ABSTRACT
Chemokine-induced T lymphocyte recruitment to the lung is critical for allergic inflammation, but chemokine signaling pathways are incompletely understood. Regulator of G protein signaling (RGS)16, a GTPase accelerator (GTPase-activating protein) for Galpha subunits, attenuates signaling by chemokine receptors in T lymphocytes, suggesting a role in the regulation of lymphocyte trafficking. To explore the role of RGS16 in T lymphocyte-dependent immune responses in a whole-organism model, we generated transgenic (Tg) mice expressing RGS16 in CD4(+) and CD8(+) cells. rgs16 Tg T lymphocytes migrated to CC chemokine ligand 21 or CC chemokine ligand 12 injection sites in the peritoneum, but not to CXC chemokine ligand 12. In a Th2-dependent model of allergic pulmonary inflammation, CD4(+) lymphocytes bearing CCR3, CCR5, and CXCR4 trafficked in reduced numbers to the lung after acute inhalation challenge with allergen (OVA). In contrast, spleens of sensitized and challenged Tg mice contained increased numbers of CD4(+)CCR3(+) cells producing more Th2-type cytokines (IL-4, IL-5, and IL-13), which were associated with increased airway hyperreactivity. Migration of Tg lymphocytes to the lung parenchyma after adoptive transfer was significantly reduced compared with wild-type lymphocytes. Naive lymphocytes displayed normal CCR3 and CXCR4 expression and cytokine responses, and compartmentation in secondary lymphoid organs was normal without allergen challenge. These results suggest that RGS16 may regulate T lymphocyte activation in response to inflammatory stimuli and migration induced by CXCR4, CCR3, and CCR5, but not CCR2 or CCR7.
Subject(s)
Chemotaxis, Leukocyte/immunology , Inflammation/immunology , Lung/pathology , Lymphocyte Activation/immunology , Proteins/physiology , RGS Proteins/physiology , Signal Transduction/immunology , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/pathology , Acute Disease , Adoptive Transfer , Allergens/administration & dosage , Animals , Cell Differentiation/genetics , Cell Differentiation/immunology , Cells, Cultured , Chemotaxis, Leukocyte/genetics , Crosses, Genetic , Cytokines/biosynthesis , Cytokines/metabolism , Female , Homeostasis/genetics , Homeostasis/immunology , Humans , Immunization , Inflammation/genetics , Inflammation/pathology , Lung/immunology , Lymphocyte Activation/genetics , Lymphoid Tissue/growth & development , Lymphoid Tissue/immunology , Male , Mice , Mice, Inbred BALB C , Mice, Transgenic , Ovalbumin/administration & dosage , Ovalbumin/immunology , Protein Biosynthesis , Proteins/genetics , RGS Proteins/biosynthesis , RGS Proteins/genetics , Receptors, Chemokine/antagonists & inhibitors , Receptors, Chemokine/physiology , Signal Transduction/genetics , T-Lymphocyte Subsets/metabolism , T-Lymphocyte Subsets/transplantation , Up-Regulation/genetics , Up-Regulation/immunologyABSTRACT
Airway function in health and disease as well as in response to bronchospastic stimuli (i.e., irritants, allergens, and inflammatory mediators) is controlled, in part, by cholinergic muscarinic receptor regulation of smooth muscle. In particular, the dependence of airway smooth muscle contraction/relaxation on heterotrimeric G protein-coupled receptor signaling suggests that these events underlie the responses regulating airway function. Galphaq-containing G proteins are proposed to be a prominent signaling pathway, and the availability of knockout mice deficient of this subunit has allowed for an investigation of its potential role in airway function. Airway responses in Galphaq-deficient mice (activities assessed by both tracheal tension and in vivo lung function measurements) were attenuated relative to wild-type controls. Moreover, ovalbumin sensitization/aerosol challenge of Galphaq-deficient mice also failed to elicit an allergen-induced increase in airway reactivity to methacholine. These findings indicate that cholinergic receptor-mediated responses are dependent on Galphaq-mediated signaling events and identify Galphaq as a potential target of preventative/intervening therapies for lung dysfunction.
