Thrombospondin-1 Restricts Interleukin-36γ-Mediated Neutrophilic Inflammation during Pseudomonas aeruginosa Pulmonary Infection.

Interleukin-36γ (IL-36γ), a member of the IL-1 cytokine superfamily, amplifies lung inflammation and impairs host defense during acute pulmonary Pseudomonas aeruginosa infection. To be fully active, IL-36γ is cleaved at its N-terminal region by proteases such as neutrophil elastase (NE) and cathepsin S (CatS). However, it remains unclear whether limiting extracellular proteolysis restrains the inflammatory cascade triggered by IL-36γ during P. aeruginosa infection. Thrombospondin-1 (TSP-1) is a matricellular protein with inhibitory activity against NE and the pathogen-secreted Pseudomonas elastase LasB-both proteases implicated in amplifying inflammation. We hypothesized that TSP-1 tempers the inflammatory response during lung P. aeruginosa infection by inhibiting the proteolytic environment required for IL-36γ activation. Compared to wild-type (WT) mice, TSP-1-deficient (Thbs1-/-) mice exhibited a hyperinflammatory response in the lungs during P. aeruginosa infection, with increased cytokine production and an unrestrained extracellular proteolytic environment characterized by higher free NE and LasB, but not CatS activity. LasB cleaved IL-36γ proximally to M19 at a cleavage site distinct from those generated by NE and CatS, which cleave IL-36γ proximally to Y16 and S18, respectively. N-terminal truncation experiments in silico predicted that the M19 and the S18 isoforms bind the IL-36R complex almost identically. IL-36γ neutralization ameliorated the hyperinflammatory response and improved lung immunity in Thbs1-/- mice during P. aeruginosa infection. Moreover, administration of cleaved IL-36γ induced cytokine production and neutrophil recruitment and activation that was accentuated in Thbs1-/- mice lungs. Collectively, our data show that TSP-1 regulates lung neutrophilic inflammation and facilitates host defense by restraining the extracellular proteolytic environment required for IL-36γ activation.IMPORTANCEPseudomonas aeruginosa pulmonary infection can lead to exaggerated neutrophilic inflammation and tissue destruction, yet host factors that regulate the neutrophilic response are not fully known. IL-36γ is a proinflammatory cytokine that dramatically increases in bioactivity following N-terminal processing by proteases. Here, we demonstrate that thrombospondin-1, a host matricellular protein, limits N-terminal processing of IL-36γ by neutrophil elastase and the Pseudomonas aeruginosa-secreted protease LasB. Thrombospondin-1-deficient mice (Thbs1-/-) exhibit a hyperinflammatory response following infection. Whereas IL-36γ neutralization reduces inflammatory cytokine production, limits neutrophil activation, and improves host defense in Thbs1-/- mice, cleaved IL-36γ administration amplifies neutrophilic inflammation in Thbs1-/- mice. Our findings indicate that thrombospondin-1 guards against feed-forward neutrophilic inflammation mediated by IL-36γ in the lung by restraining the extracellular proteolytic environment.

The IL-1 family cytokines and receptors in autoimmune diseases.

The role of the cytokines and receptors of the IL-1 family in inflammation is well known. Several cytokines of the family have a powerful inflammatory activity, with IL-1ß being the best-characterized factor. The inflammatory activity of IL-1 cytokines is regulated by other factors of the family, including receptor antagonists, soluble receptors and anti-inflammatory cytokines. The causative role of IL-1ß is well-established in autoinflammatory diseases, mainly due to gain-of-function mutations in genes encoding the IL-1ß-maturing inflammasome. Exaggerated production of IL-1ß and IL-18 correlates with disease and disease severity also in several autoimmune and chronic inflammatory and degenerative pathologies, although it is not clear whether they have a causative role or are only involved in the downstream disease symptoms. A better understanding of the pathological role of IL-1 family cytokines in autoimmunity involves a deeper evaluation, in the pathological situations, of the possible anomalies in the feed-back anti-inflammatory mechanisms that in physiological reactions control and dump IL-1-mediated inflammation. Thus, we expect that IL-1 cytokines may be pathogenic only when, in addition to enhanced production, there is a concomitant failure of their control mechanisms. In this review we will examine the current knowledge on the role of IL-1 family cytokines in autoimmune and chronic inflammatory and degenerative diseases, with a particular focus on their endogenous control mechanisms, mainly based on soluble receptors/inhibitors and receptor antagonists. This will allow us to formulate a knowledge-based hypothesis on the involvement of IL-1 cytokines in the pathogenesis vs. the clinical features of these diseases.

