Extrafollicular IgD−CD27−CXCR5−CD11c− DN3 B cells infiltrate inflamed tissues in autoimmune fibrosis and in severe COVID-19

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Effect of insulin on glucose oxidation and amino isobutyric acid transport and binding of insulin in chicken thymocytes

In chicken thymocytes isolated from 15–40 day-old chickens, after a 2 h incubation at 37°C, insulin stimulated amino isobutyric acid uptake (maximal response: 40–50% of increase at 1 μg insulin/ml and half maximal response at 60 ng/ml) by specifically stimulating the influx without altering the efflux. Insulin also stimulated glucose oxidation (maximal response: 11% of increase at 1 μg insulin/ml). Binding of ¹²⁵I-labelled chicken insulin to thymocytes was rapid and higher at 15°C than at 37°C. At steady state, (90 min at 15°C), chicken, porcine and goose insulins were equipotent in inhibiting the binding of ¹²⁵I-labelled chicken insulin. Maximal binding capacity was estimated at 1250 pg insulin/10⁸ cells, i.e., 1250 binding sites/cell with an apparent dissociation constant of 200 ng insulin/ml at 15°C. Degradation of ¹²⁵I-labelled chicken insulin in the incubation medium was negligible at 15°C but very noticeable at 37°C. Therefore, the low level of insulin binding at 15°C reflects a true scarcity of insulin receptors in chicken thymocytes as compared to rat thymocytes.     

Viable mouse thymocytes as a model system for studying the onset of hormone-induced cellular refractoriness

Mouse thymocytes are characterized as a model cellular system for studying the onset of hormone-induced cellular refractoriness (desensitization). This system has the following combination of useful features. (a) The cells can be isolated without the use of digestive enzymes, avoiding possible damage to surface receptors or to other exposed membranal constituents. (b) They can be kept viable for several hours, a period during which both stimulation and desensitization get well under way. (c) They can be stimulated by a variety of hormones which function via cAMP (β-agonists, prostaglandin E₁ and specific thymic humoral factors). (d) Their desensitization is receptor-specific. (e) They can be readily ruptured under mild conditions so as to allow a physiologically relevant biochemical analysis of hormonal stimulation and desensitization. (f) The hormonal response of these cells can be monitored simultaneously by the activation of adenylate cyclase, by the intracellular level of cAMP, and by the activation of cAMP-dependent protein kinase (which functions as a metabolic sensor for cAMP). In this cellular system, desensitization does not involve processes such as the efflux of cAMP, the activation of cAMP-phosphodiesterase or the synthesis of a protein mediator. On the other hand, desensitization can be accounted for by a hormone-triggered inactivation of the adenylate cyclase system. The immediate desensitization of thymocytes is reversible and occurs without apparent loss of functional receptors. Continuous presence of hormone is shown to be required not only for triggering the chain of events which leads to the readily reversible desensitization, but also for the process which transfers the cells to the subsequent, ‘locked’ desensitized state.     

Potentiation by steroids of the β-adrenergic agent-induced stimulation of cyclic AMP in isolated mouse thymocytes

There is an increasing amount of evidence suggesting that glucocorticoids may modulate the responsiveness of various cell types to β-adrenergic agents. In some systems, it has been shown, in addition, that steroids potentiate the elevation of cAMP induced by catecholamines. Little is known however of the mechanism underlying steroid action. We have studied this ‘permissive action’ in isolated thymocytes which have specific receptor sites for both glucocorticoids and β-adrenergic agents. The glucocorticoid compound dexamethasone did not alter intracellular cAMP level but markedly enhanced the stimulation produced by isoproterenol. This effect was instantaneous and was still measurable at 10^?7 M dexamethasone. A similar potentiating action was observed in the presence of corticosterone but also in the presence of sex steroids. Determination of β-receptors after cell preincubation in the presence of dexamethasone showed that rapid alterations in β-receptors are not involved in this permissive action. Experiments done in the presence of the calcium chelator, ethyleneglycol bis(β-aminoethyl ether)-N,N′-tetraacetic acid, suggest that dexamethasone action could be related to a modification of calcium mobilization.