Subject(s)
Bronchial Hyperreactivity/physiopathology , Bronchoconstrictor Agents/pharmacology , Heterotrimeric GTP-Binding Proteins/genetics , Heterotrimeric GTP-Binding Proteins/metabolism , Methacholine Chloride/pharmacology , Airway Resistance/physiology , Allergens/pharmacology , Animals , Bronchial Hyperreactivity/chemically induced , GTP-Binding Protein alpha Subunits, Gq-G11 , Mice , Mice, Inbred C57BL , Mice, Knockout , Muscle, Smooth/drug effects , Muscle, Smooth/physiopathology , Ovalbumin/pharmacology , Signal Transduction/physiology , Trachea/drug effects , Trachea/physiopathologyABSTRACT
Asthma and mouse models of allergic respiratory inflammation are invariably associated with a pulmonary eosinophilia; however, this association has remained correlative. In this report, a causative relationship between eosinophils and allergen-provoked pathologies was established using eosinophil adoptive transfer. Eosinophils were transferred directly into the lungs of either naive or OVA-treated IL-5(-/-) mice. This strategy resulted in a pulmonary eosinophilia equivalent to that observed in OVA-treated wild-type animals. A concomitant consequence of this eosinophil transfer was an increase in Th2 bronchoalveolar lavage cytokine levels and the restoration of intracellular epithelial mucus in OVA-treated IL-5(-/-) mice equivalent to OVA-treated wild-type levels. Moreover, the transfer also resulted in the development of airway hyperresponsiveness. These pulmonary changes did not occur when eosinophils were transferred into naive IL-5(-/-) mice, eliminating nonspecific consequences of the eosinophil transfer as a possible explanation. Significantly, administration of OVA-treated IL-5(-/-) mice with GK1.5 (anti-CD4) Abs abolished the increases in mucus accumulation and airway hyperresponsiveness following adoptive transfer of eosinophils. Thus, CD4(+) T cell-mediated inflammatory signals as well as signals derived from eosinophils are each necessary, yet alone insufficient, for the development of allergic pulmonary pathology. These data support an expanded view of T cell and eosinophil activities and suggest that eosinophil effector functions impinge directly on lung function.
Subject(s)
Allergens/immunology , Eosinophils/immunology , Eosinophils/pathology , Respiratory Hypersensitivity/immunology , Respiratory Hypersensitivity/pathology , Adoptive Transfer , Aerosols , Allergens/administration & dosage , Animals , Bronchial Hyperreactivity/genetics , Bronchial Hyperreactivity/immunology , Bronchial Hyperreactivity/pathology , CD4-Positive T-Lymphocytes/immunology , Disease Models, Animal , Eosinophils/transplantation , Interleukin-5/deficiency , Interleukin-5/genetics , Intubation, Intratracheal , Lung/immunology , Lung/metabolism , Lung/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Mucus/metabolism , Ovalbumin/administration & dosage , Ovalbumin/immunology , Pulmonary Eosinophilia/genetics , Pulmonary Eosinophilia/immunology , Pulmonary Eosinophilia/pathology , Respiratory Hypersensitivity/genetics , Respiratory Mucosa/immunology , Respiratory Mucosa/metabolism , Respiratory Mucosa/pathologyABSTRACT
We are fortunate to have many species of animals that can serve as adequate models for allergic disease in humans. This review is focused mostly on models of allergic airway disease, and some major categories of animals used for asthma research are discussed, including rodents and nonhuman primates. Furthermore, evidence that supports and criticizes the use of animal models of asthma is provided. There is no animal model that exactly reproduces the pathology of human asthma. However, these models are necessary for the development of novel therapies and an understanding of the detailed pathogenesis of the response of mammals to respirable allergens.
Subject(s)
Asthma/etiology , Disease Models, Animal , Animals , Asthma/physiopathology , Bronchial Hyperreactivity/etiology , Bronchial Hyperreactivity/physiopathology , HumansABSTRACT
A strategy to deplete eosinophils from the lungs of ovalbumin (OVA)-sensitized/challenged mice was developed using antibody-mediated depletion. Concurrent administration [viz. the peritoneal cavity (systemic) and as an aerosol to the lung (local)] of a rat anti-mouse CCR3 monoclonal antibody resulted in the abolition of eosinophils from the lung such that the airway lumen was essentially devoid of eosinophils. Moreover, perivascular/peribronchial eosinophil numbers were reduced to levels indistinguishable from saline-challenged animals. This antibody-mediated depletion was not accompanied by effects on any other leukocyte population, including, but not limited to, T cells and mast cells/basophils. In addition, no effects were observed on other underlying allergic inflammatory responses in OVA-treated mice, including OVA-specific immunoglobulin production as well as T cell-dependent elaboration of Th2 cytokines. The ablation of virtually all pulmonary eosinophils in OVA-treated mice (i.e., without concurrent effects on T cell activities) resulted in a significant decrease in mucus accumulation and abolished allergen-induced airway hyperresponsiveness. These data demonstrate a direct causative relationship between allergen-mediated pulmonary pathologies and eosinophils.
