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The rationale of α1-antitrypsin (AAT) augmentation therapy to treat progressive emphysema in AAT-deficient patients is based on inhibition of neutrophil elastase; however, the benefit of this treatment remains unclear. Here we show that clinical grade AAT (with elastase inhibitory activity) and a recombinant form of AAT (rAAT) without anti-elastase activity reduces lung inflammatory responses to LPS in elastase-deficient mice. WT and elastase-deficient mice treated with either native AAT or rAAT exhibited significant reductions in infiltrating neutrophils (23% and 68%), lavage fluid levels of TNF-α (70% and 80%), and the neutrophil chemokine KC (CXCL1) (64% and 90%), respectively. Lung parenchyma TNF-α, DNA damage-inducible transcript 3 and X-box binding protein-1 mRNA levels were reduced in both mouse strains treated with AAT; significantly lower levels of these genes, as well as IL-1β gene expression, were observed in lungs of AAT-deficient patients treated with AAT therapy compared with untreated patients. In vitro, LPS-induced cytokines from WT and elastase-deficient mouse neutrophils, as well as neutrophils of healthy humans, were similarly reduced by AAT or rAAT; human neutrophils adhering to endothelial cells were decreased by 60–80% (P < 0.001) with either AAT or rAAT. In mouse pancreatic islet macrophages, LPS-induced surface expression of MHC II, Toll-like receptor-2 and -4 were markedly lower (80%, P < 0.001) when exposed to either AAT or rAAT. Consistently, in vivo and in vitro, rAAT reduced inflammatory responses at concentrations 40- to 100-fold lower than native plasma-derived AAT. These data provide evidence that the anti-inflammatory and immunomodulatory properties of AAT can be independent of elastase inhibition. [ABSTRACT FROM AUTHOR]

Cytokines are crucial in host defence against pathogens such as bacteria, viruses, fungi and parasites. A newly described cytokine, interleukin-32 (IL-32), induces various proinflammatory cytokines (tumour necrosis factor-α, IL-1β, IL-6) and chemokines in both human and mouse cells through the nuclear factor-κB and p38 mitogen-activated protein kinase inflammatory signal pathway. The IL-32 primarily acts on monocytic cells rather than T cells. In an attempt to isolate the IL-32 soluble receptor, we used an IL-32 ligand-affinity column to purify neutrophil proteinase 3, which is a serine proteinase involved in many inflammatory diseases. IL-32 has biological activity associated with Mycobacterium tuberculosis and chronic proinflammatory diseases such as rheumatoid arthritis. IL-32 is transcribed as six alternative splice variants and the biological activity of each individual isoform remains unknown. Here, we cloned the complementary DNA of the four IL-32 isoforms (α, β, γ and δ) that are the most representative IL-32 transcripts. To produce recombinant protein with a high yield, the amino acids of two cysteine residues were mutated to serine residues, because serine residues are not conserved among different species. The multi-step purified recombinant IL-32 isoform proteins were assessed for their biological activities with different cytokine assays. The γ isoform of IL-32 was the most active, although all isoforms were biologically active. The present study will provide a specific target to neutralize endogenous IL-32, which may contribute to basic and clinical immunology. [ABSTRACT FROM AUTHOR]

