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Keywords:

  • CCL2;
  • H4 receptor;
  • histamine;
  • Langerhans cells

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Conflict of interest
  8. References

To cite this article: Gschwandtner M, Rossbach K, Dijkstra D, Bäumer W, Kietzmann M, Stark H, Werfel T, Gutzmer R. Murine and human Langerhans cells express a functional histamine H4 receptor: modulation of cell migration and function. Allergy 2010; 65: 840–849.

Abstract

Background:  Histamine is an important mediator of allergic reactions, and recent studies indicated that the function of different types of antigen presenting cells (APC) can be modulated by histamine, in particular via the newly described histamine H4 receptor (H4R). Therefore, we investigated possible interactions of histamine via the H4R on Langerhans cells (LC), which represent the professional APC in the skin and therefore have an important role in the initiation and maintenance of allergic skin diseases.

Methods:  The expression of the H4R was evaluated by real-time PCR, flow cytometry and immunofluorescence staining. The function of the H4R was determined by intracellular flow cytometric measurement of chemokine production and LC migration assays.

Results:  Here, we show H4R expression on in vitro generated monocyte-derived LC (mRNA and protein) and on primary LC from murine and human skin samples (protein). The immunofluorescence staining in murine and human skin samples clearly proved that LC express the H4R in situ. Stimulation with histamine or a H4R agonist downregulated the chemokine (C-C motif) ligand 2 (CCL2) in human monocyte-derived LC and primary LC. Prestimulation with a selective H4R antagonist abolished this effect. Moreover, migration of LC from the epidermis was increased after H4R agonist stimulation in ex vivo migration assays using human epidermis and murine in vivo assays.

Conclusion:  Our findings show that LC express a functional H4R and point towards a possible pathogenic relevance of the H4R in inflammatory and allergic diseases.

Abbreviations
APC

antigen presenting cells

DC

dendritic cells

H4R

histamine H4 receptor

LC

Langerhans cells

MoDC

monocyte-derived dendritic cells

MoLC

monocyte-derived langerhans cells

Langerhans cells (LC) are the sentinels of the immune system in the skin. In case of antigen or allergen encounter, LC migrate from the epidermis to the local lymph nodes to initiate a T cell response. Because of this pivotal role of LC in the establishment of cutaneous allergic immune responses, we were interested in studying effects of the allergic mediator histamine on these cells. During inflammatory and allergic reactions, high amounts of histamine are released in the skin, which acts via four distinct G-protein-coupled histamine receptor subtypes (H1R-H4R) (1). These receptors are expressed on many different cell types and therefore play diverse roles: the H1R is widely distributed and closely related to the development of allergic conditions, because of its influence on vascular permeability, smooth muscle and endothelial cell contraction. The H2R is mainly involved in the secretion of gastric acid (2) but is also present on other cell types such as smooth muscle cells and certain leucocytes. The H3R is primarily expressed in the central nervous system and was identified to be an autoreceptor that negatively regulates the release of histamine and other neurotransmitters (3). The recently discovered H4R (4, 5) is present on many cell types involved in inflammatory and allergic diseases, such as T cells, dendritic cells, mast cells and eosinophils, and immunomodulatory functions in the murine as well as the human system have already been identified. H4R stimulation was shown to induce chemotaxis of mast cells (6), monocyte-derived dendritic cells (MoDC) (7) and eosinophils (8). In monocytes, MoDC and monocyte-derived inflammatory epidermal dendritic cells, H4R-dependent suppression of IL-12 and CCL2 production were described (7, 9, 10).

These findings implicate an important role of histamine in modulating the function of antigen presenting cells (APC). Therefore, histamine might also influence LC, whose interaction with histamine has not been extensively investigated until now. Previous studies showed that LC do not express the H1R and the H2R on mRNA level (11). Thus, it is possible that histamine might modulate LC function via the H4R during allergic skin reactions. We intended to study the expression and function of the H4R on human LC generated in vitro from monocytes and on primary LC isolated from human skin (ex vivo studies). Moreover, we investigated the H4R on murine LC to get more insight into the comparability of H4R-mediated responses in human and mice.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Conflict of interest
  8. References

Human skin samples

Samples from healthy skin were obtained during plastic surgery or safety margin operations of skin tumours from anonymous donors directly after surgery. The investigation of the role of histamine receptors in allergic skin inflammation was approved by the local ethics committee of the Hannover Medical School (Vote Nr. 4253) and was conducted according to the Declaration of Helsinki Principles.

