TGF-ß interactions with IL-1 family members trigger IL-4-independent IL-9 production by mouse CD4(+) T cells.

TGF-ß and IL-4 were recently shown to selectively upregulate IL-9 production by naïve CD4(+) T cells. We report here that TGF-ß interactions with IL-1α, IL-1ß, IL-18, and IL-33 have equivalent IL-9-stimulating activities that function even in IL-4-deficient animals. This was observed after in vitro antigenic stimulation of immunized or unprimed mice and after polyclonal T-cell activation. Based on intracellular IL-9 staining, all IL-9-producing cells were CD4(+) and 80-90% had proliferated, as indicated by reduced CFSE staining. In contrast to IL-9, IL-13 and IL-17 were strongly stimulated by IL-1 and either inhibited (IL-13) or were unaffected (IL-17) by addition of TGF-ß. IL-9 and IL-17 production also differed in their dependence on IL-2 and regulation by IL-1/IL-23. As IL-9 levels were much lower in Th2 and Th17 cultures, our results identify TGF-ß/IL-1 and TGF-ß/IL-4 as the main control points of IL-9 synthesis.

MyDths and un-TOLLed truths: sensor, instructive and effector immunity to tuberculosis.

Controversy exists concerning the role of Toll-like receptors and MyD88 in immunity to tuberculosis (TB). This mini-review argues that (i) Toll-like receptors are not essential for an effective immune response against TB, (ii) MyD88 is essential, but not because it transduces signals from TLRs, (iii) adaptive immunity to TB is largely TLR/MyD88-independent. Some of the discrepancies may be resolved by cogent attribution of distinct immune functions to the individual components of the TLR/MyD88 system. In mice, TLRs and MyD88 are fully dispensable in sensing Mtb infection and instructing T cell-mediated adaptive immunity, and while TLRs are also redundant during macrophage effector immunity, MyD88 is essential for efficient killing of mycobacteria. This distinction should help to molecularly pinpoint the MyD88-dependent, yet TLR-independent critical mechanisms of macrophage activation involved in intracellular growth restriction of Mtb. Disrupted IL-1R and/or IFN-gamma signaling pathways likely play a much more prominent role in explaining the exquisite susceptibility of MyD88-deficient mice to TB than the function of MyD88 as a TLR adaptor.

Selective adenosine A receptor agonist, ATL-146e, attenuates stress-induced gastric lesions in rats.

BACKGROUND: Activation of adenosine A(2A) receptors reduces the production of various pro-inflammatory cytokines and suppresses neutrophil activation. Water-immersion restraint is well known to cause gastric mucosal lesions due to stress. The pathogenesis of stress-induced gastric mucosal lesions is characterized by activation of inflammatory cells and production of inflammatory cytokines. Agonists of adenosine A(2A) receptors are known to be anti-inflammatory, but the effects of these compounds on the development of gastric mucosal lesions has not been reported. In the present study, the effect of a potent and selective adenosine A(2A) receptor agonist, ATL-146e, on water-immersion stress-induced gastric mucosal lesions was studied. METHODS: Rats were subjected to water-immersion stress with or without pretreatment with a single intraperitoneal injection of a potent and selective agonist of the adenosine A(2A) receptor. The gastric concentrations of myeloperoxidase (MPO), as an index of neutrophil accumulation, and the pro-inflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta), were measured. RESULTS: The total length of gastric erosions (ulcer index) in control rats was 21.6 +/- 3.23 mm and was reduced by 86% to 3.1 +/- 0.83 mm by pretreatment with 5.0 microg/kg ATL146e (P < 0.001). The gastric content of MPO, TNF-alpha and IL-1beta were all increased after water-immersion stress and reduced to near normal levels by ATL-146e. CONCLUSION: A specific adenosine A(2A) agonist inhibits stress-induced gastric inflammation and damage. A(2A) agonist compounds may be useful for preventing ulcers and appear to act by blocking gastric inflammation.

Effects of an interleukin-1beta analogue [Lys-D-Pro-Thr], on incomplete cerebral ischemia-induced inhibition of long-term potentiation in rat hippocampal neurons in vivo.

