Monocyte differentiation into dendritic cells or macrophages and recruitment to peripheral organs in chronic inflammatory diseases are directed by allergen challenge via FcεRI as well as the nature of soluble factors in the microenvironment. High-affinity receptor for IgE stimulation of effector cells results in the release of histamine, which acts on various histamine receptors (HR) 1-4, expressed by immune cells.
We examined the effect of FcεRI stimulation of human monocytes on H1R expression and function of differentiating cells. The mRNA levels of H1R, H2R and histidine decarboxylase of differentiating cells were detected by quantitative real-time PCR. Expression of CD1c, CD11c, CD68 and CD163 was detected by flow cytometry. Amount of histamine, IL-6 and IL-12p70 in the cell culture was measured with the help of cytometric bead arrays or ELISA assays. Numbers of H1R-expressing macrophages were evaluated by immunofluorescence double staining of CD68 and H1R on human skin sections.
We demonstrated that FcεRI stimulation promotes the generation of H1R-expressing macrophage-like cells with enhanced histamine biosynthesis and H1R-mediated proinflammatory properties. Supporting our in vitro findings, high numbers of H1R-expressing CD68pos macrophages were detected in the dermis of atopic dermatitis (AD) skin lesions.
Our observations point to a close histamine-/HR-mediated activation of dermal macrophages, leading to modified cell differentiation and responsiveness via H1R, which might contribute to the aggravation of allergic skin inflammation in AD.
The effects of allergen exposure in allergic inflammatory diseases such as atopic dermatitis (AD) are mediated by high-affinity receptor for IgE (FcεRI)-bearing mast cells on one hand and FcεRIpos antigen-presenting cells (APCs) on the other hand . Stimulation of FcεRI on mast cells leads to the release of soluble factors such as histamine. Histamine, one of the most important mediators of allergic inflammatory reactions, is a dibasic multifunctional amine produced by the help of histidine decarboxylase (HDC) . Histamine is expressed in lung, skin and gastrointestinal tract and is responsible for vasodilatation, smooth muscle contraction or oedema formation. Further on, histamine is not only involved in allergic immune responses of the immediate type, but plays a key role in allergic inflammatory reactions [3-7]. In the skin, histamine released by mast cells acts preferentially on dermal dendritic cells (DCs) and macrophages (MФ) via histamine receptors (HRs) expressed by these cells. So far, four different human HR types have been identified (H1R-4) and the expression pattern of HRs is decisive for the nature of the outcoming immune response [6, 8]. H1R owns much proinflammatory and cell-activating properties, while H2R has been shown to be involved in some tolerogenic immune responses evolving from H2R-mediated suppressive effects . During the differentiation of monocytes into MФ (MoMФ), H1R is upregulated and H2R is downregulated; in contrast, H1R is downregulated and H2R is upregulated when cells develop into monocyte-derived DCs (MoDCs) [9, 10]. H1R activates specific G-proteins, and G-protein-coupled signalling involves phospholipase C activation, inositol phosphate production and intracellular Ca2+ flux  as well as the activation of NF-κB , STAT1 , STAT4  and mitogen-activated protein kinase (MAPK)  pathways. Furthermore, H1R stimulation increases the release of interleukin (IL)-6 and β-glucuronidase of lung MФs [15, 16]. Despite mast cells and basophils, some leucocyte subpopulations such as DCs and MФs are capable of producing and releasing histamine as well [17, 18].
Increased plasma and skin levels of histamine have been observed in patients with AD , and mast cells present in the dermal compartment have been suggested as major source . Allergen uptake, processing and presentation to T cells is a central mechanism in AD, which is supposed to be accomplished mainly by APCs such as monocytes or DCs in the skin and in the blood, which are equipped with respective receptors including FcεRI . Notably, a close interaction of antigen receptor–mediated immune responses and signalling with G-coupled H1R was observed in murine models, implying that comparable coregulatory functions of FcεRI and H1R might exist . In this study, we examined the effect of FcεRI stimulation of human monocytes on the H1R expression as well as responsiveness of H1R of differentiating cells. We demonstrate that FcεRI stimulation promotes the generation of H1R-expressing MФs with high proinflammatory properties and increased histamine biosynthesis. In correlation with our in vitro findings, H1R-expressing CD68pos MФs were detectable in high amounts in the dermal compartment of AD skin lesions. Our observations point to a close histamine-/H1R-mediated activation of MФs, which might follow allergen challenge in allergic inflammatory diseases such as AD.
