Quiescent lymphocytes express intracellular transferrin receptors

Both quiescent and concanavalin A stimulated murine splenic lymphocytes were examined for the expression of surface and intracellular binding sites for the serum glycoprotein transferrin. Transferrin binding activity was observed on the surface of mitogen stimulated cells only. When soluble detergent extracts of both populations were studied, quiescent lymphocytes were shown to contain a significant pool of non-surface exposed, intracellular receptors which was approximately 20% of the total receptor complement of proliferating cells. Because the ratio of surface to intracellular binding sites was dramatically increased following mitogen stimulation, the regulation of transferrin receptor expression during this process may involve a substantial alteration in its subcellular distribution in addition to the well documented increase in number of binding sites.     

Expression and phosphorylation of transferrin receptors in mitogen-activated peripheral blood lymphocytes

Expression of transferrin receptors of cultured human lymphocytes has been investigated by using monoclonal antibody (5E9) specific for human transferrin receptors. When isolated lymphocytes were cultured in a medium containing fetal calf serum, the biosynthesis of transferrin receptor was barely detectable. The addition of concanavalin A or human serum to the medium caused a slight stimulation of the biosynthesis. The addition of concanavalin A and human serum in combination caused the highest biosynthetic activity. Appearance of the receptor on the cell surface increased in parallel with the degree of the synthesis. Treatment of concanavalin A- and human serum-treated cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in a marked stimulation of the phosphorylation of the receptor. Enhancement of phosphorylation occurred within 20 min after the addition of TPA. The density of the receptor on the cell surface slightly increased upon TPA treatment of cells, and the treatment was without effect on iron incorporation from transferrin into the cells. The density of newly synthesized receptor in TPA-treated cells was similar to that in non-treated cells. These results indicated that TPA treatment of mitogen-activated human lymphocytes stimulated the phosphorylation of transferrin receptors, but TPA had no effect on the expression of the receptors thereafter.     

Interleukin-13 overexpression by tax transactivation: a potential autocrine stimulus in human T-cell leukemia virus-infected lymphocytes

The human T-cell leukemia virus type 1 (HTLV-1) Tax oncoprotein induces growth transformation and is critical for the pathogenesis of the HTLV-1-induced adult T-cell leukemia (ATL). It stimulates the cell cycle and transactivates cellular genes. Here we show that the expression of interleukin-13 (IL-13) is upregulated as a consequence of Tax in HTLV-1-transformed T cells and ATL-derived cultures. IL-13 exerts proliferative and antiapoptotic functions and is linked to leukemogenesis, since it stimulates Hodgkin lymphoma cells by an autocrine mechanism. Overexpression of IL-13 RNA and protein was confirmed in HTLV-1-positive and Tax-transformed cells. Induction of endogenous IL-13 levels in tax-transfected Jurkat cells and in conditional Tax-expressing transformed T lymphocytes suggested that Tax can replace signals required for IL-13 synthesis. For functional analysis, the IL-13 promoter and deletion variants were cloned into luciferase reporter plasmids. Experiments with transfected human T lymphocytes revealed a 16-fold stimulation of the IL-13 promoter by Tax. Experiments with Tax mutants indicated that none of the classical transactivation pathways (SRF, CREB, and NF-kappaB) is sufficient for the transactivation; at least two different Tax functions are required for full transactivation. The IL-13 promoter is stimulated via two elements; one is a NF-AT binding P element, and the other is a putative AP-1 site. The following observations suggest that IL-13 may stimulate HTLV-1-transformed cells by an autocrine mechanism: (i) the HTLV-1-transformed cells express the IL-13 receptor on their surface, and (ii) STAT6, a downstream effector of IL-13 signaling, is constitutively activated. Thus, in summary, Tax, by transactivating the promoter, induces IL-13 overexpression that possibly leads to an autocrine stimulation of HTLV-1-infected cells.     

Interleukin-1 and interleukin-6 synergize in preparing resting murine B cells to respond to anti-mu: correlation with c-myc expression.

