Expression of IL-1 receptors on human peripheral B cells

The expression of IL-1R on human peripheral B cells was analyzed by the binding assay with 125I-labeled human rIL-1 alpha and by the flow cytofluorometry by the use of FITC-conjugated IL-1 alpha. The proliferation and the differentiation of B cells stimulated with Staphylococcus aureus Cowan I (SAC) in the presence of T cell-derived factors were dependent on IL-1. By the binding experiment with 125I-labeled IL-1 alpha, B cells expressed only few IL-1R without any stimulations. When they were stimulated with SAC, IL-1R on B cells began to increase by only 1 h, reached the maximum level at 6 h. The binding of 125I-labeled IL-1 alpha to B cells was inhibited by the addition of either cold IL-1 alpha or IL-1 beta suggesting that IL-1R on B cells reactive for IL-1 alpha and IL-1 beta were identical. By Scatchard plot analysis, the existence of two classes of IL-1R on B cells was found. A major class of IL-1R (320 molecules/cell) has a lower affinity (Kd = 3.8 x 10(-10) M) and a minor class of IL-1R (70 molecules/cell) has a higher affinity (Kd = 4.4 x 10(-12) M). When B cells were stimulated with SAC, both lower and higher affinity IL-1R were increased to 1960 molecules/cell and 300 molecules/cell, respectively. Furthermore, IL-1R on B cells were also detected with FITC-conjugated IL-1 alpha by a flow cytofluorometer. Only 3 to 5% of B cells expressed IL-1R without any stimulations. When B cells were stimulated with SAC, IL-1R-positive B cells were increased to 20%. The addition of anti-class II antibodies inhibited B cell proliferation and differentiation induced with SAC, IL-1, and T cell-derived factors. Anti-class II antibodies also inhibited the number of IL-1R on B cells. These results suggest that the expression of IL-1R was induced as the initial stage of B cell activation and that class II Ag play an important role for the expression of IL-1R on B cells.     

Interleukin 1 receptors on rabbit articular chondrocytes: relationship between biological activity and receptor binding kinetics

This study gives an account of the biologic and kinetic binding properties of interleukin 1 alpha (IL 1 alpha), interleukin 1 beta (IL 1 beta), and Glu-4 (an NH2-terminal mutant of IL 1 beta) to interleukin 1 (IL 1) receptors in rabbit articular chondrocytes. All three IL 1's demonstrated full agonist properties in their ability to stimulate prostaglandin E2 (PGE2) synthesis. IL 1 alpha was 23-fold more biologically active than IL 1 beta, which was around 110-fold more active than Glu-4 based on the concentration of IL 1 required for half-maximal stimulation of PGE2. The binding of all three ligands was concentration-dependent and saturable at 4 degrees C. Scatchard analysis of receptor binding data showed that the dissociation constant (KD) of IL 1 alpha was 46 +/- 12 pM, and the receptor density was 3120 sites/cell. The association of IL 1 alpha at 4 degrees C did not attain equilibrium until after 10 h at 100 pM of 125I-labeled IL 1 alpha. The dissociation of bound IL 1 alpha was very slow, t1/2 of 21 h, although only one class of high-affinity receptors was detected. The KD of IL 1 beta binding was 72 +/- 3 pM with a receptor density of 800 +/- 40 sites/cell. Dissociation of bound 125I-labeled IL 1 beta at 4 degrees C appeared to indicate the presence of two receptor subsets, a fast and a slower component with a t1/2 of 2 min and 5 h, respectively. The receptor binding affinity of Glu-4 was 324 +/- 3 pM, in line with its reduced biologic activity. Both IL 1 alpha and IL 1 beta are rapidly internalized in chondrocytes in a time- and temperature-dependent manner.     

