Histamine up‐regulates oncostatin M expression in human M1 macrophages

Background and Purpose Human monocyte‐derived M1 macrophages develop in relation to growth factors, bacterial products, and cytokines in a local micro‐environment. M1 macrophages produce pro‐inflammatory mediators, in particular, oncostatin M (OSM), which is secreted from the cells in response to the active complement component C5a. As C5a also releases histamine from human mast cells and shows immune modulatory functions similar to histamine in regulating expression of the IL‐12 cytokine family, we investigated the effects of histamine on OSM expression in human M1 macrophages. Experimental Approach Cytokine expression was analysed by real‐time quantitative PCR and elisa techniques. Normal human epidermal keratinocytes were stimulated with supernatants from activated M1 macrophages, and phosphorylation of STAT3 was assessed by flow cytometry. Key Results OSM mRNA expression was highly up‐regulated by histamine and agonists targeting the histamine H1 H2, and H4 receptors in human M1 macrophages and by C5a, which was used as control stimulus. Protein levels of OSM and IL‐6 were up‐regulated by histamine. Supernatants from histamine‐stimulated, fully differentiated M1 macrophages were able to phosphorylate STAT3 in normal human epidermal keratinocytes. Conclusions and Implications The up‐regulation of OSM expression in response to histamine and C5a shown in this study provides further evidence that histamine and C5a, acting through their GPCRs, have almost equal functional effects in cells of the monocyte lineage. Both mediators OSM and IL‐6 have the capability to activate human keratinocytes. This effect may have an influence on the course of inflammatory skin diseases. 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


| INTRODUCTION
Human macrophages in skin or in different tissues derive from resident or circulating monocytes, which differentiate according to the micro-environment at their local niche either in more proinflammatory, classically activated M1 macrophages or in more antiinflammatory alternatively activated M2 macrophages. Beyond the expression of characteristic surface markers, the subtypes differ in gene expression of M1-or M2-specific chemokines and cytokines.
In vitro stimulation of normal human epidermal keratinocytes (NHEKs) with OSM in combination with other pro-inflammatory cytokines showed a transcriptional profile of antimicrobial peptides and of chemokine expressions that resembles in part that of lesional psoriatic skin (Guilloteau et al., 2010). OSM alone also regulates mRNA transcripts of antimicrobial peptides, in particular the expression of S100A7 and human beta defensin 2 (also defensin 4A) in NHEKs (Boniface et al., 2007;Guilloteau et al., 2010).
OSM production is increased in human dendritic cells or macrophages by lipopolysaccharide (LPS), fixed Staphylococcus aureus, or after stimulation by PGE 2 respectively (Repovic & Benveniste, 2002;Suda et al., 2002). Up-regulation of OSM expression in response to the potent anaphylatoxin C5a, which is generated during complement activation through cleavage of C5, had also been described for human macrophages (Kastl et al., 2008).
In contrast, stimulation with C5a inhibits TLR4-induced upregulation of IL-12, IL-23, and IL-27 mRNA and protein expression in human macrophages and monocytes (Hawlisch et al., 2005;Wittmann et al., 1999). Surprisingly, the TLR4-induced up-regulation of IL-27 expression as well as the TLR3-induced expression of  were also down-regulated by histamine via the histamine H 2 and H 4 receptors in monocytes and antigen-presenting cells (Gschwandtner et al., 2012;Gschwandtner, Schakel, Werfel, & Gutzmer, 2011;Gutzmer et al., 2005), pointing out that both C5a and histamine regulate expression of the IL-12 family cytokines in a similar fashion.
Histamine is a pleiotropic mediator that is present in elevated concentrations in human skin in inflammatory skin diseases such as psoriasis (Krogstad, Lonnroth, Larson, & Wallin, 1997) or atopic dermatitis (Ruzicka & Gluck, 1983) mediating its effect through four G-protein coupled receptors (Schaper-Gerhardt et al., 2018).
