ITIH5 inhibits proliferation, adipogenic differentiation, and secretion of inflammatory cytokines of human adipose stem cells-A new key in treating obesity?

Inter-α-trypsin inhibitor heavy chain 5 (ITIH5) is widely expressed in the human body, and it is detected to be particularly abundant in adipose tissue. ITIH5 expression is increased in people with obesity compared to lean persons and is decreased by diet-induced weight loss. This suggests that ITIH5 may be involved in the development of adiposity and clinical metabolic variables, although its exact function remains unknown. We measured the protein concentration of ITIH5 in adipose samples from patients undergoing abdominoplasty and tested for correlation with the subjects' BMI as well as inflammatory mediators. We stimulated human adipose stem cells (ASCs) with recombinant (r)ITIH5 protein and tested for an effect on proliferation, differentiation, and immunosuppressive properties when the cells were exposed to an artificial inflammatory environment. We found positive correlations between ITIH5 levels and the BMI (p < .001) as well as concentrations of inflammatory cytokines (TNF-α, IL-6, and MCP-1) in adipose tissue (p < .01). Application of the rITIH5 protein inhibited both proliferation (p < .001) and differentiation of ASCs. Especially, the development of mature adipocytes was reduced by over 50%. Moreover, rITIH5 decreased the release of IL-6 and MCP-1 when the cells were exposed to TNF-α and IL-1ß (p < .001). Our data suggest that ITIH5 is an adipokine that is increasingly released during human adipose tissue development, acting as a regulator that inhibits proliferation and adipogenic differentiation of ASCs. ITIH5 thus presents itself as a positive regulator of adipose tissue homeostasis, possibly protecting against both hyperplasia and hypertrophy of adipose tissue and the associated chronic inflammation.

Diversity oriented clicking delivers ß-substituted alkenyl sulfonyl fluorides as covalent human neutrophil elastase inhibitors.

Diversity Oriented Clicking (DOC) is a discovery method geared toward the rapid synthesis of functional libraries. It combines the best attributes of both classical and modern click chemistries. DOC strategies center upon the chemical diversification of core "SuFExable" hubs-exemplified by 2-Substituted-Alkynyl-1-Sulfonyl Fluorides (SASFs)-enabling the modular assembly of compounds through multiple reaction pathways. We report here a range of stereoselective Michael-type addition pathways from SASF hubs including reactions with secondary amines, carboxylates, 1H-1,2,3-triazole, and halides. These high yielding conjugate addition pathways deliver unprecedented ß-substituted alkenyl sulfonyl fluorides as single isomers with minimal purification, greatly enriching the repertoire of DOC and holding true to the fundamentals of modular click chemistry. Further, we demonstrate the potential for biological function - a key objective of click chemistry - of this family of SASF-derived molecules as covalent inhibitors of human neutrophil elastase.

Design and synthesis of sirtinol analogs as human neutrophil elastase inhibitors.

Human neutrophil elastase (HNE) overexpression has a crucial role in most acute inflammation and alpha1-antitrypsin deficiency syndromes observed in humans, triggering neutrophil invasion and activation of macrophage inflammatory and proteolytic effects, leading to tissue damage. Manipulating HNE level homeostasis could potentially help treat neutrophilic inflammation. Previous studies have shown that sirtinol (1) has a specific influence on HNE and potently attenuates acute lung injury and hepatic injury mediated by lipopolysaccharide or trauma hemorrhage. Therefore, 1 was chosen as the model structure to obtain more potent anti-HNE agents. In the present study, we synthesized a series of sirtinol analogues and determined their inhibitory effects on HNE. Structure-activity relationship (SAR) studies showed that swapping the imine and methyl groups of the sirtinol scaffold with diazene and carboxyl groups, respectively, enhances the HNE inhibiting potency. Compound 29 exhibited the highest potency in the SAR study and showed dual inhibitory effects on HNE and proteinase 3 with IC50 values of 4.91 and 20.69 µM, respectively. Furthermore, 29 was confirmed to have dual impacts on inhibiting O2•- generation and elastase release in cell-based assays with IC50 values of 0.90 and 1.86 µM, respectively. These findings suggest that 29 is a promising candidate for developing HNE inhibitors in the treatment of neutrophilic inflammatory diseases.

Thalidomide derivatives as nanomolar human neutrophil elastase inhibitors: Rational design, synthesis, antiproliferative activity and mechanism of action.

