Cyclic AMP-dependent modulation of interleukin-1 receptors in the mouse AtT-20 pituitary tumor cell line.

Previous studies have demonstrated an upregulation of interleukin-1 (IL-1) receptors following treatment of mouse AtT-20 pituitary tumor cells with corticotropin-releasing factor (CRF). In the present study, we determined the modulation of IL-1 receptors and adenylate cyclase activity in AtT-20 cultures following treatment with CRF, isoproterenol, forskolin, somatostatin and dexamethasone. CRF, isoproterenol and forskolin dose-dependently increased cAMP production and [125I]IL-1 alpha binding. In contrast, somatostatin and dexamethasone significantly inhibited CRF-stimulated cAMP production and decreased both basal and CRF-mediated increases in [125I]IL-1 alpha binding. Parallel modulation of IL-1 receptors by agents that stimulate (CRF, isoproterenol and forskolin) or inhibit (somatostatin) cAMP production in AtT-20 cells suggest the importance of this second messenger in regulating IL-1 receptors.

Role of interleukin-1 in endotoxin-induced lung injury in the rat.

The effects of the recombinant interleukin-1 receptor antagonist (rIL-1ra) on the systemic vascular and lung injury following intraperitoneal Salmonella enteritidis lipopolysaccharide (LPS) were determined in male Sprague-Dawley rats. Initial experiments identified that maximal mortality occurred with an intraperitoneal LPS dose of 20 mg/kg, and this dose was used in subsequent experiments. Albumin permeability, measured in an ex vivo perfused heart-lung preparation from the rats 2 h after injection of LPS, was increased with endotoxin as was the wet:dry weight ratio. Pretreatment of the rats with intravenous rIL-1ra, 1 to 10 mg/kg, followed by a continuous intravenous infusion at 30 to 50 micrograms/kg/min resulted in restoration of blood pressure at 100 min following endotoxin administration. Moreover, coadministration of rIL-1ra with endotoxin totally prevented the rise in albumin permeability of the pulmonary vasculature and the increase in wet:dry lung weight ratios observed in rats treated with LPS alone. LPS injected intraperitoneally caused a marked decrease in circulating leukocyte count, an effect not reversed by rIL-1ra. RNA extraction of whole-lung homogenates revealed that mRNA for IL-1 beta was constitutively expressed in the absence of endotoxin, but transcripts increased progressively from 0.5 to 2 h after endotoxin administration. Increases in mRNAs for tumor necrosis factor-alpha (TNF-alpha) and for macrophage inflammatory protein-2 (MIP-2), a potent neutrophil chemoattractant, were also observed from 0.5 until 2 h after endotoxin administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Treatment with an interleukin-1 receptor antagonist protein prolongs mouse islet allograft survival.

An interleukin-1 receptor antagonist protein was evaluated with regard to its efficacy in allogeneic and xenogeneic islet transplantation. Alloxan-induced diabetic C57BL/6 (H-2b) mice were transplanted under the kidney capsule with 500 C57BL/Ks (H-2d) mouse islets. Alzet osmotic pumps, which release their content over an 11- to 13-day period, were implanted subcutaneously for continuous infusion of interleukin-1 receptor antagonist protein (1.0, 5.0, 8.0 mg.kg-1 x day-1) or phosphate-buffered saline. Blood glucose determinations were performed every second or third day; at death, the islet-bearing kidneys were morphologically evaluated. Mice treated initially with the higher interleukin-1 receptor antagonist protein concentrations were followed for an additional period after cessation of the drug release to evaluate whether a transitory interleukin-1 receptor antagonist protein treatment would induce tolerance to the graft. All phosphate-buffered saline-treated mice were hyperglycemic 11 days after islet allotransplantation. Most of their grafts were heavily infiltrated with mononuclear cells. In the various interleukin-1 receptor antagonist protein-treated groups, 60-80% of the mice were normoglycemic after 11 days. Moreover, light microscopic examinations showed that most mice treated with interleukin-1 receptor antagonist protein had normal islet grafts or grafts infiltrated with only a few mononuclear cells. After interruption of interleukin-1 receptor antagonist protein infusion (8.0 mg.kg-1 x day-1), all animals developed hyperglycemia within 2-9 days. In xenogeneic experiments, 500-750 fetal porcine islet-like cell clusters were transplanted under the kidney capsule of normoglycemic C57BL/6 mice. These animals were treated either with interleukin-1 receptor antagonist protein (8.0 mg.kg-1 x day-1) or phosphate-buffered saline.(ABSTRACT TRUNCATED AT 250 WORDS)

IL-1 receptor antagonist protein production and gene expression in rheumatoid arthritis and osteoarthritis synovium.

