ABSTRACT
Aging is associated with a gradual loss of naïve T cells and a reciprocal increase in the proportion of memory T cells. While reduced thymic output is important, age-dependent changes in factors supporting naïve T cells homeostasis may also be involved. Indeed, we noted a dramatic decrease in the ability of aged mice to support survival and homeostatic proliferation of naïve T cells. The defect was not due to a reduction in IL-7 expression, but from a combination of changes in the secondary lymphoid environment that impaired naïve T cell entry and access to key survival factors. We observed an age-related shift in the expression of homing chemokines and structural deterioration of the stromal network in T cell zones. Treatment with IL-7/mAb complexes can restore naïve T cell homeostatic proliferation in aged mice. Our data suggests that homeostatic mechanisms that support the naïve T cell pool deteriorate with age.
Subject(s)
Homeostasis/physiology , Immunologic Memory/immunology , Lymphocyte Activation/immunology , Lymphoid Tissue/immunology , T-Lymphocyte Subsets/immunology , Animals , Cell Proliferation , Mice , Mice, Inbred C57BLABSTRACT
Understanding vascular growth and maturation in developing tumors has important implications for tumor progression, spread, and ultimately host survival. Modulating the signaling of endothelial G protein-coupled receptors (GPCRs) in blood and lymphatic vessels can enhance or limit tumor progression. Sphingosine 1-phosphate receptor 1 (S1PR1) is a GPCR for circulating lysophospholipid S1P that is highly expressed in blood and lymphatic vessels. Using the S1PR1- enhanced green fluorescent protein (eGFP) mouse model in combination with intravital imaging and pharmacologic modulation of S1PR1 signaling, we show that boundary conditions of high and low S1PR1 signaling retard tumor progression by enhancing or destabilizing neovasculature integrity, respectively. In contrast, midrange S1PR1 signaling, achieved by receptor antagonist titration, promotes abundant growth of small, organized vessels and thereby enhances tumor progression. Furthermore, in vivo S1PR1 antagonism supports lung colonization by circulating tumor cells. Regulation of endothelial S1PR1 dynamically controls vascular integrity and maturation and thus modulates angiogenesis, tumor growth, and hematogenous metastasis.
Subject(s)
Breast Neoplasms/blood supply , Breast Neoplasms/metabolism , Capillary Permeability , Endothelial Cells/metabolism , Neovascularization, Pathologic , Receptors, Lysosphingolipid/metabolism , Anilides/pharmacology , Animals , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Capillary Permeability/drug effects , Endothelial Cells/drug effects , Endothelial Cells/pathology , Female , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Lung Neoplasms/metabolism , Lung Neoplasms/secondary , Mice , Mice, Inbred C57BL , Mice, Transgenic , Neoplastic Cells, Circulating/metabolism , Neoplastic Cells, Circulating/pathology , Organophosphonates/pharmacology , Receptors, Lysosphingolipid/drug effects , Receptors, Lysosphingolipid/genetics , Signal Transduction , Sphingosine-1-Phosphate Receptors , Time Factors , Tumor BurdenABSTRACT
γδ T cells respond rapidly to keratinocyte damage, providing essential contributions to the skin wound healing process. The molecular interactions regulating their response are unknown. Here, we identify a role for interaction of plexin B2 with the CD100 receptor in epithelial repair. In vitro blocking of plexin B2 or CD100 inhibited γδ T cell activation. Furthermore, CD100 deficiency in vivo resulted in delayed repair of cutaneous wounds due to a disrupted γδ T cell response to keratinocyte damage. Ligation of CD100 in γδ T cells induced cellular rounding via signals through ERK kinase and cofilin. Defects in this rounding process were evident in the absence of CD100-mediated signals, thereby providing a mechanistic explanation for the defective wound healing in CD100-deficient animals. The discovery of immune functions for plexin B2 and CD100 provides insight into the complex cell-cell interactions between epithelial resident γδ T cells and the neighboring cells they support.
