Timothy D.G. Lee

Cutting Edge: The Dendritic Cell Cytoskeleton Is Critical for the Formation of the Immunological Synapse

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.

Fascin Is Involved in the Antigen Presentation Activity of Mature Dendritic 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.

Prevention of experimental postoperative peritoneal adhesions by N,O-carboxymethyl chitosan

BACKGROUND Postsurgical adhesion formation can result in significant morbidity and, to a lesser extent, death. The purpose of this experiment was to assess the ability of N,O-carboxymethyl chitosan (NOCC) to prevent postsurgical adhesion formation in vivo. METHODS Randomized groups of Sprague-Dawley rats were studied under two abdominal surgery models, the uterine horn model and the small bowel laceration model, for the ability of NOCC to reduce the incidence and severity of adhesion formation. Adhesions in animals were assessed after death by a blinded observer 10 to 14 days after surgical manipulation. RESULTS NOCC consistently reduced the size, strength, and number of adhesions in both rat models. NOCC was also found to be more effective than hyaluronic acid at inhibiting adhesion formation. CONCLUSIONS NOCC is a more effective antiadhesion agent than is the more expensive hyaluronic acid. Although the exact mechanism of NOCCs antiadhesion activity is as yet unclear, the novel chemical is of particular interest for clinical use.

Oral Exposure to Alloantigen Generates Intragraft CD8+ Regulatory Cells

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.

Prevention of postsurgical adhesions with N,O-carboxymethyl chitosan: Examination of the most efficacious preparation and the effect of N,O-carboxymethyl chitosan on postsurgical healing☆

BACKGROUND Adhesion formation after operation can result in major complications. We have previously demonstrated that N,O-carboxymethyl chitosan (NOCC) is an effective inhibitor of postsurgical peritoneal adhesion formation. However, the optimal form of NOCC (i.e., cross-linked gel versus solution), as well as the best time of administration for optimal reduction in adhesion development, was not investigated. In addition, because adhesion formation and normal wound healing are related events and weakening of wound healing would be a serious drawback to the use of NOCC clinically, we wished to assess the effect of NOCC on the healing of surgical incisions. METHODS Three surgical models were used: (1) an abdominal aortic anastomosis, (2) a large bowel anastomosis, and (3) an abdominal skin incision. In the first model Sprague-Dawley rats received an abdominal aortic transection and repair. NOCC solution or gel was administered at different time points throughout the procedure. Control and NOCC-treated animals were killed 14 days after operation. The condition of the anastomosed vessel was examined, and adhesion frequency and intensity in the abdomen were scored. In the second model Sprague-Dawley rats underwent large bowel transection and repair. Control and NOCC-treated animals were killed on postoperative days 4, 7, and 14, and strength of repair was assessed by removal of the large bowel and measurement of the bursting strength of the repaired incision. In the third model rats received an abdominal incision and were immediately closed. Control and NOCC-treated animals were killed 14 days after operation, and the skin tensile strength of the wound was measured with a tensiometer. RESULTS In all three models studied, NOCC treatment did not adversely affect the strength of the repaired incision. NOCC solution administered before operation did not greatly reduce adhesion formation, whereas the delivery of both NOCC gel and solution after operation was most efficacious. CONCLUSIONS The administration of both NOCC gel and solution after operation is most efficacious, and NOCC does not compromise postsurgical healing in rats at doses that prevent peritoneal adhesion formation.

Myocardial fibrosis in response to Angiotensin II is preceded by the recruitment of mesenchymal progenitor cells

Myocardial fibrosis is characterized by significant extracellular matrix (ECM) deposition. The specific cellular mediators that contribute to the development of fibrosis are not well understood. Using a model of fibrosis with Angiotensin II (AngII) infusion, our aim was to characterize the cellular elements involved in the development of myocardial fibrosis. Male C57Bl/6 and Tie2-GFP mice were given AngII (2.0 mg/kg/min) or saline (control) via mini osmotic pumps for up to 7 days. Hearts were harvested, weighed and processed for analysis. Cellular infiltration and collagen deposition were quantified. Immunostaining was performed for specific markers of leukocytes (CD45, CD11b), myofibroblasts (SMA), endothelial cells (vWF) and hematopoietic progenitor cells (CD133). Bone marrow (BM) origin of infiltrating cells was assessed using GFP+ chimeric animals. Relative qRT–PCR was performed for pro-fibrotic cytokines (transforming growth factor (TGF)-β1, CTGF) as well as the chemokine stromal-derived factor (SDF)-1α. Myocardial-infiltrating cells were grown in vitro. AngII exposure resulted in multifocal myocardial cellular infiltration, which preceded extensive ECM deposition. A limited number of myocardial-infiltrating cells were positive for leukocyte markers but were significantly positive for myofibroblast (SMA) and endothelial cell (vWF) markers. However, using Tie2-GFP mice, where endothelial cells are GFP+, myocardial-infiltrating cells were not GFP+. Transcript levels for SDF-1α were significantly elevated at 1 day of AngII exposure suggesting that hematopoietic progenitor cells may be recruited. This was confirmed by positive CD133 staining of infiltrating cells and evident GFP+ cellular infiltration when exposing GFP+ BM chimeras to AngII. Furthermore, a significant number of CD133+/SMA+ cells were grown in vitro from the myocardium of AngII-exposed animals (P<0.01). Myocardial ECM deposition is preceded by the infiltration of the myocardium with hematopoietic cells that express mesenchymal markers. These data suggest that mesenchymal progenitor cells are recruited, and may have a primary role, in the initiation of myocardial fibrosis.

