RESUMO
Signaling via the T cell antigen receptor (TCR) is initiated by Src-family kinases (SFKs). To understand how the kinase Csk, a negative regulator of SFKs, controls the basal state and the initiation of TCR signaling, we generated mice that express a Csk variant sensitive to an analog of the common kinase inhibitor PP1 (Csk(AS)). Inhibition of Csk(AS) in thymocytes, without engagement of the TCR, induced potent activation of SFKs and proximal TCR signaling up to phospholipase C-γ1 (PLC-γ1). Unexpectedly, increases in inositol phosphates, intracellular calcium and phosphorylation of the kinase Erk were impaired. Altering the actin cytoskeleton pharmacologically or providing costimulation via CD28 'rescued' those defects. Thus, Csk has a critical role in preventing TCR signaling. However, our studies also revealed a requirement for actin remodeling, initiated by costimulation, for full TCR signaling.
Assuntos
Actinas/metabolismo , Citoesqueleto/metabolismo , Proteínas Mutantes/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Timócitos/imunologia , Quinases da Família src/metabolismo , Animais , Antígenos CD28/imunologia , Proteína Tirosina Quinase CSK , Células Cultivadas , Citocalasina D/administração & dosagem , Citoesqueleto/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Mutantes/genética , Polimerização/efeitos dos fármacos , Engenharia de Proteínas , Pirazóis/administração & dosagem , Pirimidinas/administração & dosagem , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Timócitos/efeitos dos fármacos , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/genéticaRESUMO
The coordinated organization of cell membrane receptors into diverse micrometre-scale spatial patterns is emerging as an important theme of intercellular signalling, as exemplified by immunological synapses. Key characteristics of these patterns are that they transcend direct protein-protein interactions, emerge transiently and modulate signal transduction. Such cooperativity over multiple length scales presents new and intriguing challenges for the study and ultimate understanding of cellular signalling. As a result, new experimental strategies have emerged to manipulate the spatial organization of molecules inside living cells. The resulting spatial mutations yield insights into the interweaving of the spatial, mechanical and chemical aspects of intercellular signalling.
Assuntos
Junções Intercelulares/metabolismo , Transdução de Sinais , Animais , Humanos , Sinapses Imunológicas/metabolismoRESUMO
T cells react to extremely small numbers of activating agonist peptides. Spatial organization of T-cell receptors (TCR) and their peptide-major histocompatibility complex (pMHC) ligands into microclusters is correlated with T-cell activation. Here we have designed an experimental strategy that enables control over the number of agonist peptides per TCR cluster, without altering the total number engaged by the cell. Supported membranes, partitioned with grids of barriers to lateral mobility, provide an effective way of limiting the total number of pMHC ligands that may be assembled within a single TCR cluster. Observations directly reveal that restriction of pMHC content within individual TCR clusters can decrease T-cell sensitivity for triggering initial calcium flux at fixed total pMHC density. Further analysis suggests that triggering thresholds are determined by the number of activating ligands available to individual TCR clusters, not by the total number encountered by the cell. Results from a series of experiments in which the overall agonist density and the maximum number of agonist per TCR cluster are independently varied in primary T cells indicate that the most probable minimal triggering unit for calcium signaling is at least four pMHC in a single cluster for this system. This threshold is unchanged by inclusion of coagonist pMHC, but costimulation of CD28 by CD80 can modulate the threshold lower.
Assuntos
Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Animais , Biofísica/métodos , Linfócitos T CD4-Positivos/citologia , Cálcio/metabolismo , Sinalização do Cálcio , Sistema Imunitário , Molécula 1 de Adesão Intercelular/metabolismo , Ligantes , Bicamadas Lipídicas/química , Lipídeos/química , Ativação Linfocitária , Complexo Principal de Histocompatibilidade , CamundongosRESUMO
BACKGROUND: Although hospitalization for acute decompensated heart failure (HF) is common and associated with poor outcomes and high costs, few evidence-based recommendations are available to guide patient management. Thus, management of inpatient HF remains heterogeneous. We evaluated if physician-specific self-reported HF practice patterns were associated with 2 important contributors to resource utilization: length of stay (LOS) and 30-day readmission. METHODS AND RESULTS: A 5-point Likert scale survey was created to assess physician-specific HF discharge strategies and administered to all cardiologists and hospitalists at a single large academic teaching hospital. Practice patterns potentially impacting LOS and discharge decisions were queried, including use of physical examination findings, approaches to diuretic use and influence of kidney function. Likert scale responses are reported as means with any value above 3.00 considered more influential and any value below 3.00 considered less influential. Physician-specific LOS and 30-day readmission rates from July 1, 2015, to June 30, 2016, were extracted from the electronic record. We received survey responses and HF utilization metrics from 58 of 69 surveyed physicians (32 hospitalists and 26 cardiologists), encompassing 753 HF discharges over a 1-year period. Median LOS was 4.5 days (interquartile range, 4.0-5.8) and total 30-day readmission rate was 17.0% (128 unique readmissions). Physicians with below-median LOS placed less importance on observing a patient on oral diuretics for 24 hours before discharge (Likert 2.54 versus 3.30, P=0.01), reaching documented dry weight (Likert 2.93 versus 3.60, P=0.02), and complete resolution of dyspnea on exertion (Likert 3.64 versus 4.10, P=0.03) when compared with those above-median LOS. In contrast, no surveyed discharge practices were associated with physician-specific 30-day readmission. CONCLUSIONS: We identified specific inpatient HF discharge practice patterns that associated with shorter LOS but not with readmission rates. These may be targets for future interventions aimed at cost reduction; additional larger studies are needed for further exploration.
