Injection time of interleukin-6 determines fatal outcome in experimental endotoxin shock.

Circulating interleukin-6 (IL-6) levels are directly correlated to fatal outcome in both patients and animal models with endotoxin shock. However, whether IL-6 is deleterious or protective in regard to survival is obscure. We investigated the action of IL-6 in the pathogenic progress of endotoxin shock. C3H/HeN mice received 10 micrograms of natural human IL-6 (Hu-IL-6) s.c. at various times before or after challenge with Escherichia coli lipopolysaccharide (LPS) at a lethal dose. Pretreatment with Hu-IL-6 0, 1, and 4 h before LPS administration improved the survival rate of the mice. However, no protection was observed when Hu-IL-6 was administered 24 h before or 1 h after the LPS injection. The protective mechanism of Hu-IL-6 pretreatment was not explained on the changes in the circulating levels of tumor necrosis factor and endogenous murine IL-6 (Mu-IL-6). The induction of fibrinogen and immunosuppressive acidic protein, a type of acute-phase proteins, may have contributed to the protection. The results show that the order and the time interval in which the administered Hu-IL-6 and the Mu-IL-6 induced by LPS act are the key to the determination of fatal outcome.

Intervention of crescentic glomerulonephritis by antibodies to monocyte chemotactic and activating factor (MCAF/MCP-1).

We investigated the pathophysiological role of a potent macrophage (M(phi)) chemotactic cytokine (chemokine), monocyte chemotactic and activating factor/monocyte chemoattractant protein-1 (MCAF/MCP-1), in an animal model of crescentic glomerulonephritis. Administration of a small dose of nephrotoxic sera induced severe proliferative and necrotizing glomerulonephritis, with crescentic formation in the early phase and glomerulosclerosis in the later phase, in Wistar-Kyoto rats. MCAF/MCP-1 protein was detected immunohistochemically in glomeruli, vascular endothelial cells, and tubular epithelial cells in the early phase of injured kidney tissues but not in normal ones. Anti-MCAF/MCP-1 antibodies decreased the number of M(phi) in glomeruli, and prevented crescentic formation and the fusion of epithelial cell foot process in nephritic rats, thereby decreasing the excreted amounts of protein to normal levels on days 3 and 6. Furthermore, anti-MCAF/MCP-1 antibodies remarkably reduced glomerulosclerosis and improved renal dysfunction as well as proteinuria in the later phase (56 days). These results indicate that MCAF/MCP-1 essentially participates in the impairment of renal functions associated with crescentic glomerulonephritis by recruiting and activating M(phi).

Molecular cloning of APRF, a novel IFN-stimulated gene factor 3 p91-related transcription factor involved in the gp130-mediated signaling pathway.

Acute-phase response factor (APRF) is a transcription factor that binds to the interleukin-6 (IL-6)-responsive elements identified in the promoters of various acute-phase protein genes. We report here the purification and cloning of APRF. APRF exhibits a 52.5% overall homology at the amino acid level with p91, a component of the interferon (IFN)-stimulated gene factor 3 complexes. The cloned APRF protein is tyrosine phosphorylated and translocated into the nucleus in response to IL-6, but not in response to IFN-gamma. Tyrosine phosphorylation was also observed in response to other cytokines, such as leukemia inhibitory factor, oncostatin M, and ciliary neurotrophic factor, whose receptors share the IL-6 receptor signal transducer gp130. In contrast, we observed that p91 is not tyrosine phosphorylated in response to IL-6. These results suggest that this novel p91-related protein may play a major role in the gp130-mediated signaling pathway and that selective activation of p91-related factors may explain the diversity of cellular responses to different cytokines.

Effects of natural human interleukin-6 on thrombopoiesis and tumor progression in tumor-bearing mice.

The growth of inoculated colon 26 adenocarcinoma (colon 26) in mice gradually increased the platelet count owing to murine IL-6 secreted from the tumor, while Lewis lung carcinoma (LLC) decreased the platelet count in the hosts, depending on the tumor growth. Natural human IL-6 injections (hIL-6), 280 micrograms/kg/day, stimulated the platelet production in both types of carcinoma-bearing mice. When the administration of mitomycin C or cisplatin decreased the platelet number as a side reaction with a concomitant of suppressing the growth of colon 26 and LLC, respectively, hIL-6 could also increase the platelet count without the augmentation of tumor growth. However, loss of carcass weight was observed in colon 26-bearing mice treated with hIL-6, suggesting the development of cachexia is associated with hIL-6 administration. Despite the possibility of inducing cachexia in some types of tumors, our results suggest that IL-6 could be a useful means of restoring the decreased platelet number in cancer patients after intensive chemotherapy.

[Early detection of chronic obstructive pulmonary disease by following individual annual changes in lung function].