Subject(s)
Allergens/immunology , Eosinophils/physiology , Lung/immunology , Lung/physiopathology , Ovalbumin/immunology , Animals , Antibodies, Monoclonal/pharmacology , Antibody Formation , Bronchial Hyperreactivity/immunology , Bronchial Hyperreactivity/physiopathology , Cell Count , Cytokines/metabolism , Eosinophils/drug effects , Goblet Cells/pathology , Immunoglobulins/biosynthesis , Lung/pathology , Lymphocytes/physiology , Mice , Mice, Inbred C57BL , Mucus/metabolism , Rats , Receptors, CCR3 , Receptors, Chemokine/immunology , Th2 Cells/metabolismABSTRACT
The potential role of airway interleukin-5 (IL-5) expression in eliciting mucus production was demonstrated in a pulmonary IL-5 transgenic mouse model (NJ.1726) in which naive transgenic mice display comparable levels of airway mucus relative to allergen-sensitized and -challenged wild-type mice. The intrinsic mucus accumulation of NJ.1726 was abolished in compound transgenic-gene knockout mice deficient of either CD4(+) cells [NJ.1726/CD4(-/-)] or alphabeta T cell receptor-positive (TCR(+)) cells [NJ.1726/alphabeta TCR(-/-)]. In addition, mucus production in naive NJ.1726 was inhibited by >90% after administration of the soluble anti-IL-4 receptor alpha-subunit antagonist. The loss of mucus production in NJ.1726/CD4(-/-), NJ.1726/alphabeta TCR(-/-), and anti-IL-4 receptor alpha-subunit antagonist-treated mice occurred notwithstanding the significant pulmonary eosinophilia and expansion of airway B cells induced by ectopic IL-5 expression. Furthermore, the loss of mucus accumulation occurred in these mice despite elevated levels of airway and peripheral IL-5, indicating that IL-5 does not directly induce goblet cell metaplasia and mucus production. Thus pulmonary expression of IL-5 alone is capable of inducing CD4(+) T cell-dependent goblet cell metaplasia, apparently mediated by IL-4 receptor alpha-subunit-ligand interactions, and represents a previously unrecognized novel pathway for augmenting allergen-induced mucus production.
Subject(s)
Asthma/pathology , CD4-Positive T-Lymphocytes/metabolism , Interleukin-5/genetics , Mucus/metabolism , Allergens/pharmacology , Animals , Asthma/immunology , Asthma/metabolism , Eosinophils/immunology , Gene Expression/immunology , Goblet Cells/metabolism , Interleukin-5/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Mucus/immunology , Ovalbumin/immunology , Respiratory Mucosa/metabolismABSTRACT
Allergen-induced recruitment of T lymphocytes and eosinophils to the airways is associated with increased expression of the transcription factor GATA-3. In this study, the relationship between airway inflammation and GATA-3 expression in the lungs was investigated using ragweed-sensitized C57BL/6J mice. Intratracheal ragweed challenge increased both the number of GATA-3-expressing cells in the perivascular and peribronchial regions and the amount of expression per cell. Interleukin (IL)-4 and IL-5 levels in bronchoalveolar lavage fluid were upregulated in parallel with GATA-3 expression. GATA-3 mRNA and protein colocalized to eosinophils. Eosinophils isolated from the lungs and stimulated with phorbol 12-myristate 13-acetate and/or A-23187 released IL-5. The release was inhibited by actinomycin D, which indicates that de novo synthesis of the cytokine was involved. Western blot analysis of proteins from isolated eosinophils demonstrated expression of the p50 subunit of nuclear factor-kappaB, a transcription factor that is implicated in control of GATA-3 expression. These data provide evidence that allergen challenge increases GATA-3 and proinflammatory cytokine expression by pulmonary eosinophils, which could provide positive feedback for the inflammatory response.
Subject(s)
DNA-Binding Proteins/genetics , Eosinophils/immunology , Interleukin-4/analysis , Interleukin-5/analysis , Respiratory Hypersensitivity/immunology , Ribonucleases , Trans-Activators/genetics , Allergens/immunology , Animals , Asthma/immunology , Blood Proteins/analysis , Bronchoalveolar Lavage Fluid/chemistry , Bronchoalveolar Lavage Fluid/immunology , Chemotaxis, Leukocyte/immunology , DNA-Binding Proteins/analysis , Eosinophil Granule Proteins , Eosinophils/chemistry , Eosinophils/cytology , GATA3 Transcription Factor , Gene Expression/immunology , In Situ Hybridization , In Vitro Techniques , Interleukin-4/biosynthesis , Interleukin-5/biosynthesis , Male , Mice , Mice, Inbred C57BL , Plant Proteins/immunology , RNA, Messenger/analysis , Th2 Cells/immunology , Trans-Activators/analysis , Transcription, Genetic/immunologyABSTRACT
CD4(+) T cells have a critical role in the development of allergic pulmonary inflammation, including the recruitment of eosinophils to the airway lumen and interstitium. The expression of interleukin (IL)-5 by CD4(+) cells has, in particular, often been lionized as the central link between allergic inflammation and the concomitant expansion or recruitment of eosinophils. The mechanism(s) by which CD4(+) T cells mediates eosinophil recruitment was assessed with gene knockout mice deficient for T cells or T cell subtypes and a unique IL-5 transgenic mouse (line NJ.1726) that constitutively overexpresses this cytokine in the lung epithelium. Pulmonary IL-5 expression is significantly attenuated in T cell- and CD4(+) but not CD8(+) cell-deficient animals, suggesting an obvious explanation for the lack of eosinophils in the lungs of T cell-deficient and CD4(-/-) mice. However, although the constitutive expression of IL-5 in the lung epithelium of NJ.1726 mice elicited an eosinophilia in the airway lumen of both naive and ovalbumin-treated mice, in the absence of CD4(+) cells, allergen-mediated eosinophil recruitment to the bronchoalveolar lavage fluid was abolished. Moreover, intranasal instillation of the potent eosinophil-specific chemokine eotaxin-2 was incapable of eliciting eosinophil recruitment in naive and ovalbumin-treated NJ.1726 CD4(-/-) mice, suggesting that eosinophil trafficking during allergic inflammatory responses is a consequence of a CD4(+) cell-mediated event(s) in addition to IL-5 expression and the establishment of a pulmonary chemokine gradient.