Mice

BALB/c and NMRI mice were obtained from Charles River (Sulzfeld, Germany) at the age of 6–8 weeks. The H4R-deficient (H4R−/−) mouse was kindly provided by Robin Thurmond (Johnson & Johnson, La Jolla, CA, USA). Mice were bred in the animal facility of the Veterinary University Hannover. All animals were healthy and were housed in groups of six mice per cage at 22°C with a 12-h light/dark cycle. Water and standard diet (Altromin, Lage/Lippe, Germany) were available ad libitum. The animal experiments have been approved by Bezirksregierung Hannover, Germany (AZ G33-42502-06/1126).

Preparation of epidermal single cell suspensions

Single cell suspensions were prepared as described previously (12). The cells were cultured in RPMI 1640 supplemented with 2 mM l-glutamine, 100 mg/ml penicillin/streptomycin, 12 mM Hepes and 5% v/v foetal calf serum (PAN-Biotech; all other media components from Biochrom, Berlin, Germany) at 37°C in a humidified atmosphere containing 5% CO2.

Single cell suspensions from mouse ear skin were prepared as follows: Mice were killed, and ears were cut and split into dorsal and ventral halves. The epidermis was separated from the dermis after incubation of the ear halves in 0.5 M ammonium thiocyanate (Riedel de Haën, Hannover, Germany) at 37°C for 10 min. After washing of the epidermis in phosphate buffered saline (PBS), single cell suspensions were prepared analogously to human samples.

Preparation of spleen cell suspensions

Mice were killed; the spleen was taken out and minced. The spleen homogenate was filtered through a sterile gauze (40 μm), and the cells were washed in PBS. Red blood cells were lysed using RBC lysis buffer (eBioscience, San Diego, CA, USA). After another washing step, the cells were cultured in Iscove medium supplemented with 2 mM l-glutamine, 100 mg/ml penicillin/streptomycin, 0.05 mg/ml gentamycin, 1× nonessential amino acids and 10% v/v foetal calf serum (PAN-Biotech, all other components from Biochrom AG) at 37°C in a humidified atmosphere at 5% CO2.

Generation of monocyte-derived LC

Buffy coats from anonymous healthy donors were obtained from the local blood bank. Peripheral blood mononuclear cells were separated from buffy coats by density centrifugation on lymphoprep (Fresenius Kabi Norge AS, Oslo, Norway). Adherent cells were obtained by plastic adherence: 5 × 107 peripheral blood mononuclear cells were plated in 80-cm2 culture flasks (NuclonTMD Nunc GmbH & Co. AG, Wiesbaden, Germany) in supplemented RPMI 1640 medium for 1 h (37°C, 5% CO2, humidified atmosphere). The nonadherent cells were removed by vigorous washing with PBS. The adherent cells (enriched monocytes, purity at least 85%) were further cultured in supplemented RPMI 1640 and for the generation of MoLC, 10 ng/ml IL-4 (R&D Systems, Wiesbaden, Germany), 10 ng/ml granulocyte–monocyte colony-stimulating factor (Berlex Pharmaceutical Company, Montville, NJ, USA), 10 ng/ml TGF-β (Promokine, Heidelberg, Germany) and 10 mM β-mercaptoethanol (Sigma-Aldrich, Taufkirchen, Germany) were added. The medium was changed every 2–3 days, and the cells were harvested on day 8. The cells were characterized as MoLC by staining for the following surface markers: CD1a (BD Biosciences, Bedford, MA, USA), CD14, CD36, CD40, CD54, CD80, CD83, CD86 (all from Beckman Coulter, Krefeld, Germany), CD206 (BD Biosciences), CD207 (Beckman Coulter) and HLA-DR (BD Biosciences) or the respective isotypes (Beckman Coulter and Sigma-Aldrich).

