Soluble Interleukin 2 Receptors Released from Mitogen Stimulated Human Peripheral Blood Lymphocytes Bind Interleukin 2 and Inhibit IL2 Dependent Cell Proliferation

In this communication the binding characteristics and possible regulatory role of sIL2R were investigated. Soluble IL2R are released or secreted in high concentrations by phytohemagglutinin (PHA) stimulated human lymphoid cells. The addition of sIL2R, purified by gel filtration chromatography, to cultures of PHA stimulated lymphoblasts resulted in a dosedependent inhibition of [³H]TdR incorporation that could be overcome by the addition of exogenous IL2. Scatchard analysis of IL2 binding demonstrated that the presence of sIL2R did not inhibit ligand interaction with the high affinity IL2R. Immunoprecipitation studies utilizing [¹²⁵I]IL2 and the non-inhibitory anti-Tac protein antibody 7G7/B6 revealed that most of the ¹²⁵I-labeled IL2 migrated with a protein of approximately 45–50 kDa on SDS/PAGE. Together, these results provide evidence that the sIL2R limits the availability of free IL2 to proliferating cells and down-regulates their response without directly affecting the number or function of the cell bound high affinity IL2R.     

35S]tert.-butylbicyclophosphorothionate and avermectin bind to different sites associated with the gamma- aminobutyric acid-benzodiazepine receptor complex

Low concentrations of avermectin B₁a (AVM) stimulated the specific high affinity binding of [³⁵S]tert.-butylbicyclophosphorothionate ([³⁵S]TBPT) to membranes from rat cerebral cortex in the absence or presence of chloride or bromide ions. In contrast, TBPT either weakly stimulates or does not significantly influence the specific high affinity binding of [³H]AVM to the same membranes in the absence or presence of chloride ions, respectively. These results indicate that [³H]AVM and [³⁵S]TBPT bind to different but closely associated binding sites.     

Reduced density of dopamine D2-like receptors on peripheral blood lymphocytes in Alzheimer's disease

Clinical and pathological evidence points to an involvement of dopamine in Alzheimer's disease (AD). The present study was designed to assay dopamine D1-like and D2-like receptors on peripheral blood lymphocytes (PBL) in 20 patients with AD and in 25 healthy controls by radioligand binding assay techniques with [³H][R]-(+)-(−)chloro-2,3,4,5 tetrahydro-5-phenyl-1H-3-benzazepin-al-hemimaleate (SCH 23390) and [³H]7-hydroxy-N,N-di-n-propyl-2-aminotetraline (7OH-DPAT) as radioligands. The density of dopamine D1-like receptors and the affinity of [³H]SCH 23390 and [³H]7OH-DPAT binding to PBL were similar in both groups investigated. AD patients revealed a lower density of dopamine D2-like receptors on PBL than controls (P=0.0016). The pharmacological profile of [³H]SCH 23390 and [³H]7OH-DPAT binding to PBL was consistent with the labeling of dopamine D5 and D3 receptor subtypes, respectively. The reduced density of dopamine D2-like receptors on PBL is consistent with the observation of changes in the expression of D2-like receptors in dopaminergic brain areas in AD. Our findings support the hypothesis of an involvement of dopamine in AD, even in those patients with no evidence of Parkinsonism, behavioral abnormalities or psychosis.     

Binding and GTPgammaS autoradiographic analysis of preproorphanin precursor peptide products at the ORL1 and opioid receptors

