A short synthetic peptide fragment of human interleukin-1 beta increases both human and murine natural killer activity

The effect of a short synthetic fragment of human interleukin-1 beta (hu IL-1 beta) on natural killer (NK) activity was examined. Peripheral-blood mononuclear cells (PBMC) from normal donors showed a significant increase in NK activity against K562 leukemia cells after preincubation for 18 h with the IL-1 peptide. A similar augmentation was not observed after culturing the cells in the presence of hu IL-1 beta. The increase in tumor cell lysis could not be ascribed to a cytolytic activity of the synthetic fragment on target cells, since the peptide caused no direct lysis of various tumor cell lines. Although the peptide enhanced NK cytotoxicity of PBMC, highly purified large granular lymphocytes were not susceptible to its stimulatory effect. The addition to the cultures of antibodies to human interleukin-2 (hu IL-2) completely blocked the peptide-induced boost of NK cytotoxicity, suggesting that IL-2 is mainly involved in the activation process. The ability of the IL-1 peptide to increase NK activity was further confirmed in vivo in the mouse. Cytotoxicity against YAC-1 lymphoma cells, which was very low in the spleen of untreated BALB/c mice, was in fact significantly increased after a single inoculation of the peptide. These data thus indicate that a short synthetic peptide fragment of hu IL-1 beta is able to increase both human and murine NK activity.     

A mutant protein of human interleukin-1 beta with immunostimulatory but not pyrogenic potency

Interleukin-1 (IL-1) mediates a variety of immune and inflammatory responses. In order to understand the mechanisms involved in multiple biological functions, it is important to define the active sites of IL-1. Using the technique for site-specific mutagenesis, we tested whether the arginine residue at the 4th position in human IL-1 beta is essential for multiple biological activities. In our experiments, the fourth position is replaced by a non-basic amino acid--either glycine or aspartic acid. The resulting mutant protein shows both immunostimulatory activity and the ability to induce hematopoietic growth factors similar to native IL-1 beta, but has a markedly reduced pyrogenic potency. Therefore, the mutant protein of IL-1 beta may represent a good candidate for use in vivo as an adjuvant for poor immunogenic vaccines.     

Alpha melanocyte stimulating hormone inhibits immunostimulatory and inflammatory actions of interleukin 1

The ability of interleukin 1 (IL 1) to augment the proliferation of murine thymocytes in vitro was inhibited in a dose-dependent manner by the neuropeptide alpha-melanocyte-stimulating hormone (alpha MSH). The minimal effective concentration of alpha MSH was 10(-11) M. Maximal effect occurred between 10(-8) and 10(-7) M, with diminishing effectiveness at higher concentrations. IL 1-induced production of prostaglandin E (PGE) by fibroblasts was also inhibited by alpha MSH with a biphasic dose response. The minimal effective concentration was 10(-11) M, and maximum effect was achieved at 10(-10) M. alpha MSH appeared to affect the interaction of IL 1 with its target cells in a specific manner, because it did not inhibit basal mitogen-induced thymocyte proliferation or IL 2-induced proliferation of a cytotoxic T lymphocyte line. Furthermore, production of IL 1 by endotoxin-stimulated monocytes was not affected by alpha MSH. An analog of alpha MSH (Nle4, D-Phe7 alpha MSH), which is highly potent in other melanotropin-sensitive systems, did not affect the action of IL 1 on thymocytes, suggesting that the immunomodulatory effects of alpha MSH may not be mediated by the classic melanocyte alpha MSH receptor. The influence of alpha MSH on thymocytes and fibroblasts suggests that alpha MSH is an endogenous antagonist of IL 1, perhaps important for limiting inflammatory damage to host tissues.     