Differential binding of IL-1 alpha and IL-1 beta to receptors on B and T cells

The interleukin 1 receptors (IL-1R) on the human B lymphoma RAJI and on the murine thymoma EL4-6.1 have been characterized. Equilibrium binding analysis using both 125I-labeled IL-1 alpha and IL-1 beta showed that RAJI cells have a higher number of binding sites/cell for IL-1 beta (2400, Kd 2.2 nM) than for IL-1 alpha (316, Kd 0.13 nM). On the other hand, EL4-6.1 cells have more receptors/cell for IL-1 alpha (22 656, Kd 1 nM) than for IL-1 beta (2988, Kd 0.36 nM). Dexamethasone (DXM) induced on RAJI cells a time-dependent increase in binding sites for both IL-1 beta and IL-1 alpha without affecting their binding affinities. However, while receptor-bound 125I-IL-1 alpha was displaced with equal efficiency by both IL-1 forms, only unlabeled IL-1 beta could effectively displace 125I-IL-1 beta. Cross-linking experiments indicated that RAJI cells have a predominant IL-1R of about 68 kDa, while EL4-6.1 cells have an IL-1-binding polypeptide of 80 kDa. These results suggest that B and T cells possess structurally different IL-1R with distinct binding properties for IL-1 alpha and IL-1 beta.     

Production of interleukin 1 by adult T cell leukemia (ATL) cell lines

The accessory function for T cell activation and the production of interleukin 1 (IL 1) of adult T cell leukemia (ATL) cell lines were studied in vitro. ATL cell lines such as Hut-102, MT-1, and MT-2 functioned as accessory cells for the stimulation of human T cell proliferative response induced with concanavalin A (Con A) and induced allogeneic mixed lymphocyte reaction. Cell lysates of three ATL cell lines and the culture supernatant of MT-2 cells had activities to stimulate murine thymocyte proliferative response. Then we studied physicochemical properties of the factors produced by MT-2 cells. The m.w. of the factors were approximately 15,000 by Sephacryl S-200 column chromatography, and their isoelectric point values were 5.4 and 4.8 by chromatofocussing technique. No fraction contained interleukin 2 (IL 2) activities to stimulate IL 2-dependent murine cytotoxic T cell line. The thymocyte-stimulating activities of the factors were absorbed with rabbit anti-IL 1 alpha antiserum, but not with anti-IL 1 beta antiserum. Furthermore, messenger RNA extracted from MT-2 cells hybridized to complementary DNA of IL 1 alpha, but not of IL 1 beta, by Northern blot hybridization analysis. The factors from MT-2 cells could stimulate the production of IL 2 and the expression of IL 2 receptors of human T cells in the presence of Con A as well as recombinant IL 1 alpha and IL 1 beta did, and these activities were also blocked by rabbit anti-IL 1 alpha antiserum, but not by anti-IL 1 beta antiserum. These results suggest that the factors produced by MT-2 cells correspond to IL 1 alpha. However, the accessory function of MT-2 cells for T cell activation was not blocked by rabbit anti-IL 1 antiserum. These results suggest that ATL cell lines produce IL 1-like factors, but the accessory function of ATL cell lines for T cell activation is mediated by some other mechanisms rather than by secreted IL 1-like factors.     

Concanavalin A triggers T lymphocytes by directly interacting with their receptors for activation

Anti-HLA-DR antibodies did not inhibit concanavalin A-(Con A) induced T cell proliferation or the generation of suppressor cells capable of inhibiting immunoglobulin synthesis in autologous mononuclear cells after pokeweed mitogen stimulation. Nylon-wool purified T cells (pretreated with anti-HLA-DR antibody and C) exposed to Con A acquired responsiveness to interleukin 2 (IL 2) and were able to absorb this growth factor, whereas nonlectin-treated cells did not respond to IL 2 and could not absorb it. In the presence of interleukin 1 (IL 1), Con A stimulated the synthesis of IL 2 in purified OKT4+ lymphocytes but not OKT8+ cells. However, in the absence of IL 1, neither resting OKT4+ nor Con A-treated OKT4+ cells produced IL 2. Con A by itself did not directly stimulate macrophages to synthesize IL 1, although it could do so in the presence of OKT4+ but not OKT8+ lymphocytes. In addition, Con A induced proliferation of purified T cells provided IL 1 was supplied to the cultures. Cyclosporin A rendered Con A-treated T cells unresponsive to IL 2, made lectin-stimulated OKT4+ lymphocytes unable to respond to IL 1, and inhibited the synthesis of IL 2. Furthermore, this drug abrogated the Con A-stimulated synthesis of IL 1 by acting on OKT4+ lymphocytes and not on macrophages. Finally, cyclosporin-A suppressed the proliferative response and the generation of suppressor T cells induced by Con A. The following are concluded: 1) HLA-DR antigens do not seem to play any role in the triggering of T cells by Con A, and macrophages participate in lectin-induced activation of T cells mainly by providing IL 1. 2) Cyclosporin-A inhibits activation of T cells by interfering with the mechanism by which Con A stimulates T lymphocytes. 3) Con A triggers T lymphocytes by directly interacting with their receptors for activation.     

