNA easily visualized within IL-1β>TNF-α> or IFN-γstimulated cells. Immunohistochemistry to detect IL-8 was positive only in RPE cells exposed to high dose IL-1β(20 ng ml−1) for 8 or 24 hr and was weak. Immunohistochemical staining for MCP-1 in RPE cells was more intense and was visualized within RPE cells stimulated with IL-β, TNF-α, or IFN-γ. This study demonstrates that: (1) RPE cells efficiently secrete IL-8 and MCP-1 upon stimulation with pro-inflammatory cytokines; (2) secreted IL-8 and MCP-1 account for the majority of human RPE neutrophil and monocyte chemotactic activity; (3) in situ hybridization readily detects IL-8 and MCP-1 mRNA in cytokine stimulated RPE cells; and (4) immunohistochemistry demonstrates cell-associated MCP-1 in cytokine stimulated RPE cells, but only minimal cell-associated IL-8.

Interleukin-1 beta and tumor necrosis factor-alpha stimulate synergistically the expression of monocyte chemoattractant protein-1 in fibroblastic cells derived from human periodontal ligament.

The present study demonstrates that interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) induce and synergistically stimulate monocyte chemoattractant protein-1 (MCP-1) expression in fibroblasts from human periodontal ligament. IL-1 beta and TNF-alpha induced in a dose-dependent manner the expression of the MCP-1 gene in the fibroblasts from the human periodontal ligament. However, such an inducing effect was not observed with IL-6 and interferon-gamma. The peak expression of the MCP-1 gene by IL-1 beta or TNF-alpha was observed at 3 h after initiation of their treatment. Furthermore, IL-1 beta in combination with TNF-alpha synergistically stimulated the MCP-1 gene expression in the cells. We also observed significant chemotactic activity for human monocytes in conditioned medium of fibroblasts from the human periodontal ligament treated with both cytokines. The stimulated chemotactic activity induced by these cytokines depended on both dose and treatment time. The chemotactic activity in conditioned medium of IL-1 beta-treated fibroblasts from the human periodontal ligament was neutralized by antiserum specific for MCP-1 protein. The MCP-1 gene product in conditioned medium of IL-1 beta-treated fibroblasts from the human periodontal ligament was shown to have a molecular mass of 11,000 Da by immunoprecipitation with the specific antiserum, and IL-1 beta also stimulated synergistically MCP-1 protein expression in combination with TNF-alpha. These results suggest that IL-1 beta and TNF-alpha may contribute to the infiltration of monocytes into inflammatory sites of periodontal ligament tissues via the MCP-1 gene product produced by fibroblasts from the human periodontal ligament.

Expression and secretion of transforming growth factor-beta by bleomycin-stimulated rat alveolar macrophages.

Bleomycin-induced fibrosis in rodents has been used extensively as a model of human pulmonary fibrosis. The influx of monocytes observed during the early stages of fibrosis is at least partially regulated by the elaboration of chemotactic factors in the lung. Exposure of alveolar macrophages (AM phi) to bleomycin either in vivo or in vitro stimulated secretion of monocyte chemotactic activity (MCA). This MCA has been previously characterized as being primarily due to fibronectin fragments. The present experiments revealed that bleomycin also induced AM phi to secrete a second chemotactic factor, transforming growth factor-beta (TGF-beta). However, the TGF-beta secreted by macrophages was in latent form, since no TGF-beta activity was detected unless AM phi conditioned medium (CM) was acid-activated. After acidification, chemotactic activity in CM from AM phi stimulated with bleomycin in vitro was increased by 3.6, whereas activity in AM phi CM from fibrotic rats increased by 2 and that of a bleomycin-stimulated AM phi cell line increased by 1.6. This acid-activatable chemotactic activity was inhibited by antibody to TGF-beta. Bleomycin-stimulated AM phi s secreted significantly more TGF-beta than did unstimulated controls. Further, in vitro exposure of AM phi to bleomycin induced TGF-beta mRNA expression in a time- and concentration-dependent manner, with maximal mRNA being detected following a 16-h incubation with 1 microgram/ml bleomycin.

Lnterleukin-8 Production from Cultured Human Dermal Fibroblasts by Stimulation with Supernatant of Cultured Human Epidermal Cells

There is much evidence to support the theory that keratinocytes and dermal fibroblasts actively participate in inflammatory reactions by the production of proinflammatory mediators or cytokines. We investigated the neutrophil chemotactic activity in conditioned media of cultured epidermal cells and dermal fibroblasts, and found that an epidermal cell-derived factor induced fibroblasts to produce a neutrophil chemotactic factor. This neutrophil chemotactic factor was identified as interleukin-8 (IL-8) by the elution position on HPLC and by a neutralization test that uses monoclonal anti-IL-8 antibody (14E4). The epidermal cell-derived factor was fractionated together with thymocyte-proliferating activity on Sephadex G-75 gel chromatography followed by HPLC. It was blocked specifically by anti-interleukin-1 (IL-1) alpha antibody, which indicates that this factor was IL-1 alpha. Since a variety of inflammatory dermatoses is characterized by the infiltration of neutrophils into the skin, induction of IL-8 production in fibroblasts by epidermal cells may play an important role in inflammatory skin diseases.

Generation of neutrophil chemotactic activity by phorbol ester-stimulated calf pulmonary artery endothelial cells.

Chemotactic activity for human polymorphonuclear leukocytes (PMNL) was detected in serum-free conditioned media 1 to 4 hr after monolayers of calf pulmonary artery endothelial cells were pretreated with phorbol myristate acetate (PMA). Chemotactic activity was increased in conditioned media following pretreatment with either PMA or the less lipophilic active phorbol ester, 4-beta-phorbol-12,13-dibutyrate (P(Bu)2) in a dose-dependent manner. Chemotactic activity of conditioned media from PMA-treated endothelial cells was confirmed by checkerboard analysis. The chemotactic activity in conditioned media from PMA-pretreated endothelial cells was completely inhibited by pretreating endothelial cells with either cycloheximide, actinomycin D, or the lipooxygenase inhibitor, diethylcarbamazine. Furthermore, the chemotactic activity was heat-stable, inhibited by trypsin treatment, and present in both aqueous and lipid phases after ether extraction. The data demonstrate that pulmonary artery endothelial cells exposed to active phorbol esters release potent chemotactic factor(s) for PMNL. These findings suggest a role for activators of protein kinase C in mediating endothelial cell release of chemotactic factor(s) that may be important in the directed migration of circulating leukocytes to sites of vascular injury.