Utilizing agonist-stimulated GTPγS autoradiography, we analyzed the ability of preproorphanin FQ (ppOFQ) peptides to stimulate [³⁵S]-GTPγS binding in adult rat brain. Orphanin FQ (OFQ) stimulated [³⁵S]-GTPγS binding in a pattern similar to that described for [¹²⁵I]-OFQ at the endogenous opioid receptor-like (ORL1) receptor. The ppOFQ peptides nocistatin and orphanin FQ2 (OFQ II_1–17) had no effect, suggesting that they do not mediate their reported analgesic effects via a G_i/o-coupled receptor (i.e. opioid or ORL1). Unlike OFQ II_1–17, high concentrations of its C-terminal extension, OFQ II_1–28, stimulated [³⁵S]-GTPγS binding in a mu (μ) opioid receptor-like distribution and the effect was blocked by naloxone. To explore these observations, we evaluated the receptor binding profile of OFQ II_1–28 at the cloned ORL1 and μ opioid receptors. OFQ II_1–28 had no specific binding at either ORL1 or μ opioid receptors at concentrations up to 50 μM. This lack of affinity was not consistent with a μ-mediated effect, as suggested by preliminary observation using functional autoradiography in rat brain sections. Although behavioral studies suggest that OFQ II_1–28 possesses analgesic activity, this effect does not appear to be mediated via direct binding at the μ opioid receptor. Taken together, these findings support the view that (1) OFQ is the only ppOFQ peptide that binds to and activates the ORL1 receptor and (2) OFQ II_1–28 does not bind or stimulate [³⁵S]-GTPγS binding in cells expressing the μ opioid receptor.     

Segmental, coronal and subcellular distribution of 2-[I]iodomelatonin binding sites in the chicken spinal cord

Radioreceptor and autoradiography studies using chicken spinal cords demonstrated that the binding density of 2-[¹²⁵I]iodomelationin ([¹²⁵I]MEL) was significantly higher in the lumbar segment and the specific binding of [¹²⁵I]MEL was localized in the gray matter. Subcellularly, different densities of binding sites were localized in the following order: nuclear > microsomal > mitochondrial > cytosolic. Localization of [¹²⁵I]MEL binding sites in the dorsal gray matter of the chicken spinal cord suggests that melatonin plays a role in regulating the spinal cord functions which may associate with the modulation of temperature and pain transmission and/or visceral and autonomic functions.     

Anti-CD3 antibody-induced expression of both p55 and p75 chains of the high affinity interleukin-2 receptor on human T lymphocytes is inhibited by cyclosporin A.

The inhibitory effect of cyclosporin (CsA) was investigated on human lymphocytes stimulated by anti-T-cell antibodies (anti-CD3 and -CD2) or mitogenic lectins. Whereas inhibition of cell proliferation (50%) occurred at 10 ng/ml CsA after cell activation via CD3 or CD2, higher CsA concentrations (300 ng/ml) were necessary to inhibit lectin-mediated cell activation (PHA, Con A). Exogenous recombinant interleukin-2 (rIL-2) partially reversed the inhibitory effect on antibody-stimulated cells only; however, at higher CsA concentrations (300 ng/ml) proliferation was again inhibited. Thus, CsA affected IL-2R expression and/or function at higher concentrations (300 ng/ml). CsA had no effect on receptor function as measured on IL-2-dependent cell growth of CTLL cells or preactivated lymphocytes. However, CsA inhibited both high and low affinity receptor expression as shown by [125I]IL-2 equilibrium binding studies on anti-CD3-stimulated cells. Cross-linking studies revealed that both p55 (TAC) and p75 chains of the IL-2R were not induced at low CsA concentrations (10 ng/ml). However, addition of rIL-2 reversed CsA inhibition of IL-2R expression. It is concluded that CsA, at least in anti-CD3-stimulated cells, inhibits IL-2R expression and cell proliferation with similar potency. Exogenous rIL-2 reverses CsA inhibition of IL-2R expression. This might be due to binding of rIL-2 to receptors which escape CsA inhibition, thereby up-regulating receptor expression which is drug resistant.     

Calcium Uptake During Mitogenic Stimulation of Human Lymphocytes: Characterization of Intracellular Calcium Compartments and Demonstration of the Presence of Immunoreactive Calrecticulin

Phytohemagglutinin (PHA) stimulation of human peripheral blood lymphocytes (HPBL) rapidly increases ⁴⁵Ca²⁺ uptake into intracellular pools. Detectable increase in ⁴⁵Ca²⁺ uptake occurred only on exposure to mitogenic lectins but not with non-mitogenic lectins. However, intracellular free Ca²⁺ concentration [(Ca²⁺)i] increased comparably on exposure to either mitogenic or non-mitogenic lectins.