Subject(s)
Antigens, CD/immunology , Langerhans Cells/immunology , Nerve Tissue Proteins/immunology , Receptors, Antigen, T-Cell, gamma-delta/immunology , Semaphorins/immunology , T-Lymphocytes/immunology , Actin Depolymerizing Factors/metabolism , Animals , Antigens, CD/metabolism , CHO Cells , Cell Communication/immunology , Cell Shape , Cricetinae , Epidermis/immunology , Epidermis/injuries , Extracellular Signal-Regulated MAP Kinases/metabolism , HEK293 Cells , Humans , Keratinocytes/immunology , Keratinocytes/metabolism , Langerhans Cells/metabolism , Lymphocyte Activation/immunology , Mass Spectrometry , Mice , Mice, Inbred C57BL , Mice, Transgenic , Nerve Tissue Proteins/metabolism , Phosphorylation , Protein Binding/immunology , Receptors, Antigen, T-Cell, gamma-delta/metabolism , Semaphorins/metabolism , Sequence Analysis, Protein , Surface Plasmon Resonance , T-Lymphocytes/metabolism , Wound Healing/immunologyABSTRACT
Lymph nodes are highly organized structures specialized for efficient regulation of adaptive immunity. The blood and lymphatic systems within a lymph node play essential roles by providing functionally distinct environments for lymphocyte entry and egress, respectively. Direct imaging and measurement of vascular microenvironments by intravital multiphoton microscopy provide anatomical and mechanistic insights into the essential events of lymphocyte trafficking. Lymphocytes, blood endothelial cells, and lymphatic endothelial cells express sphingosine 1-phosphate receptor 1, a key G protein-coupled receptor regulating cellular egress and a modulator of endothelial permeability. Here we report the development of a differential vascular labeling (DVL) technique in which a single intravenous injection of a fluorescent dextran, in combination with fluorescent semiconductor quantum dot particles, differentially labels multiple blood and lymphatic compartments in a manner dependent on the size of the fluorescent particle used. Thus DVL allows measurement of endothelial integrity in multiple vascular compartments and the affects or pharmacological manipulation in vascular integrity. In addition, this technique allows for real-time observation of lymphocyte trafficking across physiological barriers differentiated by DVL. Last, single-field fluid movement dynamics can be derived, allowing for the simultaneous determination of fluid flow rates in diverse blood and lymphatic compartments.
Subject(s)
Computer Systems , Endothelial Cells/physiology , Extracellular Fluid/chemistry , Lymphatic Vessels/chemistry , Regional Blood Flow , Staining and Labeling/methods , Animals , Endothelial Cells/chemistry , Extracellular Fluid/physiology , Lymph Nodes/blood supply , Lymph Nodes/chemistry , Lymph Nodes/physiology , Lymphatic Vessels/physiology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Receptors, Lysosphingolipid/biosynthesis , Receptors, Lysosphingolipid/blood , Regional Blood Flow/physiologyABSTRACT
Multiple sclerosis (MS) therapies modulate T-cell autoimmunity in the central nervous system (CNS) but may exacerbate latent infections. Fingolimod, a nonselective sphingosine-1-phosphate (S1P) receptor agonist that induces sustained lymphopenia and accumulates in the CNS, represents a new treatment modality for MS. We hypothesized that sustained lymphopenia would not be required for efficacy and that a selective, CNS-penetrant, peripherally short-acting, S1P(1) agonist would show full efficacy in a mouse MS model. Using daily treatment with 10 mg/kg 2-(4-(5-(3,4-diethoxyphenyl)-1,2,4-oxadiazol-3-yl)-2,3-dihydro-1H-inden-1-yl amino)ethanol (CYM-5442) at the onset of clinical signs in myelin oligodendrocyte glycoprotein MOG(35-55)- induced experimental allergic encephalomyelitis (EAE), we assessed clinical scores, CNS cellular infiltration, demyelination, and gliosis for 12 days with CYM-5442, vehicle, or fingolimod. CYM-5442 levels in CNS and plasma were determined at experiment termination, and blood lymphopenia was measured 3 and 24 h after the last injection. Plasma levels of cytokines were assayed at the end of the protocol. Changes in S1P(1)-enhanced green fluorescent protein expression on neurons and astrocytes during active EAE and upon CYM-5442 treatment were quantified with flow cytometry and Western blotting by using native-locus enhanced green fluorescent protein-tagged S1P(1) mice. S1P(1) agonism alone reduced pathological features as did fingolimod (maximally lymphopenic throughout), despite full reversal of lymphopenia within each dosing interval. CYM-5442 levels in CNS but not in plasma were sustained. Neuronal and astrocytic S1P(1) expression in EAE was suppressed by CYM-5442 treatment, relative to vehicle, and levels of key cytokines, such as interleukin 17A, were also significantly reduced in drug-treated mice. S1P(1)-selective agonists that induce reversible lymphopenia while persisting in the CNS may be effective MS treatments.