We demonstrate that stimulation with interleukin (IL)-1 and IL-6 prepares high-density B cells to enter the S phase more promptly in response to subsequent stimulation with anti-mu F(ab')2. The stimulatory effect of IL-1 and IL-6 is compared to the one described for IL-4. In contrast to IL-4, preculture in IL-1 and IL-6 does not induce an increase in cell volume or in expression of class II major histocompatibility complex antigens on resting B cells. Similarly, the expression of the p55 subunit of the IL-2 receptor and of the transferrin receptor was not detected on resting B cells stimulated with IL-1 and IL-6. However, the stimulatory effect of IL-1 and IL-6 is correlated with an increased expression of c-myc proto-oncogene mRNA in resting murine B cells.     

Effects of tumor necrosis factor on cell growth and expression of transferrin receptors in human fibroblasts

Human recombinant tumor necrosis factor (TNF) stimulated the growth of confluent human fibroblasts (FS-4) in the presence of fetal calf serum. Epidermal growth factor (EGF) similarly stimulated cellular growth; however other mitogenic factors such as insulin, fibroblast growth factor, 12-O-tetradecanoyl-phorbol-12-acetate and Ca2+ ionophore A23187 did not. The growth-stimulating action of TNF was not synergistic with the activity of EGF in the presence of serum. TNF induced a rapid increase in the binding of transferrin to the cell surface, followed by a return to the basal level within 5 min. A similar increase in transferrin binding was observed in FS-4 cells exposed to EGF. In contrast, insulin caused a prolonged stimulation of transferrin binding. These results suggest that TNF and EGF generate similar or identical intracellular signals for cellular growth and the regulation of transferrin receptor expression.     

Comparison of phorbol ester/calcium ionophore and phytohemagglutinin-induced signaling in human T lymphocytes. Demonstration of interleukin 2-independent transferrin receptor gene expression

The proliferation of human T lymphocytes is regulated, in part, by the coordinated expression of genes encoding T cell growth factor (interleukin 2 (IL-2), IL-2 receptors, and transferrin receptors (TFR). We examined the time course of accumulation of mRNA for these genes in T cells stimulated with the phorbol ester, phorbol 12,13-dibutyrate (PDB) and the calcium ionophore, ionomycin, and compared their expression to T cells stimulated with phytohemagglutinin. In cells treated with PDB/ionomycin, maximum expression was observed at 3 hr for IL-2 mRNA and at 6 hr for TFR mRNA, whereas the level of IL-2 receptor mRNA reached a peak 24 to 48 hr after stimulation. In phytohemagglutinin-stimulated T cells IL-2 mRNA was detectable within 3 hr but peaked later at 12 hr; the level of IL-2 receptor mRNA similarly peaked 24 to 48 hr later. Accumulation of TFR mRNA in phytohemagglutinin-stimulated T cells, however, was not detectable at 6 hr and reached a peak only between 12 to 24 hr. The early accumulation of TFR mRNA in PDB/ionomycin-stimulated T cells seemed, in part, independent of the interaction of IL-2 with its own receptor, because TFR mRNA was detectable as early as 1 hr after stimulation and addition of cycloheximide before addition of PDB/ionomycin did not abolish the PDB/ionomycin-induced accumulation of TFR mRNA. In addition, either PDB or ionomycin used alone induced the expression of TFR mRNA but not IL-2 mRNA. These results indicated that the combination of PDB/ionomycin accelerated the expression of IL-2 and TFR genes in T cells compared to phytohemagglutinin and triggered an IL-2-independent pathway for the induction of TFR mRNA.     

Distinct classes of human T-cell activation antigens

The characterization of three groups of antigens expressed by activated human T lymphocytes and detected by monoclonal antibodies is reported. Antigens defined by OKT19, OKT21, and OKT22 do not appear on in vitro activated T cells until increases in DNA synthesis become apparent and are not detected on most Interleukin 2 (IL-2)-independent cell lines and normal peripheral blood lymphocytes, monocytes, and granulocytes. Cell surface molecules reactive with the monoclonal antibodies OKT23 and OKT24 are displayed prior to any notable increase in DNA synthesis and are present on IL-2 independent cell lines, irrespective of lineage. T23 and T24 do not appear on peripheral blood cells and their distribution more closely resembles that of the T9 antigen (the receptor for transferrin) than antigens of the other groups. The third group of antigens, T14 and T20, have been classified as "early" antigens relative to DNA synthesis. They are expressed by distinct populations of normal lymphoid cells as well as by some IL-2-independent cell lines. Display of each group of activation antigens on T lymphocytes can be induced by either phytohemagglutinin, purified protein derivative from tuberculin, or allogeneic non-T cells, is not restricted to the OKT4+ or OKT8+ subsets, and is predominant on cells exhibiting the light-scattering properties of blast cells. The relative lack of expression of these antigens among normal peripheral blood cells make them attractive candidates for identifying changes in the status of immune activation.     