Subject(s)
CD4-Positive T-Lymphocytes/physiology , Interleukin-5/metabolism , Administration, Intranasal , Allergens/immunology , Animals , Chemokine CCL24 , Chemokines, CC/administration & dosage , Chemokines, CC/pharmacology , Chemotaxis, Leukocyte/physiology , Eosinophils/drug effects , Eosinophils/pathology , Eosinophils/physiology , Epithelium/metabolism , Immunization , Interleukin-5/genetics , Lung/metabolism , Lung/pathology , Mice , Mice, Knockout , Mice, Transgenic/genetics , Ovalbumin/immunology , Pulmonary Eosinophilia/etiology , Pulmonary Eosinophilia/immunology , Pulmonary Eosinophilia/pathologyABSTRACT
3alpha-(diphenylmethoxy)tropane (benztropine) and its analogues are tropane ring-containing dopamine uptake inhibitors that display binding and behavioral profiles that are distinct from cocaine. We previously prepared a benztropine-based photoaffinity label [125I]-(N-[4-(4'-azido-3'-iodophenyl)butyl]-3alpha-[bis(4'-fluorophenyl)methoxy]tropane, [125I]1, that covalently attached to the 1-2 transmembrane spanning region of the dopamine transporter (DAT). This was in contrast to the 4-7 transmembrane spanning region labeled by a cocaine-based photoaffinity label, [125I] 2 (RTI 82). To characterize further these different binding domains, photoaffinity ligands that had the 4'-azido-3'-iodophenyl substituent extended from the same position on the tropane ring were desirable. Thus, identification of the optimal alkyl linker between this substituent and the tropane nitrogen in the benztropine series was investigated to ultimately prepare the identical N-substituted analogue of 2. In this pursuit, the N-[4-(4'-azido-3'-iodophenyl)propyl] analogue of 3alpha-[bis(4'-fluorophenyl)methoxy]tropane (9a) was synthesized as well as two isothiocyanate analogues that do not require photoactivation (10a,b) for irreversible binding. The synthesis of these target compounds was achieved using a modification of the strategy developed for 1. Evaluation of these compounds for displacing [3H]WIN 35 428 binding at DAT in rat caudate putamen revealed that the 4'-azido-3'-iodophenylbutyl substituent, found in 1, provided optimal binding affinity and was chosen to replace the N-CH3 group on 2. Both the 4'-azido-3'-iodophenyl- and the 4'-isothiocyanatophenylbutyl analogues of 2 (25 and 26, respectively) were synthesized. Both products bound to DAT with comparable potency (IC(50) = 30 nM) to RTI 82 (2). In addition, compound 26 demonstrated wash-resistant displacement of [3H]WIN 35 428 in HEK 293 cells stably transfected with hDAT. These ligands will provide important tools for further characterizing the binding domains for tropane-based dopamine uptake inhibitors at the DAT.
