Galectin-9 is a high affinity IgE-binding lectin with anti-allergic effect by blocking IgE-antigen complex formation.

Galectin (Gal)-9 was first described as an eosinophil chemoattractant. With the progress in research, Gal-9 has come to be known as a versatile immunomodulator that is involved in various aspects of immune regulations, and the entire picture of the function still remains elusive. To uncover as-yet unknown activity of Gal-9, we have been examining the effect of the protein in various disease animal models. Here we show that Gal-9 attenuated asthmatic reaction in guinea pigs and suppressed passive-cutaneous anaphylaxis in mice. These results indicate the mast cell stabilizing effect of Gal-9. In vitro studies of mast cell degranulation involving RBL-2H3 cells demonstrated that Gal-9 suppressed degranulation from the cells stimulated by IgE plus antigen and that the inhibitory effect was completely abrogated in the presence of lactose, indicating lectin activity of Gal-9 is critical. We found that Gal-9 strongly and specifically bound IgE, which is a heavily glycosylated immunoglobulin, and that the interaction prevented IgE-antigen complex formation, clarifying the mode of action of the anti-degranulation effect. Gal-9 is expressed by several mast cells including mouse mast cell line MC/9. The fact that immunological stimuli of MC/9 cells augmented Gal-9 secretion from the cells implies that Gal-9 is an autocrine regulator of mast cell function to suppress excessive degranulation. Collectively, these findings shed light on a novel function of Gal-9 in mast cells and suggest a beneficial utility of Gal-9 for the treatment of allergic disorders including asthma.

Galectin-9 accelerates transforming growth factor beta3-induced differentiation of human mesenchymal stem cells to chondrocytes.

Galectin-9 (Gal-9), a beta-galactoside binding lectin, plays a crucial role in innate and adaptive immunity. In the rat collagen-induced arthritis model, administration of Gal-9 induced repair of existing cartilage injury even when joints were already swollen with cartilage destruction. We thus attempted to explore the role of Gal-9 in chondrocyte differentiation utilizing human mesenchymal stem cell (MSC) pellet cultures. During chondrogenesis induced by transforming growth factor beta3 (TGFbeta3), MSCs strongly expressed endogenous Gal-9. Expression of Gal-9 peaked on day 14 and the neutralization of endogenous Gal-9 resulted in the reduced chondrogenesis, indicating possible involvement of Gal-9 in TGFbeta-mediated chondrogenesis. In pellets, addition of Gal-9 significantly enhanced TGFbeta3-induced chondrogenesis, as evidenced by increasing proteoglycan content, but not cell proliferation. In the absence of Gal-9, collagen expression by MSCs switched from type I to type II on 28 days after stimulation with TGFbeta3. When MSCs were co-stimulated with Gal-9, the class switch occurred on day 21. In addition, Gal-9 synergistically enhanced TGFbeta3-induced phosphorylation of Smad2, though Gal-9 did not itself induce detectable Smad2 phosphorylation. These results suggest that Gal-9 has a beneficial effect on cartilage repair in injured joints by induction of differentiation of MSCs into chondrocytes.

NbCl(3)-catalyzed [2+2+2] intermolecular cycloaddition of alkynes and alkenes to 1,3-cyclohexadiene derivatives.

NbCl(3)(DME)-catalyzed [2+2+2] intermolecular cycloaddition of alkynes and alkenes was successfully achieved to give 1,4,5-trisubstituted-1,3-cyclohexadiene derivatives in good yields.

Galectin-9 expands unique macrophages exhibiting plasmacytoid dendritic cell-like phenotypes that activate NK cells in tumor-bearing mice.

Galectin-9 (Gal-9) inhibits the metastasis of tumor cells by blocking their adhesion to endothelium and the extracellular matrix. In this study, we addressed the involvement of Gal-9 in anti-tumor activity. Gal-9 significantly prolonged the survival of B16F10 melanoma-bearing mice. Gal-9 increased the numbers of NK cells, CD8 T cells and macrophages in tumor-bearing mice. Gal-9-mediated anti-tumor activity was not induced in NK cell-, macrophage- and CD8 T cell-depleted mice. NK cells from Gal-9-treated mice, compared to PBS-treated mice, exhibited significantly higher cytolytic activity. Co-culture of naïve NK cells with macrophages from Gal-9-treated mice resulted in enhanced NK activity, although Gal-9 itself did not enhance the NK activity. We also found that Ly-6C(+)CD11b(+)F4/80(+) macrophages with plasmacytoid cell (pDC)-like phenotypes (PDCA-1 and B220) were responsible for the enhanced NK activity. These results provide evidence that Gal-9 promotes NK cell-mediated anti-tumor activity by expanding unique macrophages with a pDC-like phenotype.