Here, we rationally designed a human neutrophil elastase (HNE) inhibitors 4a-4f derived from thalidomide. The HNE inhibition assay showed that synthesized compounds 4a, 4b, 4e and 4f demonstrated strong HNE inhibiton properties with IC50 values of 21.78-42.30 nM. Compounds 4a, 4c, 4d and 4f showed a competitive mode of action. The most potent compound 4f shows almost the same HNE inhibition as sivelestat. The molecular docking analysis revealed that the strongest interactions occur between the azetidine-2,4-dione group and the following three aminoacids: Ser195, Arg217 and His57. A high correlation between the binding energies and the experimentally determined IC50 values was also demonstrated. The study of antiproliferative activity against human T47D (breast carcinoma), RPMI 8226 (multiple myeloma), and A549 (non-small-cell lung carcinoma) revealed that designed compounds were more active compared to thalidomide, pomalidomide and lenalidomide used as the standard drugs. Additionally, the most active compound 4f derived from lenalidomide induces cell cycle arrest at the G2/M phase and apoptosis in T47D cells.

Peptide Human Neutrophil Elastase Inhibitors from Natural Sources: An Overview.

Elastases are a broad group of enzymes involved in the lysis of elastin, the main component of elastic fibres. They are produced and released in the human body, mainly by neutrophils and the pancreas. The imbalance between elastase activity and its endogenous inhibitors can cause different illnesses due to their excessive activity. The main aim of this review is to provide an overview of the latest advancements on the identification, structures and mechanisms of action of peptide human neutrophil elastase inhibitors isolated from natural sources, such as plants, animals, fungi, bacteria and sponges. The discovery of new elastase inhibitors could have a great impact on the pharmaceutical development of novel drugs through the optimization of the natural lead compounds. Bacteria produce mainly cyclic peptides, while animals provide for long and linear amino acid sequences. Despite their diverse natural sources, these elastase inhibitors show remarkable IC50 values in a range from nM to µM values, thus representing an interesting starting point for the further development of potent bioactive compounds on human elastase enzymes.

Lobelia chinensis Extract and Its Active Compound, Diosmetin, Improve Atopic Dermatitis by Reinforcing Skin Barrier Function through SPINK5/LEKTI Regulation.

The skin acts as a mechanical barrier that protects the body from the exterior environment, and skin barrier function is attributed to the stratum corneum (SC), which is composed of keratinocytes and skin lipids. Skin barrier homeostasis is maintained by a delicate balance between the differentiation and exfoliation of keratinocytes, and keratinocyte desquamation is regulated by members of the serine protease kalikrein (KLK) family and their endogenous inhibitor SPINK5/LEKTI (serine protease inhibitor Kazal type 5/lympho-epithelial Kazal-type-related inhibitor). Furthermore, SPINK5/LEKTI deficiency is involved in impaired skin barrier function caused by KLK over-activation. We sought to determine whether increased SPINK5/LEKTI expression ameliorates atopic dermatitis (AD) by strengthening skin barrier function using the ethanol extract of Lobelia chinensis (LCE) and its active compound, diosmetin, by treating human keratinocytes with UVB and using a DNCB-induced murine model of atopic dermatitis. LCE or diosmetin dose-dependently increased the transcriptional activation of SPINK5 promoter and prevented DNCB-induced skin barrier damage by modulating events downstream of SPINK5, that is, KLK, PAR2 (protease activated receptor 2), and TSLP (thymic stromal lymphopoietin). LCE or diosmetin normalized immune response in DNCB treated SKH-1 hairless mice as determined by reductions in serum immunoglobulin E and interleukin-4 levels and numbers of lesion-infiltrating mast cells. Our results suggest that LCE and diosmetin are good candidates for the treatment of skin barrier-disrupting diseases such as Netherton syndrome or AD, and that they do so by regulating SPINK5/LEKTI.

Human Antibody Domains and Fragments Targeting Neutrophil Elastase as Candidate Therapeutics for Cancer and Inflammation-Related Diseases.