Subject(s)
Benztropine/analogs & derivatives , Benztropine/chemical synthesis , Cocaine/analogs & derivatives , Dopamine Uptake Inhibitors/chemical synthesis , Dopamine/metabolism , Isothiocyanates/chemical synthesis , Membrane Glycoproteins , Membrane Transport Proteins/metabolism , Nerve Tissue Proteins , Tropanes/chemical synthesis , Animals , Azides/metabolism , Benztropine/chemistry , Benztropine/metabolism , Binding, Competitive , Cell Line , Cocaine/metabolism , Dopamine Plasma Membrane Transport Proteins , Dopamine Uptake Inhibitors/chemistry , Dopamine Uptake Inhibitors/metabolism , Humans , In Vitro Techniques , Isothiocyanates/chemistry , Isothiocyanates/metabolism , Ligands , Male , Putamen/metabolism , Radioligand Assay , Rats , Rats, Sprague-Dawley , Structure-Activity Relationship , Tropanes/chemistry , Tropanes/metabolismABSTRACT
Treatment of mice with heat-killed (HK) Mycobacterium bovis BCG or 1- to 10-microm chitin particles (nonantigenic N-acetyl-D-glucosamine polymers) is known to induce innate immune responses, including gamma interferon (IFN-gamma) production, which plays a Th1 adjuvant role. However, HK BCG further induces prostaglandin E2-releasing spleen macrophages (Mphi) (PGE2-Mphi), which potentially inhibit Th1 adjuvant activities. We found that chitin particles did not induce PGE2-Mphi formation. To further assess whether chitin has Th1 adjuvant effects, interleukin-10 (IL-10)-knockout (KO) mice and their wild-type (WT, C57BL/6) controls were immunized with a 30-kDa MPB-59 mycobacterial protein mixed with chitin. Immunization with MPB-59 alone induced Th2 responses, characterized by increases in total serum immunoglobulin E (IgE) and specific serum IgG1 levels and spleen Th2 cells producing IL-4, IL-5, and IL-10. No IFN-gamma-producing spleen Th1 cells, specific serum IgG2a, or delayed-type hypersensitivity (DTH) footpad reactions were detected. On the other hand, chitin-MPB-59 immunization significantly increased spleen Th1 responses, DTH reaction, and serum IgG2a levels along with decreases of Th2 responses. The magnitude of these Th1 adjuvant effects was greater in IL-10-KO mice than in WT mice. In contrast, immunization with HK BCG-MPB-59 showed little or no Th1 adjuvant effect. These data indicate that chitin has a unique Th1 adjuvant effect on the development of Th1 immunity against a mycobacterial antigen. IL-10 down-regulates the adjuvant effect of chitin.
Subject(s)
Acetylglucosamine/immunology , Adjuvants, Immunologic , Antigens, Bacterial/immunology , Down-Regulation/immunology , Interleukin-10/immunology , Th1 Cells/immunology , Th2 Cells/immunology , Up-Regulation/immunology , Animals , Cells, Cultured , Chitin/immunology , Dinoprostone/immunology , Female , Hypersensitivity, Delayed/immunology , Immunoglobulin E/blood , Immunoglobulin G/blood , Interleukin-10/genetics , Macrophages, Alveolar/cytology , Macrophages, Alveolar/immunology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mycobacterium bovis/immunology , Polymers , Spleen/cytology , Spleen/immunologyABSTRACT
The short-term stability of Adderall in three extemporaneously compounded oral liquids was studied. Three suspensions of Adderall 1 mg/mL were prepared from commercially available 10-mg Adderall tablets with Ora-Sweet, Ora-Plus, and a 1:1 mixture of Ora-Sweet and Ora-Plus. Each suspension was stored in the dark in a stability chamber at 25 degrees C and 60% relative humidity for 30 days. The stability of the active drug (a mixture of levoamphetamine and dextroamphetamine salts) in each of the three vehicles was determined immediately after preparation and at 10, 20, and 30 days by using gas chromatography-mass spectrometry (GCMS). No significant changes in concentrations of either amphetamine isomer occurred during the 30-day study period. Visual inspection of samples revealed no changes in color or odor. Extemporaneously compounded liquid oral formulations of Adderall 1 mg/mL in Ora-Sweet, Ora-Plus, or a 1:1 mixture of Ora-Sweet and Ora-Plus were stable for at least 30 days at 25 degrees C and 60% relative humidity.
Subject(s)
Amphetamines/chemistry , Central Nervous System Stimulants/chemistry , Dextroamphetamine/chemistry , Administration, Oral , Amphetamines/administration & dosage , Attention Deficit Disorder with Hyperactivity/drug therapy , Central Nervous System Stimulants/administration & dosage , Child , Child, Preschool , Dextroamphetamine/administration & dosage , Drug Compounding , Drug Stability , Gas Chromatography-Mass Spectrometry , HumansABSTRACT
Paradigms of eosinophil effector function in the lungs of asthma patients invariably depend on activities mediated by cationic proteins released from secondary granules during a process collectively referred to as degranulation. In this study, we generated knockout mice deficient for eosinophil peroxidase (EPO) to assess the role(s) of this abundant secondary granule protein in an OVA-challenge model. The loss of EPO had no effect on the development of OVA-induced pathologies in the mouse. The absence of phenotypic consequences in these knockout animals extended beyond pulmonary histopathologies and airway changes, as EPO-deficient animals also displayed OVA-induced airway hyperresponsiveness after provocation with methacholine. In addition, EPO-mediated oxidative damage of proteins (e.g., bromination of tyrosine residues) recovered in bronchoalveolar lavage from OVA-treated wild-type mice was <10% of the levels observed in bronchoalveolar lavage recovered from asthma patients. These data demonstrate that EPO activities are inconsequential to the development of allergic pulmonary pathologies in the mouse and suggest that degranulation of eosinophils recruited to the lung in this model does not occur at levels comparable to those observed in humans with asthma.