Assuntos
Cardiologistas , Insuficiência Cardíaca/epidemiologia , Alta do Paciente/estatística & dados numéricos , Padrões de Prática Médica/estatística & dados numéricos , Doença Aguda , Análise Custo-Benefício , Feminino , Insuficiência Cardíaca/economia , Humanos , Tempo de Internação , Masculino , Readmissão do Paciente , Melhoria de Qualidade , Inquéritos e Questionários , Resultado do Tratamento , Estados Unidos/epidemiologiaRESUMO
Clustering of receptors associated with immunoreceptor tyrosine-based activation motifs (ITAMs) initiates the macrophage antimicrobial response. ITAM receptors engage Src-family tyrosine kinases (SFKs) to initiate phagocytosis and macrophage activation. Macrophages also encounter nonpathogenic molecules that cluster receptors weakly and must tune their sensitivity to avoid inappropriate responses. To investigate this response threshold, we compared signaling in the presence and absence of receptor clustering using a small-molecule inhibitor of Csk, which increased SFK activation and produced robust membrane-proximal signaling. Surprisingly, receptor-independent SFK activation led to a downstream signaling blockade associated with rapid degradation of the SFK LynA. Inflammatory priming of macrophages upregulated LynA and promoted receptor-independent signaling. In contrast, clustering the hemi-ITAM receptor Dectin-1 induced signaling that did not require LynA or inflammatory priming. Together, the basal-state signaling checkpoint regulated by LynA expression and degradation and the signaling reorganization initiated by receptor clustering allow cells to discriminate optimally between pathogens and nonpathogens.
Assuntos
Macrófagos/imunologia , Transdução de Sinais , Quinases da Família src/metabolismo , Animais , Ativação de Macrófagos , Camundongos Endogâmicos C57BL , FagocitoseRESUMO
The C-terminal Src kinase (Csk), the primary negative regulator of Src-family kinases (SFK), plays a crucial role in controlling basal and inducible receptor signaling. To investigate how Csk activity regulates T cell antigen receptor (TCR) signaling, we utilized a mouse expressing mutated Csk (Csk(AS)) whose catalytic activity is specifically and rapidly inhibited by a small molecule. Inhibition of Csk(AS) during TCR stimulation led to stronger and more prolonged TCR signaling and to increased proliferation. Inhibition of Csk(AS) enhanced activation by weak but strictly cognate agonists. Titration of Csk inhibition revealed that a very small increase in SFK activity was sufficient to potentiate T cell responses to weak agonists. Csk plays an important role, not only in basal signaling, but also in setting the TCR signaling threshold and affinity recognition.
Assuntos
Inibidores Enzimáticos/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Quinases da Família src/antagonistas & inibidores , Animais , Proteína Tirosina Quinase CSK , Proliferação de Células , Camundongos , Transdução de Sinais/efeitos dos fármacosRESUMO
Increased airway smooth muscle (ASM) contractility and the development of airway hyperresponsiveness (AHR) are cardinal features of asthma, but the signaling pathways that promote these changes are poorly understood. Tyrosine phosphorylation is tightly regulated by the opposing actions of protein tyrosine kinases and phosphatases, but little is known about whether tyrosine phosphatases influence AHR. Here, we demonstrate that genetic inactivation of receptor-like protein tyrosine phosphatase J (Ptprj), which encodes CD148, protected mice from the development of increased AHR in two different asthma models. Surprisingly, CD148 deficiency minimally affected the inflammatory response to allergen, but significantly altered baseline pulmonary resistance. Mice specifically lacking CD148 in smooth muscle had decreased AHR, and the frequency of calcium oscillations in CD148-deficient ASM was substantially attenuated, suggesting that signaling pathway alterations may underlie ASM contractility. Biochemical analysis of CD148-deficient ASM revealed hyperphosphorylation of the C-terminal inhibitory tyrosine of SRC family kinases (SFKs), implicating CD148 as a critical positive regulator of SFK signaling in ASM. The effect of CD148 deficiency on ASM contractility could be mimicked by treatment of both mouse trachea and human bronchi with specific SFK inhibitors. Our studies identify CD148 and the SFKs it regulates in ASM as potential targets for the treatment of AHR.