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.

/pdf/secretory-products-of-macrophages-5cf7rlxshh.pdf

Secretory products of macrophages.

Interleukin-1 beta (IL-1 beta)-converting enzyme cleaves the IL-1 beta precursor to mature IL-1 beta, an important mediator of inflammation. The identification of the enzyme as a unique cysteine protease and the design of potent peptide aldehyde inhibitors are described. Purification and cloning of the complementary DNA indicates that IL-1 beta-converting enzyme is composed of two nonidentical subunits that are derived from a single proenzyme, possibly by autoproteolysis. Selective inhibition of the enzyme in human blood monocytes blocks production of mature IL-1 beta, indicating that it is a potential therapeutic target.

A novel heterodimeric cysteine protease is required for interleukin-1 beta processing in monocytes.

A method for the quantitation of specific mRNA species by the polymerase chain reaction (PCR) has been developed by using a synthetic RNA as an internal standard. The specific target mRNA and the internal standard are coamplified in one reaction in which the same primers are used. The amount of mRNA is then quantitated by extrapolating against the standard curve generated with the internal standard. The synthetic internal standard RNA consists of a linear array of the sequences of upstream primers of multiple target genes followed by the complementary sequences to their downstream primers in the same order. This quantitative PCR method provides a rapid and reliable way to quantify the amount of a specific mRNA in a sample of less than 0.1 ng of total RNA. In addition, the same internal standard RNA is used, with appropriate primer pairs, to quantitate multiple different mRNA species.

https://www.pnas.org/content/pnas/86/24/9717.full.pdf

Quantitation of mRNA by the Polymerase Chain Reaction

I. Introduction BASIC AND acidic fibroblast growth factors (FGFs) are closely related molecules that show a similar range of biological activities. They differ, however, in some of their physical and chemical properties and in their tissue distribution (1). Basic FGF [bFGF isoelectric point (pi) 9.6] was first identified by its ability to cause the proliferation and phenotypic transformation of BALB/c 3T3 fibroblasts (2, 3). Acidic FGF (aFGF, pi 5.6) was first identified by its ability to cause proliferation and delayed differentiation of myoblasts (4); it was later rediscovered on the basis of its ability to stimulate endothelial cell proliferation (5, 6). As expected from their structural relationship, both FGF and aFGF interact with the same receptor (7), thereby having similar, if not identical, properties. In contrast to aFGF, which has a cellular distribution more restricted than bFGF, many different cells synthesize bFGF, and essentially all have a specific high affinity receptor for this peptide. ...

Structural characterization and biological functions of fibroblast growth factor

Two distinct but distantly related complementary DNAs encoding proteins sharing human interleukin-1 (IL-1) activity (termed IL-1 alpha and IL-1 beta), were isolated from a macrophage cDNA library. The primary translation products of the genes are 271 and 269 amino acids long, although expression in Escherichia coli of the carboxy-terminal 159 and 153 amino acids produces IL-1 biological activity.

Cloning, sequence and expression of two distinct human interleukin-1 complementary DNAs

Interleukin 1 (IL-1) is a two-member family of proteins (IL-1 alpha and IL-1 beta) that mediates a diverse series of immune and inflammatory responses. These two proteins have only 26% amino acid homology yet bind to the same receptor. It is of importance to define the active sites of these molecules in order to understand their receptor interactions and the mechanisms involved in their multiple biological functions. We report here the localization of the biologically active portions within the initial polypeptide translation products. An in vitro transcription and translation system was used to generate specific fragments of each of the IL-1 molecules, which then were assayed for receptor binding capability and biological activity. Using this system, we have demonstrated that core sequences of amino acids for IL-1 beta (numbers 120-266) and 140 amino acids for IL-1 alpha (numbers 128-267) must be left intact to retain full biological activity and further that the biological activities of the IL-1 polypeptides parallel their receptor binding capabilities.

https://www.pnas.org/content/pnas/84/13/4572.full.pdf

Bruce Mosley

Papers

Cloning, sequence and expression of two distinct human interleukin-1 complementary DNAs

Determination of the minimum polypeptide lengths of the functionally active sites of human interleukins 1 alpha and 1 beta.