Subject(s)
Eosinophils/enzymology , Eosinophils/immunology , Lung/metabolism , Lung/pathology , Ovalbumin/immunology , Peroxidases/metabolism , Proteins/metabolism , Allergens/administration & dosage , Allergens/immunology , Animals , Bronchial Hyperreactivity/genetics , Bronchial Hyperreactivity/immunology , Bronchial Hyperreactivity/pathology , Cell Degranulation/immunology , Cell Movement/genetics , Cell Movement/immunology , Crosses, Genetic , Cytoplasmic Granules/metabolism , Cytoplasmic Granules/ultrastructure , Disease Models, Animal , Eosinophil Peroxidase , Eosinophils/metabolism , Eosinophils/ultrastructure , Injections, Intraperitoneal , Lung/enzymology , Mice , Mice, Inbred C57BL , Mice, Knockout , Ovalbumin/administration & dosage , Oxidation-Reduction , Peroxidases/deficiency , Peroxidases/genetics , Respiratory Hypersensitivity/enzymology , Respiratory Hypersensitivity/genetics , Respiratory Hypersensitivity/pathology , Sequence DeletionABSTRACT
Intratracheal administration of interleukin-10 (IL-10) has been reported to inhibit allergic inflammation but augment airway hyperresponsiveness (AHR). In the present study, airway and smooth muscle responsiveness to methacholine (MCh) were compared in wild-type (WT) and IL-10-deficient (IL-10-KO) mice to investigate the role of endogenous IL-10 in AHR development. Naive WT and IL-10-KO mice exhibited similar dose-dependent increases in airway resistance (Raw) to intravenous MCh. Sensitization and challenge with ragweed (RW) induced a twofold increase in responsiveness to intravenous MCh in WT mice, but hyperresponsiveness was not observed in similarly treated IL-10-KO mice. Likewise, tracheal rings from RW-sensitized and -challenged WT mice exhibited a fourfold greater responsiveness to MCh than IL-10-KO tracheal preparations. Measurements of airway constriction by whole body plethysmography further supported the Raw and tracheal ring data (i.e., AHR was not observed in the absence of IL-10). Interestingly, factors previously implicated in the development of AHR, including IL-4, IL-5, IL-13, IgA, IgG1, IgE, eosinophilia, and lymphocyte recruitment to the airways, were upregulated in the IL-10-KO mice. Treatment with recombinant murine IL-10 at the time of allergen challenge reduced the magnitude of inflammation but reinstated AHR development in IL-10-KO mice. Adoptive transfer of mononuclear splenocytes to IL-10-sufficient severe combined immunodeficient mice indicated that lymphocytes were an important source of the IL-10 impacting AHR development. These results provide evidence that IL-10 expression promotes the development of allergen-induced smooth muscle hyperresponsiveness.
Subject(s)
Allergens/immunology , Interleukin-10/deficiency , Interleukin-10/immunology , Respiratory Hypersensitivity/immunology , Adoptive Transfer , Animals , Bronchial Provocation Tests , Bronchoalveolar Lavage Fluid/chemistry , Bronchoalveolar Lavage Fluid/immunology , Dose-Response Relationship, Drug , Immunoglobulins/blood , In Vitro Techniques , Inflammation/immunology , Inflammation/metabolism , Interleukin-10/administration & dosage , Interleukin-10/genetics , Interleukin-13/analysis , Interleukin-4/analysis , Interleukin-5/analysis , Male , Methacholine Chloride , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, SCID , Recombinant Proteins/administration & dosage , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Respiratory Hypersensitivity/chemically induced , Respiratory Hypersensitivity/genetics , Spleen/cytology , Spleen/immunology , Spleen/transplantation , Th2 Cells/immunologyABSTRACT
There is evidence to suggest that dopamine (DA) oxidizes to form dopamine ortho-quinone (DAQ), which binds covalently to nucleophilic sulfhydryl groups on protein cysteinyl residues. This reaction has been shown to inhibit dopamine uptake, as well as other biological processes. We have identified specific cysteine residues in the human dopamine transporter (hDAT) that are modified by this electron-deficient substrate analog. DAQ reactivity was inferred from its effects on the binding of [(3)H]2-beta-carbomethoxy-3-beta-(4-fluorophenyl)tropane (beta-CFT) to hDAT cysteine mutant constructs. One construct, X5C, had four cysteines mutated to alanine and one to phenylalanine (Cys(90)A, Cys(135)A, C306A, C319F and Cys(342)A). In membrane preparations 1 mM DAQ did not affect [(3)H]beta-CFT binding to X5C hDAT, in contrast to its effect in wild-type hDAT in which it reduced the B:(max) value by more than half. Wild-type cysteines were substituted back into X5C, one at a time, and the ability of DAQ to inhibit [(3)H]beta-CFT binding was assessed. Reactivity of DAQ with Cys(90) increased the affinity of [(3)H]beta-CFT for the transporter, whereas reactivity with Cys(135) decreased the affinity of [(3)H]beta-CFT. DAQ did not change the K:(D) for [(3)H]beta-CFT binding to wild-type. The reactivity of DAQ at Cys(342) decreased B:(max) to the same degree as wild-type. The latter result suggests that Cys(342) is the wild-type residue most responsible for DAQ-induced inhibition of [(3)H]beta-CFT binding.
