Direct activation of human resting T cells by IL 2: the role of an IL 2 receptor distinct from the Tac protein

High concentrations of interleukin 2 (IL 2) were shown to produce a delayed but pronounced proliferation of purified resting T cells in the apparent absence of other activation signals. Because these stimulatory effects of IL 2 occurred in the absence of detectable Tac+ cells, the possibility that IL 2 might be initially interacting with an IL 2 binding protein distinct from the Tac protein was studied. Chemical cross-linking studies with 125I-IL 2 revealed the presence of an IL 2 binding protein distinct from the Tac protein on the surface of these unstimulated T cells. This second IL 2 receptor has an estimated molecular size of 70,000 daltons, lacks reactivity with the anti-Tac antibody, and appears to be identical to the p70 protein recently proposed as a component of the high affinity IL 2 receptor. Scatchard analysis of IL 2 binding assays performed with the unactivated T cells revealed approximately 600 to 700 p70 sites per cell and an apparent Kd of 340 pM. These data indicate that the p70 protein present on resting T cells binds IL 2 with an intermediate affinity compared with the previously recognized high and low affinity forms of the receptor and may account for the high concentration of IL 2 needed to induce resting T cell proliferation. To investigate the early biologic consequences of IL 2 binding to the p70 protein, potential changes in the expression of genes involved in T cell activation were examined. Northern blotting revealed the rapid induction of c-myc, c-myb, and Tac mRNA after stimulation of resting T cells with a high concentration of IL 2. The anti-Tac antibody did not inhibit IL 2 induced expression of these genes, suggesting that the p70 protein rather than the Tac antigen or the high affinity IL 2 receptor complex mediated this signal. However, in contrast to these early activation events, the anti-Tac antibody significantly inhibited IL 2 induced T cell proliferation. This finding implicates the high affinity form of the IL 2 receptor in the proliferative response of the IL 2 activated T cells. Thus these data support a two step model for the induction of resting T cell proliferation by high doses of IL 2 involving the initial generation of an activation or "competence" signal through the p70 protein and a subsequent proliferation or "progression" signal through the high affinity form of the receptor.     

Presence of a p70 IL-2-binding peptide on leukemic cells from various hemopoietic lineages

Recent studies have shown that IL-2R are composed of at least two polypeptide chains of 55 kDa (Tac or alpha-chain) and 70 to 75 kDa (p70 or beta-chain). The association of both chains forms high affinity IL-2R, whereas each chain alone binds IL-2 with a low (alpha-chain) or intermediate (beta-chain) affinity. So far, the p70 peptide has been found, in the absence of the Tac peptide, on the surface of lymphoid cells of T, B, or NK lineage. In this study, we investigated whether leukemic cells of various hemopoietic lineages expressed the p70 IL-2-binding protein. We found that both fresh leukemic cells obtained from patients, and cells from established leukemic lines of T cells, B cell, and myeloid origin constitutively expressed a p70 IL-2-binding protein on their surface, as detected by affinity cross-linking of radioiodinated IL-2. IL-2 binding and cross-linking to these cells was completely inhibited in the presence of an excess unlabeled rIL-2, but not with an anti-Tac mAb. Binding experiments on pre-B and myeloid cell lines revealed intermediate affinity IL-2R, whereas both high and intermediate affinity IL-2R were detected in T leukemic cells. The intermediate affinity binding of 125I-rIL-2 to the leukemic cell lines MOLT4 and Reh6 was inhibited by the TU27 mAb, which recognized the p75 chain of IL-2R. Moreover, the TU27 mAb could stain the K562, KM3, and MOLT4 (weakly) cell lines by indirect immunofluorescence. A high dose of rIL-2 (400 U/ml) enhanced the proliferation of cells from one out of three patients with acute myeloblastic leukemia, but it did not induce differentiation of the cells in any of three cases. Thus the finding of p70 IL-2-binding molecules on immature lymphoid and nonlymphoid hemopoietic cells should disclose new biologic functions for IL-2.     

