jnj-7777120 and 4-methylhistamine

Mast cell (MC) activation through H4R releases various inflammatory mediators which are associated with allergic asthma.. To investigate the siRNA-mediated gene silencing effect of H4R on human mast cells (HMCs) functions and the activation of stress-activated protein kinases (SAPK)/jun amino-terminal kinases (JNK) signaling pathways for the release of ineterleukin-1β (IL-1β) in HMCs.. Taken together, the silencing of H4R inhibited the H4R mediated MC functions and SAPK/JNK phosphorylation. Furthermore, the H4R activation utilized SAPK/JNK signaling pathway for IL-1β release in HMC-1 cells.

Topics: Calcium; Cell Line; Gene Expression Regulation; Gene Silencing; Histamine; Humans; Indoles; Interleukin-1beta; MAP Kinase Kinase 4; MAP Kinase Signaling System; Mast Cells; Methylhistamines; Piperazines; Pyrilamine; Receptors, Histamine H4; RNA, Small Interfering

It is a well-known fact that histamine is involved in eosinophil-dependent inflammatory responses including cellular chemotaxis and migration. Nevertheless, the relative role of histamine receptors in the mechanisms of eosinophils adhesion to endothelial cells is not known. Therefore the aim of presented study was to examine the effect of selective histamine receptors ligands on eosinophils adhesion to endothelium. For that purpose the highly purified human eosinophils have been isolated from the peripheral blood. The viability and functional integrity of isolated eosinophils have been validated in several tests. Histamine as well as 4-methylhistamine (selective H4 agonist) in concentration-dependent manner significantly increased number of eosinophils that adhere to endothelium. Among the selective histamine receptors antagonist or H1 inverse agonist only JNJ7777120 (histamine H4 antagonist) and thioperamide (dual histamine H3/H4 antagonist) had direct effect on eosinophils adhesion to endothelial cells. Antagonists of H1 (diphenhydramine, mepyramine) H2 (ranitidine and famotidine) and H3 (pitolisant) histamine receptors were ineffective. To the best of our knowledge, this is the first study to demonstrate that histamine receptor H4 plays a dominant role in histamine-induced eosinophils adhesion to endothelium.

Topics: Cell Adhesion; Cell Communication; Cell Line; Cell Separation; Cell Survival; Drug Inverse Agonism; Endothelium; Eosinophils; Histamine; Histamine Agonists; Histamine H1 Antagonists; Humans; Indoles; Ligands; Methylhistamines; N-Formylmethionine Leucyl-Phenylalanine; Piperazines; Piperidines; Receptors, Histamine

Histamine 4 receptor (H4R) is a novel target for the pharmacological modulation of histamine-mediated immune signals during inflammatory diseases. The purpose of this study was to assess the effects of the H4R agonist 4-methylhistamine dihydrochloride (4-MeH) and antagonist JNJ7777120 (JNJ) in the inflamed rat knee. Animals were fasted for 18h before a single dose of 4-MeH or JNJ (30mg/kg) was administered intraperitoneally (i.p.), both followed by intra-articular (i.a.) injection of LPS 2h later. Blood and synovial fluid were collected after a short incubation period and TNF-α, NF-κB, and IkB-α levels were measured via flow cytometry. Additionally, we assessed the effects of H4R engagement on the expression of IL-1β, TNF-α, and NF-κB mRNAs and the protein levels of TNF-α, NF-κB, JAK-1, and STAT-3 in the inflamed knee tissue. These results revealed increased TNF-α and NF-κB expression and decreased IkB-α levels in both the LPS alone and 4-MeH treated groups in whole blood and synovial fluid. Further, IL-1β, TNF-α, and NF-κB mRNA levels were significantly increased and western blot analysis confirmed increased expression of TNF-α, NF-κB, JAK-1, and STAT-3 in both LPS and 4-MeH treatment groups. Furthermore, these increases were completely inhibited in the inflamed knee tissue of the JNJ-treated group. Thus, the inhibition of inflammatory mediators and signaling pathways by the H4R antagonist JNJ suggests the anti-arthritic importance of this molecule.

