The effect of donor age on human fibroblast beta-adrenergic receptor response and agonist-induced desensitization.

Aging has been associated with changes in beta-adrenergic receptor (beta-receptor) function in several tissues. The relative contribution of cellular aging and age-related changes in homeostatic regulation of receptor function is unknown. We have examined beta-receptor function in fibroblasts of young and old donors (young: mean age 31.2 +/- 0.8 years +/- S.E., n = 6; old: mean age 81.8 +/- 0.6 years +/- S.E., n = 6). Beta-receptor responses to isoproterenol (ISO), (1 microM) were similar in the two groups. The concentration of ISO required for 50% maximal beta-receptor-mediated cyclic AMP production (EC50) was similar in both groups. Fibroblast beta-receptor density was also similar in young and old groups. ISO-induced beta-receptor desensitization was both dose- and time-dependent. Submaximal desensitization by acute exposure (30 min) to ISO (1 mM) caused similar levels of beta-receptor desensitization in young (42.5 +/- 2.5%) and old (42.8 +/- 2.8%) groups and a similar increase in ISO EC50. These findings demonstrate that aging in vivo does not cause changes in fibroblast beta-receptor regulation that are retained in culture.     

Spontaneous apoptosis in human thymocytes.

Apoptosis seems to be involved in different stages of immune cell development. In particular, experimental evidence suggests that it is a major form of cell death in the thymus. The present analysis of human thymocytes reveals that a fraction of these cells, cultured in vitro, undergoes spontaneous apoptosis. This observation is based both on molecular (DNA fragmentation) and morphological (electron microscopic) investigations of the cells. The apoptotic thymocytes are CD3- or CD3lo, CD4lo, and CD8lo and do not express Bcl-2 protein. Furthermore, thymocytes die by apoptosis when exposed to pharmacological stimuli, such as tumor necrosis factor-alpha, dexamethasone, ATP, or Ca++ ionophore. Thus the apoptotic machinery in thymocytes can be triggered by an imbalance in growth factors in the in vitro culture media and can be modulated by various biochemical signals. The process of spontaneous apoptosis is independent of mRNA or protein synthesis, as actinomycin D and cycloheximide fail to inhibit this phenomenon. Furthermore, apoptosis seems to require active oxidative phosphorylation, as it is prevented by incubation of the cells with inhibitors of the respiratory chain.     

Measuring emigration of human thymocytes by T-cell receptor excision circles

Recent advances in characterizing thymic function confirm the importance of thymus to T-cell diversity in the periphery of both children and adults during both health and disease. Lack of a marker to identify human recent thymic emigrants (RTEs) is the biggest hurdle to accurately characterizing and quantifying thymic output. T-cell receptor excision circles (TRECs) are used as an assay to measure RTE levels. Controversy exists, however, as to whether TREC concentrations reflect the number of RTEs or are mainly altered by peripheral T-cell division and death. In this review, we first summarize recent data on the human thymus and RTEs. On the basis of both experimental and mathematical analyses, we characterize factors that influence TREC dynamics in the periphery and elucidate primary elements that induce a decline in TREC concentrations during normal aging and HIV-1 infection. Our findings suggest that T-cell dynamics are key to the accuracy of TREC concentrations as a useful measurement of human RTEs.     

Regulation of IL-2 beta receptor expression and beta-chain mRNA by human thymocytes.

The high affinity form of the human IL-2 receptor (IL-2R) has two known components, the IL-2R alpha (p55) and the IL-2R beta chain (p75). We have previously shown that recombinant IL-2 (rIL-2) could induce the expression of the alpha-chain (p55) on T cells and thymocytes, and increase this expression following suboptimal activation with concanavalin A (Con A) in combination with IL-2. An increase in the accumulation of IL-2R alpha-specific mRNA induced by rIL-2 in T cells and thymocytes had also been documented. We report here that the expression of IL-2R beta on the cell surface can be demonstrated on human thymocytes by the binding of Mik beta1, a MoAb directed against an epitope of the beta-chain. The IL-2R beta chain is constitutively expressed on freshly isolated thymocytes; this expression can be increased in thymocytes activated with Con A in combination with IL-2 or tetradecanoylphorbol 13-acetate (TPA). Blocking the formation of high affinity receptors with a MoAb directed against the alpha-chain of the receptor results in an increase in the display of IL-2R beta as evidenced by binding of MoAb Mik beta1. The accumulation of IL-2R-beta-specific mRNA is observed in freshly isolated thymocytes and it is increased in thymocytes cultured with rIL-2 alone, with Con A, and further enhanced by the addition of rIL-2 in combination with Con A or with TPA. Cyclosporine (CsA), which inhibits the accumulation of lymphokine-specific mRNA of thymocytes, does not inhibit the induction of the accumulation of IL-2R beta-specific mRNA. This is analogous to its effect on the expression of the alpha-chain (p55), and the accumulation of alpha-chain-specific mRNA.     