Interleukin 2 functions through novel interleukin 2 binding molecules in T cells

In this report, we examined whether novel interleukin 2 (IL-2) binding molecules (p70/75) are responsible for signal transduction and internalization of IL-2 in T cells by using a monoclonal antibody H-31 to Tac antigens. We found that H-31 inhibited the binding of IL-2 to Tac antigens but not novel IL-2 binding molecules. Scatchard plot analysis revealed that in the presence of H-31, intermediate affinity sites (Kd = 1 to 1.5 nM) were detectable and the number of them was similar to that of high affinity IL-2 receptor (IL-2R) (Kd = 10 to 15 pM) in the absence of H-31. Furthermore, the kinetics of endocytosis of IL-2 via p70/75 showed the same pattern as via high affinity IL-2R. Finally, high doses of IL-2 (100 to 10,000 U/ml) are required for the proliferation of T cells in the presence of H-31, whereas in the absence of H-31, physiologic doses of IL-2 (1 to 100 U/ml) induced the proliferation. These results taken together suggest that novel IL-2 binding molecules are related to signal transduction of IL-2 and that Tac antigens are essential for constructing of high affinity IL-2R, although Tac antigens may not be responsible for signal transduction.     

Structural analysis of high- versus low-affinity interleukin-2 receptors by means of selective expression of distinct receptor classes.

Activated T cells express at least two distinct affinity classes of interleukin-2 (IL-2) receptors. The number of low-affinity receptors per cell is normally 10-30 times greater than that of the high-affinity receptors, and the difference in the dissociation constant between the two classes of receptors is in the order of 1,000-fold. In this report normal human T cells are used in a cellular system in which the number of low-affinity receptors can be manipulated. It is demonstrated that a cell population could be achieved with such low levels of low-affinity IL-2 receptors that almost half of the surface pool of anti-IL-2 receptor antibody (anti-Tac) binding sites represented high-affinity receptors. By using this cellular system it was possible to show that anti-Tac recognizes both receptor classes with similar affinity and that IL-2 inhibits Tac binding to both receptor classes in a competitive fashion. Tac antigens were purified from surface 125I-labeled cells expressing high levels of high-affinity IL-2 receptors, but low levels of the low-affinity receptor class, and this preparation was compared with another pool of Tac antigens obtained from cells expressing the normal 10- to 20-fold excess of low-affinity IL-2 binding sites over high-affinity IL-2 receptors. Biochemical characterization by peptide mapping by limited proteolysis and two-dimensional gel analysis revealed that these distinct preparations of Tac antigens were indistinguishable.(ABSTRACT TRUNCATED AT 250 WORDS)     

Demonstration of high-affinity interleukin-2 receptors on B-chronic lymphocytic leukemia cells: functional and structural characterization

Functional and structural characteristics of interleukin 2 (IL-2) receptors on B-cell chronic lymphocytic leukemia (B-CLL) cells were analyzed by a proliferation assay, IL-2 binding assay and cross-linking study. In the 3H-thymidine incorporation assay, purified B-CLL cells from four out of sixteen cases, in which the percentage of Tac antigen (Tac Ag) positive cells in peripheral blood lymphocytes ranged from 0 to 48.8%, responded to IL-2 (100 U/ml) after both 3- and 6-day incubation. No relationship was found between the responsiveness to IL-2 and the percentage of Tac Ag positive cells. In the radiolabeled IL-2 binding assay, however, B-CLL cells from all seven cases examined, including three cases with mitogenic response to IL-2 and four cases without mitogenic response, were shown to have both high- and low-affinity receptors. The number of high- and low-affinity receptors per cell ranged from 29-186 and from 420 to 1,800, respectively. Furthermore, with the affinity cross-linking method p55 (Tac Ag) and p70/75 were found even in cases without mitogenic response in their B-CLL cells. In conclusion, the B-CLL cells so far examined possessed high-affinity IL-2 receptors consisting of p55 and p70/75; nevertheless, this was not sufficient to respond to the mitogenic signal of IL-2.     