Subject(s)
Carrier Proteins/chemistry , Cocaine/analogs & derivatives , Cysteine/chemistry , Dopamine/analogs & derivatives , Dopamine/chemistry , Membrane Glycoproteins , Membrane Transport Proteins , Nerve Tissue Proteins , Amino Acid Substitution/genetics , Binding, Competitive/drug effects , Carrier Proteins/genetics , Cell Line , Cell Membrane/chemistry , Cell Membrane/metabolism , Cocaine/metabolism , Dopamine/pharmacology , Dopamine Plasma Membrane Transport Proteins , Dose-Response Relationship, Drug , Humans , Mutagenesis, Site-Directed , Protein Binding/drug effectsABSTRACT
Coadministration of kappa-opioid receptor agonists (kappa-agonists) with cocaine prevents alterations in dialysate dopamine (DA) concentration in the nucleus accumbens (Acb) that occur during abstinence from repeated cocaine treatment. Quantitative microdialysis was used to determine the mechanism producing these effects. Rats were injected with cocaine (20 mg/kg, i.p.), or saline, and the selective kappa-agonist U-69593 (0.32 mg/kg, s.c.), or vehicle, once daily for 5 d. Extracellular DA concentration (DA(ext)) and extraction fraction (E(d)), an indirect measure of DA uptake, were determined 3 d later. Repeated cocaine treatment increased E(d), whereas repeated U-69593 treatment decreased E(d), relative to controls. Coadministration of both drugs yielded intermediate E(d) values not different from controls. In vitro DA uptake assays confirmed that repeated U-69593 treatment produces a dose-related, region-specific decrease in DA uptake and showed that acute U-69593 administration increases DA uptake in a nor-binaltorphimine reversible manner. Repeated U-69593 also led to a decrease in [(125)I]RTI-55 binding to the DA transporter (DAT), but did not decrease total DAT protein. These results demonstrate that kappa-opioid receptor activation modulates DA uptake in the Acb in a manner opposite to that of cocaine: repeated U-69593 administration decreases the basal rate of DA uptake, and acute U-69593 administration transiently increases DA uptake. kappa-agonist treatment also alters DAT function. The action of kappa-agonists on DA uptake or DAT binding, or both, may be the mechanism(s) mediating the previously reported "cocaine-antagonist" effect of kappa-opioid receptor agonists.
Subject(s)
Benzeneacetamides , Cocaine/antagonists & inhibitors , Dopamine/pharmacokinetics , Membrane Glycoproteins , Membrane Transport Proteins , Naltrexone/analogs & derivatives , Nerve Tissue Proteins , Nucleus Accumbens/drug effects , Nucleus Accumbens/metabolism , Receptors, Opioid, kappa/metabolism , Analysis of Variance , Animals , Autoradiography , Carrier Proteins/metabolism , Cocaine/administration & dosage , Corpus Striatum/chemistry , Corpus Striatum/metabolism , Dopamine/analysis , Dopamine Plasma Membrane Transport Proteins , Drug Administration Schedule , Drug Antagonism , Extracellular Space/chemistry , Extracellular Space/metabolism , Injections, Intraperitoneal , Injections, Subcutaneous , Iodine Radioisotopes , Linear Models , Male , Microdialysis , Naltrexone/administration & dosage , Nucleus Accumbens/chemistry , Pyrrolidines/administration & dosage , Rats , Rats, Sprague-Dawley , Receptors, Opioid, kappa/agonistsABSTRACT
We investigated the effects of interleukin (IL)-10 administration on allergen-induced Th2 cytokine production, eosinophilic inflammation, and airway reactivity. Mice were sensitized by intraperitoneal injection of ragweed (RW) adsorbed to Alum and challenged by intratracheal instillation of the allergen. Sensitization and challenge with RW increased concentrations of IL-10 in bronchoalveolar lavage (BAL) fluid from undetectable levels to 60 pg/ml over 72 h. Intratracheal instillation of 25 ng of recombinant murine IL-10 at the time of RW challenge further elevated BAL fluid IL-10 concentration to 440 pg/ml but decreased BAL fluid IL-4, IL-5, and interferon-gamma levels by 40-85% and eosinophil numbers by 70% (P < 0.0001). Unexpectedly, the same IL-10 treatment increased airway reactivity to methacholine in spontaneously breathing mice that had been sensitized and challenged with RW (P < 0.001). IL-10 treatment in naive animals or RW-sensitized mice challenged with PBS failed to increase airway reactivity, demonstrating that IL-10 induces an increase in airway reactivity only when it is administered in conjunction with allergic sensitization and challenge. The results demonstrate that IL-10 reduces Th2 cytokine levels and eosinophilic inflammation but augments airway hyperreactivity. Thus, despite its potent anti-inflammatory activity, IL-10 could contribute to the decline in pulmonary function observed in asthma.
