Modulation of the IL-1 cytokine network in keratinocytes by intracellular IL-1 alpha and IL-1 receptor antagonist.

The IL-1 cytokine network in epidermal cells was studied in vitro, using the spontaneously transformed HaCAT human keratinocyte line. Intracellular (ic) IL-1 alpha and IL-1 receptor antagonist protein (IL-1Ra) following cell lysis were readily identified assayed using a capture ELISA; whereas in culture supernatants IL-1Ra was not detected, and IL-1 alpha was present at only very low levels. Confluent cultures of HaCAT cells were shown to provide optimal conditions for the study, since confluence increased the icIL-1Ra:IL-1 alpha ratio to a level as seen in vivo, which was independent of Ca2+ concentration in the culture medium. The IL-1Ra extracted from HaCAT cell lysates was functionally active, as demonstrated in the mouse thymocyte co-proliferation assay which could be blocked using a rabbit anti-IL-1Ra antibody. Transforming growth factor-beta (TGF-beta 1) stimulated a dose-dependent increase in HaCAT cell IL-1 alpha without changing IL-1Ra concentration, with a resultant reduction in the icIL-1Ra: IL-1 alpha ratio from 320:1 to 100:1. Similarly, TGF-alpha, interferon-gamma (IFN-gamma), IL-6, and tumour necrosis factor-alpha (TNF-alpha) substantially increased HaCAT cell IL-1 alpha, but had no effect on the IL-1Ra, with a concomitant reduction in the icIL-1Ra:IL-1 alpha ratio. In contrast to their effects on monocytes, IL-4 and IL-10 at biologically active levels had no effect on IL-1 alpha, IL-1Ra or the icIL-1Ra: IL-1 alpha ratio in confluent HaCAT cells. Hydrocortisone reduced IL-1 alpha to below the limit of sensitivity of the ELISA, and induced a small increase in IL-1Ra of questionable biological significance. Thus, regulation of the IL-1 cytokine network in keratinocytes involves modulation of icIL-1 alpha rather than of icIL-1Ra levels, and is markedly different from that noted in monocytes.     

A tissue specific IL-1 receptor antagonist homolog from the IL-1 cluster lacks IL-1, IL-1ra, IL-18 and IL-18 antagonist activities

Interleukin (IL)-1-like protein 1 (IL-1L1) is a 155-amino acid protein that shares 27% identity with IL-1beta and 47% with IL-1 receptor antagonist (IL-1ra). A 2.7-kb IL-1L1 mRNA was cloned from human placenta and is detectable in the trophoblastic cell line JEG-3, in macrophages and in endotoxin-stimulated monocytes. Expression of IL-1L1 is much less abundant and less widespread than IL-1ra. We have determined the human and mouse IL-1L1 cDNA sequences and the complete sequence of the human gene, IL1L1. IL1L1 consists of four coding exons, has two alternative non-coding first exons, lies between IL1B and IL1RN, is orientated in the same direction as IL1RN and is separated from it by approximately 53 kb. The predicted IL-1L1 protein lacks both signal sequence and glycosylation signals. A 17-kDa protein was recovered by immunoprecipitation with IL-1L1-specific antibodies from JEG-3. IL-1L1 did not stimulate IL-6 production from primary human fibroblasts or human umbilical vein endothelial cells nor did it block the IL-1alpha or IL-1beta-dependent activation of IL-6 expression. We conclude, contrary to a recent suggestion made by others, that IL-1L1 is not a functional IL-1ra. IL-1L1 also had no detectable agonistic or antagonistic effect on IFN-gamma production in response to IL-18 in KG-1 cells.     

Regulatory immune responses induced by IL-1 receptor antagonist in rheumatoid arthritis

Anakinra, a human recombinant IL-1 receptor antagonist, is approved for the treatment of RA. In this study, 12 patients received the placebo plus MTX treatment, 38 patients received Anakinra combined with MTX treatment. Compared with the placebo plus MTX group, serum levels of IL-17, IFN-γ, IL-21 and IL-1β significantly decreased, the percentages of Th17 cells and Th1 cells were lower and the percentage of Treg cells was higher after receiving Anakinra combined with MTX treatment. The observed regulatory immune responses collectively correlated with clinical improvement in treated patients. A substantial response, ACR 20 at 24 w were consistent with those at 12 w, 16 w and 20 w, and was accompanied by a marked improvement in RA related laboratory parameters. The study reveals that the combination of Anakinra and MTX is safe and well tolerated, which induces regulatory immune responses and significantly provides greater clinical benefit than the placebo plus MTX group.     