IL-32, a recently discovered proinflammatory cytokine with four isoforms, induces lL-1β, TNF-α, lL-6, and chemokines. Here, we used ligand (IL-32α) affinity chromatography in an attempt to isolate an IL-32α soluble receptor or binding protein. Recombinant IL-32α was covalently immobilized on agarose, and preparations of concentrated crude human urinary proteins were applied for chromatographic separation. A specific 30-kDa protein eluted from the column during acid washing and was identified by mass spectrometry as proteinase 3 (PR3) and confirmed by N-terminal microsequencing. PR3, a neutrophil granule serine protease, exists in a soluble or membrane form and is the major autoantigen for autoantibodies in the systemic vasculitic disease, Wegener's granulomatosis. The affinity of IL-32α to PR3 was determined by surface plasmon resonance. The dissociation constants were 2.65 ± 0.4 nM for urinary PR3 and 1.2 ± 0.05 nM for neutrophil-derived PR3. However, irreversible inactivation of PR3 enzymatic activity did not significantly change binding to the cytokine. Nevertheless, limited cleavage of IL-32 yielded products consistent with PR3 enzyme activity. Moreover, after limited cleavage by PR3, lL-32α was more active than intact lL-32α in inducing macrophage inflammatory protein-2 in mouse macrophages and IL-8 in human peripheral blood mononuclear cells. We suggest that PR3 is a specific lL-32α binding protein, independent of its enzymatic activity. However, limited cleavage of lL-32α by PR3 enhances activities of the cytokine. Therefore, specific inhibition of PR3 activity to process IL-32 or neutralization of IL-32 by inactive PR3 or its fragments may reduce the consequences of IL-32 in immune regulated diseases. [ABSTRACT FROM AUTHOR]

IL-32 is a recently discovered cytokine that induces TNFα, IL-1β,IL-6, and chemokines. We investigated whether IL-32 is expressed in the synovia of patients with rheumatoid arthritis (RA) and studied associations with disease severity and the presence of other cytokines. Immunohistochemistry revealed that IL-32 is highly expressed in RA synovial tissue biopsies, whereas IL-32 was not observed in synovial tissues from patients with osteoarthritis. Moreover, in synovial biopsies from 29 RA patients with active disease, the level of IL-32 staining correlated with erythrocyte sedimentation rate, a marker of systemic inflammation (R = 0.63 and P < 0.0003). Synovial staining of lL-32 also correlated with indices of synovial inflammation (R = 0.80 and P < 0.0001) as well as synovial presence of TNFα (R = 0.68 and P < 0.004), IL-1β (R = 0.79 and P < 0.0001), and IL-18 (R = 0.82 and P < 0.001). IL-32 was a potent inducer of prostaglandin E2 release in mouse macrophages and human blood monocytes, an important property for inflammation. After the injection of human lL-32γ into the knee joints of naïve mice, joint swelling, with pronounced influx of inflammatory cells and cartilage damage, was observed. In TNFα- deficient mice, IL-32-driven joint swelling was absent and cell influx was markedly reduced, but loss of proteoglycan was unaffected, suggesting that IL-32 activity is, in part, TNFα-dependent. 11-32, strongly associated with TNFα, IL-1β, and IL-18, appears to play a role in human RA and may be a novel target in autoimmune diseases. [ABSTRACT FROM AUTHOR]

The activation of innate immunity requires the amplification of signals induced by pattern-recognition receptors for bacterial products. We have investigated the role of the newly described cytokine IL-32 in the amplification of cytokine production induced by the two most clinically relevant families of microbial receptors, the cell-surface Toll-like receptors (TLRs) and the intracellular nuclear oligomerization domain (NOD) receptor family. IL-32 synergized with the NOD1- and NOD2-specific muropeptides of peptidoglycans for the release of lL-1β and IL-6 (a 3- to 10-fold increase). In contrast, IL-32 did not influence the cytokine production induced via TLRs. The synergistic effect of IL-32 and synthetic muramyl dipeptide (MDP) on cytokine production was absent in the cells of patients with Crohn's disease bearing the NOD2 frameshift mutation 3020insC, demonstrating that the lL-32/MDP synergism depends on NOD2. This in vitro synergism between IL-32 and NOD2 ligands was consistent with a marked constitutive expression of IL-32 in human colon epithelial tissue. In addition, the potentiating effect of IL-32 on the cytokine production induced by the synthetic muropeptide FK-156 was absent in NOD1-deficient macrophages, supporting the interaction between IL-32 and NOD1 pathways. When specific caspase inhibitors were used, the synergism between IL-32 and MDP/NOD2 depended on the activation of caspase 1. Only additive effects of IL-32 and muropeptides were observed for TNF-α production. The modulation of intracellular NOD2 pathways by IL-32, but not cell-surface TLRs, and the marked expression of IL-32 in colon mucosa suggest a role of IL-32 in the pathogenesis of Crohn's disease. [ABSTRACT FROM AUTHOR]

