Interleukin-1 Receptor-Like 2: One Receptor, Three Agonists, and Many Implications.

The interleukin (IL)-1 superfamily of cytokines comprises 11 pro- and anti-inflammatory cytokines, which play essential roles during the immune response. Several pathogenic pathways are initiated by IL-1RL2 (interleukin 1 receptor-like 2) signaling, also known as IL-36R, in the skin, lungs, and gut. IL-36 cytokines promote the secretion of proinflammatory cytokines and chemokines, upregulation of antimicrobial peptides, proliferation mediators, and adhesion molecules on endothelial cells. In addition, the IL-36-IL-1RL2 axis has an essential role against viral infections, including a potential role in COVID-19 pathology. The evidence presented in this review highlights the importance of the axis IL-36-IL-1RL2 in the development of several inflammation-related diseases and the healing process. It suggests that IL-1RL2 ligands have specific roles depending on the tissue or cell source. However, there is still much to discover about this cytokine family, their functions in other organs, and how they accomplish a dual effect in inflammation and healing.

Functional characterization of interleukin (IL)-22 and its inhibitor, IL-22 binding protein (IL-22BP) in Mandarin fish, Siniperca chuatsi.

As an important immune regulatory molecule, interleukin (IL)-22 has been reported in several species of fish, but its soluble receptor, IL-22 binding protein (IL-22BP), discovered as a natural antagonist of IL-22 in mammals, has not been functionally characterized in fish to date. In the present study, IL-22 and IL-22BP genes were cloned in mandarin fish Siniperca chuatsi. They all exhibited a high basal expression level in mucosa-enriched tissues, implying their possible roles in mucosal immunity. The IL-22 was found to show a potent response to LPS stimulation, acting as an inducer of antimicrobial peptide (AMP) genes, such as hepcidin and Liver-expressed antimicrobial peptide-2 (LEAP-2) in intestinal cells. IL-22BP, via co-incubation with IL-22, inhibited completely the induction of downstream genes by IL-22. Through a yeast two-hybrid assay, the interaction between IL-22BP and IL-22 was confirmed, which may account for the inhibitory effect of IL-22BP. Moreover, two hot spot residues for IL-22 binding, as reported in mammalian IL-22BP, were found to be conserved both in sequence location and function in mandarin fish IL-22BP, indicating that the interaction mode between IL-22 and IL-22BP may be also conserved in fish and mammals. In conclusion, the mandarin fish IL-22 and IL-22BP are conserved in their interaction and function with their mammalian orthologues, and these findings provide basis for future research on IL-22-IL-22BP axis in fish immunity.

An unexpected discovery of two interferon gamma-like genes along with interleukin (IL)-22 and -26 from teleost: IL-22 and -26 genes have been described for the first time outside mammals.

By comparative genomics based on chromosome synteny between human and fish, IL-22, IL-26 and two IFN-gamma-like genes have been isolated in fish. Interleukin-22 and -26 have been identified for the first time outside of mammals and their organization and synteny demonstrates that this cluster of cytokines is well conserved during evolution. The cloned zebrafish IL-22 and -26 homologs are 161 and 169-aa long, respectively. Both the genes harbor a well-conserved IL-10-family signature in their respective C-terminus. The coding regions of these genes are spread in five exons and at the same position as in human homologs. Furthermore, surprisingly two IFN-gamma-like genes are present in tandem, 7.0 kb apart from each other, along this locus. IFN-gamma1 and 2 genes are composed of 170- and 185-aa and share a low amino acid identity of 17.0%. The genomic structures of the isolated fish IFN-gamma genes were similar to its mammalian homologs, sharing a four exon and three phase 0 introns. The presence two IFN-gamma genes in both zebra and pufferfishes demonstrate that the duplication of IFN-gamma might have occurred prior to the teleostean split. The structural homology is further validated by phylogenetic analysis showing that the isolated cytokine genes are homologous to mammalian counterparts. Expression analysis of IL-22, IL-26 and two IFN-gamma genes suggests an active role of these genes in immune responses in fish.

Full house: 12 receptors for 27 cytokines.

With the sequencing of the human genome nearing completion, it appears that all members of the class II cytokine receptor family (CRF2) have been identified and partially characterized. The entire family is composed of exactly one dozen members. Eleven of them combine as various heterodimers to transduce signals across the cellular membrane for 27 cytokines divided into four structurally related groups: 6 cytokines of the IL-10 family, 17 type I IFNs, 1 type II IFN and 3 IFN-lambdas. The last CRF2 member is the soluble receptor which can neutralize the action of one of the cytokines of the IL-10 family, IL-22. Although the extracellular domains of all CRF2 proteins reveal primary and structural homology, their intracellular domains are very dissimilar. Nevertheless, signaling events induced through various combinations of CRF2 subunits partially overlap, leading to the induction of overlapping but cytokine-specific biological activities.