Permeabilization of ⁴⁵Ca²⁺ loaded cells revealed distinct pools of Ca²⁺ uptake. The highly digitonin sensitive pool #1 (permeabilized by 0.02% digitonin) exchanged slowly and included a part that represented endoplasmic reticulum. Pool II was defined by lower digitonin sensitivity, had a much faster initial uptake. Pool III was digitonin-resistant and predominantly non-vesicular. During the first 120 min of PHA stimulation, significant increase in ⁴⁵Ca²⁺ uptake occurred only into pool II. Progressive increase in uptake into pool I then occurred so that by 24 hours, this pool constituted the major fraction of PHA induced increment in total ⁴⁵Ca²⁺ uptake. Using specific antibody to the calcium binding protein calreticulin, an analogous immunoreactive protein was detectable in resting HPBL. PHA stimulation led to a striking increase in abundance of immunoreactive calreticulin so that 24 hrs after PHA stimulation, there was a 28 and 3.4 fold increase in the amount of immunoreactive calreticulin present in the non-particulate fraction and the total particulate membrane fraction, respectively. A major part (72%) of the total cellular immunoreactive calreticulin in PHA stimulated cells at 24 hrs was released into the medium after permeabilization of lymphocytes with 0.02% digitonin, corresponding to the location of calcium uptake pool I.     

Mechanism of action of maternal serum on the interleukin2 receptor expression.

Neoplastic Jurkat cells were submitted to a PHA stimulation test after a preincubation in maternal or nulliparous serum (10% dilution). The Il2R expression was significantly downregulated among maternal serum treated cells. Retroplacental serum was significantly more inhibitive than peripheral maternal serum (P less than 0.01). The maternal IgG fractions and mostly the retroplacental IgG fraction proved to contain a factor mainly responsible for the Il2R expression inhibitive property. The molecular mechanism of this phenomenon was further studied. It was shown that H7 (acting as a protein kinase inhibitor) could not influence the Il2R modulation. E.G.T.A., a calcium chelator, was not able to interfere with the inhibitive influence of maternal serum. It was suggested that the maternal serum mediated inhibition of the IL2R expression is not influenced by the hydrolysis of membrane bound phosphatidyl inositol. In contrast, pertussis toxin markedly enhanced, in a dose dependent way, the suppressive influence of maternal serum as compared to nulliparous serum. At low concentrations, pertussis toxin lost its stimulating property and retained its ability to ADP ribosylate the alpha subunit of G proteins inducing a release of adenylcyclase mediating cAMP synthesis. This mechanism has been further studied by the addition of dbc AMP or dbc GMP to Jurkat cells preparations stimulated by PHA. dbc AMP, in a dose-related way, induced a downregulation of the IL2R expression of stimulated neoplastic cells preincubated in nulliparous or maternal serum. dbc GMP did not influence the IL2R expression in the same experimental conditions. The maternal serum mediated cells showed the most pronounced IL2R inhibition. Finally, it was shown that the cAMP synthesis by PHA stimulated Jurkat cells was upregulated in a dose dependent way, after a previous cellular incubation in progressive concentrations of maternal serum. In contrast, among nulliparous serum pretreated cells, cAMP synthesis remained significantly lower, after a lectin stimulation, as compared to the cAMP production derived from retroplacental serum treated and stimulated cells. Taken together, these experiments suggest that the maternal serum dependent suppression of the IL2R expression is related to a protein G stimulation followed by an enhanced cAMP synthesis.     