The functional similarities between C5a and histamine, for example, showing a down-regulation of Th1 promoting cytokines in inflammatory conditions, prompted us to investigate if histamine also increases OSM production in the same fashion as described for C5a in human M1 macrophages (Kastl et al., 2008).

| Isolation of monocyte derived macrophages
Residual blood samples from platelet apheresis disposables used for routine platelet collection and of regular anonymous healthy donors (buffy coats) served as source material for the isolation of human peripheral blood mononuclear cells (PBMCs).
The blood samples were randomly selected by the Transfusion Medicine MH-Hannover fulfilling the criteria of a bias-free representation of the population.
We have no access to individual data such as demographic or anamnestic characteristics that may influence the response to histamine or to the other histamine receptor agonists. Therefore, no inclusion/ exclusion criteria of the human donors are defined in advance.

What this study adds
• Histamine also up-regulates expression of oncostatin M and thereby may activate human keratinocytes.

What is the clinical significance
• Elevation of oncostatin M levels by histamine may critically regulate pathological skin immune responses.
• These findings may help to identify novel approaches to treatments for human inflammatory skin diseases. morphology, we had to exclude some experiments with macrophages from few donors. Therefore, in some figures, we show less than 12 experiments, but in every case, the sample size is larger as 5.
PBMCs were separated by density gradient centrifugation on lymphoprep (Fresenius Kabi Norge AS, Oslo, Norway). With a seeding density of 1 × 10 6 cells per well, PBMCs were plated in a 24-well plate in Iscoves Medium supplemented with AB serum (2.5% v/v). To attach the monocytes, cells were incubated for 2 hr at 5% CO 2 and 37°C.
2.2 | Cell culture, differentiation of M1 and M2 macrophages Cells were differentiated to M1 and M2 macrophages under the following experimental settings: Cells were cultured in the presence of GM-CSF during the differentiation process to M1 macrophages. M2 macrophages were differentiated from primary human monocytes in the presence of macrophage colony-stimulating factor (M-CSF) (10 ng·ml −1 ). M2 macrophages were activated by IL-4 (20 ng·ml −1 ) for 24 hr. At Day 5, another 50% by volume of fresh medium containing GM-CSF or M-CSF, respectively, was added. At Day 8, the medium was completely changed. At Day 10, the differentiation process was controlled: M1 macrophages appeared as adherent cells showing a typical morphology with a prominent nucleus, spread out cytoplasm, and a couple of pseudopodia. Fully differentiated M1 macrophages were positive for the intracellularly expressed macrophage differentiation marker CD68. They did not express the scavenger receptor CD163 that is specific for M2 macrophages and was expressed on the M2 macrophages cultured with M-CSF. When the cells were activated with IFN-γ (200 ng·ml −1 ) and LPS (50 ng·ml −1 ), we observed a moderate down-regulation of CD68 and a high up-regulation of CD80 reflecting the commonly accepted marker profile for activated M1 macrophages (Biswas & Mantovani, 2010). The expression of CD163 was not induced in response to the activating cytokines  (a) Primary human monocytes were obtained from peripheral blood mononuclear cells after 2-hr adherence. M1 macrophages were differentiated from primary human monocytes in the presence of GM-CSF (10 ng·ml −1 ) for 10 days and then stimulated with C5a (10 ng·ml −1 ) or histamine (10 μM) for different time periods as indicated. (b) OSM mRNA expression after treatment for different time periods as indicated with C5a (n = 18 independent donors and experiments). (c) OSM mRNA expression after treatment for different time periods as indicated with histamine (Hist; n = 9 independent donors and experiments). The amount of the target mRNA relative to the amount of the reference gene, rps 20 mRNA in each stimulated sample was normalized to the amount of the target mRNA relative to the amount of the reference gene in the non-stimulated sample (calibrator) and expressed as