Flow cytometric analysis of H4R expression

For H4R staining, the cells isolated from human epidermis were either used directly after single cell preparation or after overnight culture. The cells were washed twice in PBS and then incubated for 20 min in PBS containing human immunoglobulin G (IgG) (FcγR-blocking buffer). To distinguish LC from the other cells present in the suspension, they were stained with anti-CD207-PE (Langerin; Beckman Coulter) or mIgG1-PE as isotype control (Sigma, Taufkirchen, Germany). Subsequently, cells were fixed and permeabilized (Fixation/Permeabilization Kit, eBioscience). Intracellular staining was performed with H4R antibody recognizing amino acids 194-303 (SantaCruz Biotechnology, Santa Cruz, CA, USA) or polyclonal rabbit isotype control (R&D Systems), followed by labelling with goat anti-rabbit-FITC (Beckman Coulter). CD207 and H4R positivity of the cells was assessed by flow cytometry (FACS Calibur; Becton Dickinson, Heidelberg, Germany).

For H4R evaluation on murine LC, the cross reactivity of the human anti-H4R antibody for the murine system was confirmed by western blot experiments with murine spleen cells (data not shown). The analysis by flow cytometry was performed analogously to human cells, using anti-CD207-Alexa488 clone 929F3 (AbCys, Paris, France) or ratIgG2a-FITC (Beckman Coulter) as isotype control for surface staining. H4R antibody (SantaCruz Biotechnology) or isotype control (R&D Systems), goat anti-rabbit-biotin (Jackson Immuno Research, Suffolk, UK) and streptavidin-PerCP (BD Pharmingen) were used for the intracellular detection of H4R. In mouse spleen cell suspensions, intracellular staining was performed with H4R antibody (SantaCruz Biotechnology) or polyclonal rabbit isotype control (R&D Systems), followed by labelling with goat anti-rabbit-FITC (Beckman Coulter).

Immunofluorescence staining of the H4R on human LC

Frozen sections (5 μm) from skin biopsies were transferred to SuperFrost Plus microscope slides (Menzel-Gläser, Braunschweig, Germany). After drying, the sections were fixed in ice-cold 99% ethanol, then the sections were incubated in horse serum (Vectastain ABC kit, alkaline phosphate mouse IgG; Vector Laboratories, Burlingame, CA, USA) for 1 h at room temperature (RT). Following this, the slides were stained with anti-CD207 (Beckman Coulter) or mIgG1 (R&D Systems) overnight at 4°C, secondary antibody (biotinylated horse anti-mouse antibody, Vectastain ABC kit, alkaline phosphatase mouse IgG; Vector Laboratories) for 1 h at RT and avidin-fluorescin (Vector Laboratories) for 5 min at RT. After washing, the sections were incubated in goat serum for 1 h at RT and then stained with anti-H4R (SantaCruz Biotechnology) for 3–4 h and goat anti-rabbit-APC (Beckman Coulter) for 30 min. After a washing step, the stained sections were covered with mounting medium (Vectashield; Vector Laboratories) and coverslips. Fluorescence images were obtained with a Zeiss Axiolab microscope with AxioCam MRm, and images were acquired with the program AxioVS40 (version 4.6.1.0) (Carl Zeiss MicroImaging GmbH, Göttingen, Germany).

Immunofluorescence staining of the H4R on murine LC

Epidermal sheets were prepared and evaluated as described previously (13). In short, ear skin was floated on 0.5 M ammonium thiocyanate for 10 min at 37°C. LC were detected with biotinylated monoclonal rat anti-mouse MHC-II (Becton Dickinson) and streptavidin-carbocyanin2 (Dianova, Hamburg, Germany). H4R was stained with anti-H4R (SantaCruz Biotechnology) and goat anti-rabbit-carbocyanin3 (Dianova).

Intracellular staining of CCL2

Single cell suspensions or in vitro differentiated MoLC were stimulated with 10 μM histamine (Alk-Scherax, Wedel, Germany) or clobenpropit (Sigma) for 48 h before intracellular staining of CCL2 was performed. For blocking experiments, cells were incubated with JNJ7777120 [synthesized as described previously (14)] 30 min before stimulation. The cells of the skin suspension were washed in PBS, and after incubation with FcγR-blocking buffer, the surface was stained with anti-CD207-PE (Beckman Coulter) or mIgG1-PE (Sigma). After fixation and permeabilization (Fixation/Permeabilization Kit, eBioscience), intracellular staining was performed with anti-CCL2-APC (R&D Systems) or mIgG2b-APC (R&D Systems). CD207 and CCL2 positivity of the cells was quantified by flow cytometry. In vitro differentiated MoLC were permeabilized and stained for intracellular CCL2.