Human lung myofibroblasts as effectors of the inflammatory process: the common receptor gamma chain is induced by Th2 cytokines, and CD40 ligand is induced by lipopolysaccharide, thrombin and TNF-alpha.

The common gamma (gamma c) chain, shared by Th1 and Th2 cytokines, is fundamental for the activation of hematopoietic cells, but its role in non-hematopoietic tissues has not been explored. Here we show that in normal lung fibroblasts IL-4 and IL-13 induce the expression of the gamma c chain and its association with Janus kinase (JAK) 3, while lung myofibroblasts constitutively express a gamma c chain displaying a limited association with JAK3. In the latter cells, without exogenous cytokines, gamma c/JAK3 controls, through autocrine loops, tyrosine kinase (TYK) 2 phosphorylation and the balance between functional (IL-4Ralpha, IL-13Ralpha 1) and decoy (IL-13Ralpha 2) high-affinity receptors. Moreover, JAK3 is also associated with a pre-phosphorylated IL-4Ralpha and CD40. This novel "heterotrimer" (p-IL-4Ralpha, CD40/JAK3) is functional and controls STAT3 phosphorylation and CD40 expression, as shown by use of the specific JAK3 inhibitor WHI-P31. In basal culture conditions, CD40 signaling could be induced by the transient establishment of inter-fibroblastic CD40/CD40 ligand (CD40L) functional bridges. Indeed, powerful pro-inflammatory stimuli such as lipopolysaccharide and thrombin can rapidly mobilize CD40L at the surface of lung myofibroblasts. These interactions are modified by IL-13, which triggers the formation of a new type of functional receptor (p-IL-4Ralpha /IL-13Ralpha 1/gamma c) and also the recruitment and the phosphorylation of JAK3. Treatment with JAK3 inhibitors blocks IL-13-induced phosphorylation of JAK2, TYK2 and STAT3, but not of JAK1 and STAT6. These data underline (1) the pivotal role of the gamma c chain, CD40/CD40L, JAK3 and IL-13 in the inflammatory-like activation of lung myofibroblasts, (2) the cell-type restraint effects of IL-13 on these cells, and (3) the potential usefulness of JAK3 inhibitors in the treatment of asthma.

Cytokines: IL-21 joins the gamma(c)-dependent network?

The discovery of the cytokine IL-21 adds another member to the ever-growing list of small secreted molecules that have potent effects on lymphoid cells. Initial characterization of the IL-21 receptor complex suggests that IL-21 may belong to the cytokine family whose receptors share the common gamma chain, gamma(c).

The structure and function of gamma c-dependent cytokines and receptors: regulation of T lymphocyte development and homeostasis.

Five cytokines, IL-2, IL-4, IL-7, IL-9, and IL-15, form one group that is characterized by utilizing the common gamma chain (gamma c) as a receptor subunit. Examination of the phenotype of various cytokine or cytokine receptor "knockout" mice demonstrates that these cytokines are critical for normal lymphocyte development and subsequent functional activity of the peripheral immune compartment. This review summarizes the structural and functional properties of each of these five cytokines and their receptors, including the known redundant pathways for each cytokine or receptor. The contribution of these cytokines and receptors will then be considered in detail with respect to regulation of T lymphocyte development and homeostasis of the peripheral T cell compartment.

Chemoattractant cross-desensitization of the human neutrophil IL-8 receptor involves receptor internalization and differential receptor subtype regulation.

Human neutrophils undergo rapid homologous receptor desensitization following repeated stimulation with chemoattractants such as IL-8, C5a, and FMLP. It has also been demonstrated that cross-desensitization among these chemoattractant receptors occurs. We investigated the mechanisms underlying the cross-desensitization of responses to IL-8 induced by pretreatment with FMLP or C5a. In [125I]-labeled IL-8 binding studies we found that the cross-desensitization induced by FMLP or C5a was associated with a subsequent reduction in IL-8 binding to neutrophils. There was no recovery of [125I]-labeled IL-8 binding on removal of the C5a or FMLP pretreatment. FACS analysis using mAbs specific for the two IL-8R subtypes showed differential regulation of IL-8R A and IL-8R B cell surface expression after chemoattractant pretreatment. Homologous desensitization by IL-8 resulted in internalization of IL-8R A and IL-8R B, but only IL-8R A was completely re-expressed after removal of agonist. FMLP stimulation led to a substantial loss of IL-8R B from the cell surface, whereas C5a stimulation induced only a partial loss. In both cases there was no re-expression of IL-8R B on removal of the chemoattractant stimulation. C5a and FMLP did not affect IL-8R A expression. Calcium mobilization studies using melanoma growth stimulatory activity and IL-8 suggest that a sustained loss of IL-8R B may play a part in maintaining FMLP-induced IL-8R cross-desensitization. Chemoattractant-induced cross-desensitization of neutrophils may be of importance in regulating neutrophil accumulation during the inflammatory response in vivo.