Rabbit anti-human IgE and unconjugated mAb against CD68 (mIgG3, clone: PG-M1) were purchased from Dako (Glostrup, Denmark). Monomeric human myeloma IgE (hIgE) was from Calbiochem-Novabiochem Corp. (San Diego, CA, USA). FITC-conjugated goat-anti-mouse IgG (GaM/FITC) was from Jackson Immuno-Research Laboratories (West Grove, PA, USA). Rabbit monoclonal anti-CD1a (IgG1, clone: EP3622) antibody was from Abcam (Cambridge, MA, USA). Monoclonal antibodies (mAbs) against CD68 (mIgG2b, clone: Y1/82A), CD11c (mIgG1κ, clone: B-ly6), and allophycocyanin (APC)-labelled mAb against CD14 (mIgG2a, clone: M5E2) were from BD Biosciences Pharmingen (Heidelberg, Germany); unconjugated mAb against CD163 (mIgG1, clone: 5C6-FAT) was from Acris (Herford, Germany); unconjugated mAb against CD54 (mIgG1, clone: 84H10) was from Immunotech (Marseille Cedex, France); polyclonal rabbit anti-H1R antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA); unconjugated mAb against CD1c (mIgG1, clone: L161) and normal mouse serum for blocking purposes were obtained from Dianova (Hamburg, Germany); and PE-labelled T6RD1 (mIgG1) mAb from Beckman Coulter (Krefeld, Germany) was directed against CD1a. All other reagents were obtained from Sigma Aldrich (Taufkirchen, Germany) unless otherwise indicated.
Preparations and cultures
After the written informed consent and local ethical committee approval were obtained, the blood for monocyte isolation was taken from allergic volunteers at the Department of Dermatology and Allergy of the University of Bonn. Monocytes were isolated from peripheral blood as described before  and cultured for 6 days in very low endotoxin medium RPMI 1640 (Biochrom, Berlin, Germany) with 1% antibiotic and antimycotics, 10% inactivated foetal calf serum (FCS) with 500 U (53.82 ng)/ml IL-4 (Gentaur GmbH, Aachen, Germany) and 500 U (89.28 ng)/ml GM-CSF (Berlex Laboratories, Inc., Richmond, CA, USA).
Stimulation of FcεRI was achieved as previously described . Briefly, cells were harvested and washed with culture medium twice, and then incubated with 1 μg/ml hIgE for 1 h at 37°C. After washing with culture medium, 10 μg/ml anti-human IgE was added to perform aggregation of FcεRI-bound IgE.
Cell surface and intracellular phenotyping of MoDCs/MoMФs by flow cytometry
After 6 days in culture, intercellular and extracellular staining was performed as reported in detail elsewhere . Briefly, flow cytometry was performed using a FACS-Canto (BD Biosciences) and analysed by FACSDiva (BD Biosciences) and FlowJo (TreeStar Inc., Ashland, OR, USA) software.
Measurement of histamine, IL-6 and IL-12p70 in the cell culture supernatant
At day 6 of culture, MoDC/MoMФ were left unstimulated or stimulated with betahistidine 10−4 M or histamine 10−4 M for 6 h to determine IL-6 production and with betahistidine 10−4 M or histamine 10−4 M together with interferon (IFN)-γ (200 U/ml; R&D systems, Wiesbaden, Germany) and poly I:C (Sigma Aldrich) 10 μg/ml for 24 h to determine IL-12p70 secretion. Supernatants were collected in separator tubes, centrifuged for 10 min at approximately 1000 g and stored at −20°C. IL-6 and IL-12p70 in the supernatant were detected by cytokine bead array from BD Biosciences following instruction manual. To determine histamine secretion of differentiating cells, at day 1 of culture cells were simulated for 30 min with LPS (10 ng/ml; Sigma Aldrich) and the cell culture supernatant was analysed with the help of ELISA assay (Neogen Corporation, Scotland, UK) as described in the instruction manual.