Although interleukin-1beta (IL-1beta) has recently been implicated in neuronal cell death in vitro and in vivo after global forebrain ischemia, the role of IL-1beta in the functional injuries, i.e. impairment of synaptic transmission, after cerebral ischemia that does not cause neuronal death in the nervous system remains unknown. To address this question, we investigated the effect of short-term incomplete ischemia without apparent neural death on hippocampal long-term potentiation (LTP) in anesthetized rats, and examined the possible role of IL-1beta as an intermediary in this effect. Short-term incomplete cerebral ischemia (10 min) was induced in halothane-anesthetized rats by bilaterally clamping the common carotid arteries. Four days after ischemia, functional injuries in neuronal transmission in the hippocampal formation were observed without significant changes in pathological studies such as neuronal cell death. The LTP elicited in both Shaffer-CA1 synapses and perforant path-dentate gyrus synapses was significantly inhibited by the short-term incomplete ischemia. This inhibition of LTP was blocked by IL-1beta tripeptide antagonist (Lys-D-Pro-Thr), suggesting that the inhibitory effect of mild ischemia on synaptic potentials and LTP may be mediated by the generation of IL-1beta. These findings have important implications for the role of IL-1beta in not only neuronal cell death but also functional injuries without cell loss, perhaps elicited by transient cerebral ischemia.

Upregulation of CD44 in the inflamed mouse air pouch injected with synthetic lipid A.

OBJECTIVE: To investigate aspects of the inflammatory process of the mouse subcutaneous air pouch -- a facsimile synovial cavity -- induced by injection of lipid A, and to determine the expression and upregulation of CD44 in the lining cell layer of the inflamed air pouch. METHODS: Histological changes of inner walls in the mouse air pouch were evaluated 1, 3, and 7 days after injection of lipid A. RESULTS: Polymorphonuclear cell infiltration in the lining layer reached the maximum one day after injection of 10 microg of lipid A (10/10 mice in Grade 3; p < 0.01), while mononuclear cell infiltration and lining cell hyperplasia reached the maximum 3 days after injection (5/10 mice in Grade 2; 5/10 in Grade 3; p < 0.05; 39+/-11 layers, p < 0.05, respectively). The number of cell depth of CD44 positive lining layers and interleukin 1alpha (IL-1alpha) positive lining layers reached the maximum 3 days after injection (39+/-7 layers, p < 0.01; 35+/-12 layers, p < 0.05, respectively). CONCLUSION: These findings suggest that CD44 may have some connection with the proinflammatory cytokine IL-1alpha and induce inflammatory responses in the air pouch injected with lipid A.

Development of glycosylated human interleukin-1alpha, neoglyco IL-1alpha, coupled with D-galactose monosaccharide: biological activities in vivo.

In our previous study, a galactose monosaccharide with C9 spacer was chemically coupled to recombinant human interleukin 1alpha (rhIL-1alpha) in order to study the effect of glycosylation on its activities, and to develop IL-1 with less deleterious effects. The glycosylated IL-la exhibited reduced activities in vitro by 10 to 10000-fold depending upon different aspects of activities addressed. The affinity to type I and II IL-1 receptors were also reduced. In this study we examined a variety of IL-1 activities in vivo, including upregulation of serum levels of IL-6, alpha1-acid glycoprotein, NOx, corticosterone, downregulation of serum level of glucose, and recovery of peripheral white blood cells (WBCs) from myelosuppression in 5-fluorouracil-treated mice. In contrast to the biological activities in vitro, these activities in vivo were uniformly reduced by only about 10 to 20-fold compared to untreated IL-1alpha.

The internalized interleukin-1 activation complex in fibroblasts localizes to the Golgi apparatus.

In order to investigate binding and internalization of interleukin-1 (IL-1) by confocal laser scanning microscopy, we established a model system comprising an IL-1 receptor type I (IL-1R1) overexpressing transfectant of the murine fibrosarcoma cell line L929 (L929R1) and an N-terminal FLAG-tagged human recombinant IL-1 alpha (FLAG-IL-1 alpha). The function of the transfected receptors was shown by their IL-1-induced association with a kinase activity. The biological activity of the purified FLAG-IL-1 alpha was comparable to the unmodified molecule. L929RI cells were exposed to saturating concentrations of FLAG-IL-1 alpha. Two-color fluorescence analysis revealed increasing cell surface binding of FLAG-IL-1 alpha to the receptor over 30 min. This was followed by internalization and accumulation of the ligand/receptor complex at the Golgi apparatus. After 3 hr the receptor signal significantly decreased and patches of FLAG-epitopes reappeared on the cell surface, no longer colocalized with IL-1R1. Thus, in this model, the previously assumed nuclear accumulation of IL-1 was not detected but rather localization of the internalized IL-1/IL-1R1-complex to the Golgi apparatus was found. Direct effects of IL-1 on the nucleus or the nuclear membrane therefore are unlikely.