Expression of functional IL 2 receptors by lipopolysaccharide and interferon-gamma stimulated human monocytes

Human peripheral blood monocytes were stimulated with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) alone or in combination. Stimulated but not resting monocytes displayed the Tac peptide of the interleukin 2 (IL 2) receptor within 24 hr as measured by immunofluorescence staining and [3H] Tac binding. The total number of anti-Tac binding sites on co-stimulated monocytes was 13,700. By using scatchard analysis with radiolabeled IL 2, the activated cells were shown to express low numbers (below 100 sites/cell) of high affinity binding sites with a KD of approximately 15 pM. LPS and IFN-gamma were additive in augmenting the number of IL 2 and anti-Tac binding sites. By using an ELISA assay specific for the soluble released form of the Tac peptide we identified 112 U/ml of IL 2 receptors in the supernatant of monocytes stimulated for 24 hr with IFN-gamma, 233 U/ml after stimulation with LPS, and 519 U/ml after the addition of both stimulating agents. Both the membrane form (55,000 daltons), as well as the soluble form (45,000 to 50,000 daltons) of the Tac, IL 2 receptor, peptide from monocytes were shown by immunoprecipitation and gel electrophoresis to be similar size to the comparable forms of these receptors derived from activated T cells. In addition, monocytes stimulated for 8 hr contained mRNA specifically hybridizing to a cDNA probe coding for the Tac peptide. Finally, activated monocytes responded to the addition of recombinant IL 2 by an increase in H2O2 production that was measured by using fluorescent indicator 2,7-dichlorofluorescein. This response as well as the observed induction of monocytic IL 2 receptors by LPS may point to a functional role for this receptor during monocyte/macrophage responses to microbial infections.     

Immunoregulatory activity of recombinant human tumor necrosis factor (TNF)-alpha: induction of TNF receptors on human T cells and TNF-alpha-mediated enhancement of T cell responses

The expression of specific tumor necrosis factor (TNF) membrane receptors and biological effects of recombinant TNF (rTNF)-alpha on normal human T lymphocytes were studied. Although resting T cells lacked specific binding capacity for rTNF-alpha, high affinity (Kd 70 pM) TNF receptors were de novo induced upon primary activation of T cells. Comparison of TNF receptor expression with that of high affinity interleukin 2 (IL-2) and interferon-gamma (IFN-gamma) receptors, respectively, revealed similarities to IL 2-receptor expression with respect to kinetics of induction. However, maximum expression of TNF receptors (approximately equal to 5000/cell at day 6) and subsequent decline occurred approximately 3 days after the peak of IL 2-receptor expression. In contrast, no change in the expression of IFN-gamma receptors (Kd 10 pM, 300 to 400 receptors/cell) was found in the course of T cell activation. On activated TNF receptor positive T cells, TNF-alpha exerted multiple stimulatory activities. Thus TNF increased the expression of HLA-DR antigens and high affinity IL 2 receptors. As a consequence, TNF-treated T cells showed an enhanced proliferative response to IL 2. Moreover, TNF-alpha was effective as a co-stimulator of IL 2-dependent IFN-gamma production. These data indicate that TNF-alpha may regulate growth and functional activities of normal T cells.     

Expression of low-, intermediate-, and high-affinity IL-2 receptors on B cell lines derived from patients with undifferentiated lymphoma of Burkitt's and non-Burkitt's types

IL-2 receptors on T cells exist in at least three forms which differ in their ligand-binding affinity. The low-affinity IL-2 receptor (IL-2R) consists of the 55-kDa Tac protein (p55 alpha), the intermediate-affinity site corresponds to the 70-kDa molecule (p70 beta), and the high-affinity IL-2R consists of a noncovalent heterodimeric structure involving both p55 alpha and p70 beta. We studied 24 B cell lines (8 EBV-negative and 16 EBV-positive) for IL-2R expression in the presence or absence of the tumor promoter, teleocidin. 125I-IL-2 radioreceptor binding assays and crosslinking studies demonstrated the sole expression of p55 alpha in EBV-negative cell lines only, whereas p55 alpha present in EBV-positive cell lines was always associated with p70 beta to construct high-affinity IL-2R. p70 beta was not detected in any of the EBV-negative cell lines, but was expressed on most of the EBV-positive cell lines (13 of 16). Our data also indicate that the expression of p55 alpha and p70 beta by radiolabeling correlates with their expression in flow cytometry, and that a large excess of p55 alpha is required to construct high-affinity IL-2R. Coexpression of p55 alpha and p70 beta on human B cells contributed to constructing high-affinity IL-2R hybrid complex as shown by (i) rapid association rate contributed by p55 alpha and slow dissociation rate by p70 beta; (ii) teleocidin's ability to induce p55 alpha on cell lines which express p70 beta only, resulting in appearance of high-affinity IL-2R; (iii) blocking p55 alpha by anti-Tac mAb in cell lines which constitutively express high-affinity IL-2R eliminated both high- and low-affinity components. The existence of low, intermediate, and high IL-2R on human B cells bears important future implications for understanding the mechanism of IL-2 signaling and the role of IL-2 in B cell activation, proliferation, and differentiation.     