Topics: Animals; Anti-Inflammatory Agents; Enzyme Activation; Female; Gene Expression Regulation; Histamine Agonists; Histamine Antagonists; I-kappa B Proteins; Indoles; Inflammation; Interleukin-1beta; Janus Kinase 1; Knee Joint; Lipopolysaccharides; Methylhistamines; NF-kappa B; NF-KappaB Inhibitor alpha; Piperazines; Rats; Rats, Wistar; Receptors, G-Protein-Coupled; Receptors, Histamine; Receptors, Histamine H4; RNA, Messenger; STAT3 Transcription Factor; Tumor Necrosis Factor-alpha

Recently, the expression of histamine 4 receptor (H4R) on neurons was reported, however its function in cells within the central nervous system (CNS) remains poorly understood. To this end, we used the H4R agonist, 4-methylhistamine (4-MeH), and the H4R antagonist, JNJ77777120 (JNJ), to investigate the function of H4R signaling in immune cells in a murine model of chronic stress. Treatment of stressed mice with 4-MeH resulted in an increase in the proportion of lymphocyte subsets (CD3(+), CD8(+), CD28(+), and CD4(+)CD28(+)) and cells expressing the co-stimulatory molecules CD80(+) (B7.1) and CD86(+) (B7.2) in heparinized blood as compared to normal control (NC) and stressed control (SC) groups. We also observed that as compared to NC and SC mice, 4-MeH-treated mice showed greater production of IL-2(+), IL-6(+), IL-9(+), IL-21(+), and IL-27(+) cytokines in the spleen and by splenic CD4(+) T cells. Furthermore, 4-MeH treatment of stressed mice led to an increase in the levels of serum Th1/Th17 cytokines and corticosterone, and a decrease in Th2 cytokines. Treatment of chronically-stressed mice with 4-MeH also augmented expression of IL-6, IL-21, NF-κB p65, and STAT3 mRNA. Moreover, Western blot analyses confirmed increased protein expression of NF-κB, iNOS, and STAT3 expression following 4-MeH treatment of chronically-stressed mice as compared to controls. These proteins provide a novel relevant targets for the manipulation of chronic stress induced immune regulation. In striking contrast, treatment of stressed mice with the H4R antagonist, JNJ, resulted in a substantial reduction in all of the aforementioned effects upon immune cell percentages and cytokine production.

Topics: Analysis of Variance; Animals; B7-1 Antigen; CD28 Antigens; Corticosterone; Cytokines; Indoles; Male; Methylhistamines; Mice; Mice, Inbred BALB C; Piperazines; Receptors, Histamine; Restraint, Physical; RNA, Messenger; Signal Transduction; Spleen; Stress, Psychological; T-Lymphocytes

Rheumatoid arthritis (RA) is one of the major autoimmune diseases with a global prevalence. Despite significant research into this disease, no drugs with acceptable safety profiles are yet available for its treatment. We investigated the possible anti-arthritic effects of the 4-methylhistamine (4-MeH) histamine 4 receptor (H4R) agonist and the JNJ77777120 (JNJ) H4R antagonist to explore the role of H4R in a mouse model of collagen antibody-induced arthritis (CAIA). Arthritis was induced via intravenous (tail vein) injection of Balb/c mice with a 5-clone cocktail of mAbs against collagen type II, followed by LPS, and the effects of treatment with 4-MeH or JNJ (30 mg kg(-1), i.p, twice daily) for 7 days (prophylactic or therapeutic regimens) were assessed. The results revealed increased paw edema, arthritic scores, joint histological inflammatory damage and matrix metalloproteinase-3 levels and high levels of Th1 pro-inflammatory cytokine mRNA and serum proteins in CAIA mice or following H4R activation via 4-MeH. Additionally, 4-MeH efficiently increased expression levels of NF-κB p65. JNJ-treated mice showed a substantial reduction in all the previously mentioned effects, with a similar trend being observed under prophylactic and therapeutic treatment regimens. The results of the present work indicate that JNJ exhibits significant anti-inflammatory and anti-arthritic activities, demonstrating the clear involvement of H4R antagonism in the pathogenesis and progression of RA.

