Interleukin-10 (IL-10), first recognized for its ability to inhibit activation and effector function of T cells, monocytes, and macrophages, is a multifunctional cytokine with diverse effects on most hemopoietic cell types. The principal routine function of IL-10 appears to be to limit and ultimately terminate inflammatory responses. In addition to these activities, IL-10 regulates growth and/or differentiation of B cells, NK cells, cytotoxic and helper T cells, mast cells, granulocytes, dendritic cells, keratinocytes, and endothelial cells. IL-10 plays a key role in differentiation and function of a newly appreciated type of T cell, the T regulatory cell, which may figure prominently in control of immune responses and tolerance in vivo. Uniquely among hemopoietic cytokines, IL-10 has closely related homologs in several virus genomes, which testify to its crucial role in regulating immune and inflammatory responses. This review highlights findings that have advanced our understanding of IL-10 and its receptor, as well as its in vivo function in health and disease.

Interleukin-10 and the interleukin-10 receptor.

▪ Abstract At the interface between the innate and adaptive immune systems lies the high-output isoform of nitric oxide synthase (NOS2 or iNOS). This remarkable molecular machine requires at least 17 binding reactions to assemble a functional dimer. Sustained catalysis results from the ability of NOS2 to attach calmodulin without dependence on elevated Ca2+. Expression of NOS2 in macrophages is controlled by cytokines and microbial products, primarily by transcriptional induction. NOS2 has been documented in macrophages from human, horse, cow, goat, sheep, rat, mouse, and chicken. Human NOS2 is most readily observed in monocytes or macrophages from patients with infectious or inflammatory diseases. Sustained production of NO endows macrophages with cytostatic or cytotoxic activity against viruses, bacteria, fungi, protozoa, helminths, and tumor cells. The antimicrobial and cytotoxic actions of NO are enhanced by other macrophage products such as acid, glutathione, cysteine, hydrogen peroxide, or superoxid...

Nitric oxide and macrophage function

Interferons play key roles in mediating antiviral and antigrowth responses and in modulating immune response. The main signaling pathways are rapid and direct. They involve tyrosine phosphorylation and activation of signal transducers and activators of transcription factors by Janus tyrosine kinases at the cell membrane, followed by release of signal transducers and activators of transcription and their migration to the nucleus, where they induce the expression of the many gene products that determine the responses. Ancillary pathways are also activated by the interferons, but their effects on cell physiology are less clear. The Janus kinases and signal transducers and activators of transcription, and many of the interferon-induced proteins, play important alternative roles in cells, raising interesting questions as to how the responses to the interferons intersect with more general aspects of cellular physiology and how the specificity of cytokine responses is maintained.

http://www3.uah.es/farmamol/Public/AnnReviews/PDF/Biochemistry/Interferons.pdf

How cells respond to interferons

Interferon-gamma (IFN-gamma) coordinates a diverse array of cellular programs through transcriptional regulation of immunologically relevant genes. This article reviews the current understanding of IFN-gamma ligand, receptor, signal transduction, and cellular effects with a focus on macrophage responses and to a lesser extent, responses from other cell types that influence macrophage function during infection. The current model for IFN-gamma signal transduction is discussed, as well as signal regulation and factors conferring signal specificity. Cellular effects of IFN-gamma are described, including up-regulation of pathogen recognition, antigen processing and presentation, the antiviral state, inhibition of cellular proliferation and effects on apoptosis, activation of microbicidal effector functions, immunomodulation, and leukocyte trafficking. In addition, integration of signaling and response with other cytokines and pathogen-associated molecular patterns, such as tumor necrosis factor-alpha, interleukin-4, type I IFNs, and lipopolysaccharide are discussed.