Potent Inhibition of Interleukin 1β-Mediated Human Melanoma (A375.6) Lysis by Corticosteroids, Staurosporine, and Tilorone

The mechanism of human interleukin (IL)-1 β-mediated cytolysis was studied in a human melanoma cell line, A375.6. Purified recombinant human IL-1β produced 50% cytocidal activity at 50 pg/ml. A variety of compounds were tested for their ability to interfere with A375.6 lysis. Compounds were added simultaneously with IL-1β (100 pg/ml), and tumor cytolysis was measured after 72 hr of culture by release of ¹²⁵I from DNA of A375.6 cells labeled with [¹²⁵I]-dUrd. A variety of antiinflammatory/immunosuppressive agents (including auranofin, chloroquine, cyclosporin A, d-penicillamine) and several cyclo-oxygenase/lipoxygenase inhibitors (AA-861, BW755c, and indomethacin) lacked protective activity. Similarly, phospholipase inhibitors (mepacrine and 4-bromophenacyl bromide), putrescine, inhibitors of lysosomal activity (chloroquine and NH4CI), calcium channel blockers (nifedipine and verapamil), calmodulin inhibitors (W-7 and calmidazolium), and inhibitors of ADP ribosylation (nicotinamide and 3-aminobenzamide) were inactive. In contrast, corticosteroids (dexamethasone, hydrocortisone, and paramethasone acetate), tilorone, and protein kinase C inhibitors (1-[5-isoquinolinyl-sulfonyl]-2-methylpiperazine and stauro-sporine) significantly inhibited IL-1 β-mediated A375.6 cytolysis. These compounds also interfered with tumor necrosis factor-mediated lysis of A375.6, suggesting common mechanisms of tumor cytotoxicity by these monokines. This model may be useful for delineating intracellular biochemical events integral to IL-1 action.     

Four interleukin-2 surface binding proteins detected in rat spleen cells.

Four specific interleukin-2 (IL-2) surface binding proteins can be detected by covalent cross-linking of [125I]IL-2 to rat spleen cells that have been activated with various stimuli including concanavalin A (Con A), phytohaemagglutinin (PHA), calcium ionophore, and phorbol dibutyrate (PDB) with or without calcium ionophore. These four cross-linked proteins could not be demonstrated in either unstimulated T cells or in activated T cells when binding was performed in the presence of a 20-100-fold excess of unlabelled IL-2. The molecular weights of the four cross-linked proteins, after subtraction of the molecular weight contribution of IL-2 are: 53,000, 70,000, 90,000 and 118,000. The 53,000 MW protein was identified as the rat IL-2 receptor (IL-2R) alpha-chain by immune precipitation. Additionally, results suggest that the rat IL-2R alpha-chain is tightly complexed to both the 118,000 and 90,000 MW IL-2 binding proteins. Purification of surface labelled proteins from activated cells using IL-2 affinity chromatography yields four proteins with similar molecular weight to those identified by cross-linking plus an additional non-ligand cross-linked protein of 46,000 MW. The 46,000 MW band may be a non-binding associated protein since it was not seen following [125I]IL-2 binding cross-linking. Tryptic digests and two-dimensional separation of the affinity-isolated proteins indicate that unique peptide maps are generated for the 46,000, 53,000 and 70,000 MW proteins and excludes the possibility that the bands identified by cross-linking represents cross-linking of multiple ligands to the 53,000 MW subunit. However, the 90,000 and 118,000 MW bands yield peptide maps that closely resemble each other suggesting that these binding proteins may be related. These results suggest that at least four IL-2 surface binding proteins may constitute the rat IL-2R system.     

Distribution of angiotensin IV binding sites (AT4 receptor) in the human forebrain, midbrain and pons as visualised by in vitro receptor autoradiography

