https://www.cell.com/cell/pdf/0092-8674(86)90341-7.pdf

A conserved AU sequence from the 3′ untranslated region of GM-CSF mRNA mediates selective mRNA degradation

In the present study we demonstrate that human monocytes activated by lipopolysaccharides (LPS) were able to produce high levels of interleukin 10 (IL-10), previously designated cytokine synthesis inhibitory factor (CSIF), in a dose dependent fashion. IL-10 was detectable 7 h after activation of the monocytes and maximal levels of IL-10 production were observed after 24-48 h. These kinetics indicated that the production of IL-10 by human monocytes was relatively late as compared to the production of IL-1 alpha, IL-1 beta, IL-6, IL-8, tumor necrosis factor alpha (TNF alpha), and granulocyte colony-stimulating factor (G-CSF), which were all secreted at high levels 4-8 h after activation. The production of IL-10 by LPS activated monocytes was, similar to that of IL-1 alpha, IL-1 beta, IL-6, IL-8, TNF alpha, granulocyte-macrophage colony-stimulating factor (GM-CSF), and G-CSF, inhibited by IL-4. Furthermore we demonstrate here that IL-10, added to monocytes, activated by interferon gamma (IFN-gamma), LPS, or combinations of LPS and IFN-gamma at the onset of the cultures, strongly inhibited the production of IL-1 alpha, IL-1 beta, IL-6, IL-8, TNF alpha, GM-CSF, and G-CSF at the transcriptional level. Viral-IL-10, which has similar biological activities on human cells, also inhibited the production of TNF alpha and GM-CSF by monocytes following LPS activation. Activation of monocytes by LPS in the presence of neutralizing anti-IL-10 monoclonal antibodies resulted in the production of higher amounts of cytokines relative to LPS treatment alone, indicating that endogenously produced IL-10 inhibited the production of IL-1 alpha, IL-1 beta, IL-6, IL-8, TNF alpha, GM-CSF, and G-CSF. In addition, IL-10 had autoregulatory effects since it strongly inhibited IL-10 mRNA synthesis in LPS activated monocytes. Furthermore, endogenously produced IL-10 was found to be responsible for the reduction in class II major histocompatibility complex (MHC) expression following activation of monocytes with LPS. Taken together our results indicate that IL-10 has important regulatory effects on immunological and inflammatory responses because of its capacity to downregulate class II MHC expression and to inhibit the production of proinflammatory cytokines by monocytes.

/pdf/interleukin-10-il-10-inhibits-cytokine-synthesis-by-human-3eb6x6k1g4.pdf

Interleukin 10(IL-10) inhibits cytokine synthesis by human monocytes: an autoregulatory role of IL-10 produced by monocytes.

When stimulated with antigen, B cells are influenced by T cells to proliferate and differentiate into antibody-forming cells. Since it was reported that soluble factors could replace certain functions of helper T cells in the antibody response, several different kinds of lymphokines and monokines have been reported in B-cell growth and differentiation. Among these, human B-cell differentiation factor (BCDF or BSF-2) has been shown to induce the final maturation of B cells into immunoglobulin-secreting cells. BSF-2 was purified to homogeneity and its partial NH2-terminal amino-acid sequence was determined. These studies indicated that BSF-2 is functionally and structurally unlike other known proteins. Here, we report the molecular cloning, structural analysis and functional expression of the cDNA encoding human BSF-2. The primary sequence of BSF-2 deduced from the cDNA reveals that BSF-2 is a novel interleukin consisting of 184 amino acids.