Galectin-9 suppresses tumor metastasis by blocking adhesion to endothelium and extracellular matrices.

We previously described an inverse correlation between galectin-9 (Gal-9) expression and metastasis in patients with malignant melanoma and breast cancer. This study verified the ability of Gal-9 to inhibit lung metastasis in experimental mouse models using highly metastatic B16F10 melanoma and Colon26 colon cancer cells. B16F10 cells transfected with a secreted form of Gal-9 lost their metastatic potential. Intravenous Gal-9 administration reduced the number of metastases of both B16F10 and Colon26 cells in the lung, indicating that secreted Gal-9 suppresses metastasis. Analysis of adhesive molecule expression revealed that B16F10 cells highly express CD44, integrin alpha1, alpha 4, alpha V, and beta1, and that Colon26 cells express CD44, integrin alpha2, alpha 5, alpha V, and beta1, suggesting that Gal-9 may inhibit the adhesion of tumor cells to vascular endothelium and the extracellular matrix (ECM) by binding to such adhesive molecules. Indeed, Gal-9 suppressed the binding of hyaluronic acid to CD44 on both B16F10 and Colon26 cells, and also suppressed the binding of vascular cell adhesion molecule-1 to very late antigen-4 on B16F10 cells. Furthermore, Gal-9 inhibited the binding of tumor cells to ECM components, resulting in the suppression of tumor cell migration. The present results suggest that Gal-9 suppresses both attachment and invasion of tumor cells by inhibiting the binding of adhesive molecules on tumor cells to ligands on vascular endothelium and ECM.

Galectin-9 suppresses the generation of Th17, promotes the induction of regulatory T cells, and regulates experimental autoimmune arthritis.

The effects of galectin-9 on a mouse collagen-induced arthritis (CIA) model were assessed to clarify whether galectin-9 suppresses CIA by regulating T cell immune responses. Galectin-9 suppressed CIA in a dose-dependent manner, and such suppression was observed even when treatment was started on 7 days after the booster, indicating its preventive and therapeutic effects. Galectin-9 induced the decreased levels of pro-inflammatory cytokines, IL-17, IL-12, and IFNgamma in the joint. Galectin-9 induced the decreased number of CD4(+) TIM-3(+) T cells in peripheral blood. Galectin-9-deficient mice became susceptible to CIA may be by increased number of CD4(+) TIM-3(+) T cells and decreased number of Treg cells. We further found that galectin-9 induces differentiation of naive T cells to Treg cells, and it suppresses differentiation to Th17 cells in vitro. The present results suggested that galectin-9 ameliorates CIA by suppressing the generation of Th17, promoting the induction of regulatory T cells.

Galectin-9 inhibits CD44-hyaluronan interaction and suppresses a murine model of allergic asthma.

RATIONALE: Galectin-9 (Gal-9) belongs to the galectin family, which exhibits affinity for beta-galactosides. Gal-9 has a variety of biological activities; however, its role in allergic inflammation is unknown. OBJECTIVES: We evaluated the effect of a stable form of the human protein on allergic airway inflammation in a mite allergen-induced asthma model. METHODS: Human stable Gal-9 was given by intravenous injection to mice during antigen challenge. The effect of Gal-9 on airway inflammation and airway hyperresponsiveness (AHR) was then evaluated. MEASUREMENTS AND MAIN RESULTS: Gal-9 reduced AHR as well as Th2-associated airway inflammation. Furthermore, administration of Gal-9 as well as anti-CD44 monoclonal antibody inhibited the infiltration of peripheral blood Th2 cells into the airway. Interestingly, Gal-9 directly bound the CD44 adhesion molecule and inhibited interactions with hyaluronan (HA). Consistent with the concept that CD44-HA interactions mediate the migration of T cells into the lung, Gal-9 blocked CD44-dependent adhesion of BW5147 mouse T cells to HA. CONCLUSIONS: We conclude that Gal-9 inhibits allergic inflammation of the airway and AHR by modulating CD44-dependent leukocyte recognition of the extracellular matrix.

Inhibition of eosinophilia in vivo by a small molecule inhibitor of very late antigen (VLA)-4.

