Unique Proteomic Signatures Distinguish Macrophages and Dendritic Cells

Monocytes differentiate into heterogeneous populations of tissue macrophages and dendritic cells (DCs) that regulate inflammation and immunity. Identifying specific populations of myeloid cells in vivo is problematic, however, because only a limited number of proteins have been used to assign cellular phenotype. Using mass spectrometry and bone marrow-derived cells, we provided a global view of the proteomes of M-CSF-derived macrophages, classically and alternatively activated macrophages, and GM-CSF-derived DCs. Remarkably, the expression levels of half the plasma membrane proteins differed significantly in the various populations of cells derived in vitro. Moreover, the membrane proteomes of macrophages and DCs were more distinct than those of classically and alternatively activated macrophages. Hierarchical cluster and dual statistical analyses demonstrated that each cell type exhibited a robust proteomic signature that was unique. To interrogate the phenotype of myeloid cells in vivo, we subjected elicited peritoneal macrophages harvested from wild-type and GM-CSF-deficient mice to mass spectrometric and functional analysis. Unexpectedly, we found that peritoneal macrophages exhibited many features of the DCs generated in vitro. These findings demonstrate that global analysis of the membrane proteome can help define immune cell phenotypes in vivo

Cognitive-behavioral therapy for late-life generalized anxiety disorder: Who gets better?

The authors pooled data from three independently conducted treatment outcome studies to examine predictors of outcome from group-administered cognitive-behavioral therapy (CBT) for older adults with generalized anxiety disorder (GAD). Data were collected from 65 patients with a mean age of 67.7 years (SD = 6.6). Average reliable change indices (RCI) based on 3 outcome measures were calculated at posttreatment and at 6-month follow-up. Approximately half of patients achieved a significant RCI at posttreatment and two-thirds achieved a significant RCI at follow-up. Factors associated with better outcomes included better homework adherence, higher baseline GAD severity, and presence of a comorbid psychiatric diagnosis. Results suggest that at-home practice is associated with better and longer-lasting outcomes from CBT in older adults with GAD. © 2005 the Association for Advancement of Behavior Therapy. All rights reserved

The apolipoprotein-AI mimetic peptide L4F at a modest dose does not attenuate weight gain, inflammation, or atherosclerosis in LDLR-null mice.

High density lipoprotein (HDL) cholesterol levels are inversely related to cardiovascular disease risk and associated with a reduced risk of type 2 diabetes. Apolipoprotein A-I (apoA-I; major HDL protein) mimetics have been reported to reduce atherosclerosis and decrease adiposity. This study investigated the effect of L4F mimetic peptide and apoA-I overexpression on weight gain, insulin resistance, and atherosclerosis in an LDL receptor deficient (Ldlr-/-) model fed a high fat high sucrose with cholesterol (HFHSC) diet.Studies in differentiated 3T3-L1 adipocytes tested whether L4F could inhibit palmitate-induced adipocyte inflammation. In vivo studies used male Ldlr-/- mice fed a HFHSC diet for 12 weeks and were injected daily with L4F (100 µg/mouse) subcutaneously during the last 8 weeks. Wild-type and apoA-I overexpressing Ldlr-/- mice were fed HFHSC diet for 16 weeks.Neither L4F administration nor apoA-I overexpression affected weight gain, total plasma cholesterol or triglycerides in our studies. While pre-treatment of 3T3-L1 adipocytes with either L4F or HDL abolished palmitate-induced cytokine expression in vitro, L4F treatment did not affect circulating or adipose tissue inflammatory markers in vivo. Neither L4F administration nor apoA-I overexpression affected glucose tolerance. ApoA-I overexpression significantly reduced atherosclerotic lesion size, yet L4F treatment did not affect atherosclerosis.Our results suggest that neither L4F (100 µg/day/mouse) nor apoA-I overexpression affects adiposity or insulin resistance in this model. We also were unable to confirm a reduction in atherosclerosis with L4F in our particular model. Further studies on the effect of apoA-I mimetics on atherosclerosis and insulin resistance in a variety of dietary contexts are warranted

The Apolipoprotein-AI Mimetic Peptide L4F at a Modest Dose Does Not Attenuate Weight Gain, Inflammation, or Atherosclerosis in LDLR-Null Mice

