Biological activity and receptor binding of human prointerleukin-1 beta and subpeptides

We report here that the human interleukin-1 beta precursor (proIL-1 beta) protein as well as several interleukin-1 beta (IL-1 beta) subpeptides bind cellular receptors specifically and exhibit biological activity by stimulating proliferation of helper T-cells. IL-1 beta polypeptides have been synthesized by in vitro translation of mRNAs transcribed from plasmid vectors containing the bacteriophage SP6 promoter joined to the complete IL-1 beta cDNA or to deletion constructs. The quantity of IL-1 beta in vitro translation products was increased significantly by replacing the cognate IL-1 beta untranslated leader sequence with a 37-nucleotide plant viral untranslated leader. Translation of chimeric mRNAs followed by direct bioactivity assay demonstrated that mature IL-1 beta-(117-269), proIL-1 beta-(1-269), and peptide IL-1-(71-269) were all biologically active. Specific binding to cellular receptors was observed with these three IL-1 beta molecules; moreover, several peptides with minimal biological activity also bound receptor specifically. The biological activity and receptor binding properties of the IL-1 beta proteins reported here contrast with those described by Mosley et al. (Mosley, B., Urdal, D. L., Prickett, K. S., Larsen, A., Cosman, D., Conlon, P. J., Gillis, S., and Dower, S. K. (1987) J. Biol. Chem. 262, 2941-2944; Mosley, B., Dower, S. K., Gillis, S., and Cosman, D. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 4572-4576), who reported that proIL-1 beta-(1-269) had no biological activity and does not bind receptor. Our results indicate that proIL-1 beta is active at a relatively high concentration, and analysis of the proIL-1 beta-(1-269) and IL-1-(71-269) bioactivity data suggests a possible relationship with membrane-bound IL-1.     

The role of interleukin 1 in human B cell activation: inhibition of B cell proliferation and the generation of immunoglobulin-secreting cells by an antibody against human leukocytic pyrogen

The role of factors released by monocytes (M phi) in the activation of human B lymphocytes was examined by studying the effect of an antiserum against human leukocytic pyrogen (LP) on mitogen-stimulated B cell proliferation and the generation of immunoglobulin-secreting cells (ISC) by peripheral blood mononuclear cells (PBM). Antiserum against LP was obtained from rabbits immunized with LP-containing human M phi supernatants. The globulin fraction of this antiserum inhibited pokeweed mitogen- (PWM) stimulated B cell proliferation and the generation of ISC in a concentration-dependent manner, with 50% inhibition of responsiveness observed with 10 micrograms/ml. By contrast, PWM-induced T cell [3H]thymidine incorporation was not inhibited by concentrations of anti-LP as great as 2000 micrograms/ml. The F(ab')2 fraction of anti-LP also inhibited the generation of ISC in response to both PWM and formalinized Staphylococcus aureus, but required 50 micrograms/ml to achieve 50% inhibition. Anti-LP inhibited the generation of ISC only if present during the first 24 hr of a 6 to 7-day incubation; later addition was not inhibitory. Inhibition was more marked in cultures partially depleted of M phi than in whole PBM cultures. Whereas absorption of the anti-LP with PBM failed to remove the capacity to inhibit the generation of ISC, anti-LP-mediated inhibition of responsiveness could be reversed by the addition of crude M phi culture supernatants or a variety of highly purified interleukin 1 (IL 1) preparations, but not by T cell supernatants. These results indicate anti-LP inhibits human B cell activation by removing the requisite M phi-derived factor IL 1 and also confirm that IL 1 plays an essential role in B cell proliferation and the generation of ISC in man.     

Multiple amino acid substitutions suggest a structural basis for the separation of biological activity and receptor binding in a mutant interleukin-1 beta protein.

Receptor binding and biological activity properties of human interleukin-1 beta can be dissociated by mutating a single amino acid, arginine 127, to glycine (IL-1 beta R----G) [Gehrke et al. (1990) J. Biol. Chem. 265, 5922-5925]. The mechanism underlying the reduced biological activity has been examined by replacing arginine 127 with several other amino acids, followed by determination of biological activity using a T-helper cell proliferation assay. Mutant IL-1 beta proteins containing lysine, glutamic acid, tryptophan, or alanine in place of arginine 127 maintain biological activity. These data strongly suggest that IL-1 beta biological activity is not directly dependent upon the specific properties of charge, hydrophobicity/hydrophilicity, or side-chain group presented by the residue at position 127. Molecular modeling analyses indicate that the structural integrity of the antiparallel beta-strand 1/12 pair is disturbed in the glycine 127 mutant protein. Collapse of beta-strand 1 into a hydrated space between strands 1, 2, and 4 could structurally alter a cleft in IL-1 beta that contains a cluster of highly conserved amino acids, including a key aspartic acid residue [Ju et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 2658-2662]. Mutagenesis data and the differential activities of the IL-1 beta R----G and IL-1 receptor antagonist proteins in stimulating early and late gene expression [Conca et al. (1991) J. Biol. Chem. 266, 16265-16268] suggest that multiple receptor-ligand contacts, exclusive of those required for receptor binding, are required for the stimulation of full IL-1 biological activity.     