Angiotensin IV and other AT₄ receptor agonists, improve memory retention and retrieval in the passive avoidance and swim maze learning paradigms. Angiotensin IV binding sites (also known as the AT₄ receptors) are widely distributed in guinea pig and monkey (Macaca fascicularis) brains where high densities of the binding sites have been detected in the hippocampus, neocortex and motor nuclei. However, the distribution of the binding sites in the human brain is not known. We have recently localised the angiotensin IV binding sites (AT₄ receptors) in post-mortem human brain using iodinated Nle-angiotensin IV, a higher affinity and more stable analogue of angiotensin IV. This radioligand bound with relatively high affinity and specificity to angiotensin IV binding sites. In competition studies on consecutive sections through the prefrontal cortex and claustrum, angiotensin IV, Nle-angiotensin IV and LVV-hemorphin 7 competed for the binding of ¹²⁵I[Nle]-angiotensin IV with nanomolar affinities. Angiotensin II and the AT₁ and AT₂ receptor antagonists were ineffective in competing for the binding at concentrations of up to 10 μM. We found high densities of ¹²⁵I[Nle]-angiotensin IV binding sites throughout the cerebral cortex including the insular, entorhinal, prefrontal and cingulate cortices. Very high densities of the binding sites were observed in the claustrum, choroid plexus, hippocampus and pontine nucleus. Some thalamic nuclei displayed high densities of binding including the anteroprincipal, ventroanterior, anteromedial, medial dorsal and ventrolateral nuclei. The caudate nucleus, putamen, many amygdaloid nuclei and the red nucleus all displayed moderate densities of binding with a higher level detected in the substantia nigra pars compacta. In the hypothalamus, high densities binding sites were found in the ventromedial nucleus with lower levels in the dorsomedial and paraventricular nuclei. The distribution of ¹²⁵I[Nle]-angiotensin IV binding sites in the human brain is similar to that found in other species and supports multiple roles for the binding sites in the central nervous system, including facilitation of memory retention and retrieval.     

Soluble and membrane‐bound interleukin (IL)‐15 Rα/IL‐15 complexes mediate proliferation of high‐avidity central memory CD8+ T cells for adoptive immunotherapy of cancer and infections

The lack of persistence of infused T cells is a principal limitation of adoptive immunotherapy in man. Interleukin (IL)‐15 can sustain memory T cell expansion when presented in complex with IL‐15Rα (15Rα/15). We developed a novel in‐vitro system for generation of stable 15Rα/15 complexes. Immunologically quantifiable amounts of IL‐15 were obtained when both IL‐15Rα and IL‐15 genes were co‐transduced in NIH 3T3 fibroblast‐based artificial antigen‐presenting cells expressing human leucocyte antigen (HLA) A:0201, β₂ microglobulin, CD80, CD58 and CD54 [A2‐artificial antigen presenting cell (AAPC)] and a murine pro‐B cell line (Baf‐3) (A2‐AAPC^15Rα/15and Baf‐3^15Rα/15). Transduction of cells with IL‐15 alone resulted in only transient expression of IL‐15, with minimal amounts of immunologically detectable IL‐15. In comparison, cells transduced with IL‐15Rα alone (A2‐AAPC^Rα) demonstrated stable expression of IL‐15Rα; however, when loaded with soluble IL‐15 (sIL‐15), these cells sequestered 15Rα/15 intracellularly and also demonstrated minimal amounts of IL‐15. Human T cells stimulated in vitro against a viral antigen (CMVpp65) in the presence of 15Rα/15 generated superior yields of high‐avidity CMVpp65 epitope‐specific T cells [cytomegalovirus‐cytotoxic T lymphocytes (CMV‐CTLs)] responding to ≤ 10^{? 13} M peptide concentrations, and lysing targets cells at lower effector : target ratios (1 : 10 and 1 : 100), where sIL‐15, sIL‐2 or sIL‐7 CMV‐CTLs demonstrated minimal or no activity. Both soluble and surface presented 15Rα/15, but not sIL‐15, sustained in‐vitro expansion of CD62L⁺ and CCR7⁺ central memory phenotype CMV‐CTLs (T_CM). 15Rα/15 complexes represent a potent adjuvant for augmenting the efficacy of adoptive immunotherapy. Such cell‐bound or soluble 15Rα/15 complexes could be developed for use in combination immunotherapy approaches.     