The regulation by low-density lipoproteins of the activation of oxidative enzyme-primed lymphocytes is governed by transferrin

The activation of T lymphocytes was regulated in vitro by low-density lipoproteins (LDL). Not all prereplicative events induced by the oxidative enzymatic mitogens neuraminidase and galactose oxidase (NAGO) were susceptible to inhibition by LDL. The accessory cell-independent early blastogenic response was not suppressed. LDL suppressed accessory cell-dependent responses, and the extent of LDL suppression, depended on the concentration of transferrin. A gradient of transferrin determined the point in the cell cycle at which NAGO-primed lymphocytes were suppressed by LDL. When transferrin was low (0-10 micrograms/ml) and in serum-free medium (SFM), LDL suppressed the expression of cell surface receptors for interleukin-2 (IL-2R) and transferrin (TfR), the late blastogenic response prior to DNA replication (72 hr), and DNA replication. At higher levels of transferrin, about 100 micrograms/ml, the LDL-suppressed cells were IL-2R+, TfR+ and responsive to IL-2, but did not enter S phase. LDL suppression could be ablated by IL-2 and by high levels of transferrin (250-1000 micrograms/ml). In RPMI medium containing serum (FBS), the pattern of LDL suppression was different from that in SFM: fully activated IL-2R+, TfR+ lymphocytes were unresponsive to exogenous IL-2, suggesting that they were blocked at the G1/S boundary. This block was also relieved by transferrin (greater than 100 micrograms/ml). The data suggest that the interplay between transferrin and LDL is a critical factor in the NAGO-induced stimulation of T lymphocytes. LDL and transferrin exert negative and positive control of lymphocyte activation, respectively. In SFM, LDL appear to alter transferrin utilization by accessory cells; in RPMI-FBS, by fully activated T lymphocytes.     

Induction of activation antigens on human natural killer cells mediated through the Fc-gamma receptor

NK cells, defined here as lymphocytes bearing the CD16 Ag found on the NK cell Fc-gamma receptor (FcR), are known to enter a proliferative and activated state in response to stimulation with IL-2 as assessed by clonal expansion, short-term DNA synthesis, and de novo expression of lymphocyte-associated activation Ag. We have found that the FcR of NK cells acts as a signaling pathway through which IL-2-dependent activation may be greatly enhanced, allowing for more rapid induction of activation Ag and recruitment of an increased percentage of cells expressing surface markers of cellular activation. FcR-interactive agents, such as solid phase immobilized immune complexes or cross-linked CD16-specific mAb, work synergistically with rIL-2 to elicit a rapid expression of IL2R and transferrin receptors on greater than 50% of purified NK cells as early as day 3 of culture. IL-2 or FcR-interactive stimuli alone were weak or ineffective stimulators by comparison. In contrast to the induction of de novo activation Ag, DNA synthesis was elicited by IL-2 alone, but was not substantially or consistently enhanced by the subsequent addition of FcR-interactive stimuli.     

Lymphocytes stimulated by allogeneic B cell lines cleave the third component of complement and fix C3 fragments. Their nonspecific lytic capacity is elevated against complement receptor type 2-carrying targets