Interleukins 2 and 3 regulate the in vitro proliferation of two distinguishable populations of 20-alpha-hydroxysteroid dehydrogenase-positive cells

The ability of interleukin 2 (IL 2), interleukin 3 (IL 3), and granulocyte/macrophage colony-stimulating factor (GM-CSF) to induce the proliferation of cells from thymus, spleen, or bone marrow was examined and compared with their ability to induce expression of the enzyme 20-alpha-hydroxysteroid dehydrogenase (20 alpha SDH). In the thymus, the peanut agglutinin agglutinated cells (PNA+) lacked 20 alpha SDH and showed no detectable response to IL 2, IL 3, or GM-CSF in either proliferation or induction of 20 alpha SDH. In contrast, the PNA nonagglutinated (PNA-) subpopulation expressed 20 alpha SDH and proliferated in response to Con A and/or IL 2. The responding cells that could be expanded in vitro with IL 2 expressed high levels of 20 alpha SDH. Neither IL 3 nor GM-CSF in the presence or absence of Con A had a demonstrable effect on the PNA- population. In cultures of bone marrow cells, both IL 3 and GM-CSF induced proliferation, whereas IL 2 had no effect on proliferation in the presence or absence of Con A. Thy-1-depleted bone marrow cells, expanded in tissue culture with IL3, contained cells that co-expressed Thy-1 and 20 alpha SDH. In contrast, cells proliferating in vitro to GM-CSF did not expressed Thy-1 or 20 alpha SDH. In cultures of normal splenic lymphocytes, two populations of cells capable of expressing 20 alpha SDH were detected. One population could be expanded in vitro with IL 2 and Con A, whereas the second was responsive to IL 3. In spleens from athymic mice, only the latter cells were detected. These results demonstrate that IL 3 and IL 2 responsiveness distinguishes two populations of 20 alpha SDH cells. The relevance of these observations to the possible relationship of IL 3 and IL 2 in T cell differentiation is discussed.     

A soluble binding protein specific for interleukin 1 beta is produced by activated mononuclear cells

Soluble interleukin 1 (IL 1) binding proteins were identified by gel filtration and covalent cross-linking of 125I IL 1 in normal human serum and inflammatory exudate. High molecular weight 125I IL 1 protein complexes occurred with both IL 1 alpha and IL 1 beta, however, high molecular weight binding appeared to be non-specific. One specific IL 1 beta binding protein was observed to elute at approximately 100 kDa on gel filtration when bound to 125I IL 1 beta. This complex migrated as a broad band at 60 kDa when covalently cross-linked and analyzed by SDS-PAGE. The protein did not bind 125I IL 1 alpha and 125I IL 1 beta binding was only displaceable by excess cold IL-1 beta. The production of the specific IL 1 beta binding protein was assessed in a number of cell populations. Unstimulated peripheral blood mononuclear cells (PBMNC) did not produce the binding protein, but stimulation with phytohemagglutinin (PHA) caused production within 24 hr and binding protein levels remained elevated for up to 7 days. Stimulation with lipopolysaccharide (LPS) and IL 1 alpha did not consistently induce synthesis of the binding protein. Ligand-binding studies were performed to compare solubilized EL 4 NOB.1 cell membrane IL 1 receptor (sIL 1R) with semi-purified IL 1 beta binding protein from pooled synovial fluid. The sIL 1R preparation bound ligand with an affinity of 168 pM while the IL 1 beta binding protein bound 125I IL 1 beta with an affinity of 370 pM. This protein may function as an important carrier molecule for IL 1 beta and determine its distribution and kinetics in vivo.     