normalized ratio. This was calculated using the comparative Ct method also known as the ΔΔCt method provided by the Software LC 480 (Roche Molecular Biochemicals). Data shown are individual values with medians. *P < .05; significantly different as indicated; Friedman Dunn's Multiple Comparisons test selected pairs. Ct method, crossing point method; NS, non-stimulated; OSM, oncostatin M FIGURE 3 M1 macrophages activated with IFN-γ + LPS show the commonly accepted marker profile for these cells. Histamine up-regulates OSM mRNA expression in a dose-dependent manner in fully differentiated M1 macrophages. Primary human monocytes were obtained from peripheral blood mononuclear cells after 2-hr adherence. M1 macrophages were differentiated from primary human monocytes in the presence of GM-CSF (10 ng·ml −1 ) for 10 days. One part was activated with IFN-γ (200 ng·ml −1 ) and LPS (50 ng·ml −1 ) for additional 24 hr. (a) The M1 marker profile expression of CD68 and CD80 and expression of the M2 marker CD163 were determined by flow cytometry in fully differentiated nonstimulated and IFN-γ + LPS-activated cells to prove that the cells are M1 and not M2 polarized (n = 6 independent donors and experiments). Mean fluorescence intensities were calculated. One representative histogram out of six for (b) CD68, (c) CD80, and (d) CD163 is shown for fully differentiated non-stimulated M1 macrophages, IFN-γ + LPS-activated M1 macrophages and the isotype control. (e) OSM mRNA expression in fully differentiated M1 macrophages after treatment with different concentrations of histamine as indicated for 2 hr is depicted (n = 9 independent donors and experiments). (f) OSM mRNA expression in fully differentiated M1 macrophages after treatment with different concentrations of histamine as indicated for 2 hr and activated with IFN-γ (200 ng·ml −1 ) and LPS (50 ng·ml −1 ) for additional 24 hr is depicted (n = 10 independent donors and experiments). The amount of the target mRNA relative to the amount of the reference gene, rps 20 mRNA in each stimulated sample was normalized to the amount of the target mRNA relative to the amount of the reference gene in the non-stimulated sample (calibrator) and expressed as normalized ratio. This was calculated using the comparative Ct method also known as the ΔΔCt method provided by the Software LC 480 (Roche Molecular Biochemicals). Data shown are individual values with medians. In ( e) *P < .05; significantly different as indicated; Friedman Dunn's Multiple Comparisons test selected pairs. In (a) and (f), *P < .05; significantly different as indicated; Wilcoxon matched-pairs signed rank test. Hist, histamine; NS, non-stimulated; OSM, oncostatin M Our stimulation protocol represents an objective, non subjective assay and blinding is not applicable to these types of in vitro studies.
The expression of OSM was analysed at mRNA and protein level.

| Stimulation of normal human epidermal keratinocytes (NHEKs) with supernatants from histamine-stimulated M1 macrophages
The use of normal human epidermal keratinocytes (NHEKs) generated from human foreskin or outer root sheath keratinocytes (ORSKs)  NHEKs were prepared from juvenile foreskin, as described previously (Glatzer et al., 2013;Zeitvogel et al., 2012). Briefly, the foreskin was cut into pieces and incubated overnight at 37°C in 2.4 U of Dispase II (Roche, Mannheim, Germany). The next day, the epidermis was separated from the dermis and placed for 20 min at 37°C in EDTA (0.02%)trypsin (0.05%) solution (PAN-Biotech, Aidenbach, Germany). After stopping the trypsin reaction by addition of FCS (PromoCell, Heidelberg, Germany), the cell suspension was filtered through a sterile gauze (40 mm) and washed twice with PBS. The obtained single-cell suspension of NHEKs was incubated in the serum-free growth medium Keratinocyte Growth Medium 2 Kit (PromoCell) at 37°C in a humidified atmosphere containing 5% CO2. Normally, when cells in passages 3 to 7 reached 70-80% confluence, they were used for experiments or further passaged. We periodically check the typical morphology (see the photograph in Figure 7) and the expression of keratinocyte specific marker proteins at mRNA level (Zeitvogel et al., 2012).