Human blood lymphocytes stimulated in mixed cultures by allogeneic B cell lines were shown to cleave C3 molecules. The B cell lines were derived from Burkitt lymphoma patients: 1) established from their EBV-negative lymphoma, 2) the EBV-positive sublines converted in vitro, and 3) lymphoblastoid cell lines (LCL) i.e., B lymphocytes transformed in vitro by EBV. These cell lines differed considerably in their capacity to stimulate allogeneic lymphocytes. The split products of C3 were detected in the supernatants and on the surface of the activated lymphocytes at levels which correlated with the strength of stimulation. Lymphocytes cultured with LCL had the highest levels of thymidine incorporation blast transformation, C3 cleavage, and C3 fragment fixation. Lymphocytes exposed to the EBV-negative Burkitt lymphomas were stimulated weakly and their C3-activating capacity was low. Irrespective of the efficiency of lymphocyte stimulation induced in the cultures, 60 to 70% of the blasts were found to fix C3 fragments. The majority of the lymphocytes which fixed C3 fragments were T blasts that carried the CD3 marker and expressed IL-2R (CD25). CD4 and CD8 cells were represented with equal frequency in the C3-fragment fixing and C3-fragment negative populations. Pre-exposure of the MLC-activated lymphocytes to human serum increased their cytotoxic capacity toward CR type 2-carrying targets. The enhanced lysis was abrogated by F(ab)2 rabbit anti-human C3d or rabbit anti-CR type 2 antibodies. The results suggest that the C3 fragments fixed on the lymphocytes bind to CR on the targets and elevate the avidity of binding between the two interacting cells. This was also indicated by an increase in the frequency of lymphocyte-target conjugates.     

Cutaneous lymphocyte antigen expression on activated lymphocytes and its association with IL-12R (beta1 and beta2), IL-2Ralpha, and CXCR3

The majority of T lymphocytes that infiltrate psoriatic lesions express cutaneous lymphocyte antigen (CLA), a skin homing receptor involved in the influx of memory T cells to cutaneous sites. We investigated CLA expression on normal human peripheral blood mononuclear cells (PBMCs) and evaluated its association with IL-12 receptors, chemokine receptor, CXCR3, and IL-2Ralpha. PBMCs were stimulated in vitro with or without polyclonal activators (mitogen, or superantigens, or anti-CD3+anti-CD28) in the presence or absence of exogenous rhIL-12. The percentage of CLA+ T lymphocytes increased significantly after superantigen stimulation compared to anti-CD3+anti-CD28 or mitogen activation. The majority of activation induced CLA+ T lymphocytes co-expressed IL-12Rbeta1, IL-12Rbeta2, CXCR3, and CD25 in the presence of rhIL-12. Our results indicate that CLA expression on activated T lymphocytes is IL-12 and activation dependent and correlates with the expression of IL-12 receptors, IL-2Ralpha, and CXCR3. Monitoring the levels of Th1 differentiation markers such as CXCR3 and IL-12Rbeta2 along with activation marker, CD25 on skin homing CLA+ T lymphocytes may provide insight into the mechanism of action of immunotherapies directed against Th1 type skin inflammatory diseases.     

Disparity between expression of transferrin receptor ligand binding and non-ligand binding domains on human lymphocytes

We compared transferrin receptor (TfR) expression on human peripheral blood lymphocytes (PBL) activated by phorbol myristate acetate (PMA) or L-phytohemagglutinin (LPHA) using two techniques: (1) 125I-iron-saturated transferrin (FeTf) binding, (2) reactivity with monoclonal anti-TfR antibodies--OKT9 and B3/25. These monoclonal antibodies do not block FeTf binding, and therefore bind to TfR domains separate from the ligand binding site. Unstimulated PBL bound fewer than 1,000 molecules of 125I-FeTf per cell, and less than 5% of cells expressed TfR antigens detected by OKT9 or B3/25. 125I-FeTf binding and antibody binding increased in parallel on LPHA-activated PBL. After exposure to LPHA for 72 hr, 125I-FeTf binding increased 100-fold to 10(5) molecules per cell and greater than 50% of cells expressed TfR antigens. By contrast, PMA activation of PBL markedly increased binding of OKT9 and B3/25 but not the binding of 125I-FeTf. Cell surface expression of TfR antigens seen by OKT9 and B3/25 did not differ between LPHA- and PMA-activated PBL. However, after 72 hr with PMA, 125I-FeTf binding increased only 6-fold and consistently remained at less than 10(4) molecules per cell. Therefore, PMA induced a disparity between expression of TfR ligand binding domains and immunological domains at the cell surface. Cell proliferation assessed by fluorescent DNA analysis was similar in cultures stimulated by LPHA or PMA. These data indicate that lymphoid cells may possess a mechanism for modulating TfR expression in which down-regulation of FeTf binding occurs without receptor internalization. Alternatively, it is possible that this observation may reflect a membrane perturbation effect of PMA.     

