TNF-α dose response of h-GF for SAOS-2 binding.

<p>A graph shows the dose response for TNF-α of h-GF stimulated for 24 hrs with regard to subsequent SAOS-2 binding. The effect of TNF-α was seen by 0.06 nM of cytokine, and was maximal by 1.16 nM (p<0.05).</p

TNF-α increased ICAM-1 expression in h-GF and HUVEC, but only increased VCAM-1 in HUVEC.

<p>Fluorescence distribution profiles are shown proportionate to the peak incidence of fluorescence in each cell population studied. Background levels of ICAM-1 in both h-GF and HUVEC were appreciably increased following 24 hrs of stimulation with TNF-α (1.16nM). Although a similar effect of the cytokine was seen in HUVEC with regard to VCAM-1 expression, negligible VCAM-1 in h-GF was not significantly increased by TNF-α (1.16nM) stimulation.</p

TNF-α stimulation of SAOS-2 reduced binding to fibroblasts.

<p>A histogram is shown demonstrating the effect of pre-treating SAOS-2 with TNF-α (1.16nM) for 24hrs upon subsequent SAOS-2 binding to culture plastic, untreated h-GF and h-GF previously stimulated with TNF-α (1.16nM) for 24 hrs. TNF-α treatment of SAOS-2 did not affect SAOS-2 attachment to tissue culture plastic. While the expected increased binding of unstimulated SAOS-2 to h-GF pre-treated with TNF-α was seen (p<0.002), pre-treatment of SAOS-2 with the cytokine markedly reduced binding to TNF-α treated h-GF (p<0.003), such that binding was comparable to that seen in unstimulated h-GF.</p

ICAM-1 was involved with TNF-α stimulated h-GF binding of SAOS-2.

<p>FACS analysis for ICAM-1 expression in h-GF with or without TNF-α (1.16nM) pre-treatment is shown (A), as well as at increasing times over 24 hr of TNF-α (1.16nM) treatment (B), compared with graphs showing the time-course of increased SAOS-2 binding by TNF-α (1.16nM) treated h-GF (C), and a histogram of the effect of blocking antibody against ICAM-1 (D). (A) h-GF expressed ICAM-1 and this was increased by TNF-α with maximal expression by 6 hr of cytokine treatment (B). (C) In an experiment performed at the same time as that shown in (5B), and using h-GF from the same donor, Maximal ICAM-1 expression correlated with maximal binding of SAOS-2 (p<0.001). As seen in the insert (C) examining the first 5 hr of cytokine stimulation in a separate time course experiment, increased SAOS-2 binding occurred between 0.5 and 1 hr of TNF-α (1.16nM) h-GF stimulation, and was maximal by 1.5 hrs (p<0.001). (D) Blocking antibody against ICAM-1 reduced binding of SAOS-2 to both untreated and TNF-α (1.16nM) stimulated h-GF (p<0.04).</p

Decaying effect of TNF-α stimulation on h-GF binding of SAOS-2.

<p>A graph is shown of SAOS-2 binding to h-GF, in which h-GF were first stimulated with TNF-α (1.16nM) for 24 hrs, and then washed before further culture for from 0 to 24 hrs in M199 with BSA (4%), either with or without fresh TNF-α (1.16nM). There was decreased SAOS-2 attachment between 6 hr and 12 hrs post-cytokine stimulation (p<0.005), and this reduced to a plateau by 18 hrs (p<0.001).</p

SAOS-2 binding to culture plastic and h-GF.

<p>SAOS-2 appeared as dark round cells in photomicrographs (arrow heads), as opposed to the elongated paler h-GF (arrows). h-GF increased SAOS-2 binding compared with culture plastic alone (p<0.001), and this was increased by TNF-α (1.16nM) treatment of the h-GF (p<0.001). (Bars = 50 µm).</p