NHEKs were seeded in 48-well plates at a density of 1.5 × 10 4 cells per well in 100-μl Keratinocyte Growth Medium and treated with 100 μl of histamine-stimulated M1 macrophage supernatants as indicated. To show that NHEKs react with STAT3 phosphorylation upon OSM stimulation, we stimulated the NHEKs directly with rh OSM (0.1 ng·ml −1 ). To analyse the phosphorylation of STAT3, NHEKs were incubated for 20 min with respective M1 macrophage supernatants following phospho-STAT3 staining using the intracellular fixation/methanol protocol with monoclonal mouse IgG2b antibody against phospho-STAT3 (Tyr 705 ; 1 μg·ml −1 ; Thermo Fischer Scientific, Waltham, Massachusetts, USA) and respective mouse isotype (IgG2b, 1 μg·ml −1 ) control in the same concentration.
2.5 | Flow cytometric analysis of M1 marker CD68, CD80, and M2 marker CD163 The antibody-based procedures used in this study comply with the recommendations made by the British Journal of Pharmacology.
Macrophages were carefully scraped from the culture plates and seeded (5 × 10 5 cells per well) into 96-well plates. Fc receptors were blocked by incubation in a buffer containing 10 μg·ml −1 heataggregated human IgG (Sigma, Deisenhofen, Germany). An extracellular epitope of CD163 was stained with anti-human CD163-PE (mouse monoclonal IgG1, 1 μg/100 μl; BioLegend, San Diego, CA, USA; RRID: AB_893269); the respective isotype control IgG1 from BioLegend was tested in parallel in the same concentration. The marker CD80 was stained with anti-human CD80-PE (mouse monoclonal IgG1, 1 μg/100 μl; BioLegend; RRID:AB_314504); the respective isotype control IgG1 from BioLegend was tested in parallel in the same concentration. Then the cells were fixed, permeabilized using the BD Cytofix/Cytoperm fixation/permeabilization kit (BD Bioscience) and CD68, which is intracellularly expressed in cytoplasmic granules, and was stained with anti-human CD68-APC (mouse monoclonal IgG2b; 1 μg·ml −1 ; BioLegend; RRID:AB_10567107); the respective isotype control (IgG2b, 1 μg·ml −1 ; BioLegend) was tested in parallel.  3.3 | Histamine up-regulates OSM mRNA expression in a dose-dependent manner in fully differentiated M1 macrophages: IFN-γ + LPS-activated M1 macrophages express higher levels of OSM mRNA than those in non-activated cells  (Figure 3f). There was a non-significant trend for histamine (10 μM) to potentiate the IFN-γ + LPS-induced expression of OSM mRNA. M1 macrophages showed the commonly accepted marker profile for these cells, expressing CD68 in a moderate, lower level but expressing CD80 highly up-regulated, when compared with non-activated cells. The M2 marker CD163 was expressed neither in fully differentiated nor in activated M1 macrophages (Figure 3a-d).

| The up-regulation of OSM mRNA expression by histamine and specific agonists is blocked by pre-incubation with antagonists targeting H 1 , H 2 and H 4 receptors in M1 macrophages
To reliably demonstrate that the up-regulation of OSM mRNA expression in human monocyte-derived M1 macrophages is mediated via the H 1 , H 2 and H 4 receptors, as shown in Figure 2, we pre-incubated GM-CSF-treated M1 macrophages with antagonists targeting the H 1 , H 2 and H 4 receptors before stimulation. Up-regulation of OSM mRNA expression by specific H 1 and H 2 receptor agonists was in part inhibited by pre-incubation with the specific antagonists, clemastine or ranitidine for the H 1 and H 2 receptor, respectively (Figure 4b,c).
The histamine-mediated up-regulation of OSM mRNA was partly inhibited by pre-incubation with the H 4 receptor-specific antagonist JNJ7777120 (Figure 4d). The histamine-induced up-regulation of OSM mRNA expression was not consistently blocked by H 1 or H 2 receptor antagonists.