Effect of thymosin on glucocorticoid receptor activity and glucocorticoid sensitivity of human thymocytes

Incubation with thymosin fraction 5, (TMS F5 at 300 micrograms/ml) a partially purified thymic factor, reduced the steroid binding activity of human infant thymocytes from 9.6 +/- 2.1 fmole/ml to 5.0 +/- 2.0 fmole/ml. The glucocorticoid receptor activity in normal infant thymocytes was found to be 2,146 +/- 726 (s.d.) sites per cell with dissociation constant of 1.4 +/- 0.6 X 10(-8)M. TMS F5 also increased the resistance of human thymocytes to the cytolytic effect of dexamethasone (2.5 X 10(-8)M) to 168.6 +/- 30.2% of control (P less than 0.01). In animals, medullary and peripheral blood T cells are more resistant to glucocorticoids than immature thymic T cells. The results show that thymosin can induce changes consistent with differentiation in human thymocytes. These in vitro results are consistent with a physiological role of thymosin in intrathymic T cell maturation in man. Incubation of a human malignant thymus derived T cell line (MOLT 3) with TMS F5 also resulted in a significant reduction of the number of steroid binding sites to 44.2 +/- 15.3% of control (P less than 0.05), but TMS F5 did not significantly reduce the glucocorticoid sensitivity of MOLT 3 cells.     

HLA-DR expression in human fetal thymocytes.

We analyzed the expression of MHC class I (W6/32) and class II (HLA-DR) antigens on human fetal and postnatal thymocytes by fluorescence-activated cell sorting. Less than 5% of prenatal thymocytes expressed HLA-DR before week 12 of gestation. However, the number of HLA-DR-positive cells significantly increased during the late second and third trimester of gestation, when greater than 50% of prenatal thymocytes expressed HLA-DR. Such high-level expressions of HLA-DR in fetal thymocytes were also demonstrated by Northern-blot analysis and immunohistochemistry. After birth, the percentage of HLA-DR-positive cells in thymocytes decreased gradually. A high-level expression of class I antigen was also observed in thymocytes from the early stages of gestation, but, in contrast to MHC class II, a majority of postnatal thymocytes maintained high levels of class I antigen after birth.     

Distribution of beta-adrenergic receptors on human lymphocyte subpopulations.

A technique is described allowing the quantification and the characterization of specific beta-adrenergic receptors in intact living human lymphocytes. 125I-Iodohydroxybenzylpindolol, a potent beta-adrenergic antagonist was used to label specific binding sites on unfractionated lymphoid cells and on purified subpopulations of T (F1 and F2) and B cells. F1 and F2 were obtained by filtration through nylon wool column as previously described (Delespesse et al., 1976), they differ in their response to mitogens, and in their interactions with adherent cells and B cells. 125I-HYP binding to unfractionated lymphocytes was a saturable, stereospecific and rapid process with a dissociation constant of 2.5 10(-10) M and a binding capacity of 400--600 sites/cell. Bindings on unfractionated lymphocytes, purified B cells and T cells of the F2 fraction were similar. No detectable binding was noted on T cells from the F1 fraction. Enriched T cells obtained by a rosetting technique displayed 200 receptors/cell.     

The effect of in vitro aging on human lung fibroblast beta-adrenergic receptor density, coupling and response.