Ex vivo human LC migration assay

Epidermis samples were either taken from blisters of patients suffering from bullous pemphigoid or the epidermis of healthy skin was separated from the dermis after 4°C overnight incubation in 2.4 U dispase II (Roche, Mannheim, Germany). The epidermis samples were washed two times, cut into pieces of approximately equal size and placed in 2 ml of supplemented RPMI 1640 in a 6-well plate. Stimulation was performed with 10 μM histamine or 4-methylhistamine (Tocris Bioscience, Bristol, UK). For blocking experiments, cells were incubated with JNJ7777120 30 min before stimulation. The next day, the skin was transferred into a plate containing new medium with fresh stimuli to give another impulse for migration. After another 24 h of incubation, the epidermis was dried to allow measurement of the weight of the individual skin pieces. The medium was pooled, and the amount of migrated cells was counted using a haemocytometer. To evaluate the amount of cells that migrated from the epidermis to the culture medium, the cell number per mg of skin was calculated. After assay completion, the cells were washed twice in PBS and used for further analysis by real-time PCR and flow cytometry to prove the identity of LC.

mRNA isolation, reverse transcription and quantitative RT-PCR

Nonstimulated murine spleen cells and human MoLC, keratinocytes, T cells, monocytes, macrophages, eosinophils and the cells remaining after the migration assay were washed in PBS and lysed for RNA isolation using Mini RNA Isolation II Kit (Zymo Research, Orange, USA), and reverse transcription was carried out using the First-Strand cDNA Synthesis Kit (MBI Fermentas, St. Leon-Rot, Germany). Real-time quantitative PCR was performed on a LightCycler (Roche Molecular Biochemicals, Mannheim, Germany) using SYBR Green with Quantitect primer assays for human glyceraldehyde-3-phosphate dehydrogenase GAPDH (QT01192646), human CD207 (QT00045689), human H4R (QT00032326), murine glyceraldehyde-3-phosphate dehydrogenase GAPDH (QT01658692) and murine H4R (QT00135884) according to manufacturer’s instructions (Qiagen, Hilden, Germany). The following settings were used: an initial activation step of 15 min at 95°C with ramp 20°C/s was followed by three-step cycling (45 cycles): denaturation 15 s, 94°C; annealing 20 s, 55°C; extension 20 s, 72°C (all three with ramp 2°C/s). Melting curve analysis was performed from 60 to 90°C with ramp 20°C/s. The amount of target genes relative to the reference GAPDH was quantified using the Relative Quantification Software (Roche Molecular Biochemicals). To visualize the amplification products after completion of the PCR run, agarose gel electrophoresis was performed with 2% agarose (Roth, Karlsruhe, Germany) in 1× Tris-Borate-EDTA buffer (Roth). Samples were diluted in 6× loading dye (Fermentas) and Gene Ruler 100 bp plus DNA.

In vivo murine LC migration assay

BALB/c mice were narcotized by intraperitoneal injection with ketamine/xylazine (Alvetra, Neumünster and CP-pharma, Burgdorf, Germany). Twenty microlitres of 10 μM 4-methylhistamine was injected intradermally into the left ear and 20 μl PBS into the right ear. After 5 h, the procedure was repeated. 24 h after the second administration, the mice were killed, the ears were separated into dorsal and ventral halves, and staining of MHC-II was performed as described previously. Analysis was carried out using the Axiovision (Carl Zeiss, Göttingen, Germany) image analysis system. The density of LC was analysed at 20× magnification on a calibrated grid. Sixteen randomly chosen areas were counted per ear, and the number of LC/mm2 of epidermis was calculated.