Production of IL-1beta, IL-1 receptor antagonist and IL-10 by mononuclear cells from patients with SLE.

Depositions of immune-complexes are responsible for many of the pathological features of systemic lupus erythematosus (SLE). For example, immune-complex-induced tissue damage in glomerulonephritis has been shown to be mediated, at least in part, by interleukin (IL)-1. Inappropriate production or function of IL-1 may therefore contribute to disease manifestations in SLE. We investigated lipopolysaccharide (LPS)- and adherent IgG-stimulated release of IL-1beta, IL-1 receptor antagonist (IL-1ra) and IL-10, a potent modulator of IL-1, by blood mononuclear cells from patients with SLE. Mediator production was measured as ng cytokines/10(6) monocytes and compared with clinical parameters. Release of IL-1beta was only detectable in LPS-stimulated cultures and substantially reduced in patients with both active and inactive disease (P < 0.001). LPS-stimulated IL-1ra release was normal and the IL-1ra/IL-1beta ratio was therefore increased (P < 0.05) and correlated inversely to prednisolone dosage (P = 0.009). IgG-stimulated release of IL-1ra was reduced in patients with active disease compared to those with inactive disease and controls (P = 0.002). IL-10 release was similar in patients and controls. We conclude that monocytes from patients with active SLE are deficient in Fc gamma-R-mediated production of IL-1ra, whereas LPS-stimulated IL-1beta release by SLE monocytes is reduced regardless of disease activity. The former may contribute to immune-complex-mediated tissue damage in SLE.     

Immunohistochemical staining of IL-1 alpha and IL-1 receptor antagonist but not IL-1 beta in cultures of sertoli cells

The interleukin-1 (IL-1) system has been suggested to be involved in the cell cell cross talk within the testis. To identify a testicular cell source of IL-1 alpha, IL-1 beta and IL-1 receptor antagonist (IL-1ra), immature mouse Sertoli cells were isolated, purified, cultured and examined for the cellular compartment localization of these cytokines by immunohistochemical staining. Our results show that both Germ cells and Sertoli cells in unpurified Sertoli cell cultures (before hypotonic shock) and purified culture of Sertoli cells (after hypotonic shock) were stained for IL-1 alpha. The levels of this cytokine were increased in Sertoli cells when the purified cultures were stimulated with lipopolysaccharide (LPS) (5 microg/mL). However, we could not identify a positive staining for IL-1 beta when Sertoli cell cultures were stained for this cytokine, even after stimulation with various concentrations of LPS (0.1-10 microg/mL). On the other hand, immunohistochemical staining of isolated Sertoli cells without treatment with hypotonic shock (cultures containing Sertoli cells and Germ cells) for IL-1ra showed constitutive positive staining of both cell types (Sertoli cells and Germ cells). Our results, using immunohistochemical staining, may indicate the different expression of IL-1 alpha, IL-1 beta and IL-1ra in Sertoli cells. These results may suggest the involvement of IL-1 system in the autocrine and paracrine regulation of testicular cell functions.     

Inhibition of ghrelin-induced feeding in rats by pretreatment with a novel dual orexin receptor antagonist

Orexin-A and -B, and ghrelin are potent orexigenic peptides. The effects of ACT462206, a novel dual orexin receptor antagonist (DORA), on ghrelin-induced feeding were examined in adult male Wistar rats. Hyperphagia induced by the intracerebroventricular (icv) administration of ghrelin was significantly suppressed for at least 2 h by pretreatment with icv administration of DORA. A marked increase was observed in the number of neurons showing Fos immunoreactivity in the paraventricular nucleus, arcuate nucleus and lateral hypothalamic area (LHA), 90 min after icv administration of ghrelin. Pretreatment with DORA significantly decreased the number of Fos-immunoreactive (IR) neurons; however, Fos immunoreactivity remained significantly increased. Double-immunostaining for Fos and orexin-A showed that many orexin-A-IR neurons in the LHA coexisted with Fos immunoreactivity after icv administration of ghrelin, but their number was reduced significantly by DORA pretreatment. These results suggest that centrally administered ghrelin may activate the orexinergic and non-orexinergic pathways responsible for the regulation of feeding.     