IL-1 and IL-18 are members of the IL-1 family of ligands, and their receptors are members of the IL-1 receptor family. Although several biological properties overlap for these cytokines, differences exist. IL-18 uniquely induces IFN-γ from T lymphocytes and natural killer cells but does not cause fever, whereas fever is a prominent characteristic of IL-1 in humans and animals. In the present study, human epithelial cells were stably transfected with the IL-18 receptor p chain and responded to IL-18 with increased production of IL-1α, IL-6, and IL-8. Five minutes after exposure to either cytokine, phosphorylation of mitogen activated protein kinase (MAPK) p38 was present specific inhibition of p38 MAPK reduced IL-18 activity to background levels. Whereas IL-1β induced the expression of the NF-κB-reporter gene and was suppressed by competitive inhibition of NF-κB binding, IL-18 responses were weak or absent. In contrast to IL-1β IL-18 also did not activate degradation of the NF-κB inhibitor. After 4 h, both cytokines induced comparable levels of mRNA for the chemokine IL-8 but, in the same cells, steady-state levels of cyclooxygenase (COX)-2 mRNA were high after IL-1β but low or absent after IL-18. After 30 h, IL-18-induced COX-2 appeared in part to be IL-1 dependent. Similarly, low levels of prostaglandin E2 were measured in IL-18-stimulated A549 cells and freshly obtained primary human monocytes and mouse macrophages. We conclude that in epithelial cells, IL-18 signal transduction is primarily via the MAPK p38 pathway rather than NF-κB, which may explain the absence of COX-2 and the failure of IL-18 to cause fever. [ABSTRACT FROM AUTHOR]

Studies the antitumor histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA). Exhibition of antiinflammatory properties via suppression of cytokines; Increase in gene expression regulating tumor survival; Reduction of hepatic cellular injury through the administration of SAHA.

A new proinflammatory cytokine interleukin-32 (IL-32) has six isoforms. Although IL-32 can be detected in sera from patients suffering from Crohn’s disease and rheumatoid arthritis, it is unclear which isoforms are involved. To this end, we investigated the functions of the most abundant IL-32β by generating K562-IL-32β stable cell lines. This report confirms, using IL-32 small interfering RNA, that IL-32β induces an anti-inflammatory cytokine IL-10 in K562-IL-32β cells and U937 promonocytic cells, which express endogenous IL-32β upon phorbol 12-myristate 13-acetate (PMA) treatment, and monocyte-derived dendritic cells (DC) upon lipopolysaccharide (LPS) treatment. Interleukin-32β was induced in monocyte-derived macrophages by LPS and in monocyte-derived DC by LPS, poly(I:C), or anti-CD40 antibody, but was not induced by PMA. We showed that IL-32β expression was increased in a time-dependent manner in monocyte-derived DC upon LPS treatment and peaked at 24 hr. Production of IL-10 was exactly coincident with IL-32β expression, but IL-1β and tumour necrosis factor-α production peaked at 6 hr after LPS treatment, then steeply declined. Interleukin-12 p40 was induced at 9 hr and gradually increased until 48 hr, at which time IL-32β and IL-10 were no longer increased. Knock-down of IL-32β by IL-32 small interfering RNA led to the decrease of IL-10, but the increase of IL-12 in monocyte-derived DC, which means that IL-32β promotes IL-10 production, but limits IL-12 production. We also showed that IL-10 neutralization increases IL-12, IL-1β and tumour necrosis factor-α production, which implies that IL-10 suppresses such proinflammatory cytokines. Taken together, our results suggest that IL-32β upregulates the production of an anti-inflammatory cytokine IL-10, and then IL-10 suppresses proinflammatory cytokines. [ABSTRACT FROM AUTHOR]