The role of the IL-1 system in pregnancy and the use of IL-1 system markers to identify women at risk for pregnancy complications†.

The interleukin (IL)-1 system plays a major role in immune responses and inflammation. The IL-1 system components include IL-1α, IL-1ß, IL-1 receptor type 1 and IL-1 receptor type 2 (decoy receptor), IL-1 receptor accessory protein, and IL-1 receptor antagonist (IL-1Ra). These components have been shown to play a role in pregnancy, specifically in embryo-maternal communication for implantation, placenta development, and protection against infections. As gestation advances, maternal tissues experience increasing fetal demand and physical stress and IL-1ß is induced. Dependent on the levels of IL-1Ra, which regulates IL-1ß activity, a pro-inflammatory response may or may not occur. If there is an inflammatory response, prostaglandins are synthesized that may lead to myometrial contractions and the initiation of labor. Many studies have examined the role of the IL-1 system in pregnancy by independently measuring plasma, cervical, and amniotic fluid IL-1ß or IL-1Ra levels. Other studies have tested for polymorphisms in IL-1ß and IL-1Ra genes in women experiencing pregnancy complications such as early pregnancy loss, in vitro fertilization failure, pre-eclampsia and preterm delivery. Data from those studies suggest a definite role for the IL-1 system in successful pregnancy outcomes. However, as anticipated, the results varied among different experimental models, ethnicities, and disease states. Here, we review the current literature and propose that measurement of IL-1Ra in relation to IL-1 may be useful in predicting the risk of poor pregnancy outcomes.

Cytokines and soluble receptors of the interleukin-1 family in Schnitzler syndrome.

OBJECTIVES: Schnitzler syndrome (SchS) is an autoinflammatory disorder characterized by chronic urticaria, fever, and monoclonal gammopathy. The success of interleukin-1 (IL-1) blocking therapies suggests a crucial role for IL-1 in disease induction. The aim of this study is to perform a comprehensive analysis of IL-1 family cytokines and soluble receptors in a group of SchS patients. METHOD: Three patients fulfilling the criteria for the diagnosis of SchS were recruited; 80 blood donors formed the control group. IL-1 family cytokines (IL-1α, IL-1ß, IL-33, IL-18), soluble receptors (sIL-1R1, sIL-1R2, sIL-1R3, sIL-1R4), and antagonists [IL-1Ra, IL-18 binding protein (IL-18BP)] were measured by a multiarray enzyme-linked immunosorbent assay. Free IL-18 was calculated as the amount of IL-18 not inhibited by IL-18BP. Cytokine levels were compared by the Mann-Whitney test. RESULTS: IL-18 and free IL-18 were increased in patients compared with controls (p = 0.005 and p = 0.0082, respectively), while IL-18BP levels were not different. IL-1α, IL-1ß, and IL-33 were undetectable in both patients and controls. The soluble receptors sIL-1R1, sIL-1R2, and ST2/sIL-1R4, and the IL-1 antagonist IL-1Ra were all within normal ranges; sIL-1R3 was significantly lower in patients than in controls (p = 0.039). CONCLUSIONS: The data indicate that SchS is characterized by increased circulating levels of free IL-18, possibly leading to a higher activation of innate/inflammatory effector cells. At variance with other inflammatory diseases, the lack of increase in sIL-1R1 and sIL-1R2 and the decreased levels of sIL-R3 imply a failure in the counterbalancing mechanism aimed at inhibiting excessive IL-1ß in tissues.