IL-1 can participate in the perpetuation of arthritis through direct stimulation of synoviocytes and augmentation of matrix degradation. Hence, local production of the IL-1R antagonist protein (IRAP) might be an important negative feedback signal that regulates synovitis. We assessed synovial IRAP production in synovia from 30 individuals, by using a specific mAb and the immunoperoxidase staining method. IRAP was detected in 11 of 12 rheumatoid arthritis (RA) synovial tissues (ST) and was located primarily in the sublining, particularly in perivascular regions enriched for macrophages. Some staining was observed in the intimal lining of the synovium, although this was significantly less than in the sublining (p less than 0.05). Nine of 12 osteoarthritis (OA) tissues were positive for IRAP. In contrast to RA, the staining was observed primarily in the synovial lining in OA, with only minimal sublining IRAP being detected. Synovia from four patients without arthritis were negative (three autopsy specimens and one post-traumatic sample). Of the other two patients with miscellaneous diagnoses, one sample was negative (tenosynovitis) and one was positive (seronegative inflammatory arthritis) (sublining). Studies of serial sections and double-immunostaining experiments indicated that macrophages are the major cells containing immunoreactive IRAP. IRAP gene expression in vivo was determined by performing in situ hybridization on ST from 17 arthritis patients. RNA sense IRAP probes did not hybridize to any tissues. Anti-sense IRAP probes bound to two of nine RA tissues, two of six OA tissues, one of one seronegative inflammatory arthropathy tissue, and none of one flexor tenosynovitis tissue. As with immunoreactive protein, IRAP mRNA was primarily localized to cells in the synovial lining in OA but was more prominent in perivascular lymphoid aggregates in RA and seronegative inflammatory arthropathy. Northern blot analysis was performed on RNA isolated from nine ST. The appropriately sized IRAP band was identified in six of nine samples (five of six RA and one of three OA). Supernatants from cultured RA and OA ST cells contained immunoreactive and biologically active IRAP. Hence, IRAP gene expression and protein production occur in RA and OA synovium, albeit in different distributions.

Distribution of type I interleukin-1 receptor messenger RNA in testis: an in situ histochemical study in the mouse.

The cytokine interleukin-1 (IL-1) has been reported to inhibit the hypothalamic-pituitary-gonadal axis, both through actions in brain and at the gonadal level. Recently, high affinity binding sites for 125I-recombinant human IL-1 alpha have been identified in the mouse testis with characteristics similar to those of type I IL-1 receptors on T lymphocytes and fibroblasts. The present study employed in situ hybridization histochemistry with 35S-labeled antisense cRNA probes derived from a murine type I IL-1 receptor cDNA to identify type I IL-1 receptor mRNA in the mouse testis. An intense signal was observed over interstitial cells, and over the cytoplasm of the epithelium of epididymal ducts, most prominently in the head region. The signal over seminiferous tubules, and over sperm cells within tubules and epididymal ducts, was comparable to background. This distribution of type I IL-1 receptor mRNA was similar to that recently reported for 125(I)I-IL-1-alpha binding sites, and supports evidence implicating IL-1 as a direct regulator of gonadal function.

Interleukin-1 receptor antagonist protein inhibits interleukin-8 expression in lipopolysaccharide-stimulated human whole blood.