A synthetic peptide of human apoprotein E with antibacterial activity

In recent years, several endogenous mammalian antibacterial peptides have been described. An amphipathic cationicalpha-helix is a common feature in many cases; therefore, other peptides with this characteristic might also possess antibiotic activity. In fact, a 30-mer peptide of apoprotein E 133-162 (LRVRLASHLRKLRKRLLRDADDLQKRLAVY) was found to have antibiotic activity comparable to those of a classic antibiotic (Gentamicin) and a neutrophil-derived antibiotic peptide (CAP18). Calculation of cationicity, hydrophobicity, and hydrophobic moment and the helical wheel diagram of apoprotein E 133-162 revealed close similarities to CAP18.     

A urine inhibitor of interleukin 1 activity affects both interleukin 1 alpha and 1 beta but not tumor necrosis factor alpha

Urine from monocytic leukemia and other febrile patients contains an inhibitor of interleukin 1 (IL-1), as measured by prostaglandin E2 and collagenase production by human fibroblasts and synovial cells. With the use of recombinant IL-1, the IL-1 inhibitor was partially purified by using ammonium sulfate precipitation, anion-exchange, and gel filtration chromatographies. IL-1 inhibitory activity elutes with an 18,000 to 25,000 apparent molecular size. The same fractions also inhibit IL-1 assayed by the proliferation of murine thymocytes and human fibroblasts. Both forms of human recombinant IL-1, IL-1 alpha and IL-1 beta, which show only 26% homology, but nevertheless bind to the same receptor, are affected by this natural inhibitor to the same extent. In contrast, human recombinant tumor necrosis factor, which shares some of the biologic activities of IL-1, is not inhibited by the urinary IL-1 inhibitor. This study shows that the various biologic activities of both forms of human recombinant IL-1 are inhibited by a partially purified natural urine-derived factor.     

Correlation of interleukin 6 with interleukin 1alpha in human mammary tumours, but not with oestrogen receptor expression

The authors hypothesized that IL-6 production by breast tumour tissues would correlate with OR-positivity, as only those tumours that contain oestrogen receptors (OR) and use oestrogen as a mitogen would benefit from locally increased oestrogen. IL-6 increases the activity of the 17beta-oxidoreductase, which converts oestrone to oestradiol, a process that may contribute to the increased concentration of oestrogen around breast tumours. IL-1alpha upregulates IL-6 production; therefore, the correlation between IL-1alpha and IL-6 immunoreactivity and OR-positivity in paraffin-embedded human breast tumours was further investigated.The results indicate IL-6 immunoreactivity in 40 of 66 paraffin embedded breast tumour specimens, a finding which did not correlate with the clinical evaluation of oestrogen receptor positivity (P=0.32 by Fisher's exact test). However, there was a correlation between IL-6 and IL-1alpha immunoreactivity (P<0.05). To study an in vitro model for this phenomenon, the IL-6 immunoreactivity in available cell lines was tested. Surprisingly, no production of IL-6 protein or mRNA could be detected in any of the cell lines, and this did not change with IL-1alpha stimulation. Therefore, none of the cell lines apparently reflected the immunological potential observed in the majority of surgical specimens.     

A novel leader peptide which allows efficient secretion of a fragment of human interleukin 1 beta in Saccharomyces cerevisiae.

Killer strains of Kluyveromyces lactis secrete a toxin which presumably is processed during secretion from a larger precursor. Analysis of the sequence of the K. lactis killer toxin gene predicts that the first 16 amino acids at the amino terminus of the protein should represent its leader peptide. We have tested the capability of this leader peptide to direct secretion of a protein fused to it by inserting a synthetic oligonucleotide identical to the sequence of the putative leader peptide into a yeast expression vector. Subsequently, the cDNA coding for the secreted active portion of the human interleukin 1 beta (IL-1 beta) was fused to the leader peptide sequence of the killer toxin. This construction in Saccharomyces cerevisiae is capable of directing synthesis and secretion of correctly processed IL-1 beta into the culture medium.     