Topics: Animals; Arthritis, Experimental; Arthritis, Rheumatoid; Cells, Cultured; Cytokines; Disease Progression; Female; Gene Expression Regulation; Humans; Immunophenotyping; Indoles; Injections, Intraperitoneal; Lymphocyte Activation; Matrix Metalloproteinase 3; Methylhistamines; Mice; Mice, Inbred BALB C; NF-kappa B; Piperazines; Receptors, G-Protein-Coupled; Receptors, Histamine; Receptors, Histamine H4; Th1 Cells

Previous investigations found that epithelial-to-mesenchymal transition (EMT) was an important character of non-small cell lung cancer (NSCLC) and it was also suggested that histamine H4 receptors may have a role in preventing EMT progress in certain kind of tumours. However, the effect of H4 receptor activation on EMT progress of NSCLC and its potential mechanisms remain unclear. Therefore, we performed both in vitro and in vivo experiments to explore the effects of specific H4 receptor agonist 4-methylhistamine and antagonist JNJ7777120 on EMT progress. We showed the expression of H4 receptors in NSCLC and found that 4-methylhistamine increased the expression of the epithelial marker E-cadherin and decreased the expression of Vimentin, the mesenchymal marker, in both NSCLC cell lines and xenograft NSCLC tumours. Pretreatment with JNJ7777120 or H4 receptor gene silencing decreased while overexpression of H4 receptors facilitated this effect of 4-methylhistamine. Furthermore, we showed that down-regulation of cyclic adenosine monophosphate (cAMP) was the secondary signalling after H4 receptor activation, which in turn resulted in inactivation of transforming growth factor-β1 (TGF-β1) pathway and down-regulation of several important EMT inducing factors such as ZEB1, Snail and Slug. In conclusion, these findings revealed the anti-EMT effect of histamine H4 receptor activation in NSCLC, which provide novel insight into the development mechanism of NSCLC; and H4 receptors may be a new therapeutic target for NSCLC treatment.

Topics: Animals; Cadherins; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cyclic AMP; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Immunohistochemistry; Indoles; Lung Neoplasms; Methylhistamines; Mice; Mice, Nude; Piperazines; Receptors, G-Protein-Coupled; Receptors, Histamine; Receptors, Histamine H4; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Signal Transduction; Snail Family Transcription Factors; Survival Analysis; Transcription Factors; Transforming Growth Factor beta1; Vimentin; Xenograft Model Antitumor Assays; Zinc Finger E-box-Binding Homeobox 1

The histamine 4 receptor (H4R) is expressed primarily on cells involved in inflammation and immune responses. Despite much research into inflammatory diseases, no drugs with favourable safety profiles are yet available for their treatment. The aim of the present study was to determine the potential anti-inflammatory effect of 4-methylhistamine (4-MeH) or JNJ77777120 (JNJ) and to explore the role of H4R in a mouse model of carrageenan (Cg) -induced pleurisy. A single dose of 4-MeH or JNJ (30 mg/kg) was administered intraperitoneally 1 hr before Cg administration. The results illustrate that both the numbers of CD4(+) , CD25(+) , CD4(+)  CD25(+) , GITR(+) , GITR(+)  IL-17A(+) -expressing T cells and the levels of T helper type 1 (Th1)/Th17 cytokines were markedly increased in both the Cg-treated and 4-MeH-treated groups, whereas the cytokines produced by Th2 cells were significantly decreased in the same groups. However, JNJ treatment significantly decreased both the number of T-cell subsets and GITR(+) , GITR(+)  IL-17A(+) -expressing T cells, and the production of Th1/Th17 cytokines. Further, JNJ up-regulated the expression of the Th2 cytokines. RT-PCR analysis revealed an increased expression of interleukin-1β, tumour necrosis factor-α, monocyte chemoattractant protein-1 and intercellular adhesion molecule-1 in the Cg-treated and 4-MeH-treated groups, which was reduced by treatment with JNJ in lung tissues. Moreover, histological examinations revealed anti-inflammatory effects of JNJ, whereas 4-MeH worsened Cg-induced inflammation. In conclusion, the results of the present work clearly indicate that JNJ possesses important anti-inflammatory properties that are increased in 4-MeH-treated mice, suggesting that H4R are involved in pleurisy and that JNJ has an anti-inflammatory effect in associated disease conditions.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Carrageenan; Cytokines; Female; Indoles; Inflammation; Methylhistamines; Mice; Mice, Inbred BALB C; Molecular Targeted Therapy; Piperazines; Pleurisy; Receptors, Histamine; Structure-Activity Relationship