Neutrophil elastase (NE) is a serine protease released during neutrophil maturation. High levels of NE are related to lung tissue damage and poor prognosis in cancer; thus, NE is a potential target for therapeutic immunotherapy for multiple lung diseases and cancers. Here, we isolate and characterize two high-affinity, specific, and noncompetitive anti-NE antibodies Fab 1C10 and VH 1D1.43 from two large phage-displayed human Fab and VH libraries. After fusion with human IgG1 Fc, both of them (VH-Fc 1D1.43 and IgG1 1C10) inhibit NE enzymatic activity with VH-Fc 1D1.43 showing comparable inhibitory effects to that of the small molecule NE inhibitor SPCK and IgG1 1C10 exhibiting even higher (2.6-fold) activity than SPCK. Their epitopes, as mapped by peptide arrays combined with structural modeling, indicate different mechanisms for blocking NE activity. Both VH-Fc and IgG1 antibodies block NE uptake by cancer cells and fibroblast differentiation. VH-Fc 1D1.43 and IgG1 1C10 are promising for the antibody-based immunotherapy of cancer and inflammatory diseases.

New 3-unsubstituted isoxazolones as potent human neutrophil elastase inhibitors: Synthesis and molecular dynamic simulation.

Human neutrophil elastase (HNE) is a proteolytic enzyme belonging to the serine protease family and is involved in a variety of pathologies. Thus, compounds able to inhibit HNE represent promising therapeutics for the treatment of inflammatory diseases. Here, we report the further elaboration of our previously reported 3-methylisoxazolone derivatives, synthesizing a new series of 3-nor-derivatives bearing different substituents at the 4-phenyl ring. The most potent compounds 3a, 3g, and 3h, had IC50 values of 16, 11, and 18 nM, respectively. Molecular modeling studies and molecular dynamic (MD) simulations demonstrated no substantial differences between the 3-methylisoxazole derivatives previously tested and the corresponding 3-unsubstituted derivatives in the snapshot conformations sampled during the MD simulations, which is consistent with their similar levels of HNE inhibitory activity. Thus, we conclude that the isoxazolone scaffold is a good scaffold for developing HNE inhibitors, as it tolerates several modifications when adhering to basic scaffold requirements, and the resulting derivatives are quite potent HNE inhibitors.

TMPRSS11A activates the influenza A virus hemagglutinin and the MERS coronavirus spike protein and is insensitive against blockade by HAI-1.

The influenza virus hemagglutinin (HA) facilitates viral entry into target cells. Cleavage of HA by host cell proteases is essential for viral infectivity, and the responsible enzymes are potential targets for antiviral intervention. The type II transmembrane serine protease (TTSP) TMPRSS2 has been identified as an HA activator in cell culture and in the infected host. However, it is less clear whether TMPRSS2-related enzymes can also activate HA for spread in target cells. Moreover, the activity of cellular serine protease inhibitors against HA-activating TTSPs is poorly understood. Here, we show that TMPRSS11A, another member of the TTSP family, cleaves and activates the influenza A virus (FLUAV) HA and the Middle East respiratory syndrome coronavirus spike protein (MERS-S). Moreover, we demonstrate that TMPRSS11A is expressed in murine tracheal epithelium, which is a target of FLUAV infection, and in human trachea, suggesting that the protease could support FLUAV spread in patients. Finally, we show that HA activation by the TMPRSS11A-related enzymes human airway tryptase and DESC1, but not TMPRSS11A itself, is blocked by the cellular serine protease inhibitor hepatocyte growth factor activator inhibitor type-1 (HAI-1). Our results suggest that TMPRSS11A could promote FLUAV spread in target cells and that HA-activating TTSPs exhibit differential sensitivity to blockade by cellular serine protease inhibitors.

The novel deubiquitinase inhibitor b-AP15 induces direct and NK cell-mediated antitumor effects in human mantle cell lymphoma.

The first therapeutic proteasome inhibitor bortezomib has clinical efficacy in mantle cell lymphoma (MCL) which resulted in its incorporation in treatment algorithms for this disease. Impairment of proteasomal function by bortezomib is mediated via inhibition of the 20S core particle. However, proteasome function can also be modified by targeting upstream components of the ubiquitin-proteasome system. Recently, b-AP15 has been identified as a small molecule achieving proteasome inhibition by targeting the deubiquitinase (DUB) activity of the 19S regulatory subunit and was found to inhibit cancer cell growth in preclinical analyses. In the present study, both direct antitumor effects and the possibility to induce natural killer group 2 member D ligands (NKG2DL) to reinforce NK cell immunity with b-AP15 were investigated to provide a rational basis for clinical evaluation of this novel DUB inhibitor in MCL. Treatment with b-AP15 resulted in reduced viability as well as induction of apoptosis in a time- and dose-dependent manner, which could be attributed to caspase activation in MCL cells. In addition, treatment with b-AP15 differentially induced NKG2DL expression and subsequent NK cell lysis of MCL cells. These results indicate that the DUB inhibitor b-AP15 displays substantial antitumor activity in human MCL and suggest that b-AP15 might be a novel therapeutic option in the treatment of MCL that warrants clinical investigation.