Murine interleukin 1 receptor. Direct identification by ligand blotting and purification to homogeneity of an interleukin 1-binding glycoprotein

Functional receptors (IL1-R) for the proinflammatory cytokine interleukin 1 (IL1) were solubilized from plasma membranes of the NOB-1 subclone of murine EL4 6.1 thymoma cells using the zwitterionic detergent 3[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Membrane extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and "ligand blotted" with 125I-labeled recombinant human IL1 alpha in order to reveal proteins capable of specifically binding IL1. A single polydisperse polypeptide of Mr approximately equal to 80,000 was identified in this way, which bound IL1 alpha and IL1 beta with the same affinity as the IL1-R on intact NOB-1 cells (approximately equal to 10(-10) M). The IL1-binding polypeptide was only seen in membranes from IL1-R-bearing cells and did not react with interleukin 2, tumor necrosis factor alpha, or interferon. IL1-R was purified to apparent homogeneity from solubilized NOB-1 membranes by affinity chromatography on wheat germ agglutinin-Sepharose and IL1 alpha-Sepharose. Gel electrophoresis and silver staining of purified preparations revealed a single protein of Mr approximately equal to 80,000 which reacted positively in the ligand-blotting procedure and which we identify as the ligand-binding moiety of the murine IL1-R. Purified IL1-R exhibited the same affinity and specificity as the receptor on intact cells. The relationship of this protein to proteins identified by covalent cross-linking studies is discussed.     

TCGF (IL 2)-receptor inducing factor(s). I. Regulation of IL 2 receptor on a natural killer-like cell line (YT cells)

A continuous cell line (YT cells) with inducible receptor for T cell growth factor (TCGF)/interleukin 2 (IL 2) was established from a 15-yr-old boy with acute lymphoblastic lymphoma and thymoma. YT cells were tetraploid, having 4q+ chromosomal markers, and proliferated continuously in vitro without conditioned medium (CM) or IL 2. They were weakly positive for OKT9, OKT11, and Tac antigen (Ag), a determinant closely associated with the receptor for IL 2 (IL 2-R), and were negative for OKT1, OKT3, OKT4, and OKT8 Ag. YT cells also expressed HNK-1 Ag and Fc receptors for IgG, which are expressed on natural killer (NK) cells. They retained a killing activity against human cell lines, including K562 (myeloid), T, and B cell lines. Unlike Tac Ag/IL 2-R(+) cell lines derived from adult T cell leukemia (ATL), YT cells were negative for HTLV, as proved by Southern blotting with cDNA for viral DNA. The expression of Tac Ag was markedly enhanced in 18 hr, when YT cells were incubated with CM from PHA-stimulated peripheral blood leukocytes (PBL) or spleen cells, as determined by immunofluorescence by using flow cytometry and binding assay with 125I-anti-Tac antibody (Ab). The binding study with 125I-labeled recombinant IL 2 showed 3.2 X 10(4) IL 2 receptor sites on YT cells precultured with CM. PHA-P and Con A neither agglutinate nor enhance the expression of IL 2-R/Tac antigen on these non-T cell line cells. Furthermore, neither recombinant IL 2 nor gamma-interferon could induce IL 2-R on YT cells, suggesting the presence of a unique IL 2-R inducing factor in PBL or spleen CM. Unlike Tac Ag on HTLV(+), ATL-derived cell lines (Hut-102, MT-1, ATL-2), the expression of Tac Ag on YT cells was down-regulated by anti-Tac Ab. The induction of Tac Ag/IL 2-R on YT cells seemed specific, because the enhancement of Tac Ag expression was not associated with that of Ia Ag and T9/transferrin receptor.     