FIGURE 4
The up-regulation of OSM mRNA expression by histamine or specific histamine receptor agonists in M1 macrophages is blocked by pre-incubation with antagonists targeting H 1 , H 2 and H 4 receptors. (a) Primary human monocytes were obtained from peripheral blood mononuclear cells after 2-hr adherence. M1 macrophages were differentiated from primary human monocytes in the presence of GM-CSF (10 ng·ml −1 ) for 10 days. The cells were pre-incubated with antagonists targeting the H 1 receptor (clemastine; Clem), H 2 receptor (ranitidine; Ran), and H 4 receptor (JNJ7777120; JNJ) for 30 min before stimulation and then stimulated with 2-pyridylethylamine (2-Pyr; H 1 receptor agonist), amthamine (Amt; H 2 receptor agonist), and histamine for additional 2 hr. (b) OSM mRNA expression after treatment with H 1 receptor antagonist and agonist (n = 6 independent donors and experiments). (c) OSM mRNA expression after treatment with H 2 receptor antagonist and agonist (n = 5 independent donors and experiments). (d) OSM mRNA expression after treatment with H 4 receptor antagonist and histamine (Hist; n = 7 independent donors and experiments). The amount of the target mRNA relative to the amount of the reference gene, rps 20 mRNA in each stimulated sample was normalized to the amount of the target mRNA relative to the amount of the reference gene in the non-stimulated sample (calibrator) and expressed as normalized ratio. This was calculated using the comparative Ct method also known as the ΔΔCt method provided by the Software LC 480 (Roche Molecular Biochemicals). Data shown are individual values with medians. None of the antagonists significantly inhibited the response to the agonists (P values ranged from .06 to .08; Wilcoxon matched-pairs signed rank test), although there was a consistent trend towards inhibition. Ct method, crossing point method; Hist, histamine; NS, non-stimulated; OSM, oncostatin M 3.5 | Histamine up-regulates OSM protein production in fully differentiated and in IFN-γ + LPSactivated M1 macrophages We stimulated the fully differentiated M1 macrophages with different concentrations of histamine (0.1 -100-μM) for 24 hr. The cells were re-stimulated with respective stimuli. Twenty-four hours later, supernatants were taken, and OSM protein production was measured by ELISA. OSM protein production was significantly up-regulated in samples stimulated with histamine (100, 10, and 1 μM; Figure 5a). In further experiments, M1 macrophages were left non-stimulated or stimulated for 2 hr (Figure 5b) or 24 hr (Figure 5c) with histamine (0.1 -100-μM) and then activated with IFN-γ + LPS for further 24 hr. Activation with IFN-γ + LPS led to a significant up-regulation of OSM protein production that was potentiated by histamine, at 100 and 1 μM when stimulated for 2 hr before activation by IFN-γ + LPS; Figure 5b) and at 100 μM when stimulated for 24 hr before activation by IFN-γ + LPS; Figure 5c).
3.6 | Histamine up-regulates IL-6 protein production in fully differentiated M1 macrophages: IFN-γ + LPSactivated M1 macrophages express higher levels of IL-6, TNF-α, and IL-12 than those in non-stimulated M1 macrophages We stimulated fully differentiated M1 macrophages with different concentrations of histamine (0.1 -100-μM) for 24 hr. The cells were re-stimulated with respective stimuli. Twenty-four hours later, supernatants were taken. IL-6, TNF-α, and IL-12 protein production were measured by ELISA. IL-6 protein production was significantly up-regulated in samples stimulated with histamine (10 μM ; Figure 6a). IL-12 protein was barely detected in the unstimulated supernatants (Figure 6d). In another set of experiments, M1 macrophages were left non-stimulated or stimulated for 24 hr with histamine (0.1 -100-μM) and then activated with IFN-γ + LPS for further 24 hr. Activation with IFN-γ + LPS led to a significant up-regulation of IL-6, TNF-α, and IL-12 protein production, which was not further regulated by histamine (Figure 6b-d).  