Lipid-mobilizing activity of a synthetic peptide identical to 33-44 fragment of human growth hormone]

The lipid-mobilizing activity of a synthetic peptide, NH2-Phe-Glu-Glu-Ala-Tyr-Ile-Pro-Lys-Glu-Gln-Lys-Tyr-Ser-Phe-COOH, corresponding to the 31-44 amino-acid sequence of human growth hormone, was studied. The peptide stimulated lypolysis upon administration to fasted rats and during incubation with isolated epidiymal adiposed tissue of rat and perirenal adiposed tissue of rabbit. The lipid-mobilizing effect of the peptide,unlike the corresponding effect of the native growth hormone, developed fast and was markedly pronounced 15-30 min after the incubation was started. Direct dependence between the peptide dose logarithm and the effect studied was observed at concentrations of 0.01-10microng/ml during incubation with rat adipose tissue and at 0.001-0.1 microng/ml during incubation with rabbit tissue.     

Lymphocyte chemotactic activity of human interleukin 1

In the cellular immune response, there is an accumulation of mainly nonantigen-specific mononuclear cells that presumably is dependent on the local secretion of chemotactic factors. In view of the presence of large numbers of macrophages early in the delayed hypersensitivity response, the possible role of these cells in the chemotaxis of lymphocytes was investigated by studying the chemotactic activity of purified human interleukin 1 (IL 1) on T and B cells. Chemotactic activity for T and B cells was observed, the effect on B cells being greater than on T cells. At low concentrations (less than 1 U/ml), IL 1 had predominantly chemotactic activity for B cells and chemokinetic activity for T cells. At high concentrations (10 to 20 U/ml), IL 1 had pure chemotactic activity for both cell types. A relationship was found between levels of migration of T and B cells and mouse thymocyte proliferation induced by purified IL 1 and by lipopolysaccharide-stimulated monocyte supernatants. The principal peaks of both activities were found in 16,000 to 18,000 m.w. fractions. In additional studies, the chemotactic response to IL 1 was inhibited by preincubation of T and B cells with IL 1 or stimulated monocyte supernatant, demonstrating the role of binding of IL 1 in the chemotactic response.     

A short peptide insertion crucial for angiostatic activity of human tryptophanyl-tRNA synthetase

Human tryptophanyl-tRNA synthetase (TrpRS) is secreted into the extracellular region of vascular endothelial cells. The splice variant form (mini TrpRS) functions in vascular endothelial cell apoptosis as an angiostatic cytokine. In contrast, the closely related human tyrosyl-tRNA synthetase (TyrRS) functions as an angiogenic cytokine in its truncated form (mini TyrRS). Here, we determined the crystal structure of human mini TrpRS at a resolution of 2.3 A and compared the structure with those of prokaryotic TrpRS and human mini TyrRS. Deletion of the tRNA anticodon-binding (TAB) domain insertion, consisting of eight residues in the human TrpRS, abolished the enzyme's apoptotic activity for endothelial cells, whereas its translational catalysis and cell-binding activities remained unchanged. Thus, we have identified the inserted peptide motif that activates the angiostatic signaling.     

Walker A lysine mutations of TAP1 and TAP2 interfere with peptide translocation but not peptide binding

We generated mutants of the transporter associated with antigen-processing subunits TAP1 and TAP2 that were altered at the conserved lysine residue in the Walker A motifs of the nucleotide binding domains (NBD). In other ATP binding cassette transporters, mutations of the lysine have been shown to reduce or abrogate the ATP hydrolysis activity and in some cases impair nucleotide binding. Mutants TAP1(K544M) and TAP2(K509M) were expressed in insect cells, and the effects of the mutations on nucleotide binding, peptide binding, and peptide translocation were assessed. The mutant TAP1 subunit is significantly impaired for nucleotide binding relative to wild type TAP1. The identical mutation in TAP2 does not significantly impair nucleotide binding relative to wild type TAP2. Using fluorescence quenching assays to measure the binding of fluorescent peptides, we show that both mutants, in combination with their wild type partners, can bind peptides. Since the mutant TAP1 is significantly impaired for nucleotide binding, these results indicate that nucleotide binding to TAP1 is not a requirement for peptide binding to TAP complexes. Peptide translocation is undetectable for TAP1.TAP2(K509M) complexes, but low levels of translocation are detectable with TAP1(K544M).TAP2 complexes. These results suggest an impairment in nucleotide hydrolysis by TAP complexes containing either mutant TAP subunit and indicate that the presence of one intact TAP NBD is insufficient for efficient catalysis of peptide translocation. Taken together, these results also suggest the possibility of distinct functions for TAP1 and TAP2 NBD during a single translocation cycle.     