Autoradiography of angiotensin-converting enzyme in fixed and unfixed rat brain using the specific enzyme inhibitor [I]351A or a polyclonal antibody and [I]staphylococcal protein A

Angiotensin-converting enzyme (ACE, kininase II, EC 3.4.15.1) was visualized in unfixed rat brain sections by autoradiography after incubation with a polyclonal goat anti-rabbit lung converting-enzyme antibody and [¹²⁵I]protein A. Paraformaldehyde fixation interfered with the recognition of ACE by its antibody in brain nuclei but not in the choroid plexus. Conversely, incubation of brain sections with the specific ACE inhibitor [¹²⁵I]351A allowed ACE visualization in either unfixed and paraformaldehyde-fixed brain sections, since [¹²⁵I]351A binding was not affected by our fixation conditions. Our results indicate that ACE could be visualized in unfixed brain sections directly by incubation with the specific inhibitor [¹²⁵I]351A or indirectly by radioimmunohistochemistry and autoradiography. Different autoradiographic methods could be used to visualize and quantify ACE in unfixed or fixed tissues.     

Interleukin 4 receptors on normal human B lymphocytes: characterization and regulation

Human interleukin 4 (IL 4) up-regulates the expression of CD23 on both resting and "in vivo" activated B cells but induces proliferation and/or differentiation only on "in vitro" activated B lymphocytes. Resting B cells express 360 high-affinity IL 4 receptors (R) per cell (Kd = 25-75 pM). Activation of resting B cells with anti-IgM antibody or Staphylococcus aureus Cowan I (SAC) results in a two-to-threefold increase of IL 4R number without alteration of IL 4R affinity for IL 4. Flow cytometric analysis of biotinylated IL 4 binding shows that IL 4R expression is up-regulated on virtually all anti-IgM-stimulated B cells, but only on a subpopulation of larger cells among SAC-activated B lymphocytes. Culturing cells for 40 h with optimal concentrations of IL 4 does not significantly affect IL 4R levels on resting and anti-IgM-preactivated B lymphocytes but triples IL 4R levels on SAC-preactivated B cells. Removal of IL 4 from cell cultures results in a two-to-fourfold increase of IL 4R levels 2 h later, suggesting an increase in IL 4R turnover. Resting and activated B cells degrade 125I-labeled IL 4 at 37 degrees C. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of IL 4 binding molecules on resting, "in vivo" activated and anti-IgM-activated B cells reveals the same three species of 130, 80-75, 70-65 kDa. Thus, IL 4 displays its different biological activities on resting and activated B cells through IL 4R of the same affinity, gross biochemical structure and ability to mediate IL 4 degradation.     

Retinal [125I]iodomelatonin binding sites in the tree shrew (Tupaiidae).

The characteristics of melatonin-binding sites labelled by [¹²⁵I]iodomelatonin in membrane preparations from the tree shrew retina were determined. Specific binding of [¹²⁵I]iodomelatonin to the membrane preparations of tree shrew retina was rapid, stable, saturable, and reversible. Among the indoles tested only 6-chloromelatonin, melatonin and N-acetylserotonin had significant affinities to the [¹²⁵I]iodomelatonin binding site. Scatchard analysis of the membrane preparations revealed a dissociation constant (K_d) of 51.0 ± 16 pM and total number of binding sites (B_max) of 1.97 ± 0.6 fmol/mg protein.     

The mouse high affinity IL 2 receptor complex. I. Evidence for a third molecule, the putative gamma-chain, associated with the alpha- and/or beta-chain of the receptor