Histamine up-regulates IL-6 production in fully differentiated M1 macrophages. IFN-γ + LPS-activated M1 macrophages produce higher levels of IL-6, TNF-α and IL-12 protein when compared to non-activated cells. Histamine did not potentiate IL-6, TNF-α and IL-12 protein production. Primary human monocytes were obtained from peripheral blood mononuclear cells after 2-hr adherence. M1 macrophages were differentiated from primary human monocytes in the presence of GM-CSF (10 ng·ml −1 ) for 10 days. (a) M1 macrophages were left nonstimulated or stimulated with histamine in different concentrations as indicated for 24 hr and re-stimulated in the same concentrations for additional 24 hr. M1 macrophages were left non-stimulated or stimulated with histamine in different concentrations as indicated for 24 hr and then activated with IFN-γ (200 ng·ml −1 ) and LPS (50 ng·ml −1 ) for additional 24 hr. Secretion of (b) IL-6 protein, (c) TNF-α, and (d) IL-12 protein was measured by ELISA technique. Data shown are individual values with medians from (a) n = 11 independent donors and experiments; (b) n = 12 independent donors and experiments; (c) n = 7 independent donors and experiments; (d) n = 11 independent donors and experiments. In (a), *P < .05; significant differences between non-stimulated and histamine stimulated cells; Friedman Dunn's Multiple Comparisons test selected pairs. In (b), (c) and (d), *P < .05, significant differences as indicated; Wilcoxon matched-pairs signed rank test. Hist, histamine; NS, non-stimulated; OSM, oncostatin M the cytokines produced by M1 macrophages in an inflammatory context, OSM is also up-regulated in response to LPS, GM-CSF, or PGE 2 (Repovic & Benveniste, 2002;Suda et al., 2002). OSM is a potent activator of cytokine, chemokine, or antimicrobial peptide production, as well as inducing migration in NHEKs (Finelt et al., 2005). This may play a role in the course of inflammatory skin diseases where the OSM type II receptor is expressed on keratinocytes (Boniface et al., 2007).
On one hand, an earlier study demonstrated that the proinflammatory mediator OSM was up-regulated by the anaphylatoxin C5a in human monocyte-derived M1 macrophages (Kastl et al., 2008).
On the other hand, C5a shares the characteristics of histamine regarding its properties to modulate an overwhelming immune response by down-regulating the production of cytokines of the IL-12 family in a range of inflammatory conditions (Gutzmer et al., 2005;Okazaki, Hazeki, Izumi, Nigorikawa, & Hazeki, 2011;Wittmann et al., 1999). Furthermore, activation of the transcription factor AP-1 is induced by both C5a and histamine in macrophages and dendritic cells (Gutzmer et al., 2005;Kastl et al., 2008). Therefore, we suggested that histamine may have immune modulatory functions comparable to C5a in cells of the monocyte lineage. Following our hypothesis, we investigated if histamine exerted effects on OSM production, similar to those shown for C5a in monocyte derived M1 macrophages (Kastl et al., 2008).