Monoclonal antibodies, L-35 and L-36, define novel T cell activation antigens

Two novel T cell specific activation antigens were characterized and were defined by monoclonal antibodies developed against mitogen-stimulated human T cells. These antigens, designated as L-35 and L-36 were expressed on both the CD 4(Leu-3) and the CD 8(Leu-2) subsets of activated but not resting T cells. Moreover these antigens were not expressed on a number of T, B, and myeloid tumor cell lines. L-35 and L-36 were expressed on interleukin 2 (IL 2)-dependent cloned T cell lines, and were weakly expressed on the T cell tumor line, HSB-2. L-35 was expressed on granulocytes and a small subset of thymocytes. SDS-PAGE analysis of 125I-labeled lysates from phytohemagglutinin (PHA)-activated T cells demonstrated that L-35 existed as a complex of 32,000 and 97,000 dalton polypeptides under reducing and nonreducing conditions. Anti-L-36 immunoprecipitated a 90,000 dalton structure from PHA-activated cell lysates prepared with CHAPS detergent. When immunoprecipitates were analyzed from [35S]methionine labeled lysates, anti-L-35 precipitated only the 97,000 dalton component, suggesting that the 32,000 dalton subunit of L-35 complex was not synthesized by the activated cell population. Furthermore anti-L-35 did not immunoprecipitate a 32,000 dalton component from 125I-labeled lysates of anti-Leu-4 or Con A-activated cells, suggesting that the 32,000 dalton component of the L-35 complex may represent a subunit of PHA. The 32,000 dalton protein could not, however, be precipitated from cells incubated with PHA for less than 1 day. These results suggested that anti-L-35 recognizes a 97,000 dalton structure expressed on activated T cells, and that upon activation by PHA, becomes associated with a subunit of this mitogen.     

Down-regulation of interleukin 1 (IL 1) receptor expression by IL 1 and fate of internalized 125I-labeled IL 1 beta in a human large granular lymphocyte cell line

The regulation of interleukin 1 (IL 1) receptor expression on a human large granular lymphocyte cell line, YT, and fate of internalized 125I-labeled IL 1 beta (125I-IL 1 beta) were studied. YT cells were selected for this study, because this cell line expresses a large number of specific high-affinity receptor for IL 1, responds biologically to exogenously added IL 1 by expressing high-affinity IL 2 receptors, and does not produce IL 1. YT cells constitutively express approximately 7 X 10(3) IL 1 receptors/cell with a Kd approximately 10(-10) M. Neither IL 2, phorbol myristic acid, nor lipopolysaccharide affected the total binding of 125I-IL 1 beta by YT cells. In contrast, the capacity of YT cells to bind 125I-IL 1 beta when incubated at 37 degrees C for 3 to 16 hr with a low dose of purified IL 1 beta (approximately 6 U/ml) was reduced by greater than 80%. The loss of binding capability gradually recovered by 16 hr after removal of IL 1 beta from cultured YT cells. The apparent loss of IL 1 receptor expression was accompanied by the internalization of 125I-IL 1 beta into cells. Acid treatment of YT cells to remove bound 125I-IL 1 beta at 4 degrees C showed that 50% of the 125I-IL 1 beta bound to cells could no longer be recovered after 30 min at 37 degrees C, and this increased to 80% after 3 hr at 37 degrees C. Fractionation of cell extracts on Percoll gradient additionally showed 125I-IL 1 beta to appear intracellularly after receptor binding on plasma membranes, and to be successively transferred to some membranous organelles (d approximately equal to 1.037) through an intermediate density organelle (d approximately equal to 1.050), and to finally end up in lysosomal cell fractions (d approximately equal to 1.05 to 1.08) after approximately 3 hr at 37 degrees C. Only approximately 5% of internalized 125I-IL 1 beta was released into culture media by 6 hr of incubation at 37 degrees C. However, the radioactivity in the TCA soluble fraction of the culture media increased gradually by 6 hr and a lysosomotropic enzyme, ethylamine, significantly inhibited both the transfer of internalized 125I-IL 1 beta to the lysosomal fraction and the degradation of 125I-IL 1 beta. This study represents the first evidence of autoregulation of IL 1 receptors by IL 1 and internalization of IL 1 molecules after binding to receptors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interleukin 1 alpha and interleukin 1 beta bind to the same receptor on T cells