Age-related changes in regulation of receptor response have been observed in several tissues and include regulation of beta-adrenergic receptor (beta-receptor) responses. The role of cellular aging in age-related changes in receptor response is not clear. We have examined the effect of aging in vitro on human fibroblast beta-receptor function. MRC-5 (embryonic lung) fibroblasts were aged by replication to produce cells of early, middle and late stages corresponding to the following cumulative population doublings: 15-20, 35-45 and greater than 50, respectively. Fibroblast membrane beta-receptor responses to isoproterenol (ISO, 0.1 mM) did not differ between the three stages. Adenylate cyclase responses to prostaglandin E1 (PGE1, 1 microM), guanosine triphosphate (GTP, 0.1 mM) and 5'-guanylimidodiphosphate (Gpp(NH)p, 0.1 mM) were also similar between the stages. Beta-receptor density (Bmax) was unaffected by in vitro aging. Beta-receptor agonist affinity, an indication of the capacity for beta-receptor coupling to the nucleotide binding protein (NS), was also unaffected by cell aging. These findings suggest that cellular aging in fibroblasts alone is not accompanied by changes in beta-receptor function.     

Changes in adipocyte beta-adrenergic receptor of cold-acclimated rats.

The changes in the number and affinity of binding sites in the beta-adrenergic receptors of rat white adipocytes after cold exposure were studied with the aid of (p)-[3H]dihydroalprenolol. One day cold exposure did not change the number and affinity of binding sites in beta-adrenergic receptors. Chronic exposure of rats to cold (5 degrees C) for 1 and 4 weeks significantly decreased the affinity of beta-adrenergic receptors without any alteration in the number of binding sites. Such changes in the binding affinity observed in cold-acclimated rats (4 weeks, 5 degrees C) remained for 18 hr after these animals were transferred to a warm environment of 25 degrees C. The decreased affinity of binding sites in beta-adrenergic receptor induced by cold acclimation could not explain the enhanced metabolic response of cold-acclimated animals to noradrenaline.     

Populations of lymphocytes separated from human thymocytes.

Thymocytes from twenty-two human foetal and post-natal thymuses were separated according to their buoyant density. Thymocytes from eight were separated into multiple fractions by means of continuous gradients of bovine serum albumin (BSA), pH 5.1 and iso-osmolar with human cells, and thymocytes from fourteen were separated into two fractions of density less than and greater than 1.068 g/cm3. Fractions were tested for antigen-binding lymphocytes (ABL) to 125I-labelled human thyroglobulin, for response to phytohaemagglutinin (PHA) and for rosette-forming cells (RFC) using sheep red blood cells. For subjects of all ages there was a pronounced enrichment of both ABL and thymocytes responsive to PHA amoung low or 1.064-1.065 g/cm3 density thymocytes. In older subjects there was a second enrichment of ABL among high or 1.072-1.073 g/cm3 density thymocytes. RFC were distributed over a wider range of densities, and although they did not form a discrete subpopulation they predominated among high density thymocytes.     

The human promyelocytic leukemia cell (HL-60 cell) beta-adrenergic receptor

We have characterized the beta-adrenergic receptor binding site and the beta-adrenergic cAMP response of the HL-60 cell. The hydrophilic ligand [3H]-(-)-CGP-12177 was specifically and reversibly bound to one single class of binding sites (Kd 220 pM and Bmax 1,970 sites/cell). The adrenergic agonists inhibited the specific radioligand binding. The order of potency was isoproterenol greater than epinephrine greater than norepinephrine. The beta-2 selective antagonist ICI 118551 had a binding affinity 3 orders of potency higher than the beta-1 selective antagonist, atenolol. The adrenergic agonists elevated the cAMP accumulation in a concentration-dependent mode. The order of potency was isoproterenol greater than epinephrine greater than norepinephrine. Both the binding and the functional studies revealed stereospecificity and reversibility. The present data show that HL-60 cells possess beta-2 adrenergic receptors functionally coupled to adenylate cyclase.     

Effect of a human serum thymic factor on hydrocortisone-treated thymocytes.

A human serum thymic factors (SF) stimulated cyclic adenosine-3' ,5'-monophosphate (cAMP) synthesis in normal mouse thymocytes. Such stimulation was no longer observed when thymocytes were depleted of hydrocortisone (HC)-sensitive cells. It was concluded that SF selectively stimulate cAMP in HC-sensitive cells. Furthermore, incubation of thymocytes with SF enlarged the population of HC-resistant thymocytes. These results suggest that SF might act on HC-sensitive thymocytes increasing their cellular cAMP level and inducing their transformation in HC-resistant cells.     