Development of glycosylated human interleukin-1 alpha, neoglyco IL-1 alpha, coupled with D-mannose dimer: synthesis and biological activities in vitro.

Interleukin 1 (IL-1) is a nonglycosylated cytokine with pleiotropic effects on various cell types. In order to investigate the effect of carbohydrate introduction on IL-1 activity and to develop IL-1 with less deleterious effects recombinant human IL-1 alpha was chemically coupled with mannose dimers, alpha-D-Man1-6-D-Man[Man2(alpha 1,6)] and alpha-D-Man1-4-D-Man[Man2(alpha 1,4)]. About 5 molecules of mannose dimers were introduced per molecule of IL-1. Anti-IL-1 alpha antibody reacted only weakly with the glycosylated IL-1s. Conversely, antibody against the mannose dimer reacted with only glycosylated IL-1. The effect of glycosylation on IL-1 activity was evaluated by measuring a variety of IL-1 activities in vitro, including proliferative effect on T cells, antiproliferative effect on melanoma cells, stimulatory effect on IL-6 synthesis by melanoma cells, and stimulatory effect on prostaglandin E2 synthesis by fibroblast cells. Glycosylated IL-1s exhibited reduced activities, which were 10-fold to more than several hundred-fold lower than those of the original IL-1 alpha depending upon different aspects of activities addressed. Man2(alpha 1,6)-introduced IL-1 exhibited lower activity than Man2(alpha 1,4)-introduced IL-1. The competitive binding of 125I-IL-1 alpha to mouse T cells with unlabeled IL-1s suggests that the reduced activity of glycosylated IL-1s is due, at least partially, to the decrease of their receptor binding abilities.

Structure-function analysis of human IL-1 alpha: identification of residues required for binding to the human type I IL-1 receptor.

Using oligonucleotide-directed mutagenesis, the binding site on human interleukin-1 alpha (IL-1 alpha) for the human type I IL-1 receptor (IL-1R) has been analyzed. Substitution of seven amino acids (Arg12, Ile14, Asp60, Asp61, Ile64, Lys96 and Trp109) resulted in a significant loss of binding to the receptor. Based on crystallographic information, the side chains of these residues are clustered in one region of IL-1 alpha and exposed on the surface of the protein. Five of the residues in the IL-1 alpha binding site align with the binding residues previously determined in human IL-1 beta, demonstrating that the type I IL-1R recognizes homologous regions in both ligands. Unexpectedly, only three of the aligned residues are identical between IL-1 alpha and IL-1 beta. These observations suggest that the composition of contact residues in the binding site is unique for each ligand-receptor complex in the IL-1 system.

Receptor binding and biological activity of IL-1 alpha, IL-1 beta, IL-1 beta analogues and an IL-1 antagonist in A375 human melanoma cells.

A receptor binding assay for IL-1 peptides on human melanoma cells of the A 375 cell line is reported. Strains differing in their sensitivity to the cytotoxic effects of IL-1 beta were compared. In both strains, binding equilibrium at temperatures between 0 degrees and 37 degrees C was reached after 4 to 8 hours. At 37 degrees C, most of the bound ligand was rapidly internalized leaving a constant level of surface receptors. Scatchard analysis at 0 degrees C revealed a single class of high affinity receptors with a similar KD in both IL-1 resistant (0.18 +/- 0.07 nM) and sensitive strains (0.14 +/- 0.06 nM) but a 10-fold difference in the number of binding sites. Whereas > 1000 binding sites per cell were regularly observed in all resistant strains, only 100-200 sites could be detected on the IL-1 sensitive cells. In displacement assays, IL-1 beta was found to be slightly more potent than IL-1 alpha in both strains. In an attempt to further characterize the IL-1 binding site in these cells, the binding characteristics and biological activity of 20 point mutations of IL-1 beta were examined. EC50 values similar to those of the wild type peptide were found in all these analogues with the exception R11S and E128K: their EC50 was increased by a factor of 10 but the biological activity was reduced 1000-fold as compared to IL-1 beta. The relative potency of an IL-1 receptor antagonist was similar to that of IL-1 beta in the displacement binding assay but a 100-fold higher concentration was required to completely block the cytotoxic effects of IL-1 beta. These results show that A375 human melanoma cells are useful for screening the binding and biological properties of analogues of the IL-1 family of peptides.