Pure, E. coli-derived recombinant murine interleukin 1 alpha (IL 1 alpha) was labeled with 125I and used for receptor binding studies. The 125I-IL 1 binds to murine EL-4 thymoma cells in a specific and saturable manner. Scatchard plot analysis for binding studies carried out at 4 degrees C reveals a single type of high affinity binding site with an apparent dissociation constant of approximately 2.6 X 10(-10) M and the presence of approximately 1200 binding sites per cell. The rate of association of the 125I-IL 1 with EL-4 cells is slow, requiring more than 3 h to reach apparent steady state at 4 degrees C. Cell-bound 125I-IL 1 cannot be dissociated from EL-4 cells upon removal of unbound 125I-IL 1 and incubation of the cells at 4 degrees C in the presence or absence of unlabeled IL 1. Unlabeled recombinant murine IL 1 competes for 125I-IL 1 binding in a dose-dependent manner, whereas interferon-alpha A, interleukin 2 (IL 2), epidermal growth factor, and nerve growth factor have no effect. The 125I-IL 1 binding site is sensitive to trypsin, suggesting that it is localized on the cell surface. We have also examined the ability of purified recombinant human IL 1 alpha and IL 1 beta to compete for binding of the radiolabeled murine IL 1 to its receptor and to stimulate IL 2 production by EL-4 cells. Previous reports have shown that human IL 1 alpha is approximately 60% homologous in amino acid sequence with murine IL 1, but that human IL 1 beta is only about 25% homologous with either murine IL 1 or human IL 1 alpha. Despite these marked differences, however, we report here that both human IL 1 proteins are able to recognize the same binding site as mouse IL 1. In addition, murine as well as both human IL 1 proteins stimulate IL 2 production by EL-4 cells.     

Interleukin 5 up-regulates high-affinity interleukin 2 receptor expression by human resting peripheral T cells: a comparison with the effect of interleukin 4 on B cells.

The separate regulation mechanisms of cytokines on two classes of interleukin 2 receptors (IL-2R) on human peripheral T and B cells were analyzed by a flow cytometer using a double stain with IL-2R alpha (55 kilodalton Tac) and IL-2R beta (75 kilodalton mik beta 1, mik beta 3). Although the expression of IL-2R alpha by T cells was slightly enhanced by IL-2 and IL-4, expression of the beta chain was diminished by both cytokines. IL-5 by itself did not alter the expression of either IL-2R alpha or beta, but preculturing with IL-2 for 24 h followed by IL-5 for another 24 h induced an increase in IL-2R alpha expression and in simultaneous alpha/beta chain expression. Increased numbers of high-affinity IL-2R were confirmed by 125I binding assays. On B cells, IL-4 increased alpha, beta, and simultaneous alpha/beta chain expression, but IL-4-treated B cells did not show an increased number of high-affinity IL-2R.     

Receptors for toxic shock syndrome toxin-1 and staphylococcal enterotoxin A on human blood monocytes.

Staphylococcal toxic shock syndrome toxin-1 (TSST-1) as well as staphylococcal enterotoxin A (SEA) and B (SEB) have recently been shown to bind directly to the class II major histocompatibility antigen, HLA-DR. Whereas others have characterized TSST-1 and SEA binding to HLA-DR on transfected L cells or B lymphoma cell lines, we sought evidence for direct binding of TSST-1 and SEA to HLA-DR on purified human monocytes. A single class of high-affinity receptors was found for both TSST-1 (dissociation constant (Kd) 40 nM, 3.4 x 10(4) receptors per cell) and SEA (Kd 12 nM, 3.2 x 10(4) receptors per cell) on normal human monocytes. Affinity cross-linking of 125I-labeled toxins to monocytes revealed the presence of two membrane protein subunits with molecular masses consistent with the alpha and beta chains of human HLA-DR (35 and 28 kDa, respectively). The anti-HLA-DR monoclonal antibody L243, but not L203 or 2.06, inhibited radiolabeled toxin binding to human monocytes and neutralized the mitogenic response of human T lymphocytes to both toxins. However, L243 was consistently more effective in blocking radiolabeled TSST-1 than SEA binding to human monocytes from the same donors, suggesting that TSST-1 and SEA may be binding to overlapping epitopes rather than to the same epitope on HLA-DR. Because TSST-1 and SEB bind to distinct epitopes on HLA-DR and because SEA cross competes with both TSST-1 and SEB on the HLA-DR receptor, we postulate that SEA occupies a binding site within HLA-DR that overlaps both TSST-1 and SEB.(ABSTRACT TRUNCATED AT 250 WORDS)     