Production of anti-IFN-beta sera with chemically synthetic IFN-beta peptide fragment (1 - 13)

Region specific antisera for human fibroblast interferon (IFN-β) was obtained from New Zealand white rabbits immunized with the immunogen which was the synthetic tridecapeptide corresponding to the N-terminal sequence of native human IFN-β coupled to bovine serum albumin. A sensitive and specific radioimmunoassay for IFN-β was developed and these antisera was successfully applied in the purification of human IFN-β by affinity chromatography, resulting in about 100-fold purification in one step.     

Cleavage of human interleukin 1: isolation of a peptide fragment from plasma of febrile humans and activated monocytes

Interleukin 1 (IL 1) is a product(s) of mononuclear phagocytes, and has multiple biologic activities that mediate several host responses to infection and inflammation. Highly purified IL 1 activates lymphocytes, induces fever, increases hepatic acute phase protein synthesis, and increases muscle protein degradation. A 4.2 kd peptide has been purified from plasma of febrile humans which also induces muscle proteolysis in vitro (termed proteolysis-inducing factor, PIF). Because IL 1 purified from activated human monocytes induces muscle proteolysis in vitro, studies were performed to determine the relationship of human monocyte-derived IL 1 to plasma-derived PIF. Purified PIF was highly active in the IL 1 thymocyte assay. After gel filtration of plasma from febrile patients, fractions with PIF activity also induced thymocyte proliferation and fever in mice. Thus, it seems likely that the plasma peptide PIF has IL 1 properties and probably represents a small m.w. cleavage product of IL 1. Further studies confirmed this finding. Highly purified 15 kd IL 1, rechromatographed over different gel filtration media, consistently fragmented into a 4 kd peptide with both muscle proteolysis-inducing and lymphocyte-activating properties. The breakdown of the 15 kd IL 1 into biologically active smaller fragments increased with time, and could be accelerated by trypsinization. The monocyte-derived IL 1 fragments were partially destroyed by heat. Highly purified 125I-labeled 15 kd IL 1 also fragmented into subunits, and these radioactive subunits produced fever in mice and were active in the thymocyte assay. Fragmentation of 125I-labeled 15 kd IL 1 was reduced by agents that inhibit proteases. These results indicate that some of the biologic activities of human IL 1 are conserved in small m.w. fragments. These studies also provide evidence that IL 1 may circulate in humans as a 4.2 kd peptide, and that this cleavage product can function as an active mediator of IL 1 effects in the host.     

Neutrophil-activating peptide 2 and gro/melanoma growth-stimulatory activity interact with neutrophil-activating peptide 1/interleukin 8 receptors on human neutrophils