Interleukin-1 beta induces the expression of an isoform of nitric oxide synthase in insulin-producing cells, which is similar to that observed in activated macrophages.

The suppressive and cytotoxic effects of interleukin-1 beta (IL-1 beta) on rodent insulin-producing cells observed in vitro are probably mediated through formation of nitric oxide (NO). In this study we demonstrate that IL-1-induced NO formation in isolated rat islets and insulin-producing HIT cells is more sensitive to inhibition by NG-monomethyl-L-arginine than to inhibition by NG-nitro-L-arginine, thus suggesting that IL-1-exposed insulin-producing cells express an isoform of nitric oxide synthase similar to that present in activated macrophages. Furthermore, IL-1 beta markedly increased the mRNA levels of the inducible macrophage form of nitric oxide synthase in HIT cells.

HepG2 cells predominantly express the type II interleukin 1 receptor (biochemical and molecular characterization of the IL-1 receptor).

In this study we have characterized the cell surface interleukin 1 (IL-1) receptor in HepG2 hepatoma cells. We found that HepG2 cells bind both IL-1 alpha and beta with high affinity, KDs of 136 and 180 pM and receptor densities of 16,000 and 8500 binding sites/cell respectively. The binding sites appeared to be predominantly type II since phorbol ester treatment of the cells, which selectively downregulates type II IL-1 receptors, reduced binding by 68% while treatment of the cells with an inhibitory monoclonal antibody specific for the type I receptor had no significant effect on IL-1 binding. Competition studies with a modified IL-1 beta analog (Glu4) also revealed binding kinetics more consistent with binding to type II receptors than to type I. Crosslinking and ligand blotting with human 125I-IL-1 demonstrated the presence of two bands, a 78 kDa band typical of crosslinking to type II (p60) receptor, and a 98 kDa band, typical of crosslinking to the type I (p80) receptor. Low level expression of the type I receptor was consistent with molecular biological studies employing polymerase chain reaction (PCR) amplification which indicated that mRNA for the type I receptor was produced by the HepG2 cells. Functional receptors were demonstrated by the induction of IL-8 by IL-1 stimulated cells.

The effect of N-terminal extension on the structure and function of human interleukin-1 beta.

Interleukin-1 beta (IL-1 beta) and N-terminally extended Met-Glu-Ala-Glu-IL-1 beta (MEAE-IL-1 beta) were cloned and expressed in E. coli. Extension of the chain results in a limited conformational change reflected by the CD spectrum in the far ultraviolet, while the aromatic side chains responsible for the CD in the near ultraviolet are not affected. No difference in immunoreactivity between IL-1 beta and MEAE-IL-1 beta is observed in the IL-1 beta ELISA. Like IL-1 beta, MEAE-IL-1 beta exhibits biological activity tested in the costimulatory mouse thymocyte (LAF) assay. The specific biological activity of IL-1 beta is 3 x 10(8) U/mg and that of MEAE-IL-1 beta 3 x 10(6) U/mg. Like IL-1 beta, MEAE-IL-1 beta displaces [125I]IL-1 beta from mouse thymocytes and the binding affinities of the two forms differ by a factor of 10(2). Finally the inhibitory effect of the two IL-1 beta forms on in vitro insulin secretion from isolated rat islets of Langerhans was measured. Again MEAE-IL-1 beta is 10(2) times less potent than IL-1 beta. The structure-activity relationship for IL-1 beta and MEAE-IL-1 beta is discussed.

A human IL-1 alpha derivative which lacks prostaglandin E2 inducing activity and inhibits the activity of IL-1 through receptor competition.

