13 research outputs found
Endothelin-1 Stimulates Monocytes in vitro to Release Chemotactic Activity Identified as Interleukin-8 and Monocyte Chemotactic Protein-1
In the present study we examined whether endothelin-1 stimulation of
human monocytes causes release of chemotactic factors. It was found
that monocytes released neutrophil- and monocyte-chemotactic
activity in a dose- and time-dependent manner in response to ET-1.
ET-1 did not show any chemotactic activity by itself. NCA was
detected in monocyte supernatants in response to ET-1
(0.01–100 nM) after 1, 4, 8 and 24 h stimulation. MCA was
detected only after 24 h stimulation with ET-1 (0.1–100 nM).
Preincubation of the monocyte cultures with the lipoxygenase
inhibitors nordihydroguaiaretic acid (10−4 M) or
diethylcarbamazine (10−9 M) completely abolished
the appearance of NCA and MCA. NCA was neutralized by >
75% using a polyclonal antibody against human interleuktn-8.
The ET-1 induced release of IL-8 was confirmed by IL-8 ELISA. A
monoclonal antibody against human monocyte chemotactic protein-1
neutralized MCA by > 80%. It is concluded that ET-1
stimulation of monocytes in vitro causes release of
neutrophil- and monocyte-chemotactic activity identified as IL-8 and
MCP-I respectively. An intact lipoxygenase pathway is crucial for
this effect of ET-1 to occur
The TNF Receptors p55 and p75 Mediate Chemotaxis of PMN Induced by TNFα and a TNFα 36–62 Peptide
The present study was performed to examine whether residues
36–62 of TNFα contain the chemotactic domain of
TNFα, and whether the p55 and p75 TNF receptors are involved
in TNFα induced chemotaxis. The chemotactic effect of
TNFα on PMN was inhibited by the mAbs Hrt-7b and Utr-1,
against the p55 and p75 TNF receptors, respectively. Both receptors may
therefore be required for mediating the chemotactic effect of TNFcz.
The synthetic TNFα 36–62, similar to TNFα, had
chemotactic effects on both PMN and monocytes. The chemotactic
activity of the TNFα 36–62 peptide on PMN, was inhibited
by Htr-7b, Utr-1 and soluble p55 receptor, which shows that the
peptide possessed the ability to induce chemotaxis through the TNF
receptors. In contrast to TNFα, the peptide did not show a
cytotoxic activity against WEHI 164 flbrosarcoma cells. It is
suggested that different domains of the TNFα molecule induce
distinct biological effects
Neutral Red Assay Modification to PreventCytotoxicity and Improve Repeatability Using E-63 Rat Skeletal Muscle Cells
Cellular uptake of neutral red dye (NR) is currently used as an indirect measure of viable cells in cultures. We used E-63 rat skeletal muscle cells to identify causes of NR assay variability and to develop modifications that substantially reduce it. Three methods of NR preparation and/or addition to cells were used. When NR medium was prepared, incubated overnight, and filtered to remove precipitates, the amount of dye precipitated varied greatly. Coefficients of variation (CVs) in NR uptake were greater than 25% between assays. Higher NR concentrations, longer incubation times, increased pH, and decreased temperature promoted NR precipitation in media. NR media prepared and filtered just prior to use or direct addition of prefiltered NR stock solution to cell cultures resulted in much smaller CVs between assays. NR was cytotoxic to E-63 rat muscle and primary quail myoblasts in a time-and concentration-dependent manner. NR exposure to E-63 cells for greater than 1.25 and 2 hr at 157 or 127 μg/ml, respectively, was associated with swelling and rupture of lysosomes. By contrast, there was no evidence of cytotoxicity when E-63 cells were exposed to NR for 1 hr at either 127 or 157 μg/ml. Primary quail myoblasts developed lysosomal swelling and ruptured more rapidly than E-63 cells when exposed to NR at either 127 or 157 μg/ml. For confluent 10-day cultures of E-63 cells exposed to NR at 127 μg/ml for 1 hr, the CVs within assay and between assays were 3.3-3.9% and 5.1%, respectively. For similarly exposed, actively replicating 3-day cultures of E-63 cells, the CVs within and between assays were 6.2-9.6% and 2.4%, respectively. NR uptake by the E-63 cells was linear with respect to viable cell number