The purpose of this study was to determine the effect of histamine and its receptors on goblet cell secretion.. Cultured rat and human goblet cells were grown in RPMI 1640. Goblet cell secretion of high molecular weight glycoconjugate was measured by an enzyme-linked lectin assay. Cultured rat goblet cells were homogenized and either RNA was isolated for RT-PCR or proteins were isolated for Western blot analysis for presence of histamine receptors subtypes H₁ through H₄. The localization of these receptors was determined in rat and human goblet cells by immunofluorescence microscopy.. Histamine stimulated goblet cell secretion in a concentration- and time-dependent manner. All four histamine receptors were present in cultured rat and human goblet cells. Use of agonists specific to individual histamine receptor subtypes indicated that the rank order of agonist stimulation was H₁ = H₃ > H₄ > H₂. Using antagonists specific to individual histamine receptor subtypes determined that H₂ and H₃, but not the H₁ and H₄, antagonists, inhibited histamine-stimulated conjunctival goblet cell secretion.. Rat and human conjunctival goblet cells are a direct target of histamine, which induces secretion. All four histamine receptors are present in rat and human conjunctiva and are active in rat conjunctival goblet cells. These findings suggest that all four histamine receptor subtypes are important for conjunctival goblet cell secretion. Blockage of histamine receptor subtypes could prevent the excess mucus production associated with ocular allergy.

Topics: Animals; Blotting, Western; Cells, Cultured; Cimetidine; Conjunctiva; Conjunctivitis, Allergic; Dose-Response Relationship, Drug; Fluorescent Antibody Technique; Goblet Cells; Histamine; Histamine Agonists; Histamine Antagonists; Humans; Indoles; Male; Methylhistamines; Piperazines; Rats; Rats, Sprague-Dawley; Receptors, Histamine; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thiazoles

6-Sulpho LacNAc dendritic cells (slanDC) are a major population of human blood DC that are highly pro-inflammatory, as characterized by their outstanding capacity to produce tumour necrosis factor-α and interleukin-12 (IL-12) and to prime antigen-specific T-cell responses. SlanDC were found to be present in inflamed tissue such as atopic dermatitis, where high levels of histamine are also present. As histamine is an important regulator of allergic inflammation we investigated the role of histamine receptors, particularly the most recently identified histamine H(4) receptor (H(4) R), in modulating the pro-inflammatory function of slanDC. The expression of H(4) R was evaluated by real-time PCR and flow cytometry. Cytokine production in response to H(4) R stimulation was assessed by intracellular flow cytometric staining and enzyme-linked immunosorbent assay. We show that slanDC express the H(1) R, H(2) R and H(4) R on mRNA and the H(4) R on protein level. No differences were observed in basal H(4) R expression in patients with atopic dermatitis and psoriasis, but in atopic dermatitis patients the H(4) R was up-regulated by interferon-γ. When stimulated with lipopolysaccharide in the presence of histamine, slanDC produced substantially lower levels of the pro-inflammatory cytokines tumour necrosis factor-α and IL-12, mediated solely via the H(4) R and via the combined action of H(2) R and H(4) R, respectively. In contrast, the production of IL-10 was not affected by histamine receptor activation on slanDC. The slanDC express the H(4) R and its stimulation leads to reduced pro-inflammatory capacity of slanDC. Hence, H(4) R agonists might have therapeutic potential to down-regulate immune reactions, e.g. in allergic inflammatory skin diseases.

