Development of an inhibiting antibody against equine interleukin 5 to treat insect bite hypersensitivity of horses.

Insect bite hypersensitivity (IBH) is the most common allergic skin disease of horses. It is caused by insect bites of the Culicoides spp. which mediate a type I/IVb allergy with strong involvement of eosinophil cells. No specific treatment option is available so far. One concept could be the use of a therapeutic antibody targeting equine interleukin 5, the main activator and regulator of eosinophils. Therefore, antibodies were selected by phage display using the naïve human antibody gene libraries HAL9/10, tested in a cellular in vitro inhibition assay and subjected to an in vitro affinity maturation. In total, 28 antibodies were selected by phage display out of which eleven have been found to be inhibiting in the final format as chimeric immunoglobulin G with equine constant domains. The two most promising candidates were further improved by in vitro affinity maturation up to factor 2.5 regarding their binding activity and up to factor 2.0 regarding their inhibition effect. The final antibody named NOL226-2-D10 showed a strong inhibition of the interleukin 5 binding to its receptor (IC50 = 4 nM). Furthermore, a nanomolar binding activity (EC50 = 8.8 nM), stable behavior and satisfactory producibility were demonstrated. This antibody is an excellent candidate for in vivo studies for the treatment of equine IBH.

TRPV1 and TRPA1 Channels Are Both Involved Downstream of Histamine-Induced Itch.

Two histamine receptor subtypes (HR), namely H1R and H4R, are involved in the transmission of histamine-induced itch as key components. Although exact downstream signaling mechanisms are still elusive, transient receptor potential (TRP) ion channels play important roles in the sensation of histaminergic and non-histaminergic itch. The aim of this study was to investigate the involvement of TRPV1 and TRPA1 channels in the transmission of histaminergic itch. The potential of TRPV1 and TRPA1 inhibitors to modulate H1R- and H4R-induced signal transmission was tested in a scratching assay in mice in vivo as well as via Ca2+ imaging of murine sensory dorsal root ganglia (DRG) neurons in vitro. TRPV1 inhibition led to a reduction of H1R- and H4R- induced itch, whereas TRPA1 inhibition reduced H4R- but not H1R-induced itch. TRPV1 and TRPA1 inhibition resulted in a reduced Ca2+ influx into sensory neurons in vitro. In conclusion, these results indicate that both channels, TRPV1 and TRPA1, are involved in the transmission of histamine-induced pruritus.

Histamine 2 Receptor Agonism and Histamine 4 Receptor Antagonism Ameliorate Inflammation in a Model of Psoriasis.

Psoriasis is a chronic inflammatory skin disorder characterized by hyperproliferative keratinocytes and immune cell infiltration into the skin, often accompanied by itch. Histamine, acting via histamine 1-4 receptors, is known to modulate immune responses in the skin and to induce itch. The aim of this study was to test the role of histamine 2 receptors and histamine 4 receptors in the imiquimod-induced psoriasis-like skin inflammation model. BALB/c mice were treated intraperitoneally with amthamine (histamine 2 receptor agonist), JNJ-39758979 (histamine 4 receptor antagonist), a combination of both, or vehicle twice daily in a preventive manner. Imiquimod was applied once daily onto the back skin for 10 consecutive days. Stimulation of histamine 2 receptors and blockade of histamine 4 receptors ameliorated imiquimod-induced skin inflammation. The combination of amthamine and JNJ-39758979 reduced skin inflammation even more pronounced, diminished epidermal hyperproliferation, and inhibited spontaneous scratching behaviour. A combination of histamine 2 receptor agonist and histamine 4 receptor antagonists could represent a new strategy for the treatment of psoriasis.

The role of the histamine H4 receptor in atopic dermatitis and psoriasis.

