The binding of a T cell to an APC results in T cell actin cytoskeletal rearrangement leading to the formation of an immunological synapse. The APC cytoskeleton has been thought to play a passive role in this process. In this study, we demonstrate that dendritic cells (DC), unlike other APC, actively polarize their actin cytoskeleton during interaction with T cells. DC cytoskeletal rearrangement was critical for both the clustering and the activation of resting T cells. This study provides compelling evidence that the APC cytoskeleton plays an active role in the immunological synapse and may explain the unique ability of DC to activate resting T cells.

Maturation of dendritic cells (DC) is critical to their development into potent APCs. Upon maturation, DC up-regulate the expression of MHC class II as well as costimulatory and adhesion molecules, all of which are important in Ag presentation. In addition, they undergo structural changes characterized by the expression of numerous long dendrites. Fascin is an actin-bundling protein that has been reported to be important for the development of dendrites. In this study, we evaluated fascin expression and function during DC maturation into potent APC. In vitro, treatment of bone marrow-derived DC (BM-DC) with GM-CSF resulted in increased levels of fascin expression. This increase correlated directly with an increase in MHC class II and B7-2 expression. Fascin expression was decreased by the addition of TGF-β and increased by the addition TNF-α to the culture. These cytokines suppress or enhance DC maturation, respectively. Increased levels of fascin expression were found to correlate with increased APC activity in a one-way MLR. Specific inhibition of fascin expression, using antisense oligonucleotides, markedly reduced this APC allostimulatory activity. These data demonstrate that fascin expression correlates with DC maturation into APC, and it plays a significant role in the ability of DC to function as APC. This observation is the first evidence linking fascin-mediated dendrite formation with the APC activity of DC.

Exposure of rodents to angiotensin II (AngII) is a common model of fibrosis. We have previously shown that cellular infiltration of bone marrow-derived progenitor cells (fibrocytes) occurs before deposition of extracellular matrix and is associated with the production of connective tissue growth factor (CTGF). In the present study, we characterized the role of CTGF in promoting fibrocyte accumulation and regulation after AngII exposure. In animals exposed to AngII using osmotic minipumps (2.0 μg/kg per min), myocardial CTGF mRNA peaked at 6 hours (21-fold; P < 0.01), whereas transforming growth factor-β (TGF-β) peaked at 3 days (fivefold; P < 0.05) compared with saline control. Early CTGF expression occurred before fibrocyte migration (1 day) into the myocardium or ECM deposition (3 days). CTGF protein expression was evident by day 3 of AngII exposure and seemed to be localized to resident cells. Isolated cardiomyocytes and microvascular endothelial cells responded to AngII with increased CTGF production (2.1-fold and 2.8-fold, respectively; P < 0.05), which was abolished with the addition of anti-TGF-β neutralizing antibody. The effect of CTGF on isolated fibrocytes suggested a role in fibrocyte proliferation (twofold; P < 0.05) and collagen production (2.3-fold; P < 0.05). In summary, we provide strong evidence that AngII exposure first resulted in Smad2-dependent production of CTGF by resident cells (6 hours), well before the accumulation of fibrocytes or TGF-β mRNA up-regulation. In addition, CTGF contributes to fibrocyte proliferation in the myocardium and enhances fibrocyte differentiation into a myofibroblast phenotype responsible for ECM deposition.

Macrophages are increasingly recognized as a potential therapeutic target in myocardial fibrosis via interactions with fibroblasts. We have characterized macrophage depletion and inhibition of nonclassical macrophage migration, in addition to direct interactions between nonclassical macrophages and fibroblasts in angiotensin II (AngII)-mediated, hypertensive myocardial fibrosis. Macrophage depletion was achieved by daily i.v. clodronate liposomes (-1 day to +3 days) during AngII infusion. Cx3cr1(-/-) mice were used to inhibit nonclassical macrophage migration. Macrophage phenotype (F4/80, CD11b, Ly6C) was characterized by immunofluorescence and flow cytometry. Collagen was assessed by Sirius Red/Fast Green. Quantitative real-time RT-PCR was performed for transcript levels. AngII/wild-type (WT) mice displayed significant infiltrate and fibrosis compared with saline/WT, which was virtually ablated by clodronate liposomes independent of hypertension. In vitro data supported M2 macrophages promoting fibroblast differentiation and collagen production. AngII/Cx3cr1(-/-) mice, however, significantly increased macrophage infiltrate and fibrosis relative to AngII/WT. AngII/Cx3cr1(-/-) mice also showed an M1 phenotypic shift relative to WT mice in, which the predominant phenotype was Ly6C(low), CD206(+) (M2). Myocardial IL-1β was significantly up-regulated, whereas transforming growth factor β down-regulated with this M1 shift. We demonstrated that infiltrating macrophages are critical to AngII-mediated myocardial fibrosis by preventing the development of fibrosis after liposomal depletion of circulating monocytes. Our findings also suggest that some macrophages, namely M2, may confer a protective myocardial environment that may prevent excessive tissue injury.

No abstract

We have previously reported that oral administration of allogeneic rat spleen cells before kidney allotransplantation significantly prolongs graft survival. This prolongation was alloantigen specific and was associated with a decrease in graft-infiltrating cells (GIC) and an increase in transcription of IL-4 mRNA in the GIC. In this study increased splenic mixed lymphocyte responses from animals orally exposed to alloantigen before kidney transplantation suggested that the kidney allograft prolongation was not due to a masking of allorecognition, but to an immunomodulation of the immune response. We have assessed GIC T cell subsets on day 5 post-transplant and found decreased numbers of CD4+ T cells in fed animals compared with controls, but there was no change in CD8+ T cell numbers. The CD8+ GIC from fed animals transcribed substantial levels of perforin, granzyme, and Fas ligand mRNA, indicating the presence of active CTL. Direct CTL assays showed that the GIC from fed recipients exhibited higher allo-CTL activity than GIC from control unfed recipients. In addition, the CD8+ GIC exhibited high levels of IL-4 mRNA, suggesting Tc2-type regulatory cells. Prolonged graft survival in the face of active CTL and Tc2 cells suggests the presence of a CD8+ regulatory cell population in the allograft. To confirm this, cell transfer experiments were performed. Prolongation of graft survival was transferred from rats orally exposed to alloantigen to naive animals by transfer of CD8+ GIC. This is the first report that oral exposure to alloantigen prolongs kidney allograft survival by the generation of intragraft CD8+ regulatory cells.

Public interest in Echinacea is growing rapidly. Unfortunately, there is little scientific evidence to support claims of efficacy of this widely used botanical, and little information about potential mechanism of action. This study examines the ability of Echinacea to upregulate macrophage function and begins to elucidate the mechanism of Echinacea-induced macrophage activation. Murine peritoneal macrophages were cultured with E. purpurea extracts enriched for plant polysaccharide (EP). ELISA was used to measure cytokine production. MAPKs were blocked using specific inhibitors, and Western blotting used to identify phosphorylated proteins involved in signal transduction. To examine in vivo efficacy, EP was administered orally and Listeria monocytogenes given i.v. Mice were sacrificed three days post-infection to determine bacterial load in the spleen. We demonstrate that an endotoxin-free EP extract activates the innate immune response, stimulating production of IL-6, TNF, IL-12, and NO from macrophages in vitro. Along with evidence of enhanced macrophage function, we found that oral EP reduces bacterial burden during infection by Listeria monocytogenes, demonstrating its efficacy in vivo. EP initiates a signaling cascade within macrophages through both TLR4-dependent and -independent mechanisms, involving ERK, p38 and JNK, and ultimately the activation of NF-kappaB.

No abstract