Topics: Amino Sugars; Cytokines; Dendritic Cells; Dermatitis, Atopic; Flow Cytometry; Histamine; Humans; Indoles; Inflammation; Interferon-gamma; Lipopolysaccharides; Methylhistamines; Piperazines; Psoriasis; Receptors, G-Protein-Coupled; Receptors, Histamine; Receptors, Histamine H4; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

The expression of the recently cloned histamine H(4) receptor (H(4)R) by leukocytes suggests a role in immunomodulation.. The expression and function of the H(4)R on human monocytes obtained from peripheral blood was investigated.. H(4)R expression was studied by using flow cytometry. Effects of H(4)R stimulation on Ca(2+) mobilization was determined fluorometrically, CCL2 production was determined by means of ELISA, intracellular CCL2 staining was measured with flow cytometry, and CCL2 mRNA was measured by using real-time quantitative LightCycler PCR. The relevance of CCL2 production was determined in chemotaxis transmigration assays.. H(4)R protein was expressed by monocytes and upregulated by IFN-gamma. H(4)R agonists (clobenpropit and 4-methylhistamine) induce a Ca(2+) mobilization in monocytes, which could be blocked with the selective H(4)R antagonist JNJ7,777,120. Furthermore, H(4)R agonists downregulated CCL2 protein production. This effect could also be blocked by JNJ7,777,120. Supernatants of H(4)R agonist-stimulated monocytes attracted less monocytes in transmigration assays. The downregulation of CCL2 production was regulated at different levels. First, the synthesis of CCL2 mRNA was significantly decreased. Second, intracellular staining suggested an inhibition of CCL2 secretion after stimulation with H(4)R agonists.. Human monocytes express the H(4)R, and its stimulation leads to a Ca(2+) influx and an inhibition of CCL2 production, resulting in a reduction of monocyte recruitment.. The H(4)R could represent an important anti-inflammatory receptor on monocytes and could be an interesting target for drug development.

Topics: Calcium; Cells, Cultured; Chemokine CCL2; Chemotaxis, Leukocyte; Down-Regulation; Histamine; Histamine Agonists; Humans; Imidazoles; Indoles; Interferon-gamma; Matrix Metalloproteinase 1; Matrix Metalloproteinase 3; Matrix Metalloproteinase 8; Methylhistamines; Monocytes; Piperazines; Receptors, G-Protein-Coupled; Receptors, Histamine; Receptors, Histamine H4; Thiourea; Up-Regulation

The histamine H(4) receptor (H(4)R) is involved in the chemotaxis of leukocytes and mast cells to sites of inflammation and is suggested to be a potential drug target for asthma and allergy. So far, selective H(4)R agonists have not been identified. In the present study, we therefore evaluated the human H(4)R (hH(4)R) for its interaction with various known histaminergic ligands. Almost all of the tested H(1)R and H(2)R antagonists, including several important therapeutics, displaced less than 30% of specific [(3)H]histamine binding to the hH(4)R at concentrations up to 10 microM. Most of the tested H(2)R agonists and imidazole-based H(3)R ligands show micromolar-to-nanomolar range hH(4)R affinity, and these ligands exert different intrinsic hH(4)R activities, ranging from full agonists to inverse agonists. Interestingly, we identified 4-methylhistamine as a high-affinity H(4)R ligand (K(i) = 50 nM) that has a >100-fold selectivity for the hH(4)R over the other histamine receptor subtypes. Moreover, 4-methylhistamine potently activated the hH(4)R (pEC(50) = 7.4 +/- 0.1; alpha = 1), and this response was competitively antagonized by the selective H(4)R antagonist JNJ 7777120 [1-[(5-chloro-1H-indol-2-yl)-carbonyl]-4-methylpiperazine] (pA(2) = 7.8). The identification of 4-methylhistamine as a potent H(4)R agonist is of major importance for future studies to unravel the physiological roles of the H(4)R.

Topics: Cell Line; Histamine Agonists; Humans; Imidazoles; Indoles; Isothiuronium; Ligands; Methylhistamines; Piperazines; Radioligand Assay; Receptors, G-Protein-Coupled; Receptors, Histamine; Receptors, Histamine H1; Receptors, Histamine H2; Receptors, Histamine H3; Receptors, Histamine H4