Statistical analysis

For statistical evaluation, a normality test was performed. Because all data sets were normally distributed, the paired t-test was used for statistical calculations. A P-value below 0.05 was regarded as significant. P < 0.05 is depicted with * and P < 0.005 with **. The program graphpad prism version 3.02 (GraphPad Software Inc., San Diego, CA, USA) was used for statistical analysis.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Conflict of interest
  8. References

Human and murine LC express the H4R

To evaluate the expression of the H4R on LC, we initially generated LC in vitro from human monocytes (MoLC) and performed real-time PCR experiments and flow cytometric measurement. MoLC express the H4R on mRNA as well as on protein level as shown in Fig. 1. Because in vitro generated MoLC do not completely resemble naturally occuring LC, we additionally studied ex vivo LC obtained in epidermal single cells suspensions. H4R expression on ex vivo human LC was shown by double colour staining of langerin (CD207) and H4R. Langerin-positive cells expressed the H4R as shown by flow cytometric measurements of epidermal cell suspensions (Fig. 2A,B) and by immunofluorescence staining of human healthy skin (Fig. 2C–F).

image

Figure 1.  The H4R is expressed on human monocyte-derived Langerhans cell on the mRNA and protein level. A representative real-time PCR experiment with specific melting peaks for the H4R and Gapdh and a reaction without reverse transcriptase as a negative control (A). A representative agarose gel with the PCR products (B). One representative flow cytometric experiment showing H4R staining (grey) when compared to isotype staining (white) (C) out of four which are summarized in (D).

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image

Figure 2.  The H4R is expressed on human naturally occurring Langerhans cell (LC). One to two percentage of total cells in epidermal cell suspensions were CD207-positive LC as assessed by flow cytometry (A). The histogram shows H4R staining (grey) and isotype staining (white) for the CD207-positive cells of one representative experiment out of 11, which are summarized in (B). Immunofluorescence staining in the skin with anti-CD207 (langerin) (C) and anti-H4R (D) shows colocalization of the H4R on CD207-positive LC (E), enlarged in (F). One representative experiment of 4 is shown. Location of the basement membrane is indicated by the white line. Of note, keratinocytes express also H4R.

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In skin sections of BALB/c mice, we could show the expression of the H4R on LC by immunofluorescence staining, while the staining with the same antibody in the skin of H4R-deficient mice lead to no signal detection (Fig. 3A). To demonstrate specificity of our H4R detection methods, we performed flow cytometric measurements and real-time PCR assays of the H4R on spleen cell suspensions from wild-type mice and H4R−/− mice (Fig. 3B,C, respectively). Flow cytometric analysis of single cell suspensions isolated from murine ear skin showed that murine LC, identified by positive staining for langerin, express the H4R (Fig. 3D). Because it was described that the sensitivity to histamine varies between two strains – BALC/c and NMRI – as shown by different scratching behaviour in response to histamine (15), we investigated whether the level of H4R expression on LC can be linked to this response. We found that H4R expression on LC did not differ between the two investigated strains, the histamine-sensitive NMRI and nonhistamine-sensitive BALB/c mice, as summarized in Fig. 3E.

image

Figure 3.  The H4R is expressed on murine Langerhans cell (LC) and murine spleen cells. The H4R is expressed on MHC-II-positive LC in the wild-type BALB/c mouse (left panel) and absent on LC in the H4R−/− mouse (right panel) (A, one representative experiment of n = 6 and n = 2, respectively). Confirmation of H4R staining on spleen cells from wild-type BALB/c and H4R−/− mice (B, H4R: grey; isotype: white; one experiment out of three). Presence of H4R mRNA in the wild-type and absence of H4R mRNA in H4R−/− mice as shown in the agarose gel electrophoresis of RT-PCR products (C, one experiment out of three is shown). One to two percentage of total cells from mouse epidermal cell suspensions were CD207-positive LC, expressing the H4R (grey) when compared to isotype control (white) (E). LC from BALB/c and NMRI mice do not differ in H4R expression (F, = 4 and n = 3, respectively).

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Inhibition of CCL2 production in human LC by H4R activation

After showing that the H4R is expressed on human and murine LC, we were interested whether receptor stimulation influences the function of LC. Because we have already described previously that H4R stimulation decreases the level of CCL2 in human monocytes (9) and IDEC (10), we measured CCL2 levels in human LC. After 48 h of stimulation of human epidermal single cell suspensions with 10 μM histamine or the H4R agonist clobenpropit, we observed a downregulation of CCL2 levels in langerin-positive cells (Fig. 4A). The decrease in chemokine production was blocked by preincubation with the H4R selective antagonist JNJ7777120 (Fig. 4B), proving that the effect is specific for H4R activation. Also, in in vitro generated MoLC, we could detect lower levels of CCL2 after the activation of H4R (Fig. 4C).