Interleukin-8 (IL-8) is a neutrophil and lymphocyte chemoattractant and activator that may play an important role in mediating events at sites of inflammation. IL-8 is produced by many cell types in response to a variety of inducers, including interleukin-1 (IL-1). Studies were conducted to address the question of whether an inhibitor of IL-1 action, IL-1 receptor antagonist protein (IRAP), would suppress IL-8 production. Lipopolysaccharide (LPS)-stimulated human whole blood was used as an ex vivo model of local cytokine production. Preliminary time course studies showed that plasma IL-1 beta levels were fully induced by 6 hours (approximately 15 ng/ml) and persisted at this level over 24 hours. IL-8 production, in contrast, reached a plateau between 6 to 12 hours (5 ng/ml) and then increased rapidly to 17 ng/ml at 24 hours. Addition of IRAP was found to dose-dependently inhibit IL-8 expression at the levels of both protein and mRNA. A 50% reduction in IL-8 protein release occurred at an IRAP dose of 8 micrograms/ml (5 x 10(-7) mol/l) at 24 hours. A 2 hour delay in the addition of IRAP relative to LPS still permitted optimal reduction in IL-8 release, whereas delays of 4-8 hours reduced or eliminated the inhibitory effect. IRAP was found to have no effect on the LPS-stimulated production of IL-1 alpha or IL-1 beta. In addition, experiments performed with isolated peripheral blood cells demonstrated that, whereas monocytes were the major producers of IL-8, IRAP was equally effective in reducing IL-8 production in neutrophil and mononuclear cell suspensions. These studies further document the role of IL-1 in inducing the production of IL-8 and indicate that the ability of IRAP to suppress IL-8 expression may be an important mechanism of the anti-inflammatory properties of this molecule.

In situ histochemical localization of type I interleukin-1 receptor messenger RNA in the central nervous system, pituitary, and adrenal gland of the mouse.

The cytokine interleukin-1 (IL-1) has a number of biologic activities, including pronounced effects on the nervous and neuroendocrine systems. In this study, in situ histochemical techniques were used to investigate the distribution of cells expressing type I IL-1 receptor mRNA in the CNS, pituitary, and adrenal gland of the mouse. Hybridization of 35S-labeled antisense cRNA probes derived from a murine T-cell IL-1 receptor cDNA revealed a distinct regional distribution of the type I IL-1 receptor, both in brain and in the pituitary gland. In the brain, an intense signal was observed over the granule cell layer of the dentate gyrus, over the entire midline raphe system, over the choroid plexus, and over endothelial cells of postcapillary venules throughout the neuraxis. A weak to moderate signal was observed over the pyramidal cell layer of the hilus and CA3 region of the hippocampus, over the anterodorsal thalamic nucleus, over Purkinje cells of the cerebellar cortex, and in scattered clusters over the external-most layer of the median eminence. In the pituitary gland, a dense and homogeneously distributed signal was observed over the entire anterior lobe. No autoradiographic signal above background was observed over the posterior and intermediate lobes of the pituitary, or over the adrenal gland. This study therefore provides evidence for discrete receptor substrates subserving the central effects of IL-1, thus supporting the notion that IL-1 acts as a neurotransmitter/neuromodulator in brain. It also supports studies suggesting that IL-1-mediated activation of the hypothalamic-pituitary-adrenal axis occurs primarily at the level of the brain and/or pituitary gland.

Immunohistochemical demonstration of interleukin-1 receptor antagonist protein and interleukin-1 in human lymphoid tissue and granulomas.

Human interleukin 1 receptor antagonist protein (IRAP) is a specific antagonist of interleukin 1 (IL-1) action in both in vitro and in vivo experimental models. Presently, the significance of this protein in human pathophysiology is unknown. In the present study, monoclonal antibodies against IRAP were prepared and used to demonstrate IRAP expression in human tissues, immunohistochemically. Specifically, this study focused on lymphoid tissues and granulomatous inflammatory reactions since IL-1 is believed to play roles in lymphocyte development and inflammation. In addition, these tissues were also stained for IL-1 beta to compare the expression of agonist and antagonist. These findings indicate that IRAP expression is largely limited to macrophages and their derivatives. Strong IRAP expression was observed in germinal center macrophages of lymph nodes, spleen, and tonsil. In contrast, IL-1 was marginally expressed in these organs. Granulomas associated with active M. tuberculosis infection, sarcoidosis and foreign bodies all contained strongly IRAP positive cells, which included macrophages, epithelioid cells and multinucleate giant cells. Unlike reactive lymphoid tissue, tuberculous and sarcoid granulomas also contained IL-1 positive cells which included macrophages and their derivatives, as well as some stromal cells. Foreign body lesions showed minimal IL-1 expression. Interestingly, granulomas in patients with acquired immunodeficiency disease (AIDS) associated with M. avium-intracellulare contained IRAP positive cells but were negative for IL-1 expression. Taken together, these findings suggest that IRAP takes part in both physiologic and pathologic reactions. Moreover, they provide a basis to design future studies to determine the precise contribution of IRAP to these reactions.

Role of receptor binding and gene transcription for both the stimulatory and inhibitory effects of interleukin-1 in pancreatic beta-cells.