Inhibition of leukotriene B4(LTB4) by recombinant interleukin-1 receptor antagonist (IL-1RA) on human monocytes

Macrophages are a primary source of interleukin-1 (IL-1), a glycoprotein which plays an important and essential role in the immune response and inflammation. Cytokines stimulate many different cells to produce increasing amounts of arachidonic acid metabolites such as prostaglandins and leukotrienes. Recently, interleukin-1 receptor antagonist (IL-1ra), a natural inhibitor of IL-1 released by macrophages, has been reported to inhibit PGE₂. In accordance with these data our results show that the pretreatment, for 60 min, of purified human peripheral monocytes with IL-1ra at different concentrations (0.25–250 ng/ml) inhibits, in a dose-dependent manner, the generation of LTB₄ released after 10 min treatment with calcium ionophore A23187 (5 μM). The inhibition of LTB₄ synthesis by hrIL-1ra suggests the possibility that this new glycoprotein plays a modulatory role in immunity and inflammation.

     

The IL-1 system in HIV infection: peripheral concentrations of IL-1β, IL-1 receptor antagonist and soluble IL-1 receptor type II

The proinflammatory cytokine IL-1β is thought to be involved in ongoing HIV disease. Furthermore, its naturally occurring inhibitors soluble IL-1 receptor type II (sIL-1RII) and IL-1 receptor antagonist (IL-1Ra) may play a pivotal role in regulating its biological action. To investigate the involvement of the IL-1 system we determined serum levels of IL-1β, IL-1Ra and sIL-1RII in 90 HIV⁺ patients. The obtained values were compared with markers of disease progression such as CD⁺ count, 5′-neopterin, β₂-microglobulin and soluble tumour necrosis factor receptors (sTNF-R) p55 and p75 and then compared with C-reactive protein (CRP), granulocyte count, lL-6 and TNF-α. While IL-1Ra concentrations increased significantly with progressive CDC disease stages, sIL-1RII and IL-1β were not altered in our cohort. IL-1Ra showed statistical relation to decreasing CD4⁺ lymphocytes and increasing 5′-neopterin, β₂-microglobulin, sTNF-R p55, sTNF-R p75. Furthermore, IL-1Ra correlated positively with serum IL-6, TNF-α, CRP and granulocytes. In contrast, sIL-1RII and IL-1β tended to show an inverse correlation or showed no significant relationship to all these parameters. Il-1β was measurable only in a limited number of samples. IL-1Ra showed a clear relationship to acute inflammatory events as well as to the different disease stages. Our data suggest a dissociation between IL-1Ra and sIL-1RII serum levels which may indicate that the two IL-1 binding proteins have different pathophysiological roles in HIV infection.     

Production of IL-1 and IL-1 receptor antagonist and the pathological significance in lipopolysaccharide-induced arthritis in rabbits.

Injection of lipopolysaccharide (LPS) into rabbit knee joints provoked leucocyte infiltration and loss of proteoglycan (PG) from the cartilage. We investigated the role of IL-1 and IL-1 receptor antagonist (IL-1Ra) and its significance in the pathogenesis of LPS-arthritis. Production of IL-1 beta peaked at 6 h (196.7 +/- 89.4 pg/joint) after injection of 10 ng of LPS, while IL-1Ra peaked at 9 h (34.5 +/- 13.4 ng/joint). The amount of IL-1Ra was 180-200-fold molar excess of IL-1, and a large amount of IL-1Ra was sustained for 1 week. Both IL-1 beta and IL-1Ra were mainly produced by synovial exudate cells. Arthritis was reproduced by rabbit IL-1 beta. LPS-induced leucocyte infiltration was inhibited 70-75% by rabbit IL-1Ra. Loss of PG in LPS-arthritis was prevented by IL-1Ra and also by neutrophil elastase inhibitor, and superoxide dismutase. In leucopenic rabbits, injection of LPS induced neither production of IL-1 beta nor loss of PG. Direct injection of inflammatory exudated cells in leucopenic rabbits reproduced loss of PG, and there was only a partial recovery by IL-1Ra. These results suggest that LPS-initiated IL-1 acts as a key mediator in LPS-arthritis and that endogenous IL-1Ra may suppress a part of IL-1 activity at the site, but its amount was too low for suppression of the produced IL-1. Loss of PG is a sequela of infiltrated leucocytes and leucocyte-derived elastase, and superoxide anion may play a pivotal role in the destruction of cartilage.     