IgE-dependent cytokine production by human peripheral blood mononuclear phagocytes.

These studies demonstrate the IgE-dependent production of IL-1 beta and TNF-alpha by circulating blood monocytes. IL-1 beta production was demonstrated biologically as the stimulation of proliferation of the cloned IL-1-dependent murine T cell line D10.G4.1 in the presence of a submitogenic concentration of PHA. In a representative experiment, 3H-thymidine uptake increased from 57826 cpm in the presence of supernatants obtained from unstimulated cells to 200774 cpm with supernatants from monocytes stimulated by IgE/alpha IgE immune complexes. By ELISA, IgE complexes increased IL-1 beta production from 0.54 +/- 0.06 ng (per 10(6) monocytes) to 2.60 +/- 0.62 ng (p less than 0.01; mean of eight experiments) and TNF-alpha production from 0.17 +/- 0.10 ng to 3.00 +/- 0.54 ng (p less than 0.01; mean of four experiments). No IL-1 alpha secretion was observed. RNA hybridization analysis demonstrated that IL-1 beta production represented de novo synthesis of the cytokine. Stimulated RNA production was observed after a minimal 1/2-h incubation and was maximal at 2 h. The IgE-dependent secretion of these pro-inflammatory cytokines by mononuclear phagocytic cells may contribute to the inflammation characteristic of allergic responses.     

Effect of neurotensin on pancreatic function in man

The effect of neurotensin on submaximally-stimulated hepatobiliary and pancreatic secretion was studied in 6 healthy subjects. An intravenous infusion of neurotensin 1.4 ± 0.3 pmol/kg/min, designed to reproduce plasma neurotensin immunoreactivity levels within the physiological range, produced a significant increase in pancreatic bicarbonate output. Plasma concentrations of pancreatic polypeptide rose by 83 ± 16 pmol/l and were associated with a small reduction in trypsin, but no significant change in bilirubin outputs.     

Effect of Metalaxyl on the incidence and severity of Pearl millet downy mildew disease in Northern Nigeria

Pearl millet downy mildew (DM) incidence, severity and yield losses of two pearl millet varieties (local and improved) due to the disease were determined in the field. Significant differences in the disease incidence and severity were recorded in the plots sown with metalaxyl-treated seeds and those sown with non-treated seeds, indicating the efficacy of the fungicide on the fungus. Yield losses due to non-treatment of seeds with metalaxyl was 40.88 and 45.39% in a local variety and 43.00 and 18.60% in an improved variety in the 2000 and 2001 cropping seasons respectively. Significant differences between plots sown with metalaxyl-treated and those sown with non-treated seeds were obtained for other yield components such as 1000-grains weight, panicle length and weight.     

A Noradrenergic Mechanism Influences the Circadian Timing System in Rats and Hamsters

Brainstem monoaminergic projections to the suprachiasmatic nucleus (SCN), and to the intergeniculate leaflet (IGL), appear to modulate both photic and non-photic effects on the circadian system. Recent work in this laboratory has concentrated on the role of noradrenaline in the regulation of circadian period and phase. Previously, this lab has shown that chronic administration of the alpha₂ adrenergic agonist, clonidine, to rats maintained in constant light (LL) shortens free-running circadian period and promotes dissociation of rhythmicity, while acute clonidine administration to hamsters produces phase shifts similar to those observed with photic stimuli. These results suggest an interaction between clonidine and photic input on circadian rhythmicity, and so the present study was designed to examine systematically the relationship between chronic clonidine administration and photic input in both rats and hamsters. In DD and low intensity LL, clonidine did not alter free-running circadian wheel-running rhythms of rats, but under moderate to high intensity LL, clonidine significantly reduced the period-lengthening effects of LL. Chronic clonidine administration also altered several aspects of circadian phase in hamsters; phase shifts in response to light pulses of varying intensity at CT 19 were reduced; steady-state entrainment phase under a 24-h light-dark cycle (LD 14:10)was delayed; and synchronization to a 23-h light-dark cycle (LD 13:10) was impaired. Clonidine appeared to have little effect on free-running period of hamsters, but a trend towards dissociation of rhythmicity under LL was observed. These effects may reflect an action of clonidine at the photic input pathways to the circadian system, or directly at the circadian pacemaker, since alpha 2 adrenoceptors have been localized both in the suprachiasmatic nucleus (SCN) and in several of its projection areas. As both clinical and experimental studies suggest that clonidine may have depressogenic properties, chronic administration of clonidine to rodents may provide an animal model of the alterations in circadian rhythmicity seen in human depression.