A brief exposure of pancreatic islets to the cytokine interleukin-1 beta (IL-1 beta) induces an initial stimulatory phase, which is followed by inhibition of islet function and eventually beta-cell damage. In the present study we have investigated the effects of IRAP, a blocker of type I IL-1 receptor and actinomycin D, an inhibitor of DNA transcription, on both the stimulatory and inhibitory effects of IL-1 beta on rat pancreatic islets in vitro. The two test agents counteracted the initial stimulatory actions of IL-1 beta on both islet glucose-induced insulin release and glucose oxidation rates. Furthermore, cycloheximide, an inhibitor of protein synthesis, could also prevent the early IL-1 beta-induced stimulation of insulin release. When islets were exposed for 1 hr to IL-1 beta and studied after 12 hr, there was a 75% inhibition of glucose induced insulin release, a 50% decrease in glucose oxidation rates and a 30% decrease in (pro)insulin biosynthesis. These effects were completely counteracted by coincubation with IRAP or actinomycin D, but were not affected by coincubation with pertussis toxin. Islet exposure to IL-1 alpha also induced a 60-80% inhibition of glucose-induced insulin release after 12 hr. As observed with rIL-1 beta, IRAP was also able to block the suppressive effects of IL-1 alpha on islet function. Mouse islets exposed for 2 hr to IL-1 beta and studied after 12 hr presented a 50% decrease in the glucose-induced insulin release. This effect was completely blocked by coincubation with a rat monoclonal antibody generated against the type I mouse IL-1 receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Upregulation of interleukin-1 receptors in mouse AtT-20 pituitary tumor cells following treatment with corticotropin-releasing factor.

Treatment of AtT-20 cell cultures with increasing concentrations of rat/human corticotropin-releasing factor (r/hCRF) for 24 h resulted in a dose-dependent 2-3 fold increase in specific 125I-labelled recombinant human IL-1 alpha (125I-IL-1 alpha) binding that was paralleled by a 70-80% decrease in 125I-Tyro-ovine CRF binding. Saturation analysis of 125I-IL-1 alpha binding in control and CRF-treated cultures indicated that CRF produced an increase in the density (Bmax) of IL-1 receptors without altering their affinity (KD). The CRF-induced upregulation of IL-1 receptors appears to be mediated through specific membrane receptors for CRF since the CRF receptor antagonist, alpha-helical oCRF (9-41), blocked the CRF-induced upregulation of IL-1 receptors without producing any effect on 125I-IL-1 alpha binding by itself. In summary, these data demonstrate complex interactions between CRF and IL-1 at the pituitary level and identify potential novel mechanisms for cytokines to alter neuroendocrine function.

Cloning, heterologous expression and characterization of murine interleukin 1 receptor antagonist protein.

A cDNA coding for the human interleukin 1 receptor antagonist protein (IL 1Ra) was used to clone the corresponding murine cDNA. The nucleotide sequence of the open reading frame coding for the processed form of mIL 1Ra predicted a 152-residue protein that was 77% identical to human IL 1Ra. The cellular and tissue distribution of murine IL 1Ra (mIL 1Ra) transcripts showed high levels in macrophages and skin while lower levels were detected in tissues that contain significant numbers of resident macrophages. The portion of the mIL 1Ra cDNA that codes for the mature form of the protein was placed under the control of a Trp promoter and expressed in E. coli at a level of 37% of total cell protein. The expressed protein was secreted into the periplasm and was purified to homogeneity in a single step by cation-exchange chromatography. Recombinant mIL 1Ra competitively inhibited 125I-labeled IL 1 alpha binding to murine type I IL 1R present on EL4 6.1 cells (Ki value of 0.21 nM) and antagonized IL 1-stimulated co-mitogenesis in murine thymocytes (0.7 x 10(6)-1.1 x 10(6) units/mg).

An interleukin-1 receptor antagonist protein protects insulin-producing beta cells against suppressive effects of interleukin-1 beta.