Neutrophil-activating peptide 1/interleukin 8 (NAP-1/IL-8), neutrophil-activating peptide 2 (NAP-2), and gro/melanoma growth-stimulatory activity (gro/MGSA) are potent inflammatory cytokines with homologous structure and similar neutrophil-activating properties. Receptors on human neutrophils that interact with these peptides were studied. Analysis of 125I-NAP-1/IL-8 binding at 0-4 degrees C revealed 64,500 +/- 14,000 receptors/cell with an apparent Kd of 0.18 +/- 0.07 nM (mean +/- S.D. of six independent experiments). Unlabeled NAP-1/IL-8, NAP-2, and gro/MGSA competed with 125I-NAP-1/IL-8 for binding to human neutrophils. Competition with increasing concentrations of unlabeled NAP-2 and gro/MGSA resolved two classes of NAP-1/IL-8 binding sites: about 70% of them bound NAP-2 and gro/MGSA with high affinity (Kd: 0.34 +/- 0.2 and 0.14 +/- 0.02), while 30% were of low affinity (Kd: 100 +/- 20 and 130 +/- 10 nM). Different binding sites, however, were not apparent upon competition with unlabeled NAP-1/IL-8, suggesting that both classes of receptors have similar affinities for NAP-1/IL-8. The existence of two receptors was also suggested by ligand cross-linking and cross-desensitization experiments. Two neutrophil membrane proteins with apparent Mr of 66,000-74,000 and 42,000-46,000 became cross-linked to 125I-NAP-1/IL-8, and the labeling was decreased when excess NAP-1/IL-8, NAP-2, or gro/MGSA was present. Stimulation of neutrophils with NAP-1/IL-8 resulted in desensitization toward a subsequent challenge with NAP-2 or gro/MGSA as shown by the rise in cytosolic free calcium. By contrast, following primary stimulation with NAP-2 or gro/MGSA, responses to NAP-1/IL-8 were only moderately attenuated, supporting the existence of NAP-1/IL-8 receptors which bind NAP-2 or gro/MGSA with low affinity. In conclusion, our results demonstrate that NAP-2 and gro/MGSA act upon human neutrophils by directly interacting with two classes of receptors for NAP-1/IL-8.     

A crystalline synthetic peptide representing the epitope of a monoclonal antibody raised against synthetic interferon-alpha 1 fragment 111-166

The antigenic determinant recognized by the monoclonal antibody that had been raised against synthetic human interferon-alpha 1 (IFN-alpha 1) fragment 111-166 [Arnheiter, H., Thomas, R.M., Leist, T., Fountoulakis, M., and Gutte, B. (1981) Nature (Lond.) 294, 278-280] and that cross-reacted with human IFN-alpha 1, IFN-alpha 2, and IFN-alpha A made in Escherichia coli, was localized to the region between residues 151 and 166 using synthetic COOH-terminal interferon fragments. In solid-phase radioimmunoassays neither the strongly hydrophilic COOH-terminal nonapeptide IFN 158-166 nor its mixtures with IFN 151-162 or IFN 149-158 showed any measurable interaction with the antigen binding site of the monoclonal antibody. For antibody binding, the full covalent structure of IFN 151-166 was required. Quantitatively very similar results were obtained with IFN 149-166 and IFN 143-166. The synthetic COOH-terminal hexadecapeptide of human IFN-alpha 1 (IFN 151-166) could be crystallized.     

A synthetic peptide homologous to the envelope proteins of retroviruses inhibits monocyte-mediated killing by inactivating interleukin 1

The synthetic peptide CKS-17 has homology to a highly conserved region of the immunosuppressive retroviral envelope protein P15E, to envelope proteins of HTLV I, II, III, and to that encoded by an endogeneous C-type human retroviral DNA. CKS-17 inhibits the immune response of lymphocytes and the respiratory burst of human monocytes. Because P15E-related antigens are present in human malignant cell lines and cancerous effusions, we sought to determine the effect of CKS-17 on monocyte-mediated tumor cell lysis. Lysis of A375 tumor cells by lymphokine or lipopolysaccharide-activated human monocytes was inhibited by 10 microM CKS-17 (control, 79%; CKS-17-treated, 19%). Another synthesized peptide, CS-2, which has partial homology to CKS-17, failed to block monocyte-mediated killing. Thus, the inhibition by CKS-17 appeared to be specific. Because interleukin 1 (IL-1) is a cytocidal factor produced by activated monocytes, we also investigated the effect of CKS-17 on IL-1 production by monocytes and on direct IL-1-mediated cytotoxicity. CKS-17 did not block production or secretion of IL-1 by lipopolysaccharide- or interferon-gamma-activated monocytes. However, the direct cytocidal activity of both recombinant IL-1 alpha and IL-1 beta against A375 tumor cells was blocked by CKS-17. The cytotoxic activity of IL-1 was inhibited by CKS-17 if (a) IL-1 was preincubated with CKS-17 for 1 hr at 37 degrees C or (b) the A375 cells were incubated with CKS-17 for 1 hr prior to the addition of IL-1. CKS-17 also blocked IL-1-induced proliferation of murine thymocytes, the D10 T cell line, and an IL-1-responsive astrocytoma cell line. These data suggest that CKS-17 may be a potent inhibitor of IL-1.     