Expression of interleukin 2 receptor on murine fetal thymocytes

Rat monoclonal antibodies AMT-13, 3C7 and 7D4 which react to the mouse interleukin 2 (IL 2) receptors were used to define cell populations with the putative IL 2 receptors in the mouse thymus as a part of series of investigations to elucidate the mechanism of intrathymic cell proliferation and differentiation. With freshly dissociated cells from the thymus of 15 gestational days, the anti-IL 2 receptor monoclonal antibodies (anti-IL 2 receptor) reacted with about half of them. The proportion of the reactive cells decreased rapidly thereafter till birth as the numbers of thymus cells expanded. The antibodies reacted with only two to three percent of thymic cells from newborn mice and less than one percent of cells from adult thymus. Through the gestational period, the fetal liver cells did not react with the antibodies. When the thymus cells at early gestational days were subjected to a two-color analysis, one for the anti-IL 2 receptor and the other for anti-Thy-1 or anti-asialo GM1, by a fluorescence-activated cell sorter, it was found that the majority (up to 80%) of anti-IL 2 receptor-reactive cells (IL 2 receptor-positive cells) was also reactive with anti-Thy-1. Some of the IL 2 receptor-positive cells but Thy-1-negative cells reacted with anti-asialo GM1, a marker of the immature thymocytes. Immunohistochemically, the IL 2 receptor-positive cells were found mainly in the subcapsular area and in the border region of cortex and medulla. Collectively, these results suggest that the pre-T lymphocytes are stimulated immediately after their arrival to the thymus from the stem cell source such as fetal liver and bone marrow and are driven into the proliferation via the IL 2 receptor system.     

Regulation of histamine receptor concentration on human PBMC by homologous hormone

Human peripheral blood mononuclear cells (PBMC) preincubated with 10(-3)M histamine at 37 degrees C, washed and incubated with 3H-histamine showed a reduction in the binding of the labelled hormone as compared to cells which had not been pretreated with histamine. Using competitive binding assays it was further shown that the number of specific binding sites for histamine was reduced by 46-62%. However, the affinity of receptors as indicated by Kd values remained unaltered. It would thus appear that elevated levels of histamine lead to down-regulation of histamine receptors on human PBMC with a quantitative reduction in the number of binding sites without an alteration in their affinity characteristics. In view of the fact that histamine receptors have been shown to be present on suppressor lymphocytes, the down-regulation of these receptor sites may have relevance in physiological and disease states where perturbations in the levels of histamine are observed.     

T3 expression by human thymocytes in culture.

By panning procedures employing T6 and T3 monoclonal antibody, human thymocytes were fractionated into two subpopulations depleted of T6- or T3-positive (T6+, T3+) cells. Unfractionated thymocytes and T6- and T3-depleted subpopulations were separately cultured for 48 h in RPMI 1640 medium with 10% FCS or in HB 101 serum-free medium. Determining the phenotype of unfractionated thymocytes at various time intervals, a time-dependent increase of T3+ cells was observed. An inverse relationship was found between the percentage of T3+ cells and the T6 and peanut agglutinin (PNA) reactive thymocytes. When the surface antigen expression in the T3-depleted population (greater than 95% T6+ and PNA+ cells) was analysed, a strong increase of T3+ cells and a complementary reduction of T6+ and PNA+ cells was evidenced. During that time the surface phenotype of the T6-depleted population (greater than 80% T3+ cells) showed the same trend of differentiation, as the other thymocyte preparations. These results indicate that a conspicuous fraction of human thymocytes and particularly of those characterized by a cortical phenotype (PNA+ and T6+ cells), are able to express mature T-cell antigens when cultured in vitro in the absence of the thymic microenvironment influence. However, the in vitro acquisition of a mature phenotype is not accompanied by a parallel achievement of the capacity to respond to mitogens such as PHA or T3 monoclonal antibody.     

Generation of functional thymocytes in the human adult.

Reconstituting the immune response will be critical for the survival of HIV-infected individuals once viral load is brought under control. While the adult thymus was previously thought to be relatively inactive, new data suggest it may play a role in T cell reconstitution. We examined thymopoiesis in adults up to 56 years of age and found active T cell receptor (TCR) rearrangement, generating a diverse TCR Vbeta repertoire. The resulting thymocytes are functional and are capable of responding to costimulatory signals. These data demonstrate that the adult thymus remains active late in life and contributes functional T cells to the peripheral lymphoid pool.