The cytokine interleukin-1 beta may have an important role in the autoimmune mediated damage of pancreatic Beta cells in insulin-dependent diabetes mellitus. In the present study we have investigated the effects of an interleukin-1 receptor antagonist protein, a blocker of the type I interleukin-1 receptor, on the suppressive actions of recombinant interleukin-1 beta on insulin-producing cells. Brief exposure (1-2 h) of rat and mouse pancreatic islets to 10 ng/ml recombinant interleukin-1 beta induced an 70-80% inhibition of insulin response to glucose after 12 h. These effects were completely counteracted by co-incubation with 100 ng/ml interleukin-1 receptor antagonist protein. When rat islets were cultured for 48 h in the presence of recombinant interleukin-1 beta (5 ng/ml) higher concentrations of interleukin-1 receptor antagonist protein (5000 ng/ml) were required to protect Beta-cell function. Interleukin-1 receptor antagonist protein also counteracted the inhibitory effects of recombinant interleukin-1 beta on the growth of the rat insulinoma cell line RINm5F. These data suggest that interleukin-1 receptor antagonist protein can protect insulin-producing cells from the deleterious effects of recombinant interleukin-1 beta, and that these cells possess type I interleukin-1 receptors.

Localization of interleukin-1 receptor messenger RNA in murine hippocampus.

The cytokine interleukin-1 (IL-1) has numerous actions in brain, including pronounced neuroendocrine effects. Recent radioligand binding studies have identified high-affinity binding sites for 125I-recombinant human IL-1 alpha in the hippocampus with characteristics similar to those of IL-1 receptors in immune cells. The present study employed in situ hybridization histochemistry with 35S-labeled anti-sense cRNA probes derived from a full-length murine T-cell IL-1 receptor cDNA to identify cells producing IL-1 receptor mRNA in the murine hippocampus. An intense signal was observed over granule cells in the dentate gyrus. A weak to moderate signal was observed over the pyramidal cell layer of the hilus and CA3 region. Other aspects of the hippocampal formation, including the CA2 and CA1 regions, the subiculum, and the entorhinal area, displayed no signal above background. This distribution of IL-1 receptor mRNA was similar to that of 125I-IL-1 alpha binding sites and supports the growing body of evidence implicating IL-1 as a neurotransmitter/neuromodulator in brain.

Gastric cytoprotection by intracisternal interleukin-1 beta in the rat.

The effects of intracisternal (ic) injection of recombinant interleukin-1 beta (IL-1) on absolute ethanol-induced gastric necrotic lesions were studied in conscious rats. IL-1 given ic inhibited ethanol-induced gastric lesions. The cytoprotective effect was dose dependent (ED175 ng/rat), long lasting with a maximal action when given 1-3 h prior to ethanol, blocked by ic injection of a IL-1 receptor antagonist protein (IRAP), and by intraperitoneal injection of indomethacin. IL-1, injected ic, was detected in the peripheral blood. However, IL-1 serum levels were lower after IL-1 injection ic than after ip at a dose giving equal gastric protection. These data show that ic IL-1 induces long lasting gastric protection mediated by interaction with IL-1 receptors and prostaglandin pathways at central and/or peripheral sites that remain to be localized.

Interleukin-1 receptors in mouse brain: characterization and neuronal localization.

The cytokine interleukin-1 (IL-1) has a variety of effects in brain, including induction of fever, alteration of slow wave sleep, and alteration of neuroendocrine activity. To examine the potential sites of action of IL-1 in brain, we used iodine-125-labeled recombinant human interleukin-1 [( 125I]IL-1) to identify and characterize IL-1 receptors in crude membrane preparations of mouse (C57BL/6) hippocampus and to study the distribution of IL-1-binding sites in brain using autoradiography. In preliminary homogenate binding and autoradiographic studies, [125I]IL-1 alpha showed significantly higher specific binding than [125I]IL-1 beta. Thus, [125I]IL-1 alpha was used in all subsequent assays. The binding of [125I]IL-1 alpha was linear over a broad range of membrane protein concentrations, saturable, reversible, and of high affinity, with an equilibrium dissociation constant value of 114 +/- 35 pM and a maximum number of binding sites of 2.5 +/- 0.4 fmol/mg protein. In competition studies, recombinant human IL-1 alpha, recombinant human IL-1 beta, and a weak IL-1 beta analog. IL-1 beta +, inhibited [125I]IL-1 alpha binding to mouse hippocampus in parallel with their relative bioactivities in the T-cell comitogenesis assay, with inhibitory binding affinity constants of 55 +/- 18, 76 +/- 20, and 2940 +/- 742 pM, respectively; rat/human CRF and human tumor necrosis factor showed no effect on [125I]IL-1 alpha binding. Autoradiographic localization studies revealed very low densities of [125I]IL-1 alpha-binding sites throughout the brain, with highest densities present in the molecular and granular layers of the dentate gyrus of the hippocampus and in the choroid plexus. Quinolinic acid lesion studies demonstrated that the [125I]IL-1 alpha-binding sites in the hippocampus were localized to intrinsic neurons. After hypophysectomy, homogenate binding and autoradiographic studies showed that there was no apparent change in the relative density of IL-1 receptors in the hippocampus. The identification of IL-1 receptors in brain with characteristics similar to IL-1 receptors in immune and neuroendocrine tissues provides further support for a physiological role for IL-1 to regulate central nervous system activity.