In order to segregate multiple activities ascribed to IL-1, various human IL-1 alpha derivatives were produced by recombinant DNA technology. A derivative substituted at the 151st Asp with Tyr(termed to be TN-55) showed unique characteristics. TN-55 lost the PGE2 inducing activity in a human osteosarcoma cell line (MG-63) and growth promoting activity for a human dermal fibroblast cell line (CCD-27Sk), but partially remained LAF activity in mouse thymocyte, cytostatic activity against a human melanoma cell line (A-375) and IL-2 inducing activity in a human T cell line (HSB.2). Although TN-55 bound to the receptor on MG-63 cells with a similar affinity as native IL-1 alpha, TN-55 not only failed in inducing PGE2 production but also antagonized the PGE2 inducing action of IL-1 alpha or IL-1 beta. Thus, TN-55 seems to work as a receptor antagonist. Moreover, TN-55 did not stimulate ACTH secretion in rats in vivo. On the other hand, TN-55 induced PGE2 production in a rabbit dermal fibroblast cell line (RAB-9) and exhibited pyrogenicity in rabbits in vivo. These data suggest that TN-55 has different species-cross reactivity from native IL-1 alpha. In conclusion, multiple biological activities of IL-1 alpha can be segregated by substituting one amino acid. TN-55 may be an ideal IL-1 agonist which lacks inflammatory characteristics of IL-1 (e.g. PGE2-dependent activities) in human but partially retained T lymphocyte stimulating activity and tumor cytostatic activity. In addition, TN-55 may also work as an IL-1 antagonist to block PGE2 production induced by IL-1 through receptor competition.

Differential determination of recombinant human interleukin-1 alpha and its deamidated derivative by two sandwich enzyme immunoassays using monoclonal antibodies. Comparison with a polyclonal antibody-based competitive enzyme immunoassay.

Using three different monoclonal antibodies (McAb no. 374, no. 964 and no. 1190) to human interleukin-1 alpha (rHu-IL-1 alpha), we have established two sandwich enzyme immunoassays (EIA) to differentiate rHu-IL-1 alpha and its deamidated derivative (rHu-Asp36-IL-1 alpha) where the asparagine at position 36 (counting from the N-terminus) of rHu-IL-1 alpha is converted to Asp. The McAb no. 1190 reacts specifically with rHu-IL-alpha and not with the rHu-Asp36-IL-1 alpha whereas both no. 374 and no. 964 can react with the two different forms of rHu-IL-1 alpha. The first EIA (S-EIA I) which uses the McAb no. 964 labelled with horse-radish peroxidase and the McAb no. 1190 fixed to the microtiter plate, only measure rHu-IL-1 alpha. The second EIA (S-EIA II) which uses enzyme labelled no. 964 and no. 374 fixed to the plate, can detect both rHu-IL-1 alpha and rHu-Asp36-IL-1 alpha and this assay of total rHu-IL-1 alpha is comparable to a competitive EIA using an enzyme-labelled rHu-IL-1 alpha and an anti-rHu-IL-1 alpha polyclonal antibody. Thus, the level of rHu-Asp36-IL-1 alpha in the samples containing the two IL-1 alpha s can be calculated by subtracting the level measured by S-EIA I from that measured by S-EIA II. The two EIA systems with an assay range of 1.5-100 ng/ml do not recognize IL-1 beta, IL-2, rHu-TNF alpha, IFN-alpha and IFN-gamma of human origin.(ABSTRACT TRUNCATED AT 250 WORDS)

[Production of IL-1 like cytokine by cultured bone cells: inducing effect of Haemophilus actinomycetemcomitans lipopolysaccharide on the cytokine production].

We have studies the role that interleukin-1 (IL-1) and IL-1-like cytokine play in the mechanism of bone resorption that occurs in periodontal disease. To determine whether the bone cell itself produces IL-1-like cytokine, we examined bone cells migrating from fragments of newborn mouse calvaria. These bone cells were cultured in alpha-MEM with of without fetal calf serum. IL-1-like cytokine activity was measured by incorporation of [3H] thymidine into C3H/HeJ thymocytes treated with lipopolysaccharide (LPS) from Haemophilus actinomycetemcomitans, a bacterium found in juvenile periodontopathy. The bone cells produced a significant amount of IL-1-like cytokine. The maximum production of IL-1-like cytokine was observed at 24 hours with the LPS in serum-free alpha-MEM. IL-1-like cytokine production stimulated by LPS was marked in the bone cells from LPS high-responder C3H/HeN mice, but not in those from low-responder C3H/HeJ mice. Peak of IL-1-like cytokine activity in culture supernatants of the bone cells was detected in fractions with a molecular weight corresponding to 15,000 daltons.