Objective: High density lipoprotein (HDL) cholesterol levels are inversely related to cardiovascular disease risk and associated with a reduced risk of type 2 diabetes. Apolipoprotein A-I (apoA-I; major HDL protein) mimetics have been reported to reduce atherosclerosis and decrease adiposity. This study investigated the effect of L4F mimetic peptide and apoA-I overexpression on weight gain, insulin resistance, and atherosclerosis in an LDL receptor deficient (Ldlr-/-) model fed a high fat high sucrose with cholesterol (HFHSC) diet.Methods: Studies in differentiated 3T3-L1 adipocytes tested whether L4F could inhibit palmitate-induced adipocyte inflammation. In vivo studies used male Ldlr-/- mice fed a HFHSC diet for 12 weeks and were injected daily with L4F (100 µg/mouse) subcutaneously during the last 8 weeks. Wild-type and apoA-I overexpressing Ldlr-/- mice were fed HFHSC diet for 16 weeks.Results: Neither L4F administration nor apoA-I overexpression affected weight gain, total plasma cholesterol or triglycerides in our studies. While pre-treatment of 3T3-L1 adipocytes with either L4F or HDL abolished palmitate-induced cytokine expression in vitro, L4F treatment did not affect circulating or adipose tissue inflammatory markers in vivo. Neither L4F administration nor apoA-I overexpression affected glucose tolerance. ApoA-I overexpression significantly reduced atherosclerotic lesion size, yet L4F treatment did not affect atherosclerosis.Conclusion: Our results suggest that neither L4F (100 µg/day/mouse) nor apoA-I overexpression affects adiposity or insulin resistance in this model. We also were unable to confirm a reduction in atherosclerosis with L4F in our particular model. Further studies on the effect of apoA-I mimetics on atherosclerosis and insulin resistance in a variety of dietary contexts are warranted.</p

T cell activation inhibitors reduce CD8+ T cell and pro-inflammatory macrophage accumulation in adipose tissue of obese mice.

Adipose tissue inflammation and specifically, pro-inflammatory macrophages are believed to contribute to insulin resistance (IR) in obesity in humans and animal models. Recent studies have invoked T cells in the recruitment of pro-inflammatory macrophages and the development of IR. To test the role of the T cell response in adipose tissue of mice fed an obesogenic diet, we used two agents (CTLA-4 Ig and anti-CD40L antibody) that block co-stimulation, which is essential for full T cell activation. C57BL/6 mice were fed an obesogenic diet for 16 weeks, and concomitantly either treated with CTLA-4 Ig, anti-CD40L antibody or an IgG control (300 µg/week). The treatments altered the immune cell composition of adipose tissue in obese mice. Treated mice demonstrated a marked reduction in pro-inflammatory adipose tissue macrophages and activated CD8+ T cells. Mice treated with anti-CD40L exhibited reduced weight gain, which was accompanied by a trend toward improved IR. CTLA-4 Ig treatment, however, was not associated with improved IR. These data suggest that the presence of pro-inflammatory T cells and macrophages can be altered with co-stimulatory inhibitors, but may not be a significant contributor to the whole body IR phenotype

Immunocytochemical detection of plasma membrane protein markers.

<p>Expression levels of widely used plasma membrane protein markers (<b><i>Panels A–B</i></b>) and newly identified markers (<b><i>Panels C–D</i></b>) of M1 cells, M2 cells, BmMs, and BmDCs were assessed by mass spectrometry (<b><i>Panels A,C</i></b>) and immunocytochemistry (<b><i>Panels B,D</i></b>). For MS/MS, proteins were quantified by spectral counting and expressed relative to the cell type with the highest expression level for each protein. Results are means and SDs. Cells were stained with antibodies specific to each protein (red channel), counterstained with DAPI to visualize nuclei (blue-channel), and examined by confocal microscopy. Immunostaining and microscopy were performed on the same day with identical microscope settings. Results are representative of 3 independent analyses.</p

Analysis of eMPCs harvested from wild-type and GM-CSF-deficient (<i>Csf2−/−</i>) mice.

<p>eMPCs isolated from wild-type (<i>wt</i>) and <i>Csf2</i>−/− mice were interrogated for cell number, function, and protein expression. <b><i>Panel A–B:</i></b> Accumulation of eMPCs 3 days (<i>Panel A</i>) and 5 days (<i>Panel B</i>) following intraperitoneal injection with thioglycolate. Results (N = 6) are means and SEMs. <b><i>Panel C:</i></b> Plasma membrane proteomic analysis of eMPCs isolated from <i>Csf2</i>−/− and wild-type mice. Differentially-expressed proteins were identified using the <i>t-</i>test and <i>G-</i>test (<i>p</i><0.05 and <i>G-</i>statistic >1.5) and quantified using the spectral index. <b><i>Panel D:</i></b> Proteins differentially expressed by eMPCs isolated from <i>Csf2</i>−/− mice (see <i>Panel C</i>) were measured in BmMs and BmDCs and quantified using the spectral index. <b><i>Panel E:</i></b> Cell surface CD11c and F4/80 expression on eMPCs was assessed by flow cytometry. Results are presented as contour plots with 10% probability increments. <b><i>Panel F:</i></b> Phagocytosis of fluorescein-labeled <i>E. coli</i> by eMPCs. Results (arbitrary units, AU; N = 4) are means and SEMs. <b><i>Panel G–H:</i></b> Antigen cross-presentation by eMPCs. Ovalbumin (0.2 mg/mL)-treated eMPCs were incubated with CFSE-labeled spleen cells isolated from OT-I transgenic mice. Levels of CFSE were assessed in OT-I T cells selected by flow cytometry and expression levels of CD8 and Vb5 (<i>Panel G</i>). The division index was calculated using FlowJo software. Results (N = 4) are means and SEMs (<i>Panel H</i>). Where applicable, <i>p</i>-values were derived using a two-tailed Student's <i>t-</i>test. Results obtained for eMPC quantification, flow cytometry, phagocytosis and antigen cross-presentation are representative of 3 independent analyses.</p