Histamine H(4) receptor activation on human slan-dendritic cells down-regulates their pro-inflammatory capacity

Abstract

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.

Figure 2

6-Sulpho LacNAc dendritic cells (SlanDC) express the histamine receptor H₄R on protein level. CD16 and H₄R expression on M-DC8-positive slanDC were measured by flow cytometry at day 0 and after 1 day in culture. In (a) one representative experiment of four which are summarized in (b and c) is shown. (white: isotype; grey: specific staining; the numbers given in the histograms are mean fluorescence intensity; relative expression levels of CD16 and H₄R were calculated as follows: specific staining times hundred divided by isotype staining).

Figure 1

6-Sulpho LacNAc dendritic cells (SlanDC) express the histamine receptors H₁R, H₂R and H₄R at the mRNA level. Representative real-time PCR melting peaks and agarose gel bands of the PCR products out of two independent experiments are depicted (negative control is without reverse transcription, H₃R-transfected HEK cells were used as positive control for H₃R amplification).

Figure 3

Regulation of expression of the histamine receptor H₄R in disease and by inflammatory cytokines. (a) Basal H₄R expression does not vary in different disease groups [psoriasis, atopic dermatitis (AD) and healthy controls, eight individual measurements and their mean are depicted]. (b–d) PBMC were stimulated for 48 hr with 20 ng/ml interferon-γ (IFN-γ) and H₄R expression on slanDC was determined flow cytometrically. The relative mean fluorescence intensity of non-stimulated cells versus IFN-γ-stimulated cells is shown for 6-sulpho LacNAc dendritic cells (slanDC) isolated from AD patients (b), healthy controls (c) and psoriasis patients (d). (The relative expression levels of H₄R were calculated as follows: specific staining times hundred divided by isotype staining.) **P < 0·005.

Figure 6

The down-regulation of tumour necrosis factor-α (TNF-α) and interleukin-12 (IL-12) is mediated by the histamine receptor H₄R. (a)After 48 hr of stimulation histamine and the H₄R agonist 4-methylhistamine reduced the level of lipopolysaccharide (LPS) -induced TNF-α and IL-12, but not IL-10. Pre-incubation with the H₄R antagonist JNJ7777120 restored LPS-induced cytokine production (mean and SEM of 11 independent experiments is shown). (b) The H₁R agonist 2-pyrilethylamine did not reduce LPS-induced TNF-α and IL-12 production. The H₂R agonist amthamine had no effect on TNF-α secretion, but down-regulated IL-12 production. The cytokine IL-10 was not affected by histamine receptor stimulation (mean and SEM of seven independent experiments are shown). *P < 0·05; **P < 0·005.

Figure 5

Histamine decreases tumour necrosis factor-α (TNF-α) and interleukin-12 (IL-12) secretion into the cell supernatant. Isolated 6-sulpho LacNAc dendritic cells (slanDC) were stimulated for 6 hr with 10 μm histamine, then 100 ng/ml LPS was added and after 24, 48 and 72 hr the cytokine content in the cell culture supernatant was determined by ELISA (mean and SEM of 11 independent experiments are shown). *P < 0·05; **P < 0·005; ***P < 0·001.

Figure 4

Stimulation of the histamine receptor H₄R reduces the production of tumour necrosis factor-α (TNF-α) and interleukin-12 (IL-12) as determined by intracellular cytokine staining. PBMC were stimulated for 6 hr with 10 μm histamine or the H₄R agonist 4-methylhistamine, then 100 ng/ml lipopolysaccharide (LPS) and 1 μg/ml Brefeldin were added and intracellular (a) TNF-α production and (b) IL-12 production were measured by flow cytometry after overnight incubation. Pre-incubation with JNJ7777120 fully blocked the H₄R-induced down-regulation of TNF-α and partially IL-12 (mean and SEM of 11 independent experiments are shown). *P < 0·05; **P < 0·005; ***P < 0·001.