The P2X7 receptor: a key player in IL-1 processing and release.

Human IL-1 family proteins are key mediators of the host response to infections, injury, and immunologic challenges. The mechanism by which IL-1 activates proinflammatory responses in target cells, and the plasma membrane receptors involved, is fairly well known. This has led to the development of innovative drugs that block IL-1 downstream to its synthesis and secretion. On the contrary, the mechanism of IL-1 and other IL-1 family members (e.g., IL-18) maturation and release is incompletely understood. Accruing evidence points to a plasma membrane receptor for extracellular ATP, the P2X(7) receptor, as a key player in both processes. A deeper understanding of the mechanism by which the P2X(7) receptor triggers IL-1 maturation and exteriorization may suggest novel avenues for the treatment of inflammatory diseases and provide a deeper insight in the fundamental mechanism of protease activation and cellular export of proteins lacking a leader sequence.

Effects of micro-environment- and malignant cell-derived interleukin-1 in carcinogenesis, tumour invasiveness and tumour-host interactions.

Interleukin-1 (IL-1) comprises a family of closely related genes; the two major agonistic proteins, IL-1alpha and IL-1beta, are pleiotropic and affect mainly inflammation, immunity and haemopoiesis. IL-1beta is active solely in its secreted form, whereas IL-1alpha is active mainly as an intracellular precursor. IL-1 is abundant at tumour sites, where it may affect the process of carcinogenesis, tumour growth and invasiveness and the patterns of tumour-host interactions. Here, we review the effects of micro-environment- and tumour cell-derived IL-1 on malignant processes in experimental tumour models. We propose that membrane-associated IL-1alpha expressed on malignant cells stimulates anti-tumour immunity, while secretable IL-1beta derived from the micro-environment or the malignant cells, activates inflammation that promotes invasiveness and induces tumour-mediated suppression. Inhibition of the function of IL-1 by the inhibitor of IL-1, interleukin-1 receptor antagonist (IL-1Ra), reduces tumour invasiveness and alleviates tumour-mediated suppression, pointing to its feasible use in cancer therapy. Differential manipulation of IL-1alpha and IL-1beta in malignant cells or in the tumour's micro-environment may open new possibilities for using IL-1 in cancer immunotherapy.

Genomic organization, gene duplication, and expression analysis of interleukin-1beta in channel catfish (Ictalurus punctatus).

Interleukin-1beta (IL-1beta) is one of the pivotal early response pro-inflammatory cytokines that enables organisms to respond to infection and induces a cascade of reactions leading to inflammation. In spite of its importance and two decades of studies in the mammalian species, genes encoding IL-1beta were not identified from non-mammalian species until recently. Recent research, particularly with genomic approaches, has led to sequencing of IL-1beta from many species. Clinical studies also suggested IL-1beta as an immunoregulatory molecule potentially useful for enhancing vaccination. However, no IL-1beta genes have been identified from channel catfish, the primary aquaculture species from the United States. In this study, we identified two distinct cDNAs encoding catfish IL-1beta. Their encoding genes were identified, sequenced, and characterized. The catfish IL-1beta genes were assigned to bacterial artificial chromosome (BAC) clones. Genomic studies indicated that the IL-1beta genes were tandemly duplicated on the same chromosome. Phylogenetic analysis of various IL-1beta genes indicated the possibility of recent species-specific gene duplications in channel catfish, and perhaps also in swine and carp. Expression analysis indicated that both IL-1beta genes were expressed, but exhibited distinct expression profiles in various catfish tissues, and after bacterial infection with Edwardsiella ictaluri.

An IL-1 family member requires caspase-1 processing and signals through the ST2 receptor.

Activation of ST2, an orphan member of the IL-1 receptor family for 16 years, drives T helper type 2 (T(H)2) responses. The cytokine IL-33 is the specific ligand for ST2. IL-33 recapitulates much of the existing data that ST2 promotes T(H)2-type responses. The caspase-1 processing of precursor IL-33 provides a therapeutic target to control allergic diseases.