Interleukin-1 beta as a potent hyperalgesic agent antagonized by a tripeptide analogue.

Interleukin-1 (IL-1) describes two inflammatory proteins, IL-1 alpha and IL-1 beta, produced by activated macrophages and other cell types and encoded by two genes. Their amino acid sequences have only 26% similarity, but their biological activities are comparable, with a few exceptions; indeed, both molecules appear to act at the same receptor. As IL-1 release prostaglandins which sensitize nociceptors in man and in experimental animals, we tested IL-1 alpha and IL-1 beta in rats for hyperalgesic (nociceptive) activity. Our results show that IL-1 beta given systemically is an extremely potent hyperalgesic agent with a probable peripheral site of action; IL-1 alpha is approximately 3,000 times less active than IL-1 beta. We have delineated the region of IL-1 beta mediating the hyperalgesic effect and developed an analgesic tripeptide analogue of IL-1 beta which antagonizes hyperalgesia evoked by IL-1 beta and by the inflammatory agent carrageenan.

Phosphorylation of intracellular precursors of human IL-1.

The human IL-1 molecules (IL-1 alpha and IL-1 beta) are post-translationally cleaved from 31-kDa precursor to 18-kDa biologically active molecules. During the course of studies of post-translational modifications of human IL-1, we have observed that although LPS induced the production of both intracellular IL-1 alpha and IL-1 beta in human monocytes, [32P]orthophosphate labeling of these cells revealed that intracellular precursor of IL-1 alpha (pre-IL-1 alpha) to be phosphorylated at least 10-fold more than intracellular pre-IL-1 beta. However, no 32P-incorporation could be detected in the 18-kDa processed IL-1 alpha and IL-1 beta. Analysis by TLC revealed that the major phosphorylation site occurred at serine residue(s). The 32P was incorporated into multiply cleaved precursors of IL-1 alpha, which appeared in the absence of protease inhibitors. Since the smallest Mr pre-IL-1 alpha that was labeled with 32P was 22 kDa, the phosphorylated serine residue is presumably located adjacent to a sequence of four basic amino acids located in the 4-kDa region at the amino terminus of the 22-kDa precursor of IL-1 alpha. This serine residue might also be a major phosphorylation site for a cAMP-dependent protein kinase. This hypothesis was substantiated by the demonstration that a synthetic peptide analogue of this region (residue 84 to 112) could be similarly phosphorylated in vitro by a cAMP-dependent protein kinase. Furthermore, a truncated pre-IL-1 alpha (residue 64 to 271) and a "fusion" protein containing staphylococcal protein A and an amino-terminal half-portion of pre-IL-1 alpha (residue 1 to 112), but not mature IL-1 alpha (residue 113 to 271), could also be phosphorylated by cAMP-dependent protein kinase. There is no comparable amino acid sequence in IL-1 beta which could be expected to be phosphorylated by a cAMP-dependent protein kinase. The physiologic relevance of phosphorylation of pre-IL-1 alpha was investigated. The data showed that phosphorylation of truncated pre-IL-1 alpha greatly enhanced its susceptibility to digestion by trypsin and promoted the conversion of pre-IL-1 alpha to the more biologically active IL-1. Although the precise role of the rather selective phosphorylation of pre-IL-1 alpha is not known, our findings do suggest that the phosphorylation of serine close to dibasic/tetrabasic amino acid sequence functions to facilitate the processing and/or release of IL-1 alpha.

N-terminal-methionylated interleukin-1 beta has reduced receptor-binding affinity.

The receptor-binding affinity of recombinant-derived interleukin-1 beta containing unprocessed N-terminal methionine (MAPV-) was 10-fold lower than protein containing the authentic N-terminal sequence (APV-). Structural analysis of the methionylated and non-methionylated proteins by NMR spectroscopy detected no (or minor) conformational differences. The differences in binding affinity, therefore, suggest that the additional N-terminal methionine causes a small, direct or indirect, perturbation of the receptor-binding region.