https://jlb.onlinelibrary.wiley.com/doi/epdf/10.1189/jlb.0603252

Interferon-γ: an overview of signals, mechanisms and functions

This review concentrates on advances in nitric oxide synthase (NOS) structure, function and inhibition made in the last seven years, during which time substantial advances have been made in our understanding of this enzyme family. There is now information on the enzyme structure at all levels from primary (amino acid sequence) to quaternary (dimerization, association with other proteins) structure. The crystal structures of the oxygenase domains of inducible NOS (iNOS) and vascular endothelial NOS (eNOS) allow us to interpret other information in the context of this important part of the enzyme, with its binding sites for iron protoporphyrin IX (haem), biopterin, L-arginine, and the many inhibitors which interact with them. The exact nature of the NOS reaction, its mechanism and its products continue to be sources of controversy. The role of the biopterin cofactor is now becoming clearer, with emerging data implicating one-electron redox cycling as well as the multiple allosteric effects on enzyme activity. Regulation of the NOSs has been described at all levels from gene transcription to covalent modification and allosteric regulation of the enzyme itself. A wide range of NOS inhibitors have been discussed, interacting with the enzyme in diverse ways in terms of site and mechanism of inhibition, time-dependence and selectivity for individual isoforms, although there are many pitfalls and misunderstandings of these aspects. Highly selective inhibitors of iNOS versus eNOS and neuronal NOS have been identified and some of these have potential in the treatment of a range of inflammatory and other conditions in which iNOS has been implicated.

/pdf/nitric-oxide-synthases-structure-function-and-inhibition-1t31plqilr.pdf

Nitric oxide synthases: structure, function and inhibition

Crystal structures of human endothelial nitric oxide synthase (eNOS) and human inducible NOS (iNOS) catalytic domains were solved in complex with the arginine substrate and an inhibitor S-ethylisothiourea (SEITU), respectively. The small molecules bind in a narrow cleft within the larger active-site cavity containing heme and tetrahydrobiopterin. Both are hydrogen-bonded to a conserved glutamate (eNOS E361, iNOS E377). The active-site residues of iNOS and eNOS are nearly identical. Nevertheless, structural comparisons provide a basis for design of isozyme-selective inhibitors. The high-resolution, refined structures of eNOS (2.4 A resolution) and iNOS (2.25 A resolution) reveal an unexpected structural zinc situated at the intermolecular interface and coordinated by four cysteines, two from each monomer.

Structural characterization of nitric oxide synthase isoforms reveals striking active-site conservation.

The crystal structure of interferon-γ bound to the extracellular fragment of its high-affinity cellsurface receptor reveals the first view of a class-2 cytokine receptor-ligand complex. In the complex, one interferon-γ homodimer binds two receptor molecules. Unlike the class-1 growth hormone receptor complex, the two interferon-γ receptors do not interact with one another and are separated by 27 A. Upon receptor binding, the flexible AB loop of interferon-γ undergoes a conformational change that includes the formation of a 310 helix.

Crystal structure of a complex between interferon-γ and its soluble high-affinity receptor

The endocannabinoids anandamide and 2-arachidonyl glycerol (2-AG) bind to G protein-coupled central and peripheral cannabinoid receptors CB1 and CB2, respectively. Due to the relatively high expression of the CB2 isotype on peripheral immune cells, it has been hypothesized that this receptor mediates the immunosuppressive effects of cannabinoids. Unfortunately, there was a dearth of pharmacological studies with the endocannabinoids and human CB2 (hCB2). These studies compare and contrast the potency and efficacy of anandamide, 2-AG, and the synthetic cannabinoid HU210 at hCB2. Using [(35)S]guanosine-5'-O-(3-thio)triphosphate (GTPgammaS) and radioligand bindings in insect Sf9-hCB2 membranes, we showed that both endocannabinoids bound hCB2 with similar affinity and that the cannabinoids acted as full agonists in stimulating [(35)S]GTPgammaS exchange, although 2-AG was 3-fold more potent than anandamide (EC(50) = 38.9 +/- 3.1 and 121 +/- 29 nM, respectively). In a mammalian expression system (Chinese hamster ovary-hCB2 cells), HU210 and 2-AG maximally inhibited forskolin-stimulated cAMP synthesis (IC(50) = 1.61 +/- 0.42 nM and 1.30 +/- 0.37 microM, respectively) although anandamide was ineffective. In Chinese hamster ovary-hCB2 membranes, HU210 and 2-AG were also full agonists in stimulating [(35)S]GTPgammaS binding (EC(50) = 1.96 +/- 0.35 and 122 +/- 17 nM, respectively), but anandamide was a weak partial agonist (EC(50) = 261 +/- 91 nM; 34 +/- 4% of maximum). Due to its low intrinsic activity, coincubation with anandamide effectively attenuated the functional activity of 2-AG at hCB2. Collectively, the data showed that both endocannabinoids bound hCB2 with similar affinity, but only 2-AG functioned as a full agonist. Moreover, the agonistic activity of 2-AG was attenuated by anandamide.