The possibility of early detection of chronic obstructive pulmonary disease (COPD) was investigated among subjects whose lung function could be followed for more than ten years. Ten subjects aged 60 or more were selected from among about 1500 cases using the following diagnostic criteria; (1) subjects had complained of exertional dyspnea (2) chest X-ray abnormalities were suggestive of COPD (3) FEV1.0% was under 55%. Data on FVC/height, FEV1.0/height and FEV1.0% of these subjects were available for the previous ten years. Individual linear regression equations were estimated. Average annual changes of cases of obstruction were compared with those of normal cases, and individual deviations from the normal standard range were observed. Declines of the regression lines were as follows; FEV1.0/height: normal case--0.016 +/- 0.017, obstruction cases--0.038 +/- 0.018 (L/m/yr), FEV1.0%: normal cases 0.164 +/- 0.627, obstruction cases--1.522 +/- 0.638 (%/yr). Significant differences were observed between the normal and the obstruction cases (p < 0.01). FEV1.0% of all obstruction cases was under--2 SD of that of normal subjects. All individual FEV1.0% regression lines of the obstruction cases were outside +/- 2 RSD of the normal standard range more than 5 years before the onset of symptoms. Following individual annual changes in lung function was considered to be useful for the early detection of COPD.

Increased mRNA expression encoding for medullasin in peripheral blood mononuclear cells from patients with IgA nephropathy.

We investigated mRNA expression for medullasin (an inflammatory serine protease in bone marrow cells) in peripheral blood mononuclear cells (PBMC) obtained from 36 patients with primary IgA nephropathy (IgAN), 30 patients with other types of primary glomerular disease, 18 patients with secondary IgA nephritis including lupus nephritis and hepatic glomerulosclerosis and 24 healthy age-matched controls. The majority of patients with IgAN (86%) showed elevated medullasin expression in PBMC, while no medullasin mRNA expression was detected in PBMC obtained from patients with other types of primary glomerular disease, secondary IgA nephritis or normal healthy controls. A positive correlation was noted between mRNA levels and urinary protein excretion. The medullasin mRNA expression in PBMC also correlated with the severity of the histopathologic changes in renal tissue obtained from patients with IgAN. All the patients with severe proteinuria (more than 3.0 g/day) showed strong [more than (++)] medullasin mRNA expression in their PBMC. In addition, all the patients with more than (++) medullasin mRNA expression are grade III or IV histopathological findings. These studies suggest that abnormally regulated medullasin gene expression in PBMC may be associated with the progression of primary IgAN.

Phosphorylation at threonine-235 by a ras-dependent mitogen-activated protein kinase cascade is essential for transcription factor NF-IL6.

NF-IL6, a member of the basic leucine zipper (bZIP) family transcription factors, is involved in expression of inducible genes involved in immune and inflammatory responses. We observed that coexpression of oncogenic p21ras stimulated the transactivating activity of NF-IL6 and induced phosphorylation of Thr-235 located just N-terminal to the DNA binding domain of NF-IL6. Recently, mitogen-activated protein (MAP) kinases have been shown to be implicated in the cellular response to activated ras. Purified MAP kinases specifically phosphorylated Thr-235 of NF-IL6 in vitro. Mutation of Thr-235 abolished the ras-dependent activation of NF-IL6. From these results, we conclude that NF-IL6 is regulated through phosphorylation by MAP kinases in response to activated ras.

Production of multiple growth factors by a newly established human thyroid carcinoma cell line.

A multiple growth factor-producing tumor cell line (NIM-1) was newly established from a patient with thyroid cancer and remarkable neutrophilia. NIM-1 cells also caused severe neutrophilia in nude mice bearing tumors. NIM-1-conditioned medium (NIM-1CM) contained activities that supported not only granulocyte, macrophage and eosinophil colony formation of human bone marrow cells but also the growth of colony-stimulating factor (CSF)-dependent cell lines, NFS60-KX and TF-1. Northern blot hybridization analysis revealed the constitutive expression of granulocyte-CSF (G-CSF), granulocyte/macrophage-CSF (GM-CSF) and interleukin(IL)-6 mRNAs in NIM-1 cells. Enzyme-linked immunosorbent assays (ELISA) using NIM-1CM also confirmed the production of IL-1 alpha and a small amount of IL-1 beta besides G-CSF, GM-CSF and IL-6 in NIM-1 cells. In addition, unexpected production of IL-11 in NIM-1 cells was detected by northern blot hybridization analysis and by bioassay using an IL-11-dependent cell line. Therefore, NIM-1 cell line is shown to produce multiple cytokines including potentially megakaryopoietic growth factors such as GM-CSF, IL-6 and IL-11.

Isolation and expression of the full-length cDNA encoding CD59 antigen of human lymphocytes.

To identify the primary structure of CD59 antigen and to elucidate its function, a full-length cDNA clone of CD59 was isolated. The cDNA sequence contained an open reading frame that encodes an 128-amino-acid peptide. The amino-terminal 25 amino acids represented a typical signal peptide sequence and the carboxy-terminal hydrophobic amino acids were characteristic for phosphatidylinositol-anchored proteins. The predicted mature protein sequence showed 35% homology with murine Ly-6C.1 and 31% with Ly-6A.2. The number and the distribution of cysteine residues were conserved, implying that the CD59 represented a human homologue of murine Ly-6. RNA blot hybridization analysis revealed the expression of CD59 mRNA in placental, lung, and pancreatic tissues. The mRNA was not only expressed in T-cell lines but in some of monocytic, myeloid, and B-cell lines. In all of these tissues and cell lines, at least four mRNA species were detected. DNA blot hybridization analysis revealed a rather simple genomic structure, which suggested a single gene as compared with the complex multigene family of murine Ly-6.