Atopic dermatitis (AD) and psoriasis are common skin diseases with a high negative impact on patients' quality of life. Both diseases are mediated by a pro-inflammatory infiltrate consisting of several cell types, such as T-cells, antigen-presenting cells and granulocytes and display disturbed keratinocyte differentiation. Given the fact that histamine levels are also highly elevated in inflamed skin, it is likely that histamine plays a relevant role in disease pathology. However, antagonists blocking histamine H1 receptor or H2 receptors are largely ineffective in reducing chronic symptoms in AD and psoriasis. Over the last years, much research has been undertaken to shed light into the mode of action of the most recently discovered histamine H4 receptor. This research has shown that H4 receptor antagonists display antipruritic and anti-inflammatory effects not only in mouse models but also in first human clinical trials, and therefore, H4 receptors might present a novel therapeutic target. In this review, we summarize the effects of the H4 receptors on different cell types, mouse models and clinical studies in regard to AD and psoriasis respectively. LINKED ARTICLES: This article is part of a themed section on New Uses for 21st Century. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.3/issuetoc.

Combined treatment with H1 and H4 receptor antagonists reduces inflammation in a mouse model of atopic dermatitis.

BACKGROUND: Histamine 4 receptor (H4R) antagonists are considered as new therapeutics for the treatment of atopic dermatitis (AD) and first clinical trials have already shown promising results. Histamine 1 receptor (H1R) antagonists are traditionally used to treat AD although the evidence for the efficacy is weak. The combined blockade of both, H1R and H4R, might provide synergistic anti-inflammatory. OBJECTIVE: The study was performed to test the anti-inflammatory potential of a combined treatment with an H1R and an H4R antagonist in a mouse AD model. METHODS: The development of ovalbumin-induced AD-like skin lesions was analysed mice treated with the H1R inverse agonist mepyramine, the H4R antagonist JNJ-39758979 or a combination of both. RESULTS: Mice treated with mepyramine plus JNJ-39758979 showed less severe skin lesions, with a diminished influx of inflammatory cells, a reduced epidermal thickening and a lower level of IL-33 in lesional skin. Scratching behaviour was ameliorated in mice treated with the combination. Moreover, total numbers of skin-draining lymph node cells and splenocytes were significantly reduced. Both substances given alone did not elicit this strong anti-inflammatory effect. CONCLUSION: H1R and H4R antagonists provide synergistic anti-inflammatory effects in a mouse model of AD. The combined therapy with H1R and H4R antagonists might represent a new strategy for the treatment of AD.

The selective glucocorticoid receptor agonist mapracorat displays a favourable safety-efficacy ratio for the topical treatment of inflammatory skin diseases in dogs.

BACKGROUND: Mapracorat is a nonsteroidal Selective Glucocorticoid Receptor Agonist (SEGRA) that is presumed to have a better therapeutic index compared to classical glucocorticoids. OBJECTIVES: To compare the efficacy and safety of mapracorat with classical glucocorticoids used for the treatment of allergic skin diseases in dogs. ANIMALS: Six laboratory beagles. METHODS: The effect of mapracorat on lipopolysaccharide-induced TNFα secretion from canine peripheral blood derived mononuclear cells (PBMC) was tested. In vivo, mapracorat was compared to triamcinolone acetonide using a skin inflammation model. Skin fold thickness was determined after daily administration of mapracorat and triamcinolone acetonide over 14 days. RESULTS: Mapracorat concentration dependently inhibited TNFα secretion from activated canine PBMC with a half maximal inhibitory concentration (IC50 ) value of approximately 0.2 nmol/L. Intradermal injection of compound 48/80 (50 µg in 50 µL saline) resulted in a clear wheal and flare reaction over the 60 min observation period. Topical pre-treatment with mapracorat (0.1%) and triamcinolone acetonide (0.015%) led to significant reduction in the wheal and flare responses compared to vehicle (acetone) treated areas. However, once daily topical administration of triamcinolone acetonide significantly reduced skin fold thickness from day 8 to 14, whereas no such reduction was observed for mapracorat. CONCLUSION: These results demonstrate that mapracorat has comparable anti-inflammatory efficacy to classical steroidal glucocorticoids under these experimental settings and maintenance of skin fold thickness indicates a better safety profile compared to triamcinolone acetonide at equipotent concentrations. This profile further suggests that SEGRAs show promise in the management of inflammatory and pruritic skin diseases in dogs.