Complementary DNA for a novel human interleukin (BSF-2) that induces B lymphocytes to produce immunoglobulin

Polypeptide chain elongation factor l a (EF-la) is an eukaryotic counterpart of E. coli EF-Tu which promotes the GTP-dependent binding of an aminoacyl-tRNA to ribosomes. EF-la is one of the most abundant proteins in eukaryotic cells, and expressed in almost all kinds of mammalian cells. Recently, we have isolated human chromosomal gene coding for EF-la, and shown that the promoter of EF-la chromosomal gene very efficiently stimulates the in vitro transcription (1). In this report, we have constructed a powerful mammalian expression vector, pEF-BOS, using the promoter of human EF-la chromosomal gene. As shown in Fig. 1, pEF-BOS carries the SV40 replication origin (311 bp of EcoRH G fragment), the promoter region of human EF-la chromosomal gene (1.2 kb), the stuffer fragment (450 bp) from CDM8 vector (2) and poly(A) adenylation signal from human G-CSF cDNA (700 bp EcoSll ~ EcoRl DNA fragment) (3) in HindUL-EcoRl site of pUC119. The promoter region of EF-la gene is from nucleotide position 373 to 1561 (1) which includes 203 bp 5' flanking region, 33 bp first exon, 943 bp first intron and 10 bp of the part of the second exon located at 20 bp upstream of the ATG initiation codon. The size of pEFBOS is 5.8 kb, and the cDNA to be expressed can be inserted at BstXl site using BstXl adapter, or Xbal site using Xbal linker. Human G-CSF cDNA (4) was inserted into BstXl site of pEFBOS or CDM8, or into BamhU site of pKCR vector containing SV40 early promoter (5). As shown in Table 1, when these plasmids were transfected into COS cells by DEAEdextran/chloroquine method, the construct in pEF-BOS has directed the synthesis of human G-CSF about 20 times more efficiently than the construct in CDM8, and 50 ~ 200 times more efficiently than the construct in pKCR. In addition, when E. coli chloramphenicol acetyltransferase (CAT) gene was inserted into pEF-BOS, the CAT activities observed with pEF-BOS-CAT were 1.5 ~ 50 times higher than that of pSV2-CAT or pRSV-CAT after transfection into various cell lines including murine L929, human HeLa, CHU-2 and simian COS cells (Table 2). The pEFBOS vector, therefore, will be used to produce a large amount of growth factors and proteins in mammalian cells, to express a high level of anti-sense RNA. Furthermore, the pEF-BOS-CAT will be an ideal positive control for CAT assay in various cell types.

pEF-BOS, a powerful mammalian expression vector.

The complementary DNAs and genes encoding the four major human myeloid growth factors--granulocyte colony-stimulating factor, macrophage colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and interleukin-3--have all been molecularly cloned. These DNA clones have proved valuable for studying the molecular biology of these important regulatory molecules as well as for the large-scale production of the recombinant growth factor proteins. These advances have led to a much better understanding of the role of the myeloid growth factors in regulating hematopoiesis in vivo that should soon find practical application in clinical medicine.

https://science.sciencemag.org/content/sci/236/4806/1229.full.pdf

The human hematopoietic colony-stimulating factors

Granulocyte colony-stimulating factor (G-CSF) is a member of the CSF family of hormone-like glycoproteins that regulate haematopoietic cell proliferation and differentiation1,2, and G-CSF almost exclusively stimulates the colony formation of granulocytes from committed precursor cells in semi-solid agar culture2. Recently, Nomura et al.3 have established a human squamous carcinoma cell line (designated CHU-2) from a human oral cavity tumour which produces large quantities of CSF constitutively, and the CSF produced by CHU-2 cells has been purified to homogeneity from the conditioned medium. We have now determined the partial amino-acid sequence of the purified G-CSF protein, and by using oligonucleotides as probes, have isolated several clones containing G-CSF complementary DNA from the cDNA library prepared with messenger RNA from CHU-2 cells. The complete nucleotide sequences of two of these cDNAs were determined and the expression of the cDNA in monkey COS cells gave rise to a protein showing authentic G-CSF activity. Furthermore, Southern hybridization analysis of DNA from normal leukocytes and CHU-2 cells suggests that the human genome contains only one gene for G-CSF and that some rearrangement has occurred within one of the alleles of the G-CSF gene in CHU-2 cells.

Naoki Kubota

Papers

Molecular cloning and expression of cDNA for human granulocyte colony-stimulating factor.