Endocannabinoid 2-arachidonyl glycerol is a full agonist through human type 2 cannabinoid receptor: antagonism by anandamide.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/S0014-5793%2896%2901410-X

Purification of ADAM 10 from bovine spleen as a TNFα convertase

The expression of the cannabinoid peripheral cannabinoid receptor (CB(2)) receptor on peripheral immune cells suggests that compounds specific for CB(2) might be effective anti-inflammatory agents. In this report, we present the initial biological profiling of a novel triaryl bis-sulfone, Sch.336 (N-[1(S)-[4-[[4-methoxy-2-[(4-methoxyphenyl)sulfonyl]phenyl]-sulfonyl]phenyl]ethyl]methanesulfonamide), which is selective for the human cannabinoid CB(2) receptor (hCB(2)). Sch.336 is an inverse agonist at hCB(2), as shown by its ability to decrease guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding to membranes containing hCB(2), by the ability of GTPgammaS to left-shift Sch.336 binding to hCB(2) in these membranes, and by the compound's ability to increase forskolin-stimulated cAMP levels in CHO cells expressing hCB(2). In these systems, Sch.336 displays a greater potency than that reported for the CB(2)-selective dihydropyrazole, SR144528 (N-[(1S)-endo-1,3,3-trimethylbicyclo [2.2.1]heptan2-yl]-5-(4-chloro-3-methylphenyl)-1-[(4-methylphenyl)methyl]-1H-pyrazole-3-carboxamide). In vitro, Sch.336 impairs the migration of CB(2)-expressing recombinant cell lines to the cannabinoid agonist 2-arachidonylglycerol. In vivo, the compound impairs migration of cells to cannabinoid agonist HU210 [(6aR)-trans-3-(1,1-dimethylheptyl)-6a,7,10,10a-tetrahydro-1-hydroxy-6,6-dimethyl-6H-dibenzo [b,d] pyran-9-methanol]. Oral administration of the Sch.336 significantly inhibited leukocyte trafficking in several rodent in vivo models, induced either by specific chemokines or by antigen challenge. Finally, oral administration of Sch.336 blocked ovalbumin-induced lung eosinophilia in mice, a disease model for allergic asthma. We conclude that selective cannabinoid CB(2) inverse agonists may serve as novel immunomodulatory agents in the treatment of a broad range of acute and chronic inflammatory disorders in which leukocyte recruitment is a hallmark of disease pathology.

Charles A. Lunn

Papers

Structural characterization of nitric oxide synthase isoforms reveals striking active-site conservation.

Crystal structure of a complex between interferon-γ and its soluble high-affinity receptor

Endocannabinoid 2-arachidonyl glycerol is a full agonist through human type 2 cannabinoid receptor: antagonism by anandamide.

Purification of ADAM 10 from bovine spleen as a TNFα convertase

A Novel Cannabinoid Peripheral Cannabinoid Receptor-Selective Inverse Agonist Blocks Leukocyte Recruitment in Vivo