Synthesis, biological evaluation, and molecular modelling studies of potent human neutrophil elastase (HNE) inhibitors.

We report the synthesis and biological evaluation of a new series of 3- or 4-(substituted)phenylisoxazolones as HNE inhibitors. Due to tautomerism of the isoxazolone nucleus, two isomers were obtained as final compounds (2-NCO and 5-OCO) and the 2-NCO derivatives were the most potent with IC50 values in the nanomolar range (20-70 nM). Kinetic experiments indicated that 2-NCO 7d and 5-OCO 8d are both competitive HNE inhibitors. Molecular modelling on 7d and 8d suggests for the latter a more crowded region about the site of the nucleophilic attack, which could explain its lowered activity. In addition molecular dynamics (MD) simulations showed that the isomer 8d appears more prone to form H-bond interactions which, however, keep the reactive sites quite distant for the attack by Ser195. By contrast the amide 7d appears more mobile within the active pocket, since it makes single H-bond interactions affording a favourable orientation for the nucleophilic attack.

IL-17A promotes neutrophilic inflammation and disturbs acute wound healing in skin.

In the wound healing process, neutrophils are the first inflammatory cells to move to the wound tissues. They sterilize wounds by killing microbes, and they stimulate other immune cells to protect the host from infection. In contrast, neutrophil-derived proteases cause damage to host tissues, so neutrophils play dual opposite roles in wound healing. Interleukin-17A (IL-17A) is a proinflammatory cytokine that promotes the recruitment of these cells. The role of this cytokine in the wound healing process is not fully clarified. In the present study, therefore, we examined how defect in IL-17A production affected the wound healing in skin. IL-17A-knockout (KO) mice showed promoted wound closure, myofibroblast differentiation and collagen deposition and decreased the neutrophil accumulation compared with wild-type (WT) mice. In contrast, the administration of recombinant IL-17A led to delayed wound closure, low collagen deposition and accelerated neutrophilic accumulation. In addition, the treatment of IL-17A-administered mice with a neutrophil elastase inhibitor improved the wound repair to the same level as that of WT mice. These results indicated that IL-17A hampered the wound healing process and suggested that neutrophilic inflammation caused by IL-17A may be associated with impaired wound healing in skin.

Potential Anti-inflammatory Effects of the Fruits of Paulownia tomentosa.

As part of an ongoing search for new natural products from medicinal plants to treat respiratory disease, six new compounds, a dihydroflavonol (1) and five C-geranylated flavanones (3, 6, 8, 13, and 14), and 13 known compounds were isolated from mature fruits of Paulownia tomentosa. The structures of the new compounds were determined via interpretation of their spectroscopic data (1D and 2D NMR, UV, IR, ECD, and MS). In biological activity assays with human alveolar basal epithelial cells, the expression of TNF-α-induced proinflammatory cytokines (IL-8 and IL-6) was reduced significantly by the EtOAc fraction of a P. tomentosa extract as well as by the new compounds isolated from this fraction. Furthermore, the majority of the isolates (1-19 except 5-7) were found to inhibit human neutrophil elastase (HNE) activity, with IC50 values ranging from 2.4 ± 1.0 to 74.7 ± 8.5 µM. In kinetic enzymatic assays with the HNE substrate MeOSuc-AAPV-pNA, compound 17 exhibited the highest inhibitory activity (Ki = 3.2 µM) via noncompetitive inhibition. These findings suggest that the flavanone constituents of P. tomentosa fruits may be valuable for the development of new drug candidates to treat airway inflammation.

Effects of the neutrophil elastase inhibitor EL-17 in rat adjuvant-induced arthritis.