Stimulation of the histamine 4 receptor upregulates thymic stromal lymphopoietin (TSLP) in human and murine keratinocytes.

The cytokine thymic stromal lymphopoietin (TSLP) is involved in the development and the progression of allergic diseases. It is mainly released by epithelial cells at barriers such as skin and gut in response to danger signals. Overexpression of TSLP in keratinocytes (KC) can provoke the development of a type 2 inflammatory response. Additionally, TSLP directly acts on sensory neurons and thereby triggers itch. Since histamine is also increased in lesions of inflammatory skin diseases, the aim of this study was to investigate possible effects of histamine as well as different histamine receptor subtype agonists and antagonists on TSLP production in KC. We therefore stimulated human KC with histamine in the presence or absence of the known TSLP-inductor poly I:C and measured TSLP production at protein as well as mRNA level. Histamine alone did not induce TSLP production in human KC, but pre-incubation with histamine prior to challenge with poly I:C resulted in a significant increase of TSLP production compared to stimulation with poly I:C alone. Experiments with different histamine receptor agonists (H1R: 2-pyridylethylamine; H2R: amthamine; H2R/H4R: 4-methylhistamine (4MH)) revealed a dominant role for the H4R receptor, as 4-MH in combination with poly I:C displayed a significant increase of TSLP secretion, while the other agonists did not show any effect. The increase in TSLP production by 4MH was blocked with the H4R antagonist JNJ7777120. This effect was reproducible also in the murine KC cell line MSC. Taken together, our study indicates a new role for the H4 receptor in the regulation of TSLP in keratinocytes. Therefore, blocking of the H4R receptor in allergic diseases might be promising to alleviate inflammation and pruritus via TSLP.

Allergic inflammation is augmented via histamine H4 receptor activation: The role of natural killer cells in vitro and in vivo.

BACKGROUND: Natural Killer cells (NK cells) are identified as pivotal mediators in allergic skin diseases and accumulate in lesions of atopic dermatitis (AD) patients. Histamine levels are increased in these lesions and histamine is involved in chemotaxis in dendritic cells and NK cells. OBJECTIVE: The aim of this study was to determine if the histamine H4 receptor (H4R) mediates NK cell chemotaxis and whether it influences interplay between NK cells and dendritic cells during the early phase of allergic inflammation. METHODS: Chemotactic function of the H4R as well as the influence of the H4R on the cytokine profile of an NK cell-dendritic cell co-culture was studied in vitro. The effect of H4R activation on NK cell migration, NK cell-dendritic cell interaction and cytokine levels in the skin was further characterized in the murine TDI model of allergic dermatitis. Additionally, the impact of the H4R on dermal NK cells was determined in the ovalbumin (OVA)- induced allergic dermatitis model, comparing wild type and H4R knockout mice. RESULTS: The selective H4R agonist ST-1006 induced NK cell chemotaxis in vitro, which was inhibited with the H4R antagonist JNJ7777120. In vivo, mice treated with TDI plus ST-1006 topically onto the ear, showed significantly enhanced ear swelling and an increased number of NK cells compared to just allergen challenged ears. CCL17 levels in the ear were also significantly increased 8h after allergen challenge. Histology revealed that the main source for increased CCL17 were dendritic cells. These effects could be blocked using the H4R antagonist JNJ7777120. In the chronic model of allergic dermatitis, OVA induced NK cell migration into lesional skin sites. The number of NK cells was lower in OVA-sensitized H4R knockout mice compared to wild type mice. CONCLUSIONS: These results identify the H4R as a new target controlling NK cell migration and NK cell-dendritic cell interaction in the skin during early allergic inflammation. These results further suggest that blocking the H4R in the skin might be beneficial in diseases like AD.

PCR detects bands consistent with the expression of receptors associated with pruritus in canine dorsal root ganglia.