Decreased HLA-DR antigen expression on monocytes causes impaired suppressor cell activity in multiple sclerosis

Mitogen-induced Ts cell activity and (DR) expression on monocytes have recently been shown to be reduced in patients with multiple sclerosis (MS). In our study, T cells were cultured with autologous monocytes in the presence of Con A for 6 days (primary cultures). At the end of the culture T cells were isolated and were 1) tested for surface DR expression and 2) treated with mitomycin C and placed in a secondary culture of allogeneic responder cells and Con A, to test their suppressor function. Treatment of monocytes of the primary culture with an anti-HLA-DR antibody abolished the expression of DR Ag on the T cells as well as their ability to function as Ts cells in the secondary culture. Monocytes from patients with active MS expressed low levels of surface DR Ag and induced reduced amounts of DR Ag on the surface of autologous T cells in primary cultures, which in turn expressed deficient suppressor function when placed in secondary cultures. In contrast, monocytes obtained from patients with inactive disease expressed higher levels of DR and induced higher amounts of DR Ag on autologous T cells, which expressed normal suppressor function. We conclude that the deficient expression of DR Ag on MS monocytes causes reduced suppressor function in activated autologous T cells and may be a primary abnormality in the immunopathogenesis of MS.     

High-affinity interleukin 2 receptor alpha and beta chains are internalized and remain associated inside the cells after interleukin 2 endocytosis.

High-affinity interleukin 2 (IL2) receptors on human T lymphocytes are multimeric complexes containing two IL2-binding polypeptides, alpha and beta chains of 50-55 and 70-75 kDa, respectively, associated by noncovalent bonds. IL2 binds to high-affinity IL2 receptors on the surface of T lymphocytes, mediates cell growth, and is internalized. In this paper, we used a biochemical method to directly identify the receptors components internalized together with the ligand. 125I-IL2-receptor complexes were solubilized with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate, and IL2-binding polypeptides were identified by cross-linking with disuccinimidyl suberate. Under such conditions, the noncovalent association between alpha and beta is maintained. After IL2 internalization, two complexes of about 70 and 90 kDa, IL2 crosslinked to alpha and beta, respectively, were found inside the cells. Both components were immunoprecipitated with either anti-alpha or anti-beta monoclonal antibodies. This shows that the alpha and beta chains are found in an intracellular compartment after IL2 endocytosis, and remain associated as a ternary complex with IL2.     

Soluble interleukin 2 receptors are released by long term-cultured insulin-specific T cells transiently after contact with antigen

An enzyme-linked immunosorbent assay was used to quantitate soluble interleukin 2 receptors (IL 2R) released by antigen-dependent, insulin-specific murine T cells into the culture supernatant, as well as cell-associated IL 2R present in cell lysates. IL 2R were released solely after T cell activation by antigen. The release of IL 2R was transient, reaching optimal levels within 72 hr after antigen challenge and gradually declining to background levels thereafter, when the cells were subcultured in IL 2-enriched medium. The decrease in the amount of IL 2R released during culture in IL 2-containing medium paralleled the decrement in cellular IL 2R detected in cell lysates, in cell surface-expressed IL 2R as determined by cytofluorometry, as well as in high-affinity IL 2R. In contrast, IL 2R were constitutively released by an IL 2-dependent T cell clone. Soluble IL 2R might exert an immunoregulatory function by competing with cellular IL 2R for IL 2 binding.     

Interleukin 2-induced tyrosine phosphorylation. Interleukin 2 receptor beta is tyrosine phosphorylated