The IL-2 receptor alpha-chain alters the binding of IL-2 to the beta-chain

The binding of IL-2 to its high affinity receptor results in the formation of the ternary complex consisting of IL-2, alpha-chain (p55, Tac) and beta-chain (p75). We studied the role of alpha-chain in IL-2 binding to the high affinity receptor using IL-2 analog Lys20 which was made by the substitution of Lys for Asp20 of wild-type rIL-2. Lys20 bound to MT-1 cells solely expressing alpha-chain at low affinity, but did not bind to YT-2C2 cells which solely expressed beta-chain. However, direct binding of radiolabeled Lys20 to ED515-D cells, an HTLV-I-infected and IL-2-dependent T cell line, revealed both high affinity and low affinity binding although the Kd value of high affinity binding was 50 to 100 times higher than that of the high affinity binding of wild-type rIL-2. High affinity binding of Lys20 was completely blocked by 2R-B mAb recognizing IL-2R beta-chain. Anti-Tac mAb recognizing IL-2R alpha-chain abolished all of the specific Lys20 bindings. In contrast to the replacement of cell bound 2R-B mAb with wild-type rIL-2 at 37 degrees C, the addition of an excess of Lys20 did not cause the detachment of cell-bound radiolabeled or FITC-labeled 2R-B mAb. Consistent with the results of binding studies, Lys20 induced the proliferation of ED515-D cells, but not large granular lymphocyte leukemic cells. The growth of ED-515D cells was completely suppressed by either anti-Tac mAb or 2R-B mAb. These results strongly suggest that coexpression of the IL-2R alpha- and beta-chains alters the binding affinity of Lys20 and that the interaction between IL-2 and the alpha-chain is a key event in the formation of the IL-2/IL-2R ternary complex.     

The third molecule associated with interleukin 2 receptor alpha and beta chain.

It is known that the affinity cross-linking study of the human high-affinity Interleukin 2 (IL-2) receptor reveals triplet bands consisting of 70 kDa alpha chain(Tac)-IL-2 and the 90/80 kDa doublet. We found the cell lines lacking the lower band of the doublet in spite of the expression of both alpha and beta chains. No IL-2 binding was detectable in the presence of anti-Tac antibody in these cells. Immunoprecipitation from the cell extract of [125 I] IL-2-cross-linked T cells with anti-beta chain polyclonal IgG detected the upper band, but not lower band of the doublet. These data suggest that the lower band of the doublet represents an unknown IL-2-binding protein (p65) distinct from the beta chain and this molecule may be involved in the intermediate-affinity IL-2 binding together with the beta chain.     

IL-4 blocks the up-regulation of IL-2 receptors induced by IL-2 in normal human B cells

A negative influence of IL-4 on the IL-2-induced B cell proliferation and differentiation has recently been reported. In this study, we have further investigated a role of IL-4 on human tonsillar B cell proliferation and IL-2R expression. IL-4 enhanced Staphylococcus aureus Cowan 1 strain (SAC)-induced B cell proliferation, reaching the peak on day 3. However, from day 4, IL-4 inhibited IL-2-induced proliferation. In the cross-linking study, IL-4 enhanced the density of 125I-IL-2-binding protein at low affinity binding condition (2 nM of 125I-IL-2) in SAC-activated B cells. However, IL-4 blocked the enhancement in the density of 125I-IL-2-binding proteins induced by IL-2, from day 3, in both high (50 pM of 125I-IL-2) and low affinity binding conditions, suggesting that IL-4 is able to block IL-2-induced IL-2R up-regulation. This was confirmed by a binding study: B cells that cultured for 3 days with SAC plus IL-2 expressed an average of 180 +/- 20 high affinity receptors/cell with a Kd of 12 pM and 5800 +/- 500 low affinity receptors/cell with a Kd of 980 pM. By coculturing with IL-4, high affinity receptors were almost undetectable and the expression of low affinity receptors was reduced by more than 80%. IL-4-mediated inhibition of IL-2-induced IL-2R expression does not seem to be due to the direct interaction between IL-4 and cell surface receptors, inasmuch as preincubation of cells with IL-4 for 60 min at 37 degrees C did not alter the binding of 125I-IL-2 to cells previously cultured for 3 days with SAC plus IL-2. These data suggest that IL-4 has a capacity to block the up-regulation of the high as well as low affinity IL-2R-induced by IL-2 in normal human B cells, and could provide a possible explanation for the decreased responsiveness of B cells to IL-2 in the presence of IL-4.     