Subject(s)
Bronchial Hyperreactivity/physiopathology , Cytokines/antagonists & inhibitors , Eosinophilia/pathology , Hypersensitivity/metabolism , Hypersensitivity/physiopathology , Interleukin-10/pharmacology , Th2 Cells/metabolism , Animals , Bronchitis/pathology , Bronchoalveolar Lavage Fluid/chemistry , Cytokines/biosynthesis , Cytokines/metabolism , Dose-Response Relationship, Drug , Female , Hypersensitivity/pathology , Interleukin-10/administration & dosage , Interleukin-10/analysis , Interleukin-10/blood , Methacholine Chloride , Mice , Mice, Inbred BALB C , Pollen/immunology , TracheaABSTRACT
The effect of structural modification of the human dopamine transporter protein on bi-directional transport was explored using site-directed mutagenesis and rotating disk electrode voltammetry. The substrate-induced DA efflux, as inferred from the K(m) or K(i), was dependent on common structural features for uptake of the substrate inducer: reduced by beta-hydroxylation, stereoselective to alpha-methylation, and relatively insensitive to a switch of a single phenolic hydroxyl group between m- and p-positions. The potencies for substrates to compete with external DA for uptake and to induce DA efflux were similar and highly correlated. Despite these similarities, the efflux of internal DA was substantially slower than the uptake of its inducers. Mutation of serine-528 of the hDAT to alanine (S528A) did not change the structure-activity relationships, maximal uptake rates, and the cation dependence for the uptake of external substrates, although it modestly reduced K(m) or K(i) of most tested substrates. In contrast, it substantially enhanced substrate-induced DA efflux, with maximal efflux rates doubled for all tested inducers. Simultaneous monitoring of tyramine uptake and resulting DA efflux revealed that S528A accelerated the DA efflux relative to tyramine uptake. Saturation analysis suggested that the mutation significantly enhanced the efflux kinetics of internal DA but it exerted little effect on the uptake kinetics of external DA. These findings suggest that Ser-528 may play a role in stabilizing a hDAT conformation unfavorable for outward transport of internal DA, thereby contributing to the efficiency of the transporter.
Subject(s)
Carrier Proteins/metabolism , Dopamine/metabolism , Membrane Glycoproteins , Membrane Transport Proteins , Nerve Tissue Proteins , Alanine/genetics , Alanine/metabolism , Amino Acid Substitution , Biological Transport , Carrier Proteins/chemistry , Carrier Proteins/genetics , Cells, Cultured , Dopamine Plasma Membrane Transport Proteins , Humans , Mutagenesis , Protein Conformation , Serine/genetics , Serine/metabolism , Tyramine/metabolismABSTRACT
Nitric oxide (NO) synthesized by endothelial cell nitric oxide synthase (eNOS) elicits vasodilation of resistance-sized coronary microvessels. Since coronary blood flow increases during hypoxia, we tested the hypotheses that: (1) hypoxia results in increased blood flow through increased NO production mediated by the upregulation of both eNOS mRNA and protein and (2) the regulation of NO production in response to hypoxia differs in microvascular endothelial cells and nonresistance, epicardial endothelial cells. Monocultures of vascular endothelium from resistance (approximately 100 micro) and nonresistance epicardial arteries were established and characterized. Nitric oxide was quantitated using a chemiluminescence method. Hypoxia (pO(2) = 10 mmHg) significantly increased NO production in both cell lines, with less NO produced in microvascular endothelium. Western blots demonstrated that hypoxia caused a time-dependent increase in eNOS protein in both lines, with an average 2.5-fold increase in nonresistance, epicardial endothelial cells compared to an average 1.7-fold increase in protein from microvascular endothelium. Total mRNA recovery increased 2.4 +/- 0.6-fold within 30 min of hypoxia in nonresistance, epicardial endothelial cells with no increase in microvascular endothelial cells. Although hypoxia increased NO production in both populations of endothelial cells, the increase in NO production and eNOS protein in microvascular endothelium was less compared to nonresistance, epicardial endothelial cells. Furthermore, there was no significant upregulation of total mRNA for eNOS in microvascular endothelium. The data indicate that increased NO production in microvascular endothelium during hypoxia may be through translational or posttranslational modifications of the enzyme, whereas transcriptional upregulation may account for the increased NO production in nonresistance, epicardial endothelial cells. Oxygen-sensitive response mechanisms that modulate NO production may be different in endothelium from different coronary artery vascular beds.