IL-32, a newly described multifunctional cytokine, has been associated with a variety of inflammatory diseases, including rheumatoid arthritis, vasculitis, and Crohn's disease. In this study, we investigated the immunomodulatory effects of IL-32γ on bone marrow-derived dendritic cell (DC)-driven Th responses and analyzed the underlying signaling events. IL-32γ-treated DCs exhibited upregulated expression of cell-surface molecules and proinflammatory cytokines associated with DC maturation and activation. In particular, IL-32γ treatment significantly increased production of IL-12 and IL-6 in DCs, which are known as Th1- and Th17-polarizing cytokines, respectively. This increased production was inhibited by the addition of specific inhibitors of the activities of phospholipase C (PLC), JNK, and NF-κB. IL-32γ treatment increased the phosphorylation of JNK and the degradation of both IκBα and IκBβ in DCs, as well as NF-κB binding activity to the κB site. The PLC inhibitor suppressed NF-κB DNA binding activity and JNK phosphorylation increased by IL-32γ treatment, thereby indicating that IL-32γ induced IL-12 and IL-6 production in DCs via a PLC/JNK/NF-κB signaling pathway. Importantly, IL-32γ-stimulated DCs significantly induced both Th1 and Th17 responses when cocultured with CD4+ T cells. The addition of a neutralizing anti-IL-12 mAb abolished the secretion of IFN-γ in a dose-dependent manner; additionally, the blockage of IL-1β and IL-6, but not of IL-21 or IL-23p19, profoundly inhibited IL-32γ-induced IL-17 production. These results demonstrated that IL-32γ could effectively induce the maturation and activation of immature DCs, leading to enhanced Th1 and Th17 responses as the result of increased IL-12 and IL-6 production in DCs. [ABSTRACT FROM AUTHOR]

Since interleukin (IL)-18 is a proinflammatory cytokine, mice lacking IL-18 or its ligand-binding receptor (IL-18R) should exhibit decreased cytokine and chemokine production. Indeed, production of IL-1α, IL-6, and MIP-1α was reduced in IL-18 knock-out (ko) mouse embryonic fibroblast (MEF)-like cells. Unexpectedly, we observed a paradoxical 10-fold increase in IL-lβ-induced IL-6 production in MEF cells from mice deficient in the IL-18Rα-chain (IL-18Rα) compared with wild type MEF. Similar increases were observed for IL-1α, MIP-1α, and prostaglandin E2. Likewise, coincubation with a specific IL-18Rα-blocking antibody augmented IL-1β-induced cytokines in wild type and IL-18 ko MEF. Stable lines of IL-18Rα-depleted human A549 cells were generated using shRNA, resulting in an increase of IL-1β-induced IL-1α, IL-6, and IL-8 compared to scrambled small hairpin RNA. In addition, we silenced IL-18Rα with small interfering RNA in primary human blood cells and observed up to 4-fold increases in the secretion of lipopolysaccharideand IL-12/IL-18-induced IL-1β, IL-6, interferon-γ, and CD40L. Mechanistically, despite increases in Stat 1 and IL-6, induction of SOCS1 and -3 (suppressor of cytokine signaling 1 and 3) was markedly reduced in the absence of IL-18Rα. Consistent with these observations, activation of the p38α/β and ERK1/2 MAPKs and of protein kinase B/Akt increased in IL-18Rα ko MEF, whereas the negative feedback kinase MSK2 was more active in IL-18 ko cells. These data reveal a role for SOCS1 and -3 in the seemingly paradoxical hyperresponsive state in cells deficient in IL-18Rα, supporting the concept that IL-18Rα participates in both pro- and anti-inflammatory responses and that an endogenous ligand engages lL-18Rα to deliver an inhibitory signal. [ABSTRACT FROM AUTHOR]