mRNA was isolated with the help of the RNeasy Mini kit (Quiagen, Hilden, Germany) including digestion of genomic DNA and was subjected to cDNA synthesis with TaqMan reverse transcription reagents with random hexamers according to the manufacturer's instructions (Applied Biosystems, Darmstadt, Germany). The prepared cDNA was amplified using TaqMan assay Master Mix (Applied Biosystems) according to the recommendations of the manufacturer in an ABI Prism 7300 Sequence Detection System (Applied Biosystems). Primers were as follows: H1R forward primer 5′-TCT CGA ACG GAC TCA GAT ACC A-3′, H1R reverse primer 5′ CCT GTG TTA GAC CCA CTC CTC AA-3′, H1R probe FAM-ACA GAG ACA GCA CCA GGC AAA GGC AA-TAMRA; H2R forward primer 5′-GCT GGG CTA TGC CAA CTC A-3′, H2R reverse primer 5′-GGT GCG GAA GTC TCT GTT CAG-3′, and H2R probe FAM-CCC TGA ACC CCA TCC TGT ATG CTG C-TAMRA (all from Microsynth AG, Balgach, Switzerland); HDC (Hs00157914_m1) as well as endogenous controls  were from Applied Biosystems. All assays were performed according to the manufacturer′s instructions. Relative quantification and calculation of the range of confidence were performed using the comparative ΔΔCT methods . All analyses were conducted in duplicates.
Immunofluorescence staining of skin sections
Immunofluorescence staining was performed on 4-μm skin sections of paraformaldehyde-fixed and paraffin-embedded skin biopsies as described elsewhere . Sections were treated with monoclonal antibody against CD68 (Dako) at 4°C overnight. Expression of CD1a or H1R was analysed using anti-CD1a (Abcam) or anti-H1R antibody (Santa Cruz Biotechnology). Appropriate isotype-matched IgG (Sigma Aldrich) were used as experimental antibody controls. Antibody binding was detected with anti-mouse and anti-rabbit Cy-2- and Cy-3-labelled secondary antibodies (Jackson Immuno-Research Laboratories). Sections were analysed with a fluorescence microscope (Leica Microsystems, Wetzlar, Germany) using Diskus and ImageJ software (National Institute of Health website, http://rsbweb.nih.gov/ij/) for documentation and analysis.
Statistical analysis was performed with SPSS 17.0 for Windows (SPSS, Chicago, IL, USA). Quantitative values were compared by nonparametric testing (Wilcoxon). Results are given in mean ± standard error of the mean (SEM), respectively. Any P-values are two-sided and subject to a global significance level of 5%.
FcεRI stimulation promotes the differentiation of H1R-expressing cells
Stimulation of FcεRI on human monocytes shifts the differentiation of MoDCs towards MФs including high capacity of phagocytosis and CD14 expression . H1R expression has been reported to be upregulated during differentiation of MoMФs, while expression of H2R is downregulated [9, 10]. We observed that stimulation of FcεRI on monocytes upregulates H1R mRNA expression already after 24 h (Fig. 1A). As most of the donors were atopic, constitutive IgE bound to FcεRI stimulated by addition of anti-IgE alone was sufficient to significantly upregulate the H1R mRNA level, while saturation of the remaining FcεRI binding sites with exogenously added IgE prior to FcεRI stimulation led to a further increase in H1R mRNA expression. H2R mRNA expression was slightly increased, resulting in an increased H1R/H2R mRNA ratio (Fig. 1B). Higher H1R mRNA expression of FcεRI-preactivated cells was sustained during in vitro culture, and elevated H1R mRNA expression of FcεRI-activated cells was also observed on day 6 of culture (Fig. 1C).