Low and high affinity receptors for interleukin 2 were investigated on mitogen-activated mouse T lymphocytes and on interleukin 2 (IL2) receptor-bearing T cell lines by chemical crosslinking of 125I-labelled interleukin 2 (125I IL2) to its binding proteins or membrane proteins associated with the IL2 binding sites. In all cells investigated, the crosslinking produced a 65-70 kD complex thought to be one beta-chain molecule (55 kD IL2 receptor) and one IL2. Occasionally, a 120-130 kD band could be seen which is thought to be IL2 bound to beta-chain homodimer. Using low IL2 concentrations (200 pM), high affinity receptor-bearing cells showed specific additional complexes. Either bands of 85 kD, 105 kD and greater than 160 kD or of 90 kD and 115 kD and greater than 160 kD could be found when mouse T blasts or CTLL 16 cells were used, respectively. In the exclusively low affinity receptor-bearing T cell line Eb, only a single band of 70 kD (beta-chain + IL2) was detectable. These results show that in addition to a 85-90 kD complex, as shown already in humans, a third complex of 105-115 kD exists in mice that is specifically associated with high affinity receptors. This is the first indication of a putative gamma-chain of the high affinity IL2 receptor. All IL2-containing complexes of the mouse cells could be precipitated by the newly developed MAb AMT 45-20 raised against the beta-chain of the mouse IL2R and recognizing an epitope distinct from those recognized by the MAb AMT 13 or IL2. This suggests that either 1) all complexes contain the beta-chain, 2) share an antigenic determinant with the beta-chain, or 3) that the higher mw complexes become coprecipitated with the beta-chain.     

Increased serum levels of soluble interleukin 2 receptor in patients with drug-induced allergic hepatitis.

Soluble interleukin 2 receptor (sIL2R) level in the serum of 10 patients with drug-induced allergic hepatitis (DIAH) were quantified with a solid-phase enzyme immunoassay using two monoclonal antibodies against the receptor. The sIL2R levels in patients with DIAH were significantly higher than in 15 normal subjects (p less than 0.01). The high sIL2R levels observed during the florid stage of DIAH returned to normal during convalescence (p less than 0.05). SIL2R from the serum of DIAH patients was found to bind to a recombinant IL2 affinity column. It seems likely that sIL2R inhibits the hyper-immune reaction that occurs in drug allergies due to bind IL2. But, we have no data that sIL2R inhibits actually the biological activity of IL2.     

Interleukin 2 mRNA induction in human lymphocytes: analysis of the synergistic effect of a calcium ionophore A23187 and a phorbol ester

Induction of interleukin 2 (IL2) mRNA in human tonsillar lymphocytes under various conditions was examined by cytoplasmic dot hybridization using a 32P-labeled IL2 cDNA probe to study the signal transduction mechanisms which lead to IL2 gene expression. A tumor-promoting phorbol ester, 12-O-tetradecanoyl-phorbol 13-acetate (TPA), acted synergistically with a Ca2+ ionophore A23187 or phytohemagglutinin (PHA) to induce a high level of IL2 mRNA in lymphocytes, whereas each of them by itself could not induce the mRNA production. In two-step culture experiments the lymphocytes pulse-incubated with TPA for 1 h (the first culture) could efficiently initiate IL2 mRNA production by subsequent culture with A23187 or PHA (the second culture). Results obtained by removal of extracellular Ca2+ from either the first or second culture revealed that Ca2+ was not necessarily required during the first culture with TPA, but it is essential in the second culture with A23187 or PHA, regardless of the presence or absence of Ca2+ in the first culture. A reagent known to be a calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), almost completely inhibited the IL2 mRNA induction in A23187-TPA-stimulated lymphocytes at a concentration of 25 microM, whereas N-(6-aminohexyl)-1-naphthalenesulfonamide that has much lower affinity for calmodulin than W-7 did not inhibit at this concentration. The IL2 mRNA induction was also blocked by the addition of 50 microM of 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride which is known to block the release of Ca2+ from intracellular storage sites. These results show that mobilization of Ca2+ and the calmodulin-dependent regulatory system appear to work synergistically with TPA which probably activates protein kinase C in the pathway to IL2 gene expression.     

Distribution and postnatal ontogeny of adenosine A2A receptors in rat brain: comparison with dopamine receptors

In adult rat brain, adenosine A_2A receptors and dopamine D₂ receptors are known to be located on the same cells where they interact in an antagonistic manner. In the present study we wanted to examine when this situation develops and compared the postnatal ontogeny of the binding of the adenosine A_2A receptor agonist [³H]CGS 21680, the binding of the dopamine D₁ receptor antagonist [³H]SCH 23390 and the dopamine D₂ receptor antagonist [³H]raclopride.

