injection of anti-Fas markedly induced serum IL-6 levels, to an degree comparable to that of rmTNF. is definitely a cell surface receptor belonging to the tumor necrosis element (TNF)/nerve growth element (NGF) receptor superfamily that mediates apoptotic death upon activation by Fas ligand or agonist antibodies. 1,2 Lymphocyte-mediated apoptosis may play a role in a variety of autoimmune diseases, 3 and Fas manifestation was recently XR9576 reported in the brain of multiple sclerosis individuals 4 and was suggested to be involved in other diseases of the central nervous system. 5 In addition to inducing apoptosis, Fas induces standard inflammatory changes, including increase of interleukin (IL)-6 and IL-8 secretion and activation of nuclear factor-B. 6,7 administration of Fas ligand or the agonist anti-Fas monoclonal antibody (MAb) Jo2 induces fulminant hepatitis 8 and lethality in bioassay. 28 Biochemical Determinations Serum corticosterone was measured by a radioimmunoassay using an antiserum for corticosterone radioimmunoassay from Sigma (C-8784; Sigma Chemical Co., St. Louis, MO) and following XR9576 manufacturers indications. [3H]Corticosterone was purchased from Amersham (Arlington Heights, IL). SAA was measured by a previously explained ELISA (Hemagen Diagnostics, Waltham, MA). IL-6 was measured as hybridoma growth element using 7TD1 cells (a kind gift from Dr. Jacques vehicle Snick, Brussels, Belgium) as previously explained. 29 IL-6 activity is definitely indicated as co-stimulatory devices per milliliter using rIL-6 as a standard. The sensitivity of the assay was 50 U/ml. Results Mice received 1 g of anti-Fas antibody and were bled at 1.5, 3, or 24 hours for corticosterone or IL-6 and at Mouse monoclonal to PGR 24 hours for SAA, based on previous effects showing that these are the optimal time points for the effects under investigation when cytokines are injected i.c.v. It should also be mentioned that anti-Fas-injected mice did not look ill within this time framework (i.e., 24 hours). Number 1 ? shows the effect of an we.c.v. injection of anti-Fas on serum corticosterone 1.5 and 3 hours after injection. Control mice received saline i.c.v. only. For purpose of comparison, the effect of 1 1 g of rmTNF (also given we.c.v.) is also shown. It can be seen that both rmTNF and anti-Fas augmented approximately two- to three-fold serum corticosterone levels. Co-administration of the two resulted in an additive effect, although no direct assessment of anti-Fas and rmTNF can be made in terms of dose. In two independent experiments we tested an irrelevant antibody, as explained in Materials and Methods, at doses up to 6 g/mouse, and did not see any augmentation XR9576 of serum corticosterone compared with saline settings (data not demonstrated), in agreement with previous reports. 20 Open in a separate window Number 1. Central administration of anti-Fas activates the hypothalamus-pituitary-adrenal axis. Mice were treated i.c.v. with 1 g of anti-Fas, rmTNF, or both, and serum corticosterone was measured 1.5 and 3 hours later. Data are offered as mean SD (= 5). * 0.05 saline alone by Duncans test. Number 2 ? demonstrates we.c.v. injection of anti-Fas markedly induced serum IL-6 levels, to an degree comparable to that of rmTNF. The levels of IL-6 after anti-Fas were 3512 U/ml at 1.5 hours and 6432 U/ml at 3 hours; those after rmTNF were 4433 U/ml and 1505 U/ml 1.5 and 3 hours later, respectively. As for corticosterone, an additive effect was observed between anti-Fas and rmTNF. An irrelevant isotype MAb (observe Materials and Methods), at doses up to 6 g, did not induce any significant augmentation of serum IL-6 compared with saline settings (6 g of hTNFR p55 produced undetectable levels of serum IL-6, ie, 50 U/ml in one experiment and 149 104 U/ml in a second XR9576 experiment), in agreement with previous reports. 20 Open in a separate window Number 2. Central administration of anti-Fas induces peripheral IL-6. Mice were treated i.c.v. with XR9576 1 g of anti-Fas, rmTNF, or both, and serum IL-6 was measured 1.5 and 3 hours later. Data are offered as mean SD (= 5). ** 0.01 saline alone by Duncans test. Under the experimental conditions explained above, we.c.v. administration of murine NGF (2.5 g/mouse) did not elevate serum corticosterone or IL-6 (data not shown). As demonstrated in Number 3 ? , i.c.v. administration of anti-Fas antibody induced designated levels (50 g/ml) of SAA 24 hours after treatment. The levels of SAA were very low after saline injection (7 g/ml). Also, rmTNF and murine NGF induced designated levels of SAA. Open in a separate window Number 3. Central administration of anti-Fas induces SAA. Mice were treated i.c.v..