IL-12 receptor. II. Distribution and regulation of receptor expression.

IL-12 is a heterodimeric lymphokine that induces IFN-gamma production by resting PBMC, enhances the lytic activity of NK/lymphokine activated killer cells, and causes the proliferation of activated T cells and NK cells. In this report, we have investigated the expression of IL-12R on mitogen- and IL-2-activated PBMC or tonsillar lymphocytes as well as on a variety of cell lines. The results of radiolabeled IL-12-binding assays indicated that high affinity IL-12R are present on PBMC activated by various T cell mitogens or by IL-2. High affinity IL-12R were also found to be expressed constitutively on a transformed marmoset NK-like cell line HVS.SILVA 40. At the time of peak IL-12R expression, mitogen- or IL-2-activated cells displayed approximately 1000 to 9000 IL-12 binding sites/cell with an apparent Kd of 100 to 900 pM. Kinetic studies revealed that maximum expression of IL-12R occurred earlier on PHA-activated PBMC as compared with PBMC activated by IL-2, and that expression of IL-12R on these cells correlated with their ability to proliferate in response to IL-12. Although IL-2 could up-regulate IL-12R expression on resting PBMC, the ability of mitogen-activated PBMC to up-regulate IL-12R was found to be independent of IL-2. Analysis of IL-12R expression by flow cytometry revealed that receptors for IL-12 are present on activated T cells of both the CD4+ and CD8+ subsets and on activated CD56+ NK cells. In contrast, neither resting PBMC or tonsillar B cells nor tonsillar B cells activated by anti-IgM/Dx, anti-IgM/Dx + IL-2, or SAC + IL-2 displayed IL-12R detectable by flow cytometry or by the radiolabeled IL-12-binding assay. In summary, these results indicate that activation of T cells or NK cells results in up-regulation of IL-12R expression; on the other hand, B cell activation, at least under some circumstances, appears not to be associated with enhanced expression of IL-12R.     

Trypanosoma cruzi reduces the number of high-affinity IL-2 receptors on activated human lymphocytes by suppressing the expression of the p55 and p70 receptor components

We previously established that Trypanosoma cruzi, the causative agent of Chagas' disease, has the ability to suppress expression of the p55 component of the IL-2R by activated human PBMC. We explored in this work whether the parasite alters the expression of high affinity IL-2R, responsible for the internalization of IL-2 and signal transduction. Radiobinding measurements revealed that the trypanosome indeed inhibited the expression of high affinity IL-2R. Thus, a considerably smaller number of 125I-IL-2 molecules was necessary to saturate the IL-2R on PHA-stimulated PBMC cocultured with T. cruzi than those of control PBMC that had not been exposed to the organisms. Scatchard analysis of equilibrium binding data showed that, in the presence of T. cruzi, the number of high affinity IL-2R per cell was reduced by approximately 80%. The Kd for IL-2 binding to the fewer IL-2R expressed on PBMC exposed to T. cruzi was not significantly different from that of IL-2R on nonsuppressed PBMC. Independent measurements made after cross-linking 125I-IL-2 to its specific receptors with disuccinimidylsuberate showed that both the p55 and p70 components of the IL-2R were markedly suppressed and to comparable extents. These results demonstrate for the first time that T. cruzi suppresses the expression of high affinity IL-2R by human cells, including the p70 chain of the heterodimeric IL-2R. It is noteworthy that the in vitro model system we used in this work to study the mechanisms whereby T. cruzi may induce the immunosuppression that accompanies acute Chagas' disease also lends itself to the exploration of the regulatory mechanisms governing the expression of IL-2R by human PBMC.     