Inhibition of transferrin receptor expression by interferon-alpha in human lymphoblastoid cells and mitogen-induced lymphocytes

125I-Transferrin binding to lymphoblastoid K562 and Daudi cells markedly increased after exposure of the cells to culture conditions that stimulated proliferation. Treatment of these cells with interferon-alpha (IFN-alpha) resulted in concurrent inhibition of cell growth and of the rise in transferrin binding. Scatchard analyses revealed that IFN reduced the number of transferrin receptors without altering the binding constant. When 125I-transferrin binding was measured using permeabilized cells, the IFN-induced reduction of binding was comparable to that observed with intact cells, indicating that IFN diminished the total number of cellular transferrin receptors. We also found that addition of IFN-alpha to phytohemagglutinin-stimulated human lymphocytes inhibited the mitogen-induced enhancement of [3H]thymidine incorporation as well as surface binding of 125I-transferrin. Our findings suggest that the decrease in transferrin receptor expression on IFN-alpha-treated cells may be one of the mechanisms responsible for the antiproliferative action of IFN.     

Occupational levels of radiation exposure induce surface expression of interleukin-2 receptors in stimulated human peripheral blood lymphocytes

Interleukin-2 (IL-2) is a cytokine responsible for a variety of immune and non-immune stimulatory and regulatory functions, including the activation and stimulation of cytotoxic cells able to recognize and kill human tumour cells and T-cell proliferation and differentiation. We show that low doses of radiation, in the range commonly received by atomic radiation workers or as a result of minor medical diagnostic procedures (0.25 to 10 mGy), stimulate the expression of IL-2 receptors (IL-2R) on the surface of peripheral blood lymphocytes (PBL) taken from normal human donors. This stimulated surface expression after in vitro irradiation is an indirect effect, resulting from the secretion into the medium of a soluble factor from the irradiated cells. This factor can also stimulate IL-2R surface expression in unirradiated cells. Consequently, radiation stimulation of IL-2R expression in a large population of PBL shows a triggered-type response rather than being proportional to dose. These results demonstrate that normal human cells can respond to doses of radiation in the range of common occupational or medical exposures. The data also demonstrate a possible defence mechanism against environmental stress by which a radiation-exposed cell can use an indirect signalling mechanism to communicate with and influence the biological processes in an unexposed cell.     

Suppression of T lymphocyte function by measles virus is due to cell cycle arrest in G1

Measles virus suppresses T lymphocyte functions in vitro. When measles virus-infected T lymphocytes are stimulated with PHA or 12-O-tetradecanoylphorbol-13-acetate, plus calcium ionophore, the cells secrete IL-2 and express the IL-2R or Tac Ag to a similar extent as uninfected cells, yet proliferation is reduced by 50 to 90%. Stimulated infected T cells also express the cell surface activation Ag 4F2, transferrin R, and HLA-DR. The secretion of IFN-gamma by infected T cells in response to PHA is not suppressed at 24 to 72 h after stimulation. Total RNA synthesis at 48 and 72 h after stimulation is reduced in infected T lymphocytes. Infectious measles virus progeny are produced during this interval. Thus infected T lymphocytes can become activated in response to mitogenic stimuli and the cells support efficient viral replication before the block in cell proliferation.     

Phytohemagglutinin induced proliferation by aged lymphocytes: reduced expression of high affinity interleukin-2 receptors and interleukin-2 secretion

Human lymphocytes from elderly and young donors were cultured with phytohemagglutinin. Cultures from two groups of aged donors, recruited respectively from our ambulatory clinic and a nursing home, incorporated less tritiated thymidine (3H-TdR) and secreted less interleukin-2 than did young donors. Furthermore, as determined for the first time by a radioligand binding receptor assay, the aged lymphoblasts possessed significantly fewer high affinity IL-2 receptors per cell. Despite a decrease in the number of high affinity receptor cells the dissociation constant (Kd) was comparable for the three groups. It was also shown that the amounts of soluble IL-2 receptors that were released into the supernatants by mitogen stimulated cells did not differ for the aged and young donors. These data suggest that defects in IL-2 production and high affinity IL-2 receptor generation may both be responsible for immune deficiency in the elderly.     