Functional expression of human leukocyte elastase (HLE)/medullasin in eukaryotic cells.

We have cloned a full length cDNA for human leukocyte elastase (HLE, EC 3.4.21.37)/medullasin from the cDNA library of human leukemic cell line, ML3. Recombinant plasmid for the expression of HLE cDNA in eukaryotic cells was constructed in which HLE cDNA was fused in a frame to a leader sequence of human interleukin-2 (IL-2). COS-1 cells, transfected with the plasmid, secreted fusion protein consists of N-terminal 8 amino acid (aa) residues of human IL-2 and 238 aa residues of HLE. As the fusion protein was designed to be connected through lysine residue, elastase activity was generated after digestion of the fusion protein with lysyl-endopeptidase.

Characterization of four different mammalian-cell-derived recombinant human interferon-beta 1s. Identical polypeptides and non-identical carbohydrate moieties compared to natural ones.

In order to evaluate the structural identification of various recombinant human interferon-beta 1s, the recombinant proteins were produced in four different mammalian cells (human PC12 and PC8 lung adenocarcinoma cells, Chinese hamster ovary cells and mouse C127 cells) and characterized. Each mammalian-cell-derived recombinant human interferon-beta 1 represented a single band of 23 kDa on sodium dodecyl sulphate/polyacrylamide gel electrophoresis, the same molecular mass as fibroblast-derived natural human interferon-beta 1. Specific activities, amino acid compositions, amino-terminal sequences, peptide maps on C18 reversed-phase high-performance liquid chromatography and circular dichroic spectra of recombinant proteins were in good agreement with natural ones. On the other hand, the patterns of isoelectric focusing were different between mammalian-cell-derived recombinant human interferon-beta 1s and natural human interferon-beta 1. Sugar composition analysis revealed that the recombinant protein from Chinese hamster ovary cells has a similar sugar composition to that of natural protein and the other recombinant proteins have increased amounts of galactose and glucosamine in comparison to the natural protein. Furthermore, there is no galactosamine in the natural protein, while small amounts of galactosamine were detected in the oligosaccharides released from PC8- and C127-derived recombinant proteins by N-glycanase. These results indicate that mammalian-cell-derived recombinant human interferon-beta 1s have identical polypeptides to those of natural human interferon-beta 1 but their carbohydrate moieties, including unusual N-linked oligosaccharides, are individually different from natural ones and depend on the host cell.

Constitutive long-term production and characterization of recombinant human interferon-gammas from two different mammalian cells.

Two expression plasmids (pSVIFN gamma/BPV97 and pSVIFN gamma/AdDHFR) for constitutive production of human interferon-gamma (HuIFN-gamma) were constructed and introduced into the two different mammalian cell lines, mouse C127 cells and Chinese hamster ovary (CHO) cells. Genetically engineered C127 and CHO cells grew on microcarriers having high productivity of HuIFN-gammas for at least six months. Isoelectric focusing patterns and molecular weight analyses suggest that C127- and CHO-HuIFN-gammas are glycoproteins and that both HuIFN-gammas have different molecular structures. The development of a microcarrier culture system for genetically engineered mammalian cells has enabled us to prepare glycosylated HuIFN-gammas on a large scale.

Molecular cloning of complementary DNA for human medullasin: an inflammatory serine protease in bone marrow cells.

Medullasin, an inflammatory serine protease in bone marrow cells, modifies the functions of natural killer cells, monocytes, and granulocytes. We have cloned a medullasin cDNA from a human acute promyelocytic cell (ML3) cDNA library using oligonucleotide probes synthesized from the information of N-terminal amino acid sequence of natural medullasin. The cDNA contained a long open reading frame encoding 237 amino acid residues beginning from the second amino acid of natural meduallasin. The deduced amino acid sequence of medullasin shows a typical serine protease structure, with 41% homology with pig elastase 1.

Autonomous replicating sequences from intron of human ras gene in a simian virus 40 T antigen dependent system.

Of the several DNA fragments present in the human lung cancer gene, 1.1 and 2.0 kilobase (kb) fragments corresponding to the intron of this gene were hybridized to a half part of the 27 nucleotides perfect palindrome present in the initiation part of replication in simian virus 40 (SV40) DNA. These two fragments cloned in pBR322 had good template activity, and the initiation of DNA replication started from the region of these fragments in an in vitro system, in which the initiation of DNA replication occurs on cloned DNA containing SV40 origin of DNA replication as described previously. Furthermore, these two clones could replicate autonomously in nuclei of SV40 transformed Cos cells, producing SV40 T antigen constitutively when the clones were transfected into Cos cells. These results show that functional SV40 origin-like sequences are present in human genomes, and they can replicate autonomously within the cells which are producing SV40 T antigen.