The alpha4beta1 integrin (very late antigen-4, VLA-4) plays an important role in the migration of lymphocytes, monocytes, and eosinophils, but not neutrophils, to sites of inflammation. Pharmacological antagonism of VLA-4 is an attractive prospect for the treatment of predominantly eosinophil mediated diseases such as asthma and allergic rhinitis. We report here on a potent and selective, small molecule VLA-4 inhibitor, (2S)-3-(2', 5'-dichlorobiphenyl-4-yl)-2-({[1-(2-methoxybenzoyl)piperidin-3-yl]carbonyl}amino) propanoic acid, compound 1, and characterize the antagonist activities of this molecule in various cell-based assays and in an animal model of eosinophil migration. Compound 1 inhibited VLA-4/ vascular cell adhesion molecule-1(VCAM-1) interactions with in vitro potencies (IC50 value of 210 nM) in VLA-4-expressing Ramos cells, although the compound did not inhibit cell adhesion to fibronectin via alpha5beta1 integrin (very late antigen-5, VLA-5). Blockade of phorbol-12-myristate-13-acetate (PMA)- or Mn2+-stimulated VLA-4 interactions with compound 1 was observed in human T lymphocytes (IC50 value of 230 nM), human eosinophils (IC50 value of 4.0 microM) and mouse eosinophils (IC50 value of 1.6 microM). Furthermore, compound 1 administered by intraperitoneal injection inhibited eosinophil infiltration in a dose-dependent manner by up to 80% in an air pouch model. These data support the use of small molecule VLA-4 antagonists in the treatment of relevant diseases, such as asthma, atopic dermatitis, or allergic rhinitis.

A selective novel low-molecular-weight inhibitor of IkappaB kinase-beta (IKK-beta) prevents pulmonary inflammation and shows broad anti-inflammatory activity.

1 Pulmonary inflammatory diseases such as asthma are characterized by chronic, cell-mediated inflammation of the bronchial mucosa. 2 Recruitment and activation of inflammatory cells is orchestrated by a variety of mediators such as cytokines, chemokines, or adhesion molecules, the expression of which is regulated via the transcription factor nuclear factor kappa B (NF-kappaB). 3 NF-kappaB signaling is controlled by the inhibitor of kappa B kinase complex (IKK), a critical catalytic subunit of which is IKK-beta. 4 We identified COMPOUND A as a small-molecule, ATP-competitive inhibitor selectively targeting IKK-beta kinase activity with a K(i) value of 2 nM. 5 COMPOUND A inhibited stress-induced NF-kappaB transactivation, chemokine-, cytokine-, and adhesion molecule expression, and T- and B-cell proliferation. 6 COMPOUND A is orally bioavailable and inhibited the release of LPS-induced TNF-alpha in rodents. 7 In mice COMPOUND A inhibited cockroach allergen-induced airway inflammation and hyperreactivity and efficiently abrogated leukocyte trafficking induced by carrageenan in mice or by ovalbumin in a rat model of airway inflammation. 8 COMPOUND A was well tolerated by rodents over 3 weeks without affecting weight gain. 9 Furthermore, in mice COMPOUND A suppressed edema formation in response to arachidonic acid, phorbol ester, or edema induced by delayed-type hypersensitivity. 10 These data suggest that IKK-beta inhibitors offer an effective therapeutic approach for inhibiting chronic pulmonary inflammation.

CRTH2 is a prominent effector in contact hypersensitivity-induced neutrophil inflammation.

Chemoattractant receptor-homologous molecule expressed on Th2 lymphocytes, CRTH2, is a cognate receptor for prostaglandin (PG) D(2) and, in humans, is suggested to play a functional role in Th2-dependent allergic inflammation. While peripheral blood leukocytes expressing high levels of surface CRTH2 have been detected in disease, little is known of the functional significance of CRTH2 in disease etiology. We have utilized a Th2-dependent murine model of FITC-induced contact hypersensitivity to assess the role, if any, CRTH2-PGD(2) may play in the elicitation or maintenance of such pathobiology. Expression of both PGD(2) and CRTH2 in lesional skin was paralleled by the release of the chemoattractants LTB(4) and the chemokine KC, as well as a profuse dermal neutrophilic and eosinophilic infiltrate, closely paralleling the acute inflammatory pathology observed in human atopic dermatitis. A small molecule CRTH2 antagonist, but not a selective PGD(2)R (DP) receptor antagonist, was able to completely abrogate these responses. Inflammatory cascades mediated by CRTH2 ligation may therefore represent an important early step in the elicitation and maintenance of Th2-dependent skin inflammation.

Essential role of MHC II-independent CD4+ T cells, IL-4 and STAT6 in contact hypersensitivity induced by fluorescein isothiocyanate in the mouse.