FcεRI stimulation induced H1R-expressing cells express surface marker of MФs
As a next step, we evaluated the expression pattern of surface markers of MФs and dermal DCs of cells differentiated in the presence of IL-4 and GM-CSF from FcεRI-stimulated monocytes as compared to cells derived from unstimulated control monocytes. With the help of double staining of CD14 and CD1a, we observed that unstimulated human monocytes mainly differentiated into CD1apos MoDCs, while stimulation of FcεRI promoted the differentiation of an additional fraction of CD14pos cells besides CD1apos MoDCs (Fig. 2A). Furthermore, we evaluated expression of CD1c, CD11c, CD54, CD68 and CD163 on CD1apos and CD14pos cells. CD14pos cells expressed less CD1c and CD11c and more CD68 and CD163 than CD1apos cells (Fig. 2A,B). From these data, we conclude that part of the cells differentiate into MФs after FcεRI stimulation.
FcεRI stimulation induced H1R-expressing cells display characteristics of MФs with high IL-6 and low IL-12p70 releasing properties
Furthermore, we analysed the cytokine profile of the differentiating cells. Cells generated from FcεRI-stimulated precursors secreted more proinflammatory cytokine IL-6 (Fig. 3A). Secretion of IL-6 further increased significantly after stimulation of H1R with the H1R-agonist betahistidine or stimulation of the cells with histamine. In contrast, cells generated after FcεRI stimulation released significantly lower amounts of IL-12p70, which is secreted by DCs and promotes immune responses of the Th1 type (Fig. 3B). From these data, we conclude that FcεRI stimulation leads to the differentiation of MФs with high secretion of the proinflammatory cytokine IL-6 and lower production of IL-12p70.
Increased biosynthesis of histamine indicated by higher HDC mRNA expression during differentiation of DCs as well as by murine MФs and increased histamine release after stimulation of DCs with LPS has been demonstrated in previous studies [17, 18]. We wanted to know next whether FcεRI stimulation modifies endogenous histamine synthesis of the differentiating cells. Therefore, mRNA of FcεRI-stimulated and unstimulated cells was isolated at days 1 and 6 of culture. We observed significantly upregulated HDC mRNA levels on both day 1 (Fig. 4A) and day 6 (Fig. 4B) in cells, which have been stimulated via FcεRI at the onset of the culture. Interestingly, exogenous histamine at day 1 of cultures further increased HDC mRNA expression of the cells (Fig. 4B). In parallel, higher amounts of histamine were detectable in the cell culture supernatant of FcεRI-stimulated cells stimulated concomitantly with LPS to induce histamine secretion as compared to unstimulated control cells incubated with LPS (Fig. 4C). These data demonstrate that FcεRI stimulation as well as histamine in the cellular microenvironment amplifies the endogenous histamine biosynthesis of differentiating cells.
High numbers of H1R-expressing MФs are located in the dermis of atopic dermatitis lesions
Infiltration of AD lesions with distinct DC subtypes and MФ has been demonstrated . To evaluate the in vivo relevance of our findings, paraffin-embedded skin sections taken from lesional skin of patients with AD and healthy controls were analysed by immunofluorescence staining. Double staining of skin sections demonstrated that CD1apos DCs exist in epidermis while CD68pos MФs were mainly located in the dermis (Fig. 5A). Infiltrates of H1R-expressing cells with morphologic characteristics of MФs expressing CD68 were detectable in the upper part of the dermis as well as the lower dermal compartment (Fig. 5B) of lesional skin from patients with AD. In contrast, only few H1RposCD68pos MФs were detectable in sections taken from healthy skin.
FcεRI-mediated allergen challenge and soluble factors in the microenvironment determine the nature of the differentiating cells and their capacity, to amplify the allergic inflammatory immune response. Here, we demonstrate that FcεRI stimulation of human monocytes promotes the differentiation of H1R-expressing MФs with increased production of proinflammatory cytokines.
FcεRI stimulation and efficient allergen uptake and presentation to T cells are central mechanisms of allergic diseases. Stimulation of FcεRI on monocytes promotes NF-κB signalling , prevents Fas/Fas-L-mediated apoptosis , induces the production of IL-10 , upregulates the expression of indoleamine dioxygenase (IDO) and favours the release of proinflammatory mediators such as IL-6, monocytes chemoattractant protein (MCP)-1 or macrophage inflammatory protein (MIP)-1β . In addition, FcεRI stimulation and consequent IL-10 secretion profoundly promote the differentiation of MФ, while preventing the differentiation of CD1a pos DCs . Our data provide evidence for a close interaction and regulation of FcεRI with HRs, in particular H1R, and the sensitivity of differentiating cells for H1R-mediated signals during allergic immune responses.