Subject(s)
Lymphopenia/drug therapy , Multiple Sclerosis/drug therapy , Receptors, Lysosphingolipid/agonists , Animals , Central Nervous System/metabolism , Cytokines/drug effects , Disease Models, Animal , Encephalomyelitis, Autoimmune, Experimental/drug therapy , Fingolimod Hydrochloride , Immunosuppressive Agents , Indans , Mice , Oxadiazoles , Propylene Glycols/therapeutic use , Receptors, Lysosphingolipid/metabolism , Sphingosine/analogs & derivatives , Sphingosine/therapeutic useABSTRACT
Sphingosine 1-phosphate receptor 1 (S1P(1)) is critical for lymphocyte recirculation and is a clinical target for treatment of multiple sclerosis. By generating a short-duration S1P(1) agonist and mice in which fluorescently tagged S1P(1) replaces wild-type receptor, we elucidate physiological and agonist-perturbed changes in expression of S1P(1) at a subcellular level in vivo. We demonstrate differential downregulation of S1P(1) on lymphocytes and endothelia after agonist treatment.
Subject(s)
Gene Knock-In Techniques , Green Fluorescent Proteins/chemistry , Multiple Sclerosis/drug therapy , Receptors, Lysosphingolipid/agonists , Receptors, Lysosphingolipid/therapeutic use , Animals , Down-Regulation/drug effects , Endothelium/drug effects , Endothelium/metabolism , Flow Cytometry , Fluorescent Dyes/chemistry , Fluorescent Dyes/metabolism , Green Fluorescent Proteins/metabolism , Lymphocytes/drug effects , Lymphocytes/metabolism , Mice , Multiple Sclerosis/metabolism , Multiple Sclerosis/pathology , Receptors, Lysosphingolipid/metabolism , Time FactorsABSTRACT
We have determined the pharmacological profile of the new serotonin 5-HT(7) receptor agonist N-(4-cyanophenylmethyl)-4-(2-diphenyl)-1-piperazinehexanamide (LP-211). Radioligand binding assays were performed on a panel of 5-HT receptor subtypes. The compound was also evaluated in vivo by examining its effect on body temperature regulation in mice lacking the 5-HT(7) receptor (5-HT(7)(-/-)) and their 5-HT(7)(+/+) sibling controls. Disposition studies were performed in mice of both genotypes. It was found that LP-211 was brain penetrant and underwent metabolic degradation to 1-(2-diphenyl)piperazine (RA-7). In vitro binding assays revealed that RA-7 possessed higher 5-HT(7) receptor affinity than LP-211 and a better selectivity profile over a panel of 5-HT receptor subtypes. In vivo it was demonstrated that LP-211, and to a lesser degree RA-7, induced hypothermia in 5-HT(7)(+/+) but not in 5-HT(7)(-/-) mice. Our results suggest that LP-211 can be used as a 5-HT(7) receptor agonist in vivo.