All three radioligands bound to the striatum at birth and this binding increased several-fold during the postnatal period. [³H]SCH 23390 binding developed first (mostly during the first week), followed by [³H]raclopride binding (first to third week) and [³H]CGS 21680 binding (only during second and third week). For all three radioligands the binding tended to decrease between 21 days and adulthood. This occurred earlier and was more pronounced in the globus pallidus than in the other examined structures. The increase in [³H]CGS 21680 binding from newborn to adult was mainly due to four-fold increase in the number of binding sites. The pharmacology of [³H]CGS 21680 binding to caudate–putamen was similar in newborn, one-week-old and adult animals, and was indicative of A_2A receptors. The binding was inhibited by guanylyl imidodiphosphate at all ages, indicating that A_2A receptors are G-protein-coupled already at birth. In contrast to the large increase in [³H]CGS 21680 binding, there was a decrease in the levels of A_2A messenger RNA during the postnatal period in the caudate–putamen. In cerebral cortex [³H]CGS 21680 bound to a different site than the A_2A receptor. From birth to adulthood cortical binding of [³H]CGS 21680 increased four-fold and that of the adenosine A₁ agonist [³H]cyclohexyladenosine 19-fold. During early postnatal development [³H]SCH 23390 binding was higher in deep than in superficial cortical layers, but this difference disappeared in adult animals. There was binding of both [³H]CGS 21680 and [³H]cyclohexyladenosine to the olfactory bulb, suggesting a role of the two adenosine receptors in processing of olfactory information. [³H]CGS 21680 binding was present in the external plexiform layer and glomerular layer, and increased during development, but the density of binding sites was about one tenth of that seen in caudate–putamen. [³H]cyclohexyladenosine showed a very different labelling pattern, resembling that observed with [³H]SCH 23390.

Postnatal changes in adenosine receptors may explain age-dependent differences in stimulatory caffeine effects and endogenous protection against seizures. Since A_2A receptors show a co-distribution with D₂ receptors throughout development, caffeine may partly exert such actions by regulating the activity of D₂ receptor-containing striatopallidal neurons     

Two Distinct P70 Interleukin-2 Receptors on a Murine Large Granular Lymphocyte Clone Y479

A continuous cloned cell line (Y479) was established by culturing normal mouse spleen cells in a high concentration of interleukin-2 (IL-2). Y479 cells showed morphological characteristics of large granular lymphocyte with the phenotypes of Thy-1.2⁺. T3⁺, Lyt-1^-, Lyt-2^-, L3T4^-, B220^-, AsGM₁⁺, LFA-1⁺, and TcRVβ8^-. The Y479 cells required a high concentration of IL-2 for their growth but did not express detectable p55 IL-2 receptor (IL-2R) although they bound IL-2 with high and low affinities. Analysis of the IL-2 binding proteins on the Y479 cells revealed that both the high and low affinity receptors consisted only of 70 kDa protein. Analysis of the 70 kDa protein was performed using five monoclonal antibodies (L15, L20, 1,23, L34, and L61) against human recombinant IL-2. Although they recognized different epitopes, all monoclonal antibodies immunoprecipitated 70 kDa IL-2R that was cross-linked with radioiodinated IL-2. The supernatant after immunoprecipitation with L61 still contained IL-2/IL-2R complex that was L23-reactive, and the supernatant after immunoprecipitation with L23 contained L61-reactive IL-2/IL-2R complex, whereas L15 immunoprecipitated almost all the complex. Limited digestion of IL-2-cross-linked Y479 cells with trypsin caused the liberation of 45 kDa IL-2R fragment cross-linked with IL-2. This complex was immunoprecipitated by L15 or L61 but not by L23. These results suggest that there are at least two distinct 70 kDa IL-2R on the surface of Y479 cells.