Interaction of interleukin 2 (IL2) with its high affinity membrane receptor complex (IL2R) is sufficient to induce proliferation of T lymphocytes. However, the biochemical mechanisms by which IL2 induces this process remain unresolved. The IL2R complex consists of at least two distinct polypeptides that bind IL2, a 75-kDa intermediate affinity subunit (IL2R beta) and a 55-kDa low affinity subunit (IL2R alpha). As indicated by Western blotting with anti-phosphotyrosine-specific antibodies and confirmed by phosphoamino acid analysis, we now demonstrate that interaction of the T cell growth factor interleukin 2 (IL2) with its high affinity receptor on IL2-sensitive human peripheral blood lymphoblasts induces tyrosine phosphorylation of proteins of 92, 80, 78, 70-75, and 57 kDa. IL2 induced tyrosine phosphorylation in YT 2C2 cells which express only the 75-kDa intermediate affinity IL2 binding molecule (IL2R beta) but not in cells which either express only the 55-kDa low affinity IL2 receptor molecule (IL2R alpha) or no IL2-binding sites. Therefore, IL2R beta, in the absence of IL2R alpha, appears sufficient to transduce the transmembrane signal leading to tyrosine phosphorylation. Two different antibodies reactive with phosphotyrosine specifically immunoprecipitated IL2R beta cross-linked to radiolabeled IL2. These findings suggest that IL2R beta is a substrate for the tyrosine kinase which is activated by IL2 binding to its receptor. Thus, like several other growth factor receptors, activation of the IL2R results in an increase in tyrosine phosphorylation with the receptor itself serving as one substrate.     

Selective inhibition of high- but not low-affinity interleukin 2 binding by lectins and anti-interleukin 2 receptor alpha antibody

The present study demonstrated that various reagents can specifically reduce the affinity of high-affinity interleukin 2 receptor (IL-2R) but not that of low-affinity IL-2R. They included lectins such as wheat germ agglutinin (WGA), concanavalin A and phytohemagglutinin, and a chemical cross-linker, glutaraldehyde, in addition to anti-IL-2R monoclonal antibodies, HIEI and H-47. The affinity of the high-affinity IL-2R was reduced when the cells were treated with WGA or H-47 before, but not after, addition of 125I-labeled interleukin 2 (IL-2). Their inhibitory effects were also demonstrated by the chemical cross-linking method. On treatment with the reagents, the IL-2 binding to both IL-2R alpha and beta chains was inhibited and these inhibitory effects were seen only when the reagents were added before IL-2 addition, like their high-affinity reducing effects. These results support a supposition that the high affinity IL-2R is generated by assembly of the alpha and beta chains, and suggest that the IL-2 binding to IL-2R alpha and beta chains could induce stable constitution of the high-affinity state of IL-2R, but these affinity modulating reagents could perturb the optimum association between alpha and beta chains to generate the high-affinity IL-2R.     

Effect of interleukin 1 on the expression of interleukin 2 receptor (Tac antigen) on human natural killer cells and natural killer-like cell line (YT cells)

The effect of interleukin 1 (IL 1) on the expression of interleukin 2 receptor (IL 2R/Tac antigen) on human natural killer (NK) cells and the NK-like cell line, YT was studied with the use of a fluoresceinated anti-IL 2R monoclonal antibody and a Spectrum III flow cytofluorometer. IL 2R was expressed on approximately 10% of NK cells. The expression of IL 2R on NK cells was increased to approximately 25% by the in vitro culture with monocytes or IL 1 and to a less extent by the culture with IL 2 or interferon-gamma (IFN-gamma). IL 2R was expressed on approximately 50% of YT cells without any stimulations. The expression of IL 2R on YT cells was increased up to almost 100% by the culture with IL 1 or monocytes, but not with IL 2, IFN-alpha, IFN-beta, IFN-gamma, or lectins such as concanavalin A and phytohemagglutinin-P. IL 1 absorbed with YT cells or murine thymocytes lost both IL 1 activity detected by the stimulation of murine thymocyte proliferative response and enhancing activity of IL 2R expression on YT cells, suggesting that IL 1 has both activities. However, the assay system of the expression of IL 2R on YT cells is much more sensitive than the stimulation of murine thymocyte proliferative response. By the kinetic study, the enhancement of IL 2R expression was induced by only a 2-hr incubation of YT cells with IL 1. This enhancement did not proceed at 4 degrees C or by the treatment of YT cells with actinomycin D or cycloheximide, suggesting that this enhancement is energy dependent and requires the synthesis of RNA and protein but not DNA. Thus IL 1 plays an important role for the regulation of the expression of IL 2R on NK cells, and IL 1-dependent IL 2R expression on YT cells may give us a good model for the study of the molecular mechanism of the regulation of IL 2R expression.