Identification of interleukin-1 receptors in mouse testis.

The cytokine interleukin-1 (IL-1) has been reported to alter reproductive functions through both effects in brain and direct actions at the level of the gonads. To further define the role of IL-1 at the gonads, we have used 125I-labeled recombinant human IL-1 to identify and characterize IL-1 receptors in crude membrane preparations of mouse (C57BL/6) testis and to study the distribution of IL-1-binding sites using autoradiography. In preliminary homogenate binding and autoradiographic studies, [125I]IL-1 alpha showed significantly higher specific binding than [125I]IL-1 beta. Thus, [125I]IL-1 alpha was used in all subsequent assays. The binding of [125I]IL-1 alpha was linear over a broad range of membrane protein concentrations, saturable, and reversible, and on Scatchard analysis revealed an equilibrium dissociation constant (Kd) of 82 +/- 4 pM and a maximum number of binding sites (Bmax) of 10.8 +/- 1.5 fmol/mg protein. In competition studies, IL-1 alpha, IL-1 beta, and a weak IL-1 beta analog IL-1 beta+ inhibited [125I]IL-1 alpha binding to mouse testis in parallel with their relative bioactivities in immune assays, with inhibitory binding affinity constant (Ki) values of 14.2 +/- 1.7, 88.8 +/- 5.7, and 7183.3 +/- 603 pM, respectively; rat/human CRF and human tumor necrosis factor showed no effect on [125I]IL-1 alpha binding. In autoradiographic studies, IL-1 receptors were heterogeneously distributed, with highest densities present in the luminal border of the epididymis and interstitial areas of the testis. After hypophysectomy, the testes were significantly atrophied, and homogenate binding and autoradiographic studies showed that while the total number of binding sites per testis was significantly decreased in hypophysectomized mice in proportion to the reduction in testicular mass, there was no apparent change in the relative density of IL-1 receptors. These data provide the first identification of IL-1 receptors in testis and provide further support for a physiological role for IL-1 to alter reproductive functions through a direct effect at the gonads.

Corticotropin-releasing factor receptors in mouse spleen: identification of receptor-bearing cells as resident macrophages.

CRF is a primary integrator of the organism's coordinated neuroendocrine, autonomic, behavioral, and immune responses to stress. In the present study the identity of the cell type(s) expressing CRF receptors in mouse spleen was determined using a combination of cell fractionation and receptor-binding techniques. Autoradiographic studies of the distribution of [125I]Tyro-ovine CRF [( 125I]oCRF)-binding sites in spleen localized CRF receptors primarily to the red pulp and marginal zones. The distribution pattern of [125I]oCRF-binding sites closely resembled the pattern of India ink accumulated in phagocytic cells in the same sections. To identify the specific cell type(s) expressing CRF receptors, [125I]oCRF-binding activity was evaluated in splenic cell populations fractionated on the basis of their physical and functional properties. Macrophages were identified in each fraction by their phagocytosis of polystyrene beads and membrane labeling with MONTS-4, a monoclonal antibody specific for resident macrophages. Spleen cells were fractionated by adherence to glass bead or Sephadex G-10 columns, phagocytosis of carbonyl iron particles, and centrifugation on discontinuous Percoll gradients. By all fractionation methods, there was a significant correlation of [125I]oCRF binding with both phagocytic activity (r = 0.75; P less than 0.001) and MONTS-4 staining (r = 0.84; P less than 0.001), strongly suggesting that CRF receptors are primarily expressed on resident splenic macrophages. However, there was essentially no specific binding of [125I]oCRF to either resident or elicited peritoneal macrophages or to several monocyte/macrophage, B-cell, or T-cell lines. While these results suggest that the expression of CRF receptors may be restricted to a population of splenic macrophages, they do not exclude the possibility that CRF receptors may be induced on resident macrophages in spleen and other immune system-related tissues by factors present in the microenvironment.