Differential expression of functional adrenocorticotropic hormone receptors by subpopulations of lymphocytes

In an effort to investigate the presence of adrenocorticotropic hormone (ACTH) receptors on rat lymphocytes, cells were separated by a panning procedure into T and B cell populations. By using the radiolabeled ACTH agonist, (125I-Tyr23) phenylalanine2-norleucine4-ACTH1-24, substantial numbers of ACTH binding sites were detected on T and B lymphocytes, but not on thymocytes. Scatchard analysis revealed two types of binding sites on each cell population, one with Kd1 = 0.088 +/- 0.025 nM and one with Kd2 = 4.2 +/- 0.6 nM; however, the absolute number of binding sites per cell was different. B lymphocytes expressed approximately three times the number of Kd1 binding sites per cell when compared with T lymphocytes. However, ACTH receptor expression by these cell populations was not static as suggested by the ability to induce receptor expression via mitogens. B or T cells and thymocytes stimulated with the mitogens LPS or Con A, respectively, substantially increased their number of Kd1 binding sites per cell (approximately three-fold). Even more dramatic increases in Kd1 receptor expression (approximately 100-fold) were observed when comparing "normal" and stimulated thymocytes. To demonstrate that these ACTH binding sites were in fact functional, cAMP levels were measured in lymphocytes 10 min after exposure to varying concentrations of ACTH. Dose-dependent increases in cAMP levels were observed, with significant stimulation occurring with as little as 0.1 nM ACTH added. Taken together, these studies demonstrate the presence of functional ACTH receptors on normal, rat T and B lymphocytes.     

Expression of interleukin 2 and the interleukin 2 receptor in aging rats

Lymphocytes of aged animals exhibit a marked decrease in proliferative capacity in response to mitogen stimulation when compared to those of younger animals. In humans and mice the decreased proliferation is due at least in part (i) to the inability of lymphocytes to synthesize sufficient interleukin 2 (IL-2) and (ii) to decreased expression of IL-2 receptors (IL-2R) on the surface of aged lymphocytes. We compared proliferative abilities, IL-2 production, and IL-2R expression in splenocyte cultures of 4- to 5- and 22- to 24-month-old Fischer 344 rats stimulated with either concanavalin A (Con A) or A23187 and phorbol myristate acetate (PMA). Proliferation was significantly decreased in aged lymphocytes (30-50%) with both treatment protocols. However, unlike mice and humans we observed no difference in IL-2 activity, IL-2 mRNA levels, or IL-2R cell surface expression of lymphocytes from young and aged rats stimulated with either Con A or A23187 and PMA. These results indicate that factors other than decreased expression of IL-2 and IL-2R are responsible for the diminished proliferative capacity of aged rat lymphocytes following mitogen stimulation.     

Insulin receptor expression in the Burkitt lymphoma cells Daudi and Raji

The specific binding of insulin to either intact or Triton-solubilized Daudi cells (a Burkitt lymphoma cell line) was reduced by over 95% compared to that to control IM-9 lymphocytes due to a decrease in receptor number without a change in affinity. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that 125I-labeled Daudi cells had reduced amounts (approximately 1/20th) of immunoprecipitable binding (alpha) subunit [mol wt (Mr), 130,000] of the receptor and a relatively abundant 210,000 Mr form not seen in IM-9 cells. The transmembranous (beta) subunit (Mr, 90,000) of the receptor, although not detected by 125I surface labeling, could be phosphorylated and, together with the 210,000 Mr form, exhibited the same 2-fold stimulation of phosphorylation by insulin as that in IM-9 cells. Northern blot hybridization revealed a decrease in Daudi cells of all four major species of insulin receptor mRNA. The Raji cell, another Burkitt lymphoma cell line, also exhibited reduced protein and genetic expression of the insulin receptor, indicating that reduced insulin receptor expression may be representative of other Burkitt lymphoma cell lines.