Synergistic action of monoclonal antibodies directed at p55 and p75 chains of the human IL-2-receptor

TU27, a mouse IgG1 mAb directed at the p75 chain of the human IL-2R, was analyzed for its ability to interact with IL-2 binding on isolated p75 chains (YT-2C2 cells) and high affinity p55/p75 receptors (human alloreactive T cell clone 4AS), to inhibit IL-2-induced proliferation (4AS cells) and to cooperate with an anti-p55 chain mAb (33B3.1) for inhibiting IL-2 binding and proliferation. TU27 and IL-2 bound to the isolated p75 chain expressed by YT-2C2 cells with respective dissociation constants (Kd) of 1.3 and 1 nM. They cross-inhibited each other for binding with inhibition constants (Ki) in agreement with their respective Kd values. The nature of the interaction was, however, not purely competitive and suggested nonidentical epitopes for the two ligands on the p75 chain. On 4AS cells, IL-2 bound with high affinity (Kd = 50 pM) and TU27 with an affinity similar to that found on YT-2C2 cells. The binding of TU27 and IL-2 were also mutually exclusive on 4AS cells. However, the mechanism of interaction of TU27 with IL-2 was complex since the inhibitory potency of the antibody depended on temperature, antibody preincubation and time of assay. Data obtained at 4 degrees C in the presence of suboptimal, tracer-like concentrations of IL-2 indicated that the intrinsic affinity of TU27 for the high affinity configuration was 15-fold lower than for the isolated p75 chain and argued in favor of the affinity-conversion model (as opposed to the preformed complex model) in which p55 and p75 are dissociated in the absence of IL-2. At 37 degrees C, TU27 inhibited IL-2 binding only on short time assays (6 min). Longer time (30 min) of IL-2 binding resulted in an almost complete disappearance of the effect of TU27, suggesting that internalization of the high affinity p55/p75/IL2 complex enables the cells to escape from the inhibitory effect of TU27. In the presence of the 33B3.1 mAb, the interaction of TU27 with IL-2 resembled the one observed on YT-2C2 cells, suggesting that 33B3.1 is able to inhibit the IL-2-induced association of p55 and p75. Both antibody were found to synergize on 4AS cells, as a result of a cooperative mechanism in which 33B3.1 blocks the formation of the high affinity complex hence allowing TU27 to bind with higher affinity, and TU27 blocks IL-2 binding to the p75 chain. Proliferation studies corroborated the binding experiments.(ABSTRACT TRUNCATED AT 400 WORDS)     

IL 2 receptor induction on human T lymphocytes: role for IL 2 and monocytes

In this report we studied the requirements for the activation and proliferation of highly purified human T lymphocytes. Purified T cells incubated for 3 days with PHA neither proliferate nor express IL 2 receptors as detected by FACS analysis with the use of anti-Tac antibodies. However, purified T cells incubated with Con A or anti-T3 moAb do not proliferate, albeit 30 to 35% T cells express Tac epitopes. The addition of IL 2, either natural purified or recombinant, resulted in both the appearance of Tac antigen and the proliferation of PHA-activated T cells. Much to our surprise, IL 2 did not induce proliferation of Tac-positive T cells activated by Con A or soluble anti-T3 unless monocytes were added to the cultures. These data suggested that two classes of IL 2 receptors might exist on T cells, one of which was not functionally involved in T cell proliferation. In keeping with this interpretation, we have been able to demonstrate, using a radiolabeled IL 2 binding assay, that anti-T3 moAb induced almost exclusively IL 2 receptors of low affinity (Kd = 30 to 70 X 10(-9) M) and that additional signals, provided by monocytes, are required for the acquisition of high affinity receptors. IL 2 itself can induce high affinity receptors on PHA-stimulated T cells but not on cells activated by Con A or anti-T3. In this latter case the physical presence of monocytes is required and cannot be substituted by IL 1, thus indicating a previously unreported role for monocytes. It is postulated that the contact of monocytes with T cells induces a switch from an inactive low affinity conformation of the IL 2 receptor to a functional high affinity one.     