BACKGROUND: Various mediators are involved in the induction of itch, i.e. pruritus; however, the in vivo pharmacology of pruritus seems to be different in distinct species, and little is known about receptors that are involved in the induction of itch in dogs. The species differences in the mediation of pruritus might be explained by species differences in receptor expression in the sensory nerves, including the dorsal root ganglia (DRG). HYPOTHESIS/OBJECTIVES: The aim of the study was to analyse the expression of receptors for various mediators of pruritus in canine DRG. METHODS: Dorsal root ganglia of 14 dogs, which were euthanized for reasons not related to this study, were analysed. Multiple DRG per dog were dissected and, after homogenization of the DRG tissues, total RNA was isolated, reverse transcribed to complementary DNA and amplified with custom-synthesized primers. RESULTS: The following receptors were found in canine DRG: transient receptor potential cation channel subfamily V member 1, tachykinin receptor 1, Toll-like receptor 7, endothelin receptor type A, opioid receptors µ1 and κ1, histamine H1 -H4 receptors and the interleukin-31 receptor complex. CONCLUSIONS AND CLINICAL IMPORTANCE: PCR analysis detected bands consistent with the expression of receptors associated with pruritus in canine DRG.

Histamine induces proliferation in keratinocytes from patients with atopic dermatitis through the histamine 4 receptor.

BACKGROUND: Epidermal hyperproliferation resulting in acanthosis is an important clinical observation in patients with atopic dermatitis, and its underlying mechanisms are not completely understood. OBJECTIVE: Because increased levels of histamine are present in lesional skin, we investigated the effect of histamine, especially with regard to histamine 4 receptor (H4R) activation, on the proliferation of human and murine keratinocytes. METHODS: The expression of H4R on human and murine keratinocytes was detected by using real-time PCR. Keratinocyte proliferation was evaluated by using different in vitro cell proliferation assays, scratch assays, and measurement of the epidermal thickness of murine skin. RESULTS: We detected H4R mRNA on foreskin keratinocytes and on outer root sheath keratinocytes; H4R mRNA was more abundant in keratinocytes from patients with atopic dermatitis compared with those from nonatopic donors. Stimulation of foreskin keratinocytes, atopic dermatitis outer root sheath keratinocytes, and H4R-transfected HaCaT cells with histamine and H4R agonist resulted in an increase in proliferation, which was blocked with the H4R-specific antagonist JNJ7777120. Abdominal epidermis of H4R-deficient mice was significantly thinner, and the in vitro proliferation of keratinocytes derived from H4R-deficient mice was lower compared with that seen in control mice. Interestingly, we only detected H4R expression on murine keratinocytes after stimulation with LPS and peptidoglycan. CONCLUSION: H4R is highly expressed on keratinocytes from patients with atopic dermatitis, and its stimulation induces keratinocyte proliferation. This might represent a mechanism that contributes to the epidermal hyperplasia observed in patients with atopic dermatitis.

Pathogenetic and therapeutic implications of the histamine H4 receptor in inflammatory skin diseases and pruritus.

Chronic inflammatory skin diseases such as atopic dermatitis (AD) are clinically characterized by erythematous and pruritic skin lesions, immunologically mediated by an inflammatory infiltrate consisting of T-cells, antigen presenting cells (APC) and eosinophilic granulocytes. Histamine levels are increased in lesions of inflammatory skin diseases. It is likely that histamine also plays a pathogenetic role since various relevant cell types such as T-cells and APC express functional histamine receptors. However, therapeutic blockade of the histamine H1 and H2 receptor is inefficient at least in the treatment of atopic dermatitis. We summarize here current data on the role of the recently described histamine H4 receptor (H4R) in chronic inflammatory skin diseases. The H4R is functionally expressed on relevant cell types such as T-cells, APC and keratinocytes. In murine models of contact hypersensitivity and pruritus, H4R blockade had significant in vivo effects. Taken together, several lines of evidence suggest a role of the H4R in chronic inflammatory skin disease and the H4R might be a therapeutic target for diseases such as AD.

[Histamine as an immunomodulator]. / Histamin als Immunmodulator.