Corticotropin-releasing factor (CRF) and interleukin-1 (IL-1) receptors in the brain-pituitary-immune axis.

Corticotropin-releasing factor (CRF) and interleukin-1 (IL-1) are two major components of the brain-endocrine-immune response to stress. We identified, characterized, and localized CRF and IL-1 receptors in brain, pituitary, and spleen using 125I-CRF and 125I-IL-1 alpha, respectively. 125I-CRF binding had comparable kinetic (KD:200-400 pM) and pharmacological characteristics in brain, pituitary, and spleen. In studies using cross-linking techniques, 125I-CRF was incorporated in rat pituitary and mouse spleen homogenates into a complex of Mr = 75,000 and in rat brain, into a complex of Mr = 58,000. The differences observed in the molecular weights between the pituitary and splenic vs. brain CRF receptors were evident across a variety of species and appeared to be due to differential glycosylation of the receptor proteins. In autoradiographic studies, CRF receptors were localized in highest densities in anterior pituitary and in brain regions involved in cognitive function, in limbic areas involved in emotion, and in brain areas regulating autonomic and other stress-related responses. In spleen, CRF binding sites were localized in the macrophage-rich red pulp and marginal zone surrounding the white pulp regions. High affinity 125I-IL-1 alpha binding sites were identified in pituitary membranes with kinetic and pharmacological characteristics comparable to the well-characterized IL-1 receptors in the EL-4 6.1 mouse thymoma cell line. Preliminary studies have identified IL-1 receptors in discrete regions of mouse brain. These data further substantiate the physiological role of CRF and IL-1 in modulating the brain-endocrine-immune axis.

Pharmacological inhibition of interleukin-1 activity on T cells by hydrocortisone, cyclosporine, prostaglandins, and cyclic nucleotides.

The effects of a panel of hormones and pharmacological agents on the activation of T cells by a combination of interleukin-1 and phytohemagglutinin (IL-1/PHA) was studied. Pharmacological effects on various stages of IL-1/PHA-induced interleukin-2 (IL-2) production by the cloned murine thymoma cell line LBRM-33-1A5.7 were dissected using a multi-step assay procedure. A 4-h lag phase in the kinetics of IL-2 production allowed the operational definition of an early, IL-1-dependent programming stage, followed by an IL-2-production stage of the assay. A cell-washing procedure between these stages was introduced in order to distinguish IL-1 receptor antagonists from functional IL-1/PHA antagonists. Hydrocortisone and cyclosporine were potent inhibitors (active in the nM range) of both stages of IL-2 production, suggesting that neither is an IL-1 receptor antagonist. The cyclic adenosine monophosphate (cAMP)-elevating agents prostaglandin E2, dibutyryl cAMP, and theophylline inhibited IL-2 production during the early, IL-1-dependent programming stage. By contrast, prostaglandin F2 alpha and dibutyryl cyclic guanosine monophosphate did not appreciably inhibit IL-1/PHA activity. These results are discussed in relationship to the effects of these test agents in thymocyte IL-1 assays or mitogenesis assays and the implications toward understanding the mechanisms underlying IL-1/PHA activation of T cells.

Crystallization of purified recombinant human interleukin-1 beta.

The gene for human interleukin-1 beta was cloned from SK-hep-1 hepatoma cellular RNA and expressed at high levels in Escherichia coli both as the naturally processed form (rIL-1 beta) and as a variant with an additional sequence of three amino acids on the N-terminus (rIL-1 beta +). Expressed protein was purified to homogeneity by a sequence of steps, which included low pH incubation, adsorption and desorption from Procion Red Sepharose, sizing on a Superose 12 fast-performance liquid chromatography (FPLC) column, and anion exchange chromatography on QAE Sepharose. The final step provided a biologically active protein that migrates on two-dimensional (2-D) gels as a single spot with a pI of 6.7 +/- 0.2 and a molecular mass of 17,500 daltons. Concentrated solutions of rIL-1 beta have produced crystals by ammonium sulfate precipitation. The crystals are tetragonal, show the symmetry of space group P4(1) or its enantiomer, have lattice constants of a = 58.46 (1) and c = 77.02 (3) A, and scatter to at least 2 A resolution. A structure determination based on these crystals is under way.