Proper macrophage activation in a T cell-deficient F344/NJcl-rnu/rnu rat.

<p>Bone marrow macrophages (BMMs) were prepared from a T cell-deficient F344/NJcl-rnu/rnu rat and a wild type F344/Jcl rat as described in Materials and Methods in detail. Phagocytic activity (A) and response to inflammatory stimulation with LPS (B, C) of BMMs prepared were then evaluated. To examine phagocytic activity, BMMs from both strains were treated with iron-containing nanoparticles, Ferumoxytol, for 24 h and engulfed particles were detected by Berlin blue staining (A). Bar, 20 μm. Response of BMMs from both strains to LPS was assessed by Western blot analysis and RT-PCR analyses targeting NF-κB-regulated pro-inflammatory genes. For Western blotting, BMMs were stimulated with LPS (1 μg/ml) for indicated time period and total cell lysate prepared from these stimulated cells was subjected to Western blotting for IκBα, phosphorylated form of p65 subunit (s536, p-p65) and p65 subunit to evaluate NF-κB activation, using Western blotting for α-tubulin as an internal control. For RT-PCR analyses, total RNA was purified from stimulated cells (LPS 0.1, 1, 10 μg/ml, 60 min) and subjected to RT-PCR analyses for MCP-1 (<i>Ccl2</i>), TNF-α (<i>Tnf</i>) and COX-2 (<i>Ptgs2</i>) after reverse transcription. Data represents mean ± SEM (n = 4). Noted that BMMs from a T cell-deficient rat shows a similar phagocytic activity and properly responses to inflammatory stimulation as in a wild type BMMs.</p

Effect of a pharmacological inhibition of a T cell function on IA progression.

<p>(A) Dose-dependent suppression of Concanavalin A-induced IL-2 production by Cyclosporine A <i>in vivo</i>. F344/Jcl rats were treated with each dose of Concanavalin A (n = 3) or Cyclosporine A combined with 15 mg/kg Concanavaline A (n = 3). Before administration (pre) or after 3 h (post), serum samples were prepared from rats and IL-2 concentration in these samples was examined by ELISA. Noted that 15 mg/kg Cyclosporine A almost completely suppressed Concanavalin A-induced IL-2 production. (B) Failure of suppression of IA progression, degenerative changes and infiltration of macrophages in lesions by treatment with Cyclosporine A. F344/Jcl rats were subjected to IA induction and, at 14^th day after the induction, slices from IA specimen induced at ACA-OA bifurcation were prepared for further analyses. Administration of Cyclosporine A was started 1 day before the induction and lasted for a whole experimental period (15 mg/kg, once a day). Systemic blood pressure was evaluated before sacrifice without any anesthesia. Area of induced IAs (Vehicle; n = 8, Cyclosporine A; n = 8) and distance between disrupted internal elastic lamina (IEL) in lesions were evaluated after Elastica van Gieson (EvG) staining (Representative images are shown in upper left panels). Degenerative change in IA walls was assessed by thickness of medial smooth muscle cell layer after immunostaining for α-smooth muscle actin. Number of infiltrated macrophages in IA lesions was evaluated after immunostaining for a macrophage marker CD68 (per 12,100 μm²). Data represents mean ± SEM. Representative images of immunostaining for CD68 (green) and α-smooth muscle actin (α-SMA, red) and of nuclear staining DAPI (blue) are shown in lower left panels. Bar, 20 μm.</p

Accumulation of T cells in human unruptured IA walls.

<p>(A, B) Accumulation of CD3-, CD4- and CD8-positive T cells in human unruptured IA walls. Adjacent sections were prepared from branches of carotid artery (Control) or unruptured IA lesion (Aneurysm) of humans and immunostained for a T cell marker, CD3, or subset specific makers, CD4 and CD8, respectively. Box in (A) indicates the region magnified in the following panels shown in (B). Bar, 100 μm. (C) Number of CD3-, CD4- and CD8-positive T cells in control arterial walls and IA walls. Data represents mean ± SEM (n = 10 in control, n = 15 in aneurysm). *, p<0.05, **, p<0.01 in a Mann−Whitney U test. Noted the significant increase in both CD4- and CD8-positive T cells in IA walls compared with those in control arterial walls.</p