Purification of a 130-kDa T cell glycoprotein that binds human interleukin 4 with high affinity

The interleukin 4 (IL-4) receptor was purified from the gibbon T cell line MLA 144. These cells were found to express high numbers of human IL-4-binding proteins (5000-6000 sites/cell) with an affinity constant (Kd) similar to that measured in human cell lines (Kd = 40-70 pM). Affinity cross-linking of 125I-IL-4 to human cell lines and MLA 144 cells demonstrated the labeling of three proteins of approximately 130, 75, and 65 kDa. Human IL-4-binding sites were solubilized from MLA 144 cells using Triton X-100 and then purified by carboxymethyl chromatography, which removed 50% of the protein without loss of IL-4-binding activity. Then sequential affinity purification over wheat germ agglutinin and a single IL-4 Affi-Gel 10 column resulted in a final 8000-fold purification of the IL-4 receptor. When analyzed on a silver-stained sodium dodecyl sulfate-polyacrylamide gel, the purified receptor migrated as a single molecular species of 130 +/- 5 kDa. Identification of the 130-kDa protein as the IL-4 receptor was demonstrated by cross-linking experiments and specific binding of 125I-IL-4 to nitrocellulose membranes after electrophoretic transfer of the purified receptor on sodium dodecyl sulfate-polyacrylamide gel.     

IL-1 binds to high affinity receptors on human osteosarcoma cells and potentiates prostaglandin E2 stimulation of cAMP production

IL-1 is a potent bone resorbing agent. Its mechanism of action is unknown, but the presence of osteoblasts was shown to be necessary for IL-1 stimulation of bone resorption by isolated osteoclasts. This study examines the presence of IL-1R and IL-1 effects in osteoblastic cells from a clonal human osteosarcoma cell line, Saos-2/B-10. We found that the binding affinity and the number of binding sites increases substantially during the postconfluent stage. Scatchard and curve-fitting analysis revealed one class of high affinity binding sites, with Kd/Ki's of 40 +/- 17 pM (mean +/- SD) for IL-1 alpha (n = 5) and 9 +/- 7 pM for IL-1 beta (n = 5) and 2916 +/- 2438 (n = 6) receptors/cell. Incubation of the cells with 125I-IL-1 alpha (100 pM) at 4 degrees C, followed by incubation at 37 degrees C up to 4 h, revealed internalization of receptor-bound IL-1 alpha. Chemical cross-linking studies showed that the IL-1R in Saos-2/B-10 cells had a molecular mass of approximately 80 kDa. To assess the biologic effect of IL-1 in Saos-2/B-10 cells, we determined PGE2 content and adenylate cyclase activity. Although IL-1 had no effect on PGE2 synthesis, both IL-1 alpha and IL-1 beta enhanced PGE2 stimulation of adenylate cyclase two- to four-fold in a dose-dependent manner. The half-maximal effect for IL-1 alpha was seen at 8 to 10 pM and for IL-1 beta at 0.6 to 1.8 pM. IL-1 did not enhance basal adenylate cyclase or stimulation by parathyroid hormone, isoproterenol, or forskolin. IL-1 enhancement of PGE2-stimulated adenylate cyclase was detected between 1 to 2 h, was maximal at 4 to 5 h, was not prevented by cycloheximide treatment, and was seen in membranes from IL-1 pretreated cells. These data show effects of IL-1 on a human osteoblast-like cell line that are mediated by high affinity receptors. These IL-1 effects could contribute to the biologic action of IL-1 on bone.     

Expression and the functional role of a p70/75 interleukin 2-binding molecule in human B cell

Expression of p70/75 IL-2-binding molecules and their functional roles in induction of Ig secretion by IL-2 were examined in human B cells. IL-2, at high concentrations induced higher levels of Ig secretion in Staphylococcus aureus strain Cowan I (SAC)-activated B cells than at low concentrations. About 50% of SAC-activated B cells, lacking Tac antigen, were also responsive to Ig secretion by IL-2, although the required dose of IL-2 was higher than that for Tac-positive B cells. H-31 antibody which recognizes Tac antigen did not inhibit the induction of Ig secretion by high concentrations of IL-2 in both Tac-negative and Tac-positive B cells, suggesting that IL-2 might induce Ig secretion through a receptor distinct from Tac antigens. In contrast, IL-2 was ineffective in the absence of SAC stimulation even at high concentrations. Upon analysis by SDS-PAGE, p70/75 IL-2-binding molecules were detected on Tac-negative SAC-activated B cells. Similar IL-2-binding molecules distinct from Tac antigen (p55) were detected in both Tac-positive B and T cells. However, neither p55 nor p70/75 IL-2-binding molecules could be detected in the absence of SAC stimulation. These observations suggest that p70/75 IL-2 binding molecules are induced in human B-cells in the presence or absence of Tac antigen by SAC stimulation and these determinants play an important function in the transduction of IL-2 associated signal for B cell differentiation.