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Figure 4.  Downregulation of CCL2 in human Langerhans cells (LC) after H4R stimulation. Intracellular CCL2 is downregulated in LC in epidermal cell suspensions after 48 h of incubation with 10 μM histamine or the H4R agonist clobenpropit, percentages of CCL2 negative and positive cells are stated above the dot plots (A). Prestimulation with the H4R antagonist JNJ7777120 blocks the downregulation of CCL2 (B, mean and SEM of seven independent experiments are depicted). Also, in monocyte-derived LC, a decrease in CCL2 production was detected (C, mean and SEM of four independent experiments are depicted). *P < 0.05, **P < 0.005.

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Histamine-induced ex vivo migration of human LC

We investigated the influence of histamine on the migration of LC from the human epidermis, because it has recently been shown that H4R stimulation induces migration of murine bone-marrow-derived DC and skin DC (15) and human monocyte-derived DC (7). In a LC migration assay using epidermis derived from patients with bullous pemphigoid, significantly increased LC migration was induced by histamine and the H4R agonist 4-methylhistamine (Fig. 5A). Preincubation with the H4R antagonist JNJ7777120 inhibited the histamine-induced migration of LC, proving that the effect is specific for H4R (Fig. 5A). Disease-specific effects seem to play no role, because the H4R agonist 4-methylhistamine also induced migration of LC from the epidermis derived from healthy donors (Fig. 5B). The migrated cells represent LC, as shown by real-time PCR (Fig. 5C) and flow cytometry (Fig. 5D).

image

Figure 5.  H4R agonists induce emigration of human Langerhans cells (LC) from the epidermis ex vivo. Upon 48 h of incubation of epidermal sheets with histamine or H4R agonist 4-methylhistamine (10 μM), LC emigrate from epidermis derived from patients with bullous pemphigoid. This was blocked by the H4R antagonist JNJ7777120 (A, Mean and SEM of seven independent experiments, normalized to nonstimulated control, 100% equals 1200 LC per mg epidermis). H4R agonist-induced LC migration was also observed in epidermis derived from healthy skin (B, n = 6, P-value = 0.063). Real-time PCR showing that the migrated cells express CD207 and H4R mRNA, while control cells do not express CD207, GAPDH was used as reference gene (C, one experiment of three). Flow cytometric evaluation of CD207 expression (D, CD207: grey, isotype: white, one experiment out of three is shown). *P < 0.05, **P < 0.005.

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Histamine-induced in vivo migration of murine LC

In a previous study, migration of skin DC induced by H4R stimulation was shown by ex vivo migration assays (15). To show that this functions as well in vivo, we performed in vivo LC migration experiments in the mouse.

After intradermal injection of 4-methylhistamine into the mouse ear, the density of LC significantly decreased when compared to the ear of the contralateral site which was injected with PBS (Fig. 6A,B). These results showed that H4R stimulation can induce the migration of LC also in vivo and results in a lower number of LC present in the epidermis.

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Figure 6.  Intradermal injection of a H4R agonist induces emigration of murine Langerhans cell (LC) from skin in vivo. After intradermal injection of 10 μM 4-methylhistamine (4MH), less MHC-II-positive cells are detected in the epidermis when compared to PBS control. One representative experiment is shown (A), six independent experiments are summarized in (B) (Data points represent mean numbers of LC/mm2 of 16 randomly chosen areas; the line represents the mean of six experiments). *P < 0.05.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Conflict of interest
  8. References

In this study, we investigated a possible effect of histamine on Langerhans cells (LC), because they are likely to come into contact with histamine at sites of allergic skin diseases, and histamine has been shown to modulate the function of other dendritic cell subtypes. We focused on the H4R, because a previous study suggested the absence of the H1R and the H2R on LC (11), and the H3R is not likely to be expressed on LC, because it is predominantly found in the central nervous system (3).