Genotypes and haplotypes in the IL-1 gene cluster: analysis of two genetically and diagnostically distinct groups of Alzheimer patients.

Increased risk of Alzheimer's disease (AD) has been associated with polymorphisms in the IL-1 gene cluster, and in particular with the IL-1alpha-889 T/T genotype. However, this association is still unclear, and needs further investigation. In order to clarify the role of these polymorphisms in the complex pathogenesis of AD we examined genotype and haplotype frequencies of the two C-to-T SNPs at position -889 and -551 in the IL-1alpha and IL-1beta genes, respectively, and of the 86 bp VNTR intron-2 polymorphisms in the IL-1Ra gene. The analysis was performed in two genetically and diagnostically distinct groups of sporadic AD from Italy and the USA. In the Italian group a significant association between the IL-1alpha-889 T/T genotype and AD (OR=3.022, 95% CI: 1.001-9.119) was found, whereas no difference was found in the group from the USA. Results were also compared with previously published studies that analyzed the same IL-1 polymorphisms in AD. In both groups, the analysis of the estimated haplotypes shows that AD patients and controls who carry the IL-1beta-511 C allele, were also more frequently carriers of the IL-1Ra 1 allele (haplotypes -C-1). The total frequency of the two -C-1 haplotypes (C-C-1 plus T-C-1) was about one half of the total frequency of the eight estimated haplotypes. This was confirmed by significant linkage disequilibrium between these two loci in both the Italian and USA groups. In the Italian group a weak association of the T-C-2 haplotype with the disease (OR=1.648, 95% CI: 1.519-1.788) was also found, whereas in the USA group no difference was found. Although ours and other published data on different samples of Caucasian and non-Caucasian AD show a great heterogeneity in the frequencies of the IL-1alpha-889, the IL-1beta-511 and the IL-1Ra VNTR gene polymorphisms, we confirm the role of the IL-1alpha-889 T/T genotype as a risk factor for sporadic AD, and show the presence of an allelic association between IL-1beta C and IL-1Ra 1 alleles in both the Italian and the USA groups, confirmed by the presence of significant levels of linkage disequilibrium between these two loci.

Association of IL-1 RN*2 allele and methionine synthase 2756 AA genotype with dementia severity of sporadic Alzheimer's disease.

BACKGROUND: Genetic polymorphisms of APO-E, homocysteine, and the IL-1 gene cluster (IL-1A, IL-1B, receptor antagonist IL-1RN) are associated with sporadic Alzheimer's disease and may involve interdependent pathways of neuronal toxicity. OBJECTIVE: To determine whether these polymorphisms and the genetic determinants of homocysteine (methylenetetrahydrofolate reductase, MTHFR; methionine synthase, MTR; transcobalamin, TC) are associated with an increased risk of severe dementia in Alzheimer's disease. METHODS: 152 patients with Alzheimer's disease and 136 controls were studied. The association of occurrence and dementia severity (Reisberg score <6 and >or=6) of Alzheimer's disease with APO-E, IL-1A, IL-1B, IL-1RN, MTHFR677 C-->T and 1298A-->C, MTR 2756 A-->G, and TC 776 C-->G polymorphisms was evaluated by multivariate logistic regression analysis after adjustment for age, sex, and age of onset of Alzheimer's disease. RESULTS: IL-1A TT and IL-1B CT/TT associated genotypes were at risk of Alzheimer's disease (odds ratio 4.80 (95% confidence interval, 1.32 to 17.40), p = 0.017); the MTR 2756 AA genotype was at risk of severe dementia (OR 2.97 (1.23 to 7.21), p = 0.016); IL-1 RN*2 was protective (OR 0.28, (0.11 to 0.69), p = 0.006). Allele epsilon4 of the APO-E and IL-1B CC genotypes increased the risk of severe Alzheimer's disease associated with the MTR 2756 AA genotype by 3.3-fold and 1.5-fold, respectively. CONCLUSIONS: Distinct determinants of the IL-1 gene cluster are related to the generation and progression of Alzheimer's disease. MTR only influences progression of the disease, which may be enhanced by carriage of allele epsilon4 of APO-E.

