Phosphorylation of intracellular precursors of human IL-1

The human IL-1 molecules (IL-1 alpha and IL-1 beta) are post-translationally cleaved from 31-kDa precursor to 18-kDa biologically active molecules. During the course of studies of post-translational modifications of human IL-1, we have observed that although LPS induced the production of both intracellular IL-1 alpha and IL-1 beta in human monocytes, [32P]orthophosphate labeling of these cells revealed that intracellular precursor of IL-1 alpha (pre-IL-1 alpha) to be phosphorylated at least 10-fold more than intracellular pre-IL-1 beta. However, no 32P-incorporation could be detected in the 18-kDa processed IL-1 alpha and IL-1 beta. Analysis by TLC revealed that the major phosphorylation site occurred at serine residue(s). The 32P was incorporated into multiply cleaved precursors of IL-1 alpha, which appeared in the absence of protease inhibitors. Since the smallest Mr pre-IL-1 alpha that was labeled with 32P was 22 kDa, the phosphorylated serine residue is presumably located adjacent to a sequence of four basic amino acids located in the 4-kDa region at the amino terminus of the 22-kDa precursor of IL-1 alpha. This serine residue might also be a major phosphorylation site for a cAMP-dependent protein kinase. This hypothesis was substantiated by the demonstration that a synthetic peptide analogue of this region (residue 84 to 112) could be similarly phosphorylated in vitro by a cAMP-dependent protein kinase. Furthermore, a truncated pre-IL-1 alpha (residue 64 to 271) and a "fusion" protein containing staphylococcal protein A and an amino-terminal half-portion of pre-IL-1 alpha (residue 1 to 112), but not mature IL-1 alpha (residue 113 to 271), could also be phosphorylated by cAMP-dependent protein kinase. There is no comparable amino acid sequence in IL-1 beta which could be expected to be phosphorylated by a cAMP-dependent protein kinase. The physiologic relevance of phosphorylation of pre-IL-1 alpha was investigated. The data showed that phosphorylation of truncated pre-IL-1 alpha greatly enhanced its susceptibility to digestion by trypsin and promoted the conversion of pre-IL-1 alpha to the more biologically active IL-1. Although the precise role of the rather selective phosphorylation of pre-IL-1 alpha is not known, our findings do suggest that the phosphorylation of serine close to dibasic/tetrabasic amino acid sequence functions to facilitate the processing and/or release of IL-1 alpha.     

Interdependence of the radioprotective effects of human recombinant interleukin 1 alpha, tumor necrosis factor alpha, granulocyte colony-stimulating factor, and murine recombinant granulocyte-macrophage colony-stimulating factor

Interleukin 1 alpha (IL-1 alpha), tumor necrosis factor alpha (TNF alpha), granulocyte-colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF) are molecularly distinct cytokines acting on separate receptors. The release of these cytokines can be concomitantly induced by the same signal and from the same cellular source, suggesting that they may cooperate. Administered alone, human recombinant (hr)IL-1 alpha and hrTNF alpha protect lethally irradiated mice from death, whereas murine recombinant GM-CSF and hrG-CSF do not confer similar protection. On a dose basis, IL-1 alpha is a more efficient radioprotector than TNF alpha. At optimal doses, IL-1 alpha is a more radioprotective cytokine than TNF alpha in C57BL/6 and B6D2F1 mice and less effective than TNF alpha in C3H/HeN mice, suggesting that the relative effectiveness of TNF alpha and IL-1 alpha depends on the genetic makeup of the host. Administration of the two cytokines in combination results in additive radioprotection in all three strains. This suggests that the two cytokines act through different radioprotective pathways and argues against their apparent redundancy. Suboptimal, nonradioprotective doses of IL-1 alpha also synergize with GM-CSF or G-CSF to confer optimal radioprotection, suggesting that such an interaction may be necessary for radioprotection of hemopoietic progenitor cells.     

Properties of a specific interleukin 1 (IL 1) receptor on human Epstein Barr virus-transformed B lymphocytes: identity of the receptor for IL 1-alpha and IL 1-beta

The properties of specific human interleukin 1 (IL 1) receptors on human Epstein Barr virus-transformed B lymphocytes (EBV-B) were studied. Purified human IL 1-beta from a myelomonocytic cell line (THP-1) was labeled with 125I by the Bolton-Hunter method without detectable loss of biological activity. Among four EBV-B cell lines tested, a pre-B cell type (VDS-O) specifically bound the highest amount of 125I-IL 1-beta. Maximal binding was reached within 20 min at 4 degrees C. Scatchard plot analysis of the binding of 125I-IL 1-beta to VDS-O cells yielded a Kd (dissociation constant) of 2.4 to 5.9 X 10(-10) M with 110 to 220 binding (receptor) sites/cell. The binding of 125I-IL 1-beta to VDS-O cells was also inhibited by F(ab)'2 fragments of anti-human IL 1 and recombinant human IL 1-alpha, as well as by unlabeled human IL 1-beta but not by recombinant lymphotoxin, recombinant tumor necrosis factor, or phorbol myristic acid, suggesting that IL 1-alpha and IL 1-beta bind specifically to the same receptor. The m.w. of IL 1 receptor on human EBV-B cells was estimated to be 60,000 by both the chemical cross-linking method and high pressure liquid chromatography (HPLC) gel filtration analysis of receptor extracted from membrane enriched fraction by a non-ionic detergent (CHAPS). The isoelectric point of solubilized human IL 1 receptor was 7.3 on HPLC chromatofocusing. The evidence of existence of IL 1 receptor on human EBV-B cells additionally supports the hypothesis that IL 1 may be an autocrine signal for these cells.     