The role of histamine as an important proinflammatory mediator has been well known for nearly 100 years. In regards to the immediate type allergic response, there is no debate about the importance of histamine. In addition, histamine has immunomodulatory functions, some of which are related to the histamine H2 receptor. However, with the discovery of the histamine H(4) receptor in the year 2000, the role of histamine as an immunomodulator became more obvious. The histamine H(4) receptor is expressed on several hematopoietic cells; along with the chemotaxis of immune cells, this recently characterized receptor modulates also the cytokine and chemokine secretion of some hematopoietic cells. Highly selective histamine H(4) receptor antagonists display promising results in animal models of allergic inflammatory diseases and also in in vitro studies on animal and human cells. These first results indicate that the histamine H(4) receptor is an interesting new pharmacological target for the treatment of allergic inflammatory disorders. This review summarizes the most important immunomodulatory functions of histamine and points to some possible indications in dermatology.

The histamine H receptor as a new target for treatment of canine inflammatory skin diseases.

Histamine is a well known mediator of allergic skin diseases and, with the discovery of the histamine H(4) receptor, the role of histamine is re-evaluated. There are only limited published data elucidating the role of the histamine H(4) receptor in dogs. Twelve beagles intradermally injected with histamine (0.25 micromol and 2.5 micromol/site) reacted with a classical wheal and flare reaction. None of the dogs showed signs of pruritus. The dogs reacted with a wheal and flare reaction after intradermal injection of histamine H(4) receptor agonist/H(3) receptor antagonist clobenpropit (0.1 micromol) and selective histamine H(4) receptor agonist VUF 8430 (1.5 micromol). Again, no scratching occurred in any of the dogs. The highly selective histamine H(4) receptor antagonist JNJ 7777120 reduced the histamine-induced wheal reaction in nine out of 12 dogs. To determine whether canine mast cells are susceptible to histamine H(4) receptor-mediated reactions, effects of clobenpropit and VUF 8430 were tested in canine mastocytoma cells (C2). Incubation with histamine H(4) receptor agonists (up to 10 micromol/L) induced a distinct calcium(2+) influx. C2 cells also responded with enhanced chemotaxis when stimulated with histamine, VUF 8430 and clobenpropit. Neither VUF 8430, nor clobenpropit (up to 10 micromol/L) led to a modulation of histamine concentration in supernatants of canine mastocytoma cells, whereas mastoparan, used as a positive control, enhanced histamine concentration in supernatants. For treatment of allergic skin diseases in dogs, a combination of H(1)R and H(4)R antagonists might be advantageous.

Histamine H4 receptor antagonism reduces hapten-induced scratching behaviour but not inflammation.

Effects of the histamine H(4) receptor antagonist JNJ 7777120 (1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methylpiperazine) were tested in two models of allergic contact dermatitis. Dermatitis was induced by 2,4-dinitrochlorobenzene and toluene-2,4-diisocyanate, which differ in their Th1-Th2 profile in that way that 2,4-dinitrochlorobenzene is a classical contact allergen with a pronounced Th1-mediated inflammation, while the respiratory chemical allergen toluene-2,4-diisocyanate induces a Th2-dominated inflammation. JNJ 7777120 (15 mg/kg) administered 2 h and 30 min before and 1 h after challenge did not reduce the hapten-induced ear swelling determined 24 h after challenge. This was confirmed by histological evaluation of the ear skin. A repeated administration of the haptens to the rostral part of the back of sensitized animals resulted in a frequent scratching behaviour. An administration of JNJ 7777120 (15 mg/kg) 30 min before challenge reduced this hapten-induced scratching significantly. The H(1) receptor antagonist cetirizine also reduced the scratching bouts in sensitized mice. A combination of H(1) and H(4) receptor antagonists resulted in the strongest inhibition of scratching behaviour associated with allergic dermatitis. These results indicate that H(4) receptor antagonism fails to reduce the allergic inflammatory response but strongly inhibits allergen-induced itch. Thus, a combination of H(4) and H(1) receptor antagonism might be a new strategy to treat pruritus related to allergic diseases like atopic dermatitis.