Blocking IL-1: interleukin 1 receptor antagonist in vivo and in vitro.

Clinical and experimental evidence suggests that shock, arthritis, osteoporosis, colitis, leukemia, diabetes, wasting and atherosclerosis are mediated, in part, by interleukin 1 (IL-1). Inhibition of this cytokine has been a strategy for studying disease and for new drug development. A naturally-occurring IL-1 inhibitor (IL-1 receptor antagonist, IL-1ra) that blocks binding of IL-1 to its receptors has been cloned and produced in recombinant organisms. IL-1ra reduces the severity of sepsis, colitis, arthritis and diabetes in animals and is presently being tested in humans with arthritis, shock and myelogenous leukemia.     

Inhibition of leukotriene B4(LTB4) by recombinant interleukin-1 receptor antagonist (IL-1RA) on human monocytes

Macrophages are a primary source of interleukin-1 (IL-1), a glycoprotein which plays an important and essential role in the immune response and inflammation. Cytokines stimulate many different cells to produce increasing amounts of arachidonic acid metabolites such as prostaglandins and leukotrienes. Recently, interleukin-1 receptor antagonist (IL-1ra), a natural inhibitor of IL-1 released by macrophages, has been reported to inhibit PGE₂. In accordance with these data our results show that the pretreatment, for 60 min, of purified human peripheral monocytes with IL-1ra at different concentrations (0.25–250 ng/ml) inhibits, in a dose-dependent manner, the generation of LTB₄ released after 10 min treatment with calcium ionophore A23187 (5 M). The inhibition of LTB₄ synthesis by hrIL-1ra suggests the possibility that this new glycoprotein plays a modulatory role in immunity and inflammation.     

Allelic polymorphism in IL-1 beta and IL-1 receptor antagonist (IL-1Ra) genes in inflammatory bowel disease.

Recent reports have shown that allele 2 of the IL-1 receptor antagonist (IL-1Ra) gene is over-represented in ulcerative colitis (UC). Healthy individuals carrying allele 2 of this gene have increased production of IL-1Ra protein. Since the final outcome of the biological effects of IL-1 beta may depend on the relative proportion of these two cytokines, we have studied if a TaqI polymorphism in the IL-1 beta gene, which is relevant to IL-1 beta protein production, may be involved in the genetic susceptibility to UC and Crohn's disease (CD), in association with the established IL-1Ra gene polymorphism. Polymorphisms in the closely linked genes for IL-1 beta and IL-1Ra were typed in 100 unrelated Dutch patients with UC, 79 with CD, and 71 healthy controls. The polymorphic regions in exon 5 of the IL-1 beta gene and in intron 2 of the IL-1Ra gene, were studied by polymerase chain reaction (PCR)-based methods. The IL-1 beta allele frequencies in UC and CD patients did not differ from those in healthy controls. In order to study if the IL-1 beta gene polymorphism might participate synergistically with the IL-1Ra gene polymorphism in susceptibility to UC and CD, individuals were distributed into carriers and non-carriers of allele 2 of the genes encoding IL-1 beta and IL-1Ra, in each of the patient groups and controls. Results indicated a significant association of this pair of genes, estimated by the odds ratio (OR) after performing Fisher's exact test, in the UC group (P = 0.023, OR = 2.81), as well as in the CD group (P = 0.01, OR = 3.79). Thus, non-carriers of IL-1 beta allele 2 were more often present in the subgroup of patients carrying the IL-1Ra allele 2. By contrast, no association of these alleles was detected in the group of healthy controls (P = 1.00, OR = 0.92). These results suggest that the IL-1 beta/IL-1Ra allelic cluster may participate in defining the biological basis of predisposition to chronic inflammatory bowel diseases.     