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)     

Calcium ionophore (A23187) increases interleukin 1 (IL-1) production by human peripheral blood monocytes and interacts synergistically with IL-1 to augment concanavalin A stimulated thymocyte proliferation

The effects of calcium ionophore, A23187, on production of interleukin 1 (IL-1) by human peripheral blood monocytes (PEMo) and on murine thymocyte proliferation were examined. A23187 induced IL-1 production by human PBMo. The optimal dose was 10(-6) M. Although IL-1 production induced by A23187 was less than that by lipopolysaccharide (LPS) or silica, A23187 together with LPS had a synergistic effect on induction of IL-1. A23187 also had a more marked synergistic effect in concert with Concanavalin A and/or IL-1 on murine thymocyte proliferation. The optimal dose was also 10(-6) M. This represents the first report suggesting that monocytes or the monocyte product, IL-1, may contribute to the mitogenic effect of A23187 for thymocytes.     

Amino acid sequence of a proline-rich phosphoglycoprotein from parotid secretion of the subhuman primate Macaca fascicularis

The complete amino acid sequence of the macaque proline-rich phosphoglycoprotein (MPRP) was determined by automated Edman degradation of the protein, fragments F-1 and F-2 derived from the protein by an intrinsic salivary protease, and chymotryptic, tryptic, Staphylococcus aureus V8 protease, and endoproteinase lysine-C peptides. MPRP contains 115 amino acid residues including phosphorylated serine at residues 1, 2, 6, 12, and 15, and 6 O-glycosidic carbohydrate units at residues 69, 75, 87 (threonine) and 96, 103, and 106 (serine). The Mr of the polypeptide moiety of the protein is 12,656. The amino-terminal domain contains all 5 phosphoserine residues and most of the other negatively charged and hydrophilic residues, whereas the carboxyl-terminal domain contains 24 of 25 proline residues, and 6 O-glycosidic oligosaccharides. Comparison of MPRP with the four major anionic proline-rich proteins (PRPs) from human glandular secretion shows that 57% of the amino acid residues are identical if gaps are introduced to maximize homology, suggesting that these proteins are phylogenetically related. Significant structural and functional differences occur between the macaque and human proteins. MPRP has 5 phosphoserines, PRPs have 2. MPRP is a glycoprotein, PRPs are not. MPRP inhibits the spontaneous precipitation (primary precipitation) of calcium phosphate salts from supersaturated solutions in addition to inhibiting seeded crystal growth (secondary precipitation) (Oppenheim, F. G., Offner, G. D., and Troxler, R. F. (1982) J. Biol. Chem. 257, 9271-9282), whereas PRPs inhibit only secondary precipitation. MPRP is the only major anionic proline-rich protein in macaque glandular secretion; in contrast, there are four major anionic PRPs and these display a genetic polymorphism. The significance of these structural differences with respect to biological function and the possible relationship of MPRP to salivary mucins are discussed.     

Frequency distribution studies of epipelic diatoms along an intertidal shore

Live and dead cell counts of epipelic diatoms were analysed at 16 sites along a transect crossing a saltmarsh, sandflat, and mudflat at Berrow Flats, Somerset, U.K. Replicate monthly samples were taken from January 1982 to June 1984. The ratio of dead to live cells varied according to position along the transect. Temporal changes of the ratios did not display a cyclic seasonal pattern. The clustered mosaic of live cells and empty frustules on surface sediments was quantitatively assessed. Graphing variance against seasonal mean cell counts showed that the variance was proportional to the square of the mean. The Neyman Type A equation is suggested as a possible model to explain this relationship.     

The cellular basis for immune interferon production in autoimmune MRL-Ipr/Ipr mice

Disturbances in immune interferon (IFN gamma) activity have been implicated in the development of human systemic lupus erythematosus (SLE) and the spontaneous disease sustained by autoimmune-prone mice. We therefore investigated the cellular basis for IFN gamma production in MRL-Ipr/Ipr mice and examined the relationship between synthesis of interleukin 2 (IL 2) and IFN gamma. In vitro IL 2 and IFN gamma production in 3 to 6-mo-old, autoimmune MRL-Ipr/Ipr and MRL-+/+ mice was compared with that seen in age- and sex-matched, immunologically normal CBA/J mice. 5 X 10(6) spleen cells were pulsed with 5 micrograms of concanavalin A (Con A), and the cellfree supernatant was assayed for IL 2 and IFN gamma activity at various times up to 72 hr. We found that peak levels of IL 2 in MRL mice were less than 10% of those in the CBA/J. Yet, production of IFN gamma by cells from the autoimmune and normal strains was quite comparable. The addition of murine IL 2 to optimally Con A-stimulated cells from the MRL-Ipr/Ipr or normal mice did not affect the subsequent peak production of IFN gamma. Although the primary producers of IFN gamma in cultures of normal mice bear the Lyt-2+ phenotype, the Lyt-1+2- T-cell subset was found to be the principal source of IFN gamma in the aged MRL-Ipr/Ipr. These data suggest that Lyt-1+ cells from MRL-Ipr/Ipr mice may be differentially responsive to the signal delivered by the same mitogenic lectin with respect to lymphokine production and may indicate a distorted commitment of such cells toward production of IFN gamma and repression of IL 2 synthesis. The relationship between hypoproduction of IL 2, this usual source of IFN gamma, and the autoimmune disease sustained by MRL-Ipr/Ipr mice remains unclear.