CD8 T Lymphocytes Mediate Destruction of the Vascular Media in a Model of Chronic Rejection

Allograft arteriosclerosis is an important characteristic of chronic graft rejection. In allograft arteriosclerosis there is a striking loss of medial smooth muscle cells (SMCs) before the development of a concentric intimal proliferative response. In this study we evaluated the role of CD8+ T lymphocytes in this medial SMC loss. Brown Norway aortic segments were transplanted into Lewis animals for 60 days (long allo-exposure) or 20 days (short allo-exposure). After 20 days allogeneic exposure aortic segments were transplanted back into syngeneic (Brown Norway) animals for 40 days. Experimental animals were treated with mAb to CD8. Apoptosis was measured by terminal dUTP nick-end labeling at 20 days and morphometry analyzed at 60 days to evaluate medial and intimal changes. Anti-CD8 treatment significantly lowered CD8+ T cell counts in peripheral blood, reduced medial SMC apoptosis at 20 days, and increased medial SMC counts at 60 days. Both short- and long-allogeneic exposure groups confirmed these findings and demonstrated that medial SMC loss is proportional to the length of allogeneic exposure. Antibody depletion of CD8+ T cells results in reduced medial SMC apoptosis and better medial SMC preservation. This supports the hypothesis that medial SMC loss occurs by apoptotic death and is driven by CD8+ T lymphocytes.

Prolongation of rat kidney allograft survival by nematodes.

Nippostrongylus infection strongly stimulates TH2 activity in vivo. Given the evidence of cross regulation between TH2 and TH1 cells, and the link between TH1 activity and graft rejection, we examined the effects of Nippostrongylus infection on the fate of kidney allografts in rats. Both prior Nippostrongylus infection and prior treatment with a soluble worm product significantly delayed kidney allograft rejection. Control graft rejection occurred at 9.7 +/- 1.2 days whereas grafts in Nippostrongylus- or worm extract-treated recipients lasted 32.7 +/- 11.3 days and 21.5 +/- 4.6 days, respectively. At day 5 posttransplant mononuclear cell infiltration was much reduced in the Nippostrongylus-treated recipients. Flow cytometry of isolated graft-infiltrating leukocytes showed a marked decrease in infiltrating T cells (82.8% reduction) with both CD4+ cells (81.0% reduction) and CD8+ cells (84.6% reduction) being reduced. CD8+ T cells, in particular, made up a much smaller proportion of the graft-infiltrating cells (22% rather than 49%) in the Nippostrongylus-treated animals as compared with untreated controls. Immunohistochemical assay of the graft tissue confirmed the flow cytometric results. Interleukin 4 expression was clearly demonstrated by RT-PCR of the isolated graft-infiltrating leukocytes from the Nippostrongylus-treated recipients but not from the control recipients. These data are consistent with our current hypothesis that Nippostrongylus delays graft rejection by inducing a cross-regulatory suppression of TH1 activity.

Intravenous immunoglobulin G-mediated inhibition of T-cell proliferation reflects an endogenous mechanism by which IgG modulates T-cell activation

Commercial intravenous immunoglobulin G (IVIG) at high doses has therapeutic benefit in autoimmune and inflammatory diseases. It has been shown to inhibit T-cell function but the mechanisms are unclear. Inhibition could result from IVIG processing, donor pooling or intrinsic downregulatory activity of IgG. To address these points, we compared the effects on T-cell activation of IVIG, Fab(2) fragment and IgG isolated from single-donor plasma. We also investigated the role of accessory cells in the IVIG effects using highly purified T cells stimulated through CD3 and CD28 engagement. T-cell proliferation was evaluated by Oregon Green 488 dye dilution and (3)H-thymidine incorporation. IVIG, Fab(2) fragment of IVIG and autologous, single-donor IgG significantly inhibited T-cell proliferation (35-50%), even in the absence of accessory cells. Depletion of IgG from plasma used for culture significantly increased (by 50%) the T-cell proliferation. The addition of physiological concentrations of single-donor, autologous IgG or IVIG to IgG-depleted plasma reduced T-cell proliferation to levels observed in normal plasma. Therefore, donor pooling in IVIG and accessory cells are not required for inhibition of T-cell proliferation by IVIG and the Fab(2) region is sufficient to mediate this inhibition. Suppression of T-cell activation by IVIG likely reflects a physiologic, endogenous mechanism of IgG-mediated regulation of T-cell activation.

Echinacea-induced macrophage activation.

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-κB.