Salivary gland extract from Ixodes ricinus tick modulates the host immune response towards the Th2 cytokine profile.

In our previous work, the salivary gland extract (SGE) from Ixodes ricinus ticks impaired T-lymphocyte proliferation and clearly modulated the immune response towards the Th2 pattern in human peripheral blood mononuclear cell culture. In the present work, the results obtained on mouse splenocytes are compared with those on human leukocytes. ELISA (protein level) and RNAse protection assay (mRNA level) showed that SGE enhanced interleukin (IL)-1alpha, IL-1beta, IL-1Ra, IL-6, and IL-12p40 cytokines, whereas production of IL-2, IL-5, IL-10, and IL-13 was decreased. The minute levels of IL-9, IL-15 and IL-12p70 were not changed after the addition of tick saliva. IL-4 was upregulated, whereas the production of gamma interferon and migratory inhibition factor was downregulated after the addition of SGE. Tick saliva decreased concanavalin A-stimulated spleen cell proliferation and the percentage of activated T-cells. We conclude that the Th2 polarization did not involve all of the cytokines tested. However, the Th2 subset-augmenting effect of tick saliva was confirmed.

Molecular cloning and characterization of beluga whale (Delphinapterus leucas) interleukin-1beta and tumor necrosis factor-alpha.

Interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) are cytokines produced primarily by monocytes and macrophages with regulatory effects in inflammation and multiple aspects of the immune response. As yet, no molecular data have been reported for IL-1beta and TNF-alpha of the beluga whale. In this study, we cloned and determined the entire cDNA sequence encoding beluga whale IL-1beta and TNF-alpha. The genetic relationship of the cytokine sequences was then analyzed with those from several mammalian species, including the human and the pig. The homology of beluga whale IL-1beta nucleic acid and deduced amino acid sequences with those from these mammalian species ranged from 74.6 to 86.0% and 62.7 to 77.1%, respectively, whereas that of TNF-alpha varied from 79.3 to 90.8% and 75.3 to 87.7%, respectively. Phylogenetic analyses based on deduced amino acid sequences showed that the beluga whale IL-1beta and TNF-alpha were most closely related to those of the ruminant species (cattle, sheep, and deer). The beluga whale IL-1beta- and TNF-alpha-encoding sequences were thereafter successfully expressed in Escherichia coli as fusion proteins by using procaryotic expression vectors. The fusion proteins were used to produce beluga whale IL-1beta- and TNF-alpha-specific rabbit antisera.

Phylogeny of cytokines: molecular cloning and expression analysis of sea bass Dicentrarchus labrax interleukin-1beta.

In this paper the cloning of interleukin-1beta (IL-1beta) from the fish Dicentrarchus labrax (sea bass) is described. Using degenerate primers designed from known IL-1beta sequences, a cDNA fragment was amplified by PCR and elongated by 3' and 5' RACE to give the full-length coding sequence for sea bass IL-1beta. The cDNA is 1292 bp, lacks a putative ICE cut site, and codes for a deduced peptide of 29.4 kDa with a pI of 5.1. Sequence analysis showed highest amino acid similarity with rainbow trout (62%), Xenopus (46%), and carp (45.5%) IL-1beta sequences. Expression studies show that sea bass IL-1beta can be upregulated by bacterial lipopolysaccharide both in vitro and in vivo in leucocytes from blood, head-kidney, spleen, gills and liver, whereas the IL-1beta transcript was not detectable in thymus and gut-associated lymphoid tissue. Northern blot analysis with head-kidney leucocyte RNA showed a main LPS-upregulated band at 1.3 kb, and two minor bands at 0.9 and 3.0 kb, respectively. Phylogenetic comparisons with IL-1beta from other vertebrates is presented.