Contact hypersensitivity (CHS) induced by a hapten is thought to be mediated by T helper type 1 (Th1) cells. However, FITC can induce contact allergy in vivo, and in vitro studies suggest that this response is Th2-type driven. We compared CHS reactions induced by FITC or dinitrofluorobenzene (DNFB), a well-known Th1 inducing hapten, in Balb/c mice, C57/B6 mice, and several gene knock-out mice, and investigated the role of Th1/Th2 cytokines, T cell populations, eosinophils, and mast cells. Balb/c mice (Th2 dominant strain) had a stronger response to FITC than C57/B6 mice (Th1 dominant strain). The skin inflammation was characterized by edema and eosinophilia, and serum IgE levels were elevated following FITC challenge. All responses were enhanced by a second round of sensitization. Anti-TNF-alpha or anti-very late antigen-4 (VLA-4) antibody partly inhibited both FITC- and DNFB-induced CHS. Pretreatment of mice with anti-IL-4 antibody, anti-IL-5 antibody, recombinant INF-gamma, or the mast-cell depleting agent 48/80 significantly diminished edema formation, and Stat6(-/-) mice were fully protected from FITC-induced CHS, while DNFB-induced CHS was enhanced (Stat6(-/-), mast cell depletion) or not affected (anti-IL-5 antibody). Further, mice lacking CD4(+) T cells and mice lacking both CD8 and MHC II showed very little reaction at all to FITC, while the absence of CD8 T cells alone or MHC II alone conferred partial protection only. These findings indicate a contribution of MHC II-independent CD4(+) T cells and/or CD4(+) NKT cells to the Th2 response triggered by FITC in vivo, and makes FITC-induced CHS a suitable animal model for atopic dermatitis.

Critical role of L-selectin and histamine H4 receptor in zymosan-induced neutrophil recruitment from the bone marrow: comparison with carrageenan.

Zymosan and carrageenan represent two inflammatory stimuli leading to significant neutrophilia when injected into mice. Despite several similarities between the two proinflammatory agents, the mechanisms leading to neutrophil influx into the site of stimulus injection are unclear. As demonstrated by antibody (Ab) studies directed against adhesion molecules, L-selectin was pivotal for zymosan-induced but not carrageenan-induced pleurisy. Zymosan but not carrageenan injection into the pleural cavity caused blood neutrophilia and significant release of neutrophils from the bone marrow, events that were inhibited by anti-L-selectin but not anti-Mac-1 Ab pretreatment. Pertussis toxin, known to regulate cell efflux, abrogated both zymosan- and carrageenan-induced pleurisy, but only zymosan-induced neutrophil release from the bone marrow. Dexamethasone, known to inhibit pleurisy induced by either stimulus, had no effect on bone marrow neutrophil numbers. The G(i/o) G protein-coupled H4 histamine receptor is highly expressed in the bone marrow and on leukocytes and plays an important role in zymosan-induced pleurisy in vivo. Zymosan-triggered neutrophil release from bone marrow was abrogated by pretreatment of mice with thioperamide, a known H(3/4) receptor antagonist, whereas H1 and H2 receptor antagonists had no effect. Moreover, histamine itself, when injected intravenously, led to a similar time- and dose-dependent decrease of neutrophil numbers in the bone marrow that was inhibited by thioperamide. Because the H3 receptor is not expressed on neutrophils, these findings indicate that both H4 and L-selectin regulate zymosan-induced neutrophil release from bone marrow and subsequent infiltration in the pleurisy model.

Critical role of histamine H4 receptor in leukotriene B4 production and mast cell-dependent neutrophil recruitment induced by zymosan in vivo.

The recently identified histamine receptor, H4, was shown to be primarily expressed on leukocytes and has been implicated in the activation of lymphocytes, eosinophils, and mast cells in vitro. Its function in vivo, however, has not yet been characterized. We present evidence for a critical role of H4 receptor in the mast cell-dependent recruitment of neutrophils. Mice injected with zymosan into the pleural cavity developed massive neutrophilia within hours after challenge. Neutrophilia was dose-dependently reduced when mice were pretreated with thioperamide, a known H(3/4) receptor antagonist, whereas H1 and H2 receptor antagonists lacked efficacy. Similarly, a 70 to 80% reduction in neutrophils in the pleural cavity compared with wild-type animals was noted in mice lacking mast cells (W/W(v) mice); mice deficient in MyD88 (MyD88(-/-)); a critical component of the signaling cascade of the major receptor for zymosan, toll-like receptor 2 (TLR2); or in mice pretreated with a functionally antagonistic anti-TLR2 antibody. The residual 20% neutrophil infiltration seen in mast cell-deficient and MyD88(-/-) mice was not further reduced by thioperamide. Neutrophilia was completely restored by transferring wild-type bone marrow-derived mast cells into MyD88(-/-) or W/W(v) mice. Interestingly, when neutrophilia was evoked by carrageenan injection, mast cell depletion and thioperamide had no effect. Various inflammatory mediators were detectable in the pleural cavity of zymosan-challenged mice. Upon pretreatment with thioperamide, reduced levels of the neutrophil chemoattractant leukotriene B4 were observed, providing a mechanistic explanation for the prevention of neutrophilia by H4 receptor antagonism.