The CXC chemokines growth-regulated oncogene (GRO) alpha, GRObeta, GROgamma, neutrophil-activating peptide-2, and epithelial cell-derived neutrophil-activating peptide-78 are potent agonists for the type B, but not the type A, human interleukin-8 receptor.

Interleukin-8 (IL-8), growth-related oncogene (GRO) alpha, GRObeta, GROgamma, neutrophil-activating peptide-2 (NAP-2), epithelial cell-derived neutrophil activating peptide- 78 (ENA-78), and granulocyte chemoattractant protein-2 are potent neutrophil chemoattractants 40-90% identical in amino acid sequence that comprise a subgroup of human CXC chemokines defined by the conserved sequence motif glutamic acid-leucine-arginine (ELR). Two human chemotactic receptor subtypes for IL-8, named IL-8 receptors (IL8R) A and B, have been cloned. They are 78% identical in amino acid sequence, coexpressed in neutrophils, and distinguished by their different selectivities for GROalpha and NAP-2. Their selectivity for other ELR+ CXC chemokines has not been previously reported. By measuring calcium flux in human embryonic kidney 293 cells transfected with plasmids encoding IL8RA or IL8RB, we have now defined receptor selectivity for GRObeta, GROgamma, and ENA-78. The rank order of agonist potency, based on inspection of the mean effective concentration values (EC50), for IL8RB was GROgamma (1 nM) > IL-8 (4 nM) approximately GROalpha (5 nM) approximately GRObeta (4 nM) approximately NAP-2 (7 nM) > ENA-78 (11 nM), and for IL8RA was IL-8 (4 nM) >>> ENA-78 (40 nM) approximately NAP-2 (45 nM) > GROalpha (63 nM) approximately GROgamma (65 nM) >> GRObeta. The maximal response of IL8RA to IL-8 was at least 2-fold greater than the other five chemokines. All six agonists for IL8RB competed for high affinity 125I-IL-8, -GROalpha, -NAP-2, and -ENA-78 binding sites at IL8RB. GROalpha, GRObeta, GROgamma, NAP-2, and ENA-78 competed weakly for the high affinity IL-8 binding site at IL8RA. Thus, IL8RA and IL8RB are both highly selective for IL-8 and have similar sequences but differ dramatically in their selectivity for all other ELR+ CXC chemokines tested. These findings have important implications for developing novel neutrophil-specific anti-inflammatory drugs directed against the CXC chemokine signaling system.

Different functions for the interleukin 8 receptors (IL-8R) of human neutrophil leukocytes: NADPH oxidase and phospholipase D are activated through IL-8R1 but not IL-8R2.

Two monoclonal antibodies, anti-IL8R1 and anti-IL8R2, raised against both interleukin 8 receptors (IL-8R) of human neutrophils, IL-8R1 and IL-8R2, were used to study individual receptor functions after stimulation with IL-8, GRO alpha, or NAP-2. Efficacy and selectivity of the antibodies were tested in Jurkat cells transfected with cDNA coding for one or the other receptor. The binding of 125 I labeled IL-8 and IL-8-induced changes of the cytosolic free Ca2+ concentration were inhibited by anti-IL8RI in cells expressing IL-8R1 and by anti-IL8R2 in cells expressing IL-8R2. In human neutrophils, release of elastase was observed after stimulation with IL-8 or GRO alpha. The response to IL-8 was inhibited slightly by anti-IL8R1 and more substantially when both monoclonal antibodies were present, while the response to GRO alpha was inhibited by anti-IL8R2 but was not affected by anti-IL8R1. These results indicate that both IL-8 receptors can signal independently for granule enzyme release. Superoxide production, a measure of the respiratory burst, was obtained with increasing concentrations of IL-8 with maximum effects at 25 to 50 nM, but no response was observed upon challenge with GRO alpha or NAP-2 up to 1000 nM. The superoxide production induced by IL-8 was inhibited by anti-IL8R1, but was not affected by anti-IL8R2. Stimulation of neutrophils with IL-8, in contrast to GRO alpha or NAP-2, also elicited phospholipase D activity. The effect of IL-8 was again inhibited by anti-IL-8R1 but not by anti-IL8R2, indicating that this response, like the respiratory burst, was mediated by IL-8R1. Taken together, our results show that IL-8R1 and IL-8R2 are functionally different. Responses, such as cytosolic free Ca2+ changes and the release of granule enzymes, are mediated through both receptors, whereas the respiratory burst and the activation of phospholipase D depend exclusively on stimulation through IL-8R1.