Relationship of surface epithelium concentrations of IL-1 alpha and IL-1 beta to clinical inflammation during experimental gingivitis.

Experimental evidence has clearly demonstrated that IL-1 cytokine levels increase in experimental gingivitis (EG) models in response to plaque accumulation following the cessation of oral hygiene. These changes in cytokine production are reported to occur prior to visible signs of clinical inflammation, and as such may represent early markers of gingival inflammation. This clinical study used a novel dermal sampling tape as a method to collect cytokines from gingival epithelium, as opposed to the more commonly sampled gingival crevicular fluid. The primary objective of the study was to examine the relationship between changes in cytokine levels and clinical inflammation. Ten subjects participated in a 14-day EG model, where 5 days following a dental prophylaxis subjects refrained from all oral hygiene measures for 14-days. Clinical measures including the Löe-Silness Gingival Index (GI), a bleeding index derived from the GI, and the inflammation index (II) were made at baseline prior to the initiation of the EG period and following 14 days of EG. Dermal tape samples were collected from the right posterior buccal quadrant of each subject at both baseline and day 14. The tapes were extracted and the extracts analyzed for both IL-1 alpha and IL-1 beta by ELISA. Results of this study indicate that over a 14-day EG period statistically significant (p < 0.05) increases in GI, gingival bleeding, II, and IL-1 alpha were observed (tested by matched-pairs t test and Wilcoxon signed ranks test). A directional increase in IL-1 beta was also observed. Linear regression analyses demonstrated a strong positive correlation between the number of gingival bleeding sites from the region of gingiva sampled with Sebutape and IL-1 alpha (r = 0.93), as well as IL-1 beta (r = 0.90). In addition, linear regression analyses also demonstrated a strong positive correlation between the mean II score from the region of gingiva sampled with Sebutape and IL-1 alpha (r = 0.93), as well as IL-1 beta (r = 0.86). Similar correlations were observed for whole mouth scores of the number of gingival bleeding sites and mean II with respect to IL-1 alpha and IL-1 beta levels. Collectively, these data confirm the utility of the dermal tape for sampling epithelium cytokine levels from the gingiva and demonstrate a strong positive correlation between IL-1 alpha and IL-1 beta concentrations and gingival inflammation.

IL-1 alpha, IL-1 beta and psoriasis: conflicting results in the literature. Opposite behaviour of the two cytokines in lesional or non-lesional extracts of whole skin.

A still debated question in the field of the cytokine network in psoriasis is represented by contrasting data reported on the local amount of IL-1 beta amounts, of IL-1 in this dermatosis. In fact previous studies have suggested that there were decreased Il-1 alpha amounts at the lesional level but increased nonfunctional IL-1 beta concentrations as compared to the non-lesional and normal epidermis. However, recent data suggest that IL-1 alpha and, to a lesser extent, IL-1 beta amounts, are both increased and biologically active in the epidermal cell suspension of lesional psoriatic skin as compared to those of normal skin. The data reported in the present paper show that IL-1 alpha levels are decreased in psoriatic lesional extracts of whole skin (mean 2.9 +/- 2 pg/mg) as compared to non-lesional (mean 6.7 +/- 6.2 mg/mg; p = 0.02) or normal skin (mean 13.8 +/- 9.4 pg/mg; p = 0.0002). IL-1 alpha concentrations were also significantly lower in the non-lesional skin than in normal skin (p = 0.02). In contrast, the IL-1 beta levels (mean 1.2 +/- 0.74 pg/mg were higher in the lesional samples than in the non-lesional ones (mean 0.5 +/- 0.4 pg/mg; p = 0.0004) or in normal skin (mean 0.4 +/- 0.2 pg/mg; p = 0.004). No differences in IL-1 beta levels were observed between non-lesional and normal skin (p = 0.3). In addition both IL-1 alpha and IL-1 beta are directly correlated with the disease severity and each other. Our data, extending the Il-1 determination to the whole skin, seem to confirm the previously reported findings at the epidermis level and provide new light on possible interpretation of literature discrepancies.