OBJECTIVES: Neutrophil elastase (NE), a granule-associated enzyme, participates in connective tissue breakdown and promotes cytokine release and specific receptor activation during various inflammatory diseases like RA. NE is increased in the SF and cartilage of RA patients and represents a target for the development of new therapeutic possibilities. The present research aimed to evaluate the preclinical pharmacological profile of the N-benzoylpyrazole derivative EL-17, a potent and selective NE inhibitor, in a rat model of RA. METHODS: Complete Freund's Adjuvant (CFA) was injected in the tibiotarsal joint and the effect of acute or repeated treatments with EL-17 (1-30 mg/kg by mouth) were evaluated. RESULTS: On day 14 after CFA injection, a single administration of EL-17 significantly reduced CFA-dependent hypersensitivity to mechanical noxious stimuli and the postural unbalance related to spontaneous pain. To evaluate the preventive efficacy, EL-17 was administered daily starting from the day of CFA treatment. Behavioural measurements performed on days 7 and 14 showed a progressive efficacy of EL-17 against hypersensitivity to mechanical noxious and non-noxious stimuli, as well as a decrease of hind limb weight-bearing alterations. Histological evaluation of the tibiotarsal joint (day 14) demonstrated significant prevention of articular derangement after EL-17 (30 mg/kg) treatment. The protective effects of EL-17 directly correlated with a complete reversion of the plasma NE activity increase induced by CFA. CONCLUSIONS: The NE inhibitor EL-17 relieved articular pain after acute administration. Furthermore, repeated treatment reduced the development of hypersensitivity and protected joint tissue, revealing a disease-modifying profile.

Local inhibition of elastase reduces EMILIN1 cleavage reactivating lymphatic vessel function in a mouse lymphoedema model.

Lymphatic vasculature critically depends on the connections of lymphatic endothelial cells with the extracellular matrix (ECM), which are mediated by anchoring filaments (AFs). The ECM protein EMILIN1 is a component of AFs and is involved in the regulation of lymphatic vessel functions: accordingly, Emilin1(-/-) mice display lymphatic vascular morphological alterations, leading to functional defects such as mild lymphoedema, lymph leakage and compromised lymph drainage. In the present study, using a mouse post-surgical tail lymphoedema model, we show that the acute phase of acquired lymphoedema correlates with EMILIN1 degradation due to neutrophil elastase (NE) released by infiltrating neutrophils. As a consequence, the intercellular junctions of lymphatic endothelial cells are weakened and drainage to regional lymph nodes is severely affected. The local administration of sivelestat, a specific NE inhibitor, prevents EMILIN1 degradation and reduces lymphoedema, restoring a normal lymphatic functionality. The finding that, in human secondary lymphoedema samples, we also detected cleaved EMILIN1 with the typical bands of an NE-dependent pattern of fragmentation establishes a rationale for a powerful strategy that targets NE inhibition. In conclusion, the attempts to block EMILIN1 degradation locally represent the basis for a novel 'ECM' pharmacological approach to assessing new lymphoedema treatments.

Cinnoline derivatives as human neutrophil elastase inhibitors.

Compounds that can effectively inhibit the proteolytic activity of human neutrophil elastase (HNE) represent promising therapeutics for treatment of inflammatory diseases. We present here the synthesis, structure-activity relationship analysis, and biological evaluation of a new series of HNE inhibitors with a cinnoline scaffold. These compounds exhibited HNE inhibitory activity but had lower potency compared to N-benzoylindazoles previously reported by us. On the other hand, they exhibited increased stability in aqueous solution. The most potent compound, 18a, had a good balance between HNE inhibitory activity (IC50 value = 56 nM) and chemical stability (t1/2 = 114 min). Analysis of reaction kinetics revealed that these cinnoline derivatives were reversible competitive inhibitors of HNE. Furthermore, molecular docking studies of the active products into the HNE binding site revealed two types of HNE inhibitors: molecules with cinnolin-4(1H)-one scaffold, which were attacked by the HNE Ser195 hydroxyl group at the amido moiety, and cinnoline derivatives containing an ester function at C-4, which is the point of attack of Ser195.

Human neutrophil elastase inhibitory potential of flavonoids from Campylotropis hirtella and their kinetics.

Campylotropis hirtella is used as a food supplement in the subtropical region of China. In an intensive hunt for human neutrophil elastase inhibitors, we isolated eight flavonoids from C. hirtella three of which (1-3) emerged to be elastase inhibitors. Geranylated flavonoids (1-3) displayed significant inhibitory activity with IC50s between 8.5 and 30.8 µM. The most striking example was geranylated isofavanone 3 that inhibited elastase significantly (IC50 = 30.8 µM) but its parent compound (dalbergioidin) and isoflavone analog (5) were inactive (IC50 > 200 µM). Compounds (1-3) displayed different kinetic mechanisms (noncompetitive, competitive, and mixed type, respectively) that were dependent upon the parent skeleton. The competitive inhibitor, isoflavan-3-ol-4-one 2 manifested an inhibition of isomerization profile for elastase with kinetic parameters K5 = 0.0386 M-1S-1, K6 = 0.0244 µM-1S-1 and Kiapp = 16.3427 µM. The specific identification of metabolites was accomplished by LC-DAD-ESI/MS that was also used to analyze abundance of active components (1-3) within the plant.