Hematopoietic stem cells in the blood after stem cell factor and interleukin-11 administration: evidence for different mechanisms of mobilization.

Peripheral blood stem cells and progenitor cells, collected during recovery from exposure to cytotoxic agents or after cytokine administration, are being increasingly used in clinical bone marrow transplantation. To determine factors important for mobilization of both primitive stem cells and progenitor cells to the blood, we studied the blood and splenic and marrow compartments of intact and splenectomized mice after administration of recombinant human interleukin-11 (rhlL-11), recombinant rat stem cell factor (rrSCF), and IL-11 + SCF. IL-11 administration increased the number of spleen colony-forming units (CFU-S) in both the spleen and blood, but did not increase blood long-term marrow-repopulating ability (LTRA) in intact or splenectomized mice. SCF administration increased the number of CFU-S in both the spleen and blood and did not increase the blood or splenic LTRA of intact mice, but did increase blood LTRA to normal marrow levels in splenectomized mice. The combination of lL-11 + SCF syngeristically enhanced mobilization of long-term marrow-repopulating cells from the marrow to the spleen of intact mice and from the marrow to the blood of splenectomized mice. These data, combined with those of prior studies showing granulocyte colony-stimulating factor mobilization of long-term marrow repopulating cells from the marrow to the blood of mice with intact spleens, suggest different cytokine-induced pathways for mobilization of primitive stem cells.

Diverging signal transduction pathways activated by interleukin-8 and related chemokines in human neutrophils: interleukin-8, but not NAP-2 or GRO alpha, stimulates phospholipase D activity.

Interleukin-8 (IL-8) and the structurally related cytokines neutrophil-activating peptide-2 (NAP-2) and GRO alpha are powerful chemotactic agents for human neutrophils. Although these three chemokines act by binding to overlapping but not identical receptor subsets, the data available to date have stressed the similarities in their mechanisms of action. The present studies were undertaken to further our understanding of the signal transduction mechanisms associated with these neutrophil agonists. IL-8, NAP-2, and GRO alpha stimulated similar increases in the level of cytoplasmic free calcium. They were also shown to stimulate qualitatively similar increases in the levels of protein tyrosine phosphorylation. In contrast, only IL-8 enhanced the formation of phosphatidylethanol (PEt), the product catalyzed by phospholipase D (PLD) in the presence of ethanol. The formation of PEt stimulated by IL-8 was inhibited by pertussis toxin and the tyrosine kinase inhibitors erbstatin and herbimycin A. The ability of IL-8 to stimulate the activity of PLD was additively enhanced, or primed, by cytochalasin B and by tumor necrosis factor alpha. Although all three chemokines increased the level of free cytoplasmic calcium to the same extent, IL-8 was significantly more potent than either NAP-2 or GRO alpha with respect to its ability to enhance CD11b expression and to stimulate chemotactic and oxidative responses. The differences between IL-8, NAP-2, and GRO alpha in their ability to stimulate PLD is likely to be related to their respective binding affinities for the two IL-8 receptors (IL-8R-A and IL-8R-B). These results suggest that the signalling pathways activated by IL-8R-A and IL-8R-B diverge at a step preceding the activation of PLD.

Complete mutagenesis of the extracellular domain of interleukin-8 (IL-8) type A receptor identifies charged residues mediating IL-8 binding and signal transduction.

We systematically converted each of the amino acids in the extracellular domain of the interleukin-8 (IL-8) type A receptor to alanine for the purpose of identifying amino acids contributing to IL-8 binding and IL-8-mediated signal transduction. We identified 20 mutations which cause a decrease in receptor affinity from a Kd of 2 nM to a Kd > or = 25 nM. We then analyzed these receptor mutants for their ability to mobilize intracellular calcium upon stimulation with 10 nM IL-8. The majority of the mutants were able to produce calcium fluxes at levels approximating that of wild-type IL-8 receptor A, with the exception of six mutants (R199A, R203A, C30A, C110A, C187A, and C277A) which showed no significant response. In addition, we performed calcium mobilization experiments to further characterize a series of previously constructed mutants which had only been characterized by their binding affinities in our previous report and found that mutant D265A showed no response upon stimulation with 10 nM IL-8. Our study shows that, besides the extracellular domain cysteines which may be critical for the overall folding of the receptor, three residues, Arg-199, Arg-203, and Asp-265, are important for IL-8 binding and IL-8-mediated signal transduction.

Multimeric cytokine receptors: common versus specific functions.

The class I cytokine receptors consist of multiple subunits without any intrinsic enzymatic activities. Receptors for a subset of cytokines with overlapping biological activities often share a common receptor subunit with a signaling function. Each receptor regulates its specific signaling pathways, as well as common pathways depending on the target cell type.