As we could show that FcεRI stimulation further increases histamine biosynthesis of the differentiating cells by directly upregulating mRNA expression of HDC, an endogenous feedback amplification consisting of FcεRI-mediated upregulation of H1R, enhanced autologous supply of histamine and thereby increased H1R mediated signalling is very likely. Endogenous histamine synthesis and HDC activity are assumed to play a crucial role during the differentiation and proliferation of cells. As a proof of this concept, blockage of HDC profoundly disturbs DC differentiation . This implies that the modified HDC expression induced by FcεRI stimulation might in part further impact on the altered cell differentiation observed here. In vivo, the autologous supply of histamine by differentiating cells itself might be replenished by histamine provided by mast cells in the dermis, which undergo frequent allergen challenge in allergic diseases such as AD.
MФs are regarded as important immune cells for a long time and their activities are grouped into three main functions: host defence, wound healing and immune regulation . Infiltration of MФs is regarded as a hallmark of inflammatory skin diseases such as psoriasis and AD . It is assumed that monocytes recruited from the blood during inflammation represent an important source for inflammatory MФs in the peripheral tissue . MФs and DCs are outlined in the mononuclear phagocyte systems (MPS) and derive from a common progenitor that allows to some degree the recapitulation of the differentiation from DCs to MФs or back through the modification of environmental cytokines and growth factors . High H1R expression has been described in human in vitro-generated MoMФs as well as human lung MФs of asthmatics . Histidine decarboxylase-expressing CD68pos MФs are detectable at chronic inflammatory sites such as in human artherosclerotic carotid arteries , indicating that this subset of MФs is actively involved in the maintenance of allergic and inflammatory immune responses. Infiltrates of CD68pos MФs are detectable in the epidermal, but foremost in the dermal part of lesional AD skin, but there is not much knowledge about the origin of these cells as well as specific mechanisms boosting their proinflammatory properties within the skin. Here, we provide a model of enhanced differentiation and activation of H1R-expressing MФs regulated by the degree of FcεRI stimulation of their monocytic precursor cells as well as mast cells in their microenvironment.
Concerning the high density and close contact of mast cells and H1R-bearing MФs in the dermal compartment of the skin in AD or the airways of asthmatic patients , a cross-talk of mast cells and MФs via histamine as a mediator represents a conceivable in vivo scenario. Hence, the augmentation of antigen-receptor-mediated responses by H1R such as shown here might directly account for the amplification of the allergic inflammatory reaction in vivo and represent therefore a target for therapeutic intervention. So far, application of H1R antagonists for the treatment of AD has been only of limited value as most of the studies focused on the antipruritic effect of H1R antagonists in AD [35-37]. However, there are also studies that support the concept that H1R antagonists, although being not beneficial in regard to pruritus, might nevertheless ameliorate AD, mirrored by a steroid-sparing effect achievable under treatment, which indirectly provides evidence for positive effects of some antihistamines on skin inflammation . Taken together, in view of the data presented here, FcεRI-H1R cross-regulated mechanisms impacting on MФ differentiation and activation might aggravate skin inflammation in AD and should be therefore considered in the development of novel therapeutic strategies.
This work was supported by grants from the German Research Council (DFG NO454/2-4, NO454/1-4; SFB704 TPA4, A8 and A15) and a BONFOR grant of the University of Bonn. N.N. is supported by a Heisenberg-Professorship (DFG NO454/5-2). We are thankful to Juana Hart for excellent technical assistance.
N.N., C.A. and W.M.P. contributed to experimental design. N.N. and W.M.P. performed the experiments, data analysis, interpretation of the results. N.N., W.M.P. and C.A. wrote the manuscript. T.B. reviewed the manuscript. N.N. gained funding.
Conflict of interest
The authors have no conflicting financial interests.