Purification and partial biochemical characterization of a human monocyte-derived, neutrophil-activating peptide that lacks interleukin 1 activity

A novel monocyte-derived neutrophil-activating peptide (MONAP) produced by lipopolysaccharide- and phorbol myristate acetate-stimulated human peripheral blood monocytes was purified by sequential ion exchange-high performance liquid chromatography (HPLC), size exclusion HPLC, and reversed phase HPLC. Biologic activities of the purified cytokine were monitored by either an enzyme release assay or a chemotaxis assay, using peripheral human neutrophils. Purified MONAP was found to be homogeneous, giving a single peak on size-exclusion HPLC, reversed-phase HPLC, as well as a single 10-kDa band on silver-stained polyacrylamide gels. Purified MONAP stimulate human neutrophil chemotaxis at an estimated molarity of 5 x 10(-11) M. Half-maximal enzyme release of cytochalasin B pretreated neutrophils occurred at 2 to 3 x 10(-10) M, whereas superoxide anion production elicited by various concentrations of MONAP was found to be low. Isolated human peripheral monocytes, as well as human eosinophils, showed no chemotactic response to MONAP, indicating neutrophil specificity. MONAP activity was separated from thymocyte-stimulating activity by reversed-phase HPLC, indicating nonidentity with interleukin (IL)-1. This was further supported by heat resistance of MONAP, which is in contrast to the heat sensitivity of IL-1. In addition, IL-1 obtained as a by-product during isolation of MONAP did not stimulate human neutrophil chemotaxis.     

Stimulation by human interleukin 1 of cartilage breakdown and production of collagenase and proteoglycanase by human chondrocytes but not by human osteoblasts in vitro

Human articular chondrocytes in culture synthesise collagenase and neutral proteoglycanase in response to addition of a 12-17 kDa protein produced by cultured human monocytes. This factor copurifies with interleukin 1, as assessed by lymphocyte activating factor activity, on gel filtration chromatography and isoelectric focusing. The interleukin 1 and chondrocyte-stimulating activities are destroyed by pretreatment of the material with phenylglyoxal. The same materials also promote the release of glycosaminoglycans from cultures of intact bovine nasal cartilage. The proteoglycanase activity release from chondrocytes appears to be a metalloproteinase because it is inhibited by EDTA and not by phenylmethylsulphonyl fluoride (PMSF), and because detection of its activity is dependent on the presence of 4-aminophenylmercuric acetate. Human osteoblast-like cells do not respond to this factor by increased proteinase production, but are stimulated to produce prostaglandins. These results suggest that interleukin 1 has activities upon non-immune cells which promote the degradation of connective tissue matrices. Human osteoblasts do not synthesise neutral collagen- and proteoglycan-degrading enzymes and thus are unlikely to be directly responsible for the matrix degradation which occurs during bone resorption.     

Monoclonal antibody, but not synthetic peptide, targeting the ectodomain of influenza B virus M2 proton channel has antiviral activity

The proton channels of influenza A virus (A/M2) and influenza B virus (BM2) are essential for viral replication. Previously we have shown that monoclonal antibodies targeting the ectodomain of the A/M2 proton channel have antiviral activity in vitro. In this study, we generated both monoclonal antibody and phage displayed peptide against the eight amino acids comprising the ectodomain of the BM2 proton channel and investigated their antiviral activities in vitro. A cytopathic assay showed that the monoclonal antibody potently protected MDCK cells from homologous, but not heterologous, virus infections. A plaque forming assay showed that viral replication was not completely neutralized, but greatly inhibited, by the monoclonal antibody. In contrast, no antiviral activity was observed for the synthetic native or engineered peptides. These results indicate that antibody targeting the M2 proton channel is a promising therapeutic candidate for treating influenza virus infections, and that antibody structure is important for antiviral activity.