We stimulated GM-CSF differentiated M1 macrophages in parallel with C5a and histamine for different time periods. Analysing mRNA expression, we observed that actually, histamine mirrors the enhanced OSM mRNA expression of C5a after 2-and 4-hr treatment. In our hands, expression of OSM mRNA by C5a was upregulated only after a short stimulation time of 2 hr , whereas Kastl et al. showed a significant up-regulation of OSM mRNA expressions after 2 and 4 hr and with a peak at 8 hr (Kastl et al., 2008). Histamine up-regulated expression of OSM mRNA over a range of concentrations (0.1 -100-μM). As we demonstrated that the mRNA expression levels for H 1 , H 2 and H 4 receptors were up-regulated during the differentiation process of monocyte-derived human M1 macrophages in the presence of GM-CSF , we treated these cells with agonists and antagonists targeting these three histamine receptors. We observed that the OSM up-regulation FIGURE 7 Normal human epidermal keratinocytes (NHEKs) incubated with supernatants from histamine-treated M1 macrophages show increased STAT3 phosphorylation. Primary human monocytes were obtained from peripheral blood mononuclear cells after 2-hr adherence. M1 macrophages were differentiated from primary human monocytes in the presence of GM-CSF (10 ng·ml −1 ) for 10 days and stimulated with histamine (10 μM) for 24 hr and re-stimulated for additional 24 hr. The supernatants of stimulated M1 macrophages were added to the cultures of NHEKs for 20 min. (b) Phosphorylation of STAT3 was analysed in rh OSM (1 ng·ml −1 ) stimulated NHEKs (direct stimulation of NHEKs) by flow cytometry. (c) Phosphorylation of STAT3 was analysed in NHEKs stimulated with supernatants from M1 macrophages, as indicated, by flow cytometry. Data shown are individual values with medians from n = 6 independent donors and experiments. *P < .05; significantly different as indicated; Wilcoxon matched-pairs signed rank test. Hist, histamine; NS, non-stimulated; OSM, oncostatin M, NHEKs, normal human epidermal keratinocytes was mainly attributable to H 2 receptors with moderate contributions from H 1 and H 4 receptors. Furthermore, we could show an up-regulation of OSM and IL-6 at the protein level after stimulation with different concentrations of histamine. IFN-γ + LPS-activated M1 macrophages secreted higher amounts of OSM, IL-6, TNF-α, and IL-12 when compared to nonstimulated cells. However, histamine potentiated the IFN-γ + LPSinduced OSM production only.
Notably, histamine regulated OSM expression in concentrations ranging from 100 to 0.1 μM. We suggest that this wide concentration range of effective concentrations is due to the different affinities that have been described for the H 1 , H 2 and H 4 receptors towards histamine (Panula et al., 2015;Thurmond, 2010). We observed that the up-regulation of OSM production in response to histamine showed high donor-dependent variability. In contrast to cell lines, human primary cells reflect more an individual phenotype related to demographic or anamnestic characteristics of the donor. The response of M1 macrophages to histamine also strongly depends on the expression levels of the H 1 , H 2 and H 4 receptors, which were differentially and donor dependently regulated on these cells .
The presence of both the OSM receptor II and OSM was detected in lesions of cutaneous inflammatory skin diseases. Further, in these studies OSM induced STAT3 phosphorylation and translocation to the nucleus, which in turn is responsible for the OSM-mediated gene expressions followed by keratinocyte alterations (Boniface et al., 2007;Finelt et al., 2005). We stimulated NHEKs with supernatants from non-stimulated M1 macrophages and from cells that were treated with histamine. Intracellular staining of phospho-STAT3 revealed that supernatants from histamine-treated M1 macrophages have a significantly higher capacity to phosphorylate STAT3 in NHEKs, when compared with non-stimulated cells. These results may provide evidence that histamine triggers M1 macrophages to produce mediators, which in turn have functional relevance to modulate the activation of keratinocytes. Our study suggests that this effect is mainly attributed to the enhanced secretion of OSM and IL-6 by histamine.
In human skin, C5a is present in inflammatory conditions and has the ability to trigger the release of histamine from mast cells and basophils (el-Lati, Dahinden, & Church, 1994). In the present study, we showed that C5a and histamine, in parallel, induced OSM expression in a direct manner, in human M1 macrophages. Our data provide additional hints, in the context of results from previous studies, that C5a and histamine are tracking similar pathways in cells of the monocyte lineage.
Interestingly, the up-regulation of the pro-inflammatory mediator OSM or IL-6 by histamine is in contrast to previous observations where histamine played a more anti-inflammatory role by reducing the TLR-induced expression of cytokines of the IL-12 family (Gschwandtner et al., 2011;Gutzmer et al., 2005). The up-regulation of OSM production by both mediators may play a critical role in the regulation of pathological immune responses in skin and raises the possibility of novel target strategies in human inflammatory skin diseases. Systemic treatment of inflammatory skin diseases by cyclosporine or corticoids should only be used for short term interventions. Therefore, antagonists targeting the histamine receptors, in particular the histamine H 4 receptor, which is also involved in OSM up-regulation will attract increasing attention .