Subject(s)
Brain/metabolism , Phenylcarbamates/metabolism , Phenylcarbamates/pharmacology , Piperazines/metabolism , Piperazines/pharmacology , Receptors, Serotonin/metabolism , Serotonin Receptor Agonists/metabolism , Serotonin Receptor Agonists/pharmacology , Urethane/analogs & derivatives , Animals , Body Temperature/drug effects , Body Temperature/genetics , Brain/anatomy & histology , Brain/drug effects , Dose-Response Relationship, Drug , Humans , Male , Mice , Mice, Knockout , Phenylcarbamates/chemistry , Piperazines/chemistry , Protein Binding/drug effects , Protein Binding/genetics , Receptors, Serotonin/deficiency , Serotonin Receptor Agonists/chemistry , Urethane/chemistry , Urethane/metabolism , Urethane/pharmacologyABSTRACT
The 5-HT(7) receptor has been suggested as a target for treating depression since inactivation or blockade of the receptor has an antidepressant-like behavioral effect. The present study investigated possible interactions between various classes of drugs with antidepressant properties and blockade or inactivation of the 5-HT(7) receptor. Immobility despair in the tail suspension test and the forced swim test was evaluated in mice lacking the 5-HT(7) receptor (5-HT(7)(-/-)) and in wild-type controls (5-HT(7)(+/+)) following acute drug treatments. Citalopram, a selective serotonin reuptake inhibitor and widely used antidepressant, dose-dependently reduced immobility in the tail suspension test in both 5-HT(7)(+/+) and 5-HT(7)(-/-) mice. Combining doses of citalopram and the 5-HT(7) receptor antagonist SB-269970 that by themselves did not affect behavior, reduced immobility in 5-HT(7)(+/+) mice in both the tail suspension test and the forced swim test. No effect was seen in 5-HT(7)(-/-) mice. Desipramine and reboxetine, two norepinephrine reuptake inhibitors, dose-dependently reduced immobility in the tail suspension test in 5-HT(7)(+/+) mice, but had no effect in 5-HT(7)(-/-) mice. A synergistic effect between desipramine and SB-269970 was found in both behavioral tests in 5-HT(7)(+/+) mice. Reboxetine combined with SB-269970 had effect only in the forced swim test. GBR 12909, a dopamine reuptake inhibitor, dose-dependently reduced tail suspension test immobility in both genotypes. There was no interaction between GBR 12909 and SB-269970. Aripiprazole, an antipsychotic, reduced immobility in both tests in 5-HT(7)(+/+) mice, but not in 5-HT(7)(-/-) mice. The results show that the 5-HT(7) receptor is required for the observed interaction between this receptor and antidepressants such as citalopram. The data furthermore support the hypothesis that the 5-HT(7) receptor might be a suitable target for treating depression.
Subject(s)
Antidepressive Agents/pharmacology , Citalopram/pharmacology , Receptors, Serotonin/metabolism , Animals , Antipsychotic Agents/pharmacology , Aripiprazole , Corticosterone/blood , Depression/genetics , Disease Models, Animal , Dopamine Uptake Inhibitors/pharmacology , Dose-Response Relationship, Drug , Drug Synergism , Hindlimb Suspension/methods , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Motor Activity/drug effects , Phenols/pharmacology , Piperazines/pharmacology , Quinolones/pharmacology , Receptors, Serotonin/deficiency , Serotonin Antagonists/pharmacology , Sulfonamides/pharmacology , Swimming/psychologyABSTRACT
The hippocampus has been implicated in aspects of spatial memory. Its ability to generate new neurons has been suggested to play a role in memory formation. Hippocampal serotonin (5-HT) neurotransmission has also been proposed as a contributor to memory processing. Studies have shown that the 5-HT(7) receptor is present in the hippocampus in relatively high abundance. Thus the aim of the present study was to investigate the possible role of the 5-HT(7) receptor in spatial memory using 5-HT(7) receptor-deficient mice (5-HT(7)(-/-)). A hippocampus-associated spatial memory deficit in 5-HT(7)(-/-) mice was demonstrated using a novel location/novel object test. A similar reduction in novel location exploration was observed in C57BL/6J mice treated with the selective 5-HT(7) receptor antagonist SB-269970. These findings prompted an extended analysis using the Barnes maze demonstrating that 5-HT(7)(-/-) mice were less efficient in accommodating to changes in spatial arrangement than 5-HT(7)(+/+) mice. 5-HT(7)(-/-) mice had specific impairments in memory compilation required for resolving spatial tasks, which resulted in impaired allocentric spatial memory whereas egocentric spatial memory remained intact after the mice were forced to switch back from striatum-dependent egocentric to hippocampus-dependent allocentric memory. To further investigate the physiological bases underlining these behaviors we compared hippocampal neurogenesis in 5-HT(7)(+/+) and 5-HT(7)(-/-) mice employing BrdU immunohistochemistry. The rate of cell proliferation in the dentate gyrus was identical in the two genotypes. From the current data we conclude that the 5-HT(7)(-/-) mice performed by remembering a simple sequence of actions that resulted in successfully locating a hidden target in a static environment.
