ABSTRACT
Neutrophil extracellular trap (NET) formation is a recently discovered process in the field of innate immunity. It is important to have consistent standards in inducing and quantifying NET formation to compare data from different labs in this new area of investigation. Here we describe the conditions of neutrophil isolation from peripheral blood and stimulation that we find allow the study of NETosis in vitro. The criteria for conclusively identifying the process of NETosis, and the pros and cons of various quantification methods are discussed.
Subject(s)
Extracellular Space/immunology , Neutrophils/immunology , Cell Separation/methods , Humans , Microscopy, Fluorescence/methodsABSTRACT
Inflammation and stress are regarded as two important atherogenic factors. Because stress can affect leukocyte distribution, we hypothesized that stress-mediated leukocyte extravasation can modify the inflammatory environment of the arterial wall possibly contributing to atherogenesis. To test this hypothesis we evaluated the inflammatory environment of the aorta in C57Bl/6 mice subjected to 3 and 12 months of chronic stress and compared it to age matched non-stressed animals. Experiments were carried out in mice fed regular chow or atherogenic diets. Both treatments increased the expression of vascular and leukocyte adhesion molecules and leukocyte accumulation. At 3 months, stress but not an atherogenic diet elevated the number of CD4 cells, CD8 cells, macrophages, dendritic cells and neutrophils. These changes were associated with elevation of transcripts for ICAM-1 and VCAM-1, E-selectin and neuropeptide Y. At 12 months, stress or high cholesterol acted similarly to elevate the number of CD8 and macrophages, and synergistically on the number of all cell types investigated. At this time-point, strong synergism was also observed on the level of E-selectin and NPY in the aorta, but not in the circulation. Despite these effects, histological and morphological alterations of the arterial wall were severe in the atherogenic diet, but not in the stress groups. Thus, although stress and an atherogenic diet may both affect leukocyte accumulation in the aorta, they may contribute differently to atherogenesis.
Subject(s)
Aorta/physiology , Diet, Atherogenic/adverse effects , Inflammation/immunology , Inflammation/physiopathology , Stress, Psychological/immunology , Stress, Psychological/physiopathology , Animals , Atherosclerosis/chemically induced , Atherosclerosis/genetics , Biomarkers/analysis , Chronic Disease , Diet , E-Selectin/metabolism , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Immunoenzyme Techniques , Immunohistochemistry , Intercellular Adhesion Molecule-1/biosynthesis , Intercellular Adhesion Molecule-1/genetics , Leukocyte Count , Male , Mice , Mice, Inbred C57BL , Neuropeptide Y/metabolism , Plaque, Atherosclerotic/pathology , Real-Time Polymerase Chain Reaction , Vascular Cell Adhesion Molecule-1/biosynthesis , Vascular Cell Adhesion Molecule-1/geneticsABSTRACT
CC Chemokine ligand 22 (Ccl22) is a selective, high affinity ligand at the CC chemokine receptor 4 (Ccr4). We have identified cDNAs encoding both ligand and receptor of the Ccl22-Ccr4 pair in cDNA libraries of the anterior hypothalamus/pre-optic area (AH/POA) by PCR. The AH/POA is the key brain region where endogenous pyrogens have been shown to act on warm sensitive neurons to affect thermogenesis in brown adipose tissue (BAT) and other thermogenically responsive tissues. We show that functional Ccr4 receptors are present in the AH/POA neurons as injection of Ccl22 into the POA but not to other hypothalamic nuclei induces an increase in core body temperature as measured by radiotelemetry. Indomethacin (5 mg/kg s.c) pre-treatment markedly reduced the hyperthermia evoked by POA injection of Ccl22 (10 ng/0.5 ul) and thus suggests that this hyperthermia is mediated through cyclooxygenase activation and thus likely through the formation and action of the pyrogen prostaglandin E2. The temperature elevation involves a decrease in the respiratory exchange ratio and increased activation of the brown adipose tissue as demonstrated by ¹8F-FDG-PET imaging. We describe a novel role to the ligand Ccl22 and its receptor Ccr4 in the anterior hypothalamus in temperature regulation that depends on the synthesis of the endogenous pyrogen, prostaglandin E2.
Subject(s)
Adipose Tissue, Brown/metabolism , Chemokine CCL22/genetics , Fever/physiopathology , Hypothalamus, Anterior/metabolism , Adipose Tissue, Brown/drug effects , Animals , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Body Temperature/drug effects , Chemokine CCL22/metabolism , Chemokine CCL22/pharmacology , Dinoprostone/metabolism , Female , Fever/chemically induced , Fever/prevention & control , Gene Expression , Hypothalamus, Anterior/drug effects , Indomethacin/pharmacology , Male , Mice , Mice, Inbred C57BL , Positron-Emission Tomography , Preoptic Area/drug effects , Preoptic Area/metabolism , Pyrogens/metabolism , Pyrogens/pharmacology , Rats , Rats, Sprague-Dawley , Receptors, CCR4/genetics , Receptors, CCR4/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Telemetry , Tomography, X-Ray ComputedABSTRACT
The cytokine IL-18 acts on the CNS both in physiological and pathological conditions. Its action occurs through the heterodimeric receptor IL-18Ralpha\beta. To better understand IL-18 central effects, we investigated in the mouse brain the distribution of two IL-18Ralpha transcripts, a full length and an isoform lacking the intracellular domain hypothesized to be a decoy receptor. Both isoforms were expressed in neurons throughout the brain primarily with overlapping distribution but also with some unique pattern. These data suggest that IL-18 may modulate neuronal functions and that its action may be regulated through expression of a decoy receptor.