Molecular basis of the synergistic production of IL-1 receptor antagonist by human neutrophils stimulated with IL-4 and IL-10

In this study, we report that the release of IL-1 receptor antagonist (IL-1ra) from IL-4-stimulated neutrophils is markedly enhanced in the presence of IL-10. We also show that up-regulation of IL-1ra release by IL-10 in IL-4-stimulated neutrophils takes place through IL-1ra mRNA stabilization and enhancement of IL-1ra de novo synthesis. Furthermore, we report that the ability of IL-10 to up-regulate IL-1ra mRNA expression in IL-4-treated neutrophils requires 5-6 h and it is preceded by the acquisition of the capacity to activate Stat3 tyrosine phosphorylation. This latter response to IL-10 was strictly dependent on the levels of expression of IL-10R1, which were in fact significantly increased by IL-4 in cultured neutrophils via a signaling pathway sensitive to the serine/threonine kinase inhibitor H-7. Collectively, our data emphasize the central role of IL-10R1 expression in regulating cell responsiveness to IL-10. In addition, the fact that IL-10 strongly up-regulates IL-1ra production in IL-4-activated neutrophils uncovers a novel mechanism whereby IL-10 and IL-4 cooperate to negatively modulate the inflammatory responses.     

Genetic control of IL-1beta bioactivity through differential regulation of the IL-1 receptor antagonist

Regulation of IL-1 bioactivity by the IL-1 receptor antagonist (IL-1Ra) is critical for the preservation of normal vascular structure and function in mice. In humans, IL-1 bioactivity may be further fine-tuned by genetic polymorphisms. We recently proposed that an intronic polymorphism of the human gene encoding the IL-1R antagonist (IL1RN) is a genetic risk factor for the development of chronic cardiac allograft rejection. Here we aimed to establish a physiological basis for this susceptibility. Plasma and peripheral blood mononuclear cells (PBMC) were obtained from 55 healthy human volunteers, whose genotypes for four polymorphisms of IL1 family genes (IL1B, IL1R1 and IL1RN) were determined by PCR. IL-1beta and IL-1Ra production and release in PBMC cultures were studied by flow cytometry and ELISA. Functional and genotypic data were pooled and analyzed first by multivariate and, subsequently, by univariate statistical tests. We were able to confirm our observed association of IL1RN genotype with chronic cardiac allograft rejection using multivariate statistics. IL1RN genotype also emerged as the principal regulator of both constitutive and stimulated IL-1Ra and IL-1beta release. Allele IL1RN*2 was consistently associated with higher IL-1Ra and lower IL-1beta release, in a dosage-dependent manner. Conversely, IL1RN*2 carriage was associated with reduced production of the intracellular isoform of IL-1Ra. These genotypic effects were only observed with prolonged culture prior to stimulation and were not appreciably influenced by the presence of exogenous modulators of IL-1beta production. We conclude that IL1RN genotype is the principal determinant of IL-1beta bioactivity within theIL1 gene cluster in humans and discuss its putative role in disease.     

Detection of IL-5 and IL-1 receptor antagonist in bronchoalveolar lavage fluid in acute eosinophilic pneumonia

BACKGROUND: Acute eosinophilic pneumonia is an idiopathic cause of respiratory failure, characterized by very high numbers of alveolar eosinophils without significant blood eosinophilia. OBJECTIVE: The purpose of this study was to determine which cytokines are associated with acute eosinophilic pneumonia. METHODS: Soluble IL-1 type II receptor and the cytokines IL-1β, IL-1ra, IL-3, IL-5, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor-α were measured in serum and in bronchoalveolar lavage fluid from two patients with acute eosinophilic pneumonia during both acute and convalescent phases. RESULTS: Compared with patients with adult respiratory distress syndrome, the patients with acute eosinophilic pneumonia had high bronchoalveolar lavage fluid levels of IL-5, IL-1ra, and soluble type II IL-1 receptor but not IL-1β, tumor necrosis factor-α>, IL-3, or granulocyte-macrophage colony-stimulating factor. Bronchoalveolar lavage fluid levels of IL-5 and IL-1ra fell after resolution of symptoms. In the serum of patients with acute eosinophilic pneumonia, IL-5 was not detectable, and IL-1ra was initially high but fell after corticosteroid treatment. CONCLUSION: Acute eosinophilic pneumonia is characterized by locally high levels of IL-5, IL-1ra, and soluble type II IL-1 receptor in the alveolar space. (J ALLERGY CLIN IMMUNOL 1996;97:1366-74.)