We measured the expression of the H4R on human MoLC, which were in vitro generated from monocytes and on primary ex vivo LC isolated from murine and human skin samples (naturally occurring LC). We demonstrate for the first time that murine as well as human LC express the H4R on the mRNA and protein level, as shown by real-time PCR, flow cytometry and immunofluorescence staining of the skin. Immunomodulatory effects of histamine mediated via the H4R have already been described; in particular, the induction of migration of a variety of immunologically relevant cell types has been shown, such as mast cells (6), MoDC (7), eosinophils (8) and regulatory T cells (16). Because of these observations and the finding that the migration of murine skin DC can as well be induced by H4R agonists (15), we were interested in studying the migration of LC in response to histamine. We could show that H4R stimulation increases the migration of human LC ex vivo and of murine LC in vivo. Induction of LC migration via the H4R may be an explanation for the finding that the epidermis overlying lesions of urticaria pigmentosa – a type of mastocytosis with accumulation of mast cells in the dermis – contains a reduced number of LC (17). Because mast cells in the dermis can release histamine upon various stimulations, this might induce the migration of LC from the epidermis resulting in decreased LC numbers in the epidermis.

LC are not only important in the priming of T cell responses, in addition they secrete chemokines which are responsible for recruitment and functional polarization of other cell types involved in inflammatory processes. An example is the chemokine CCL2, which we investigated in this study. CCL2 leads to the recruitment of macrophages, monocytes and T cells and has been associated with Th2 responses (18). As described previously for monocytes and IDEC (9, 10), we also observed a downregulation of CCL2 production in LC mediated via H4R stimulation. It is tempting to speculate that activation of the H4R limits the cell influx of inflammatory cells in lesions of allergic skin diseases and contributes to the shift from a Th2 to a Th1 milieu as observed during the transition from acute to chronic eczematous skin lesions (19).

Another interesting observation is that CCL2 among other chemokines can activate and thereby degranulate mast cells and basophil granulocytes in an immunoglobulin E (IgE)-independent manner. This mechanism of activation is of particular importance in late-phase reactions. In the murine system, CCL2-dependent activation of mast cells and basophils was shown (20), while in human cells, induction of degranulation and histamine release by CCL2 was only observed in basophils (21). These findings may point at a feedback mechanism, in which the downregulation of CCL2 by H4R stimulation might be inhibiting the late-phase release of additional histamine from mast cells and basophils. Moreover, in response to the lack of CCL2, also other chemokines such as CCL5 and CCL3 are downregulated (18), which can also function as histamine-releasing factors. Because the decreased production of CCL2 is not only seen in LC but also was described for monocytes and IDEC (9, 10), this might be a general mechanism of dampening an immune response.

In conclusion, this study clearly shows that histamine, and in particular its action via the H4R, plays an important role in modulating the function of LC. On one hand, increased migration of LC from the epidermis was observed, probably resulting in faster transport of encountered antigens to the local lymph nodes. On the other hand, we found downregulation of the production of CCL2 presumably resulting in decreased recruitment of macrophages and T cells and impaired IgE-independent release of histamine. These findings indicate that histamine influences the local immune response by two different mechanisms: one ensuring fast presentation of antigens and one preventing an overwhelming immune response. These findings underline the relevance of the H4R in allergic skin diseases and encourage further exploration of the H4R as a therapeutic target in allergic diseases.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Conflict of interest
  8. References

We appreciate the excellent technical assistance of Gitta Köther and Susanne Mommert. We thank Robin Thurmond (Johnson & Johnson, La Jolla, CA, USA) for giving us the opportunity to use tissues from the H4R−/− mouse as specificity control for our H4R detection methods. Wolfgang Bäumer is appointed as an endowed professor in ‘Veterinary Dermatopharmacology’ granted by Bayer Animal Health GmbH. Maria Gschwandtner was awarded for this study with the Art A Hancock Young Investigator Award from the European Histamine Research Society sponsored by Abbott Laboratories.

This study was supported by grants from the Deutsche Forschungsgemeinschaft DFG: Gu434/5-1, Ba2071/2-1 and GRK1441/1 and the European Community (COST action BM0806). Maria Gschwandtner was supported by a grant from the Hannover Biomedical Research School.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Conflict of interest
  8. References
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