Synthesis and Pharmacological Evaluation of Indole Derivatives as Deaza Analogues of Potent Human Neutrophil Elastase Inhibitors.

Preclinical Research A number of N-benzoylindoles were designed and synthesized as deaza analogs of previously reported potent and selective HNE inhibitors with an indazole scaffold. The new compounds containing substituents and functions that were most active in the previous series were active in the micromolar range (the most potent had IC50 = 3.8 µM) or inactive. These results demonstrated the importance of N-2 in the indazole nucleus. Docking studies performed on several compounds containing the same substituents but with an indole or an indazole scaffold, respectively, highlight interesting aspects concerning the molecule orientation and H-bonding interactions, which could help to explain the lower activity of this new series. Drug Dev Res, 2016. © 2016 Wiley Periodicals, Inc.

Fucosylated chondroitin sulfates from the body wall of the sea cucumber Holothuria forskali: conformation, selectin binding, and biological activity.

Fucosylated chondroitin sulfate (fCS) extracted from the sea cucumber Holothuria forskali is composed of the following repeating trisaccharide unit: → 3)GalNAcß4,6S(1 → 4) [FucαX(1 → 3)]GlcAß(1 →, where X stands for different sulfation patterns of fucose (X = 3,4S (46%), 2,4S (39%), and 4S (15%)). As revealed by NMR and molecular dynamics simulations, the fCS repeating unit adopts a conformation similar to that of the Le(x) blood group determinant, bringing several sulfate groups into close proximity and creating large negative patches distributed along the helical skeleton of the CS backbone. This may explain the high affinity of fCS oligosaccharides for L- and P-selectins as determined by microarray binding of fCS oligosaccharides prepared by Cu(2+)-catalyzed Fenton-type and photochemical depolymerization. No binding to E-selectin was observed. fCS poly- and oligosaccharides display low cytotoxicity in vitro, inhibit human neutrophil elastase activity, and inhibit the migration of neutrophils through an endothelial cell layer in vitro. Although the polysaccharide showed some anti-coagulant activity, small oligosaccharide fCS fragments had much reduced anticoagulant properties, with activity mainly via heparin cofactor II. The fCS polysaccharides showed prekallikrein activation comparable with dextran sulfate, whereas the fCS oligosaccharides caused almost no effect. The H. forskali fCS oligosaccharides were also tested in a mouse peritoneal inflammation model, where they caused a reduction in neutrophil infiltration. Overall, the data presented support the action of fCS as an inhibitor of selectin interactions, which play vital roles in inflammation and metastasis progression. Future studies of fCS-selectin interaction using fCS fragments or their mimetics may open new avenues for therapeutic intervention.

Granulocyte-macrophage colony-stimulating factor primes interleukin-13 production by macrophages via protease-activated receptor-2.

Chronic inflammation is often linked to the presence of type 2-polarized macrophages, which are induced by the T helper type 2 cytokines interleukin-4 and interleukin-13 (IL-13). IL-13 is a key mediator of tissue fibrosis caused by T helper type 2-based inflammation. Human neutrophil elastase (HNE) plays a pivotal role in the pathogenesis of pulmonary fibrosis. This study investigated the priming effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on IL-13 expression by macrophages stimulated with HNE. Adherent macrophages were obtained from primary cultures of human mononuclear cells. Expression of IL-13 mRNA and protein by GM-CSF-dependent macrophages was investigated after stimulation with HNE, using the polymerase chain reaction and enzyme-linked immunosorbent assay. GM-CSF had a priming effect on IL-13 mRNA and protein expression by macrophages stimulated with HNE, while this effect was not observed for various other cytokines. GM-CSF-dependent macrophages showed a significant increase in the expression of protease activated receptor-2 (PAR-2) mRNA and protein. The response of IL-13 mRNA to HNE was significantly decreased by pretreatment with alpha1-antitrypsin, a PAR-2 antibody (SAM11), or a PAR-2 antagonist (ENMD-1068). These findings suggest that stimulation with HNE can induce IL-13 production by macrophages, especially GM-CSF-dependent macrophages. Accordingly, neutrophil elastase may have a key role in fibrosis associated with chronic inflammation.