Chemoattractant receptor-homologous molecule expressed on Th2 cells activation in vivo increases blood leukocyte counts and its blockade abrogates 13,14-dihydro-15-keto-prostaglandin D2-induced eosinophilia in rats.

We cloned, expressed, and characterized in vitro and in vivo the gene encoding the rat ortholog of chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2), a G protein-coupled receptor for prostaglandin D2 (PGD2). Quantitative reverse transcription-polymerase chain reaction analysis demonstrated highest CRTH2 expression in the lung, brain, ovary, and spleen. Pharmacologically, rat CRTH2 stably transfected in mouse preB lymphoma L1.2 cells behaved very similar compared with the mouse and human orthologs, showing a binding affinity for PGD2 of 11 nM, a functional calcium mobilization when exposed to agonist, and similar sensitivity to agonists and antagonists. In vivo, selective activation of CRTH2 by 13,14-dihydro-15-keto (DK)-PGD2 injection into rats led to a dose- and time-dependent increase of the number of leukocytes in the peripheral blood. Specifically, eosinophils, lymphocytes, and neutrophils were recruited with maximum effects seen 60 min after the injection of 300 microg of DK-PGD2 per rat. Pretreatment of the animals with the CRTH2/thromboxane A2 receptor antagonist, ramatroban, completely abrogated DK-PGD2-induced eosinophilia, suggesting that CRTH2 might have a physiological and/or pathophysiological role in controlling leukocyte migration.

The orally available spleen tyrosine kinase inhibitor 2-[7-(3,4-dimethoxyphenyl)-imidazo[1,2-c]pyrimidin-5-ylamino]nicotinamide dihydrochloride (BAY 61-3606) blocks antigen-induced airway inflammation in rodents.

Spleen tyrosine kinase (Syk) tyrosine kinase plays essential roles in receptors for Fc portion of immunoglobulins and B cell receptor complex signaling in various inflammatory cells; therefore, inhibitors of Syk kinase may show potential as antiasthmatic/allergic therapeutics. We identified 2-[7-(3,4-dimethoxyphenyl)-imidazo[1,2-c]pyrimidin-5-ylamino]-nicotinamide dihydrochloride (BAY 61-3606), a potent (Ki = 7.5 nM) and selective inhibitor of Syk kinase. BAY 61-3606 inhibited not only degranulation (IC50 values between 5 and 46 nM) but also lipid mediator and cytokine synthesis in mast cells. BAY 61-3606 was highly efficacious in basophils obtained from healthy human subjects (IC50 = 10 nM) and seems to be at least as potent in basophils obtained from atopic (high serum IgE) subjects (IC50 = 8.1 nM). B cell receptor activation and receptors for Fc portion of IgG signaling in eosinophils and monocytes were also potently suppressed by BAY 61-3606. Oral administration of BAY 61-3606 to rats significantly suppressed antigen-induced passive cutaneous anaphylactic reaction, bronchoconstriction, and bronchial edema at 3 mg/kg. Furthermore, BAY 61-3606 attenuated antigen-induced airway inflammation in rats. Based on these anti-inflammatory effects of BAY 61-3606 both in vitro and in vivo, it was demonstrated that Syk may play a very critical role in the pathogenesis of allergic reactions.

Discovery of novel and selective IKK-beta serine-threonine protein kinase inhibitors. Part 1.

IkappaB kinase beta (IKK-beta) is a serine-threonine protein kinase critically involved in the activation of the transcription factor Nuclear Factor kappa B (NF-kappaB) in response to various inflammatory stimuli. We have identified a small molecule inhibitor of IKK-beta. Optimization of the lead compound resulted in improvements in both in vitro and in vivo potency, and provided IKK-beta inhibitors exhibiting potent activity in an acute cytokine release model (LPS-induced TNFalpha).