To cite this article: Kasraie S, Niebuhr M, Kopfnagel V, Dittrich-Breiholz O, Kracht M, Werfel T. Macrophages from patients with atopic dermatitis show a reduced CXCL10 expression in response to staphylococcal α-toxin.Allergy 2012; 67: 41–49.
Background: Patients with atopic dermatitis (AD) are frequently colonized with Staphylococcus aureus (S. aureus), one-third of them producing α-toxin, which is correlated with the severity of eczema in AD. Staphylococcus aureus colonizes in patients with psoriasis as well. Distinct expression of chemokine (C-C motif) ligand (CCL) and chemokine (C-X-C motif) ligand (CXCL) chemokines has been documented in both diseases. In this study, we investigated the effects of sublytic α-toxin concentrations on human macrophages that accumulate in the skin of patients with AD and psoriasis.
Methods: IFN-γ-induced protein of 10-kDa (IP-10)/CXCL10 and macrophage-derived chemokine (MDC)/CCL22 production were evaluated at the mRNA or at the protein level using qRT–PCR or ELISA, respectively. Cell surface markers’ expression and chemotaxis were determined by flow cytometry and Boyden chamber technique, respectively.
Results: Sublytic concentrations of α-toxin strongly induced CXCL10 in macrophages at both the mRNA and the protein levels and significantly up-regulated MHC class II expression. Supernatants of α-toxin-stimulated macrophages induced the migration of human CD4+ lymphocytes via the CXCL10 receptor (CXCR3). Macrophages from patients with AD produced lower levels of CXCL10 compared to cells from patients with psoriasis as well as healthy controls in response to α-toxin. α-Toxin did not lead to a large variation in CCL22 production in macrophages from all three groups.
Conclusions: Staphylococcal α-toxin contributes to Th1 polarization by induction of CXCL10 in macrophages. Macrophages from patients with AD and psoriasis responded to α-toxin in the induction of Th1-related chemokine CXCL10 diversely, which could favour the recruitment of distinct leucocyte subsets into the skin.
chemokine (C-C motif) ligand
chemokine (C-C motif) receptor
chemokine (C-X-C motif) ligand
chemokine (C-X-C motif) receptor
human leucocyte antigen
IFN-γ-induced protein of 10 kDa
major histocompatibility complex
Psoriasis Area and Severity Index
- S. aureus
Staphylococcal enterotoxin B
tumour necrosis factor alpha
Atopic dermatitis (AD) and psoriasis are common chronic inflammatory skin disorders in which T-cell-mediated mechanisms have a crucial role in pathogenesis (1–3). Patients with AD exhibit exaggerated Th2 responses, and initiation of AD lesions is thought to be mediated by means of early skin infiltration of Th2 lymphocytes releasing high levels of IL-4, IL-5 and IL-13 (1, 4). Subsequently, the accumulation of activated monocytes, mature dendritic cells and eosinophils determines a rise in IL-12 expression and the appearance of a mixed Th2/Th1 cytokine pattern, with reduced IL-4 and IL-13 and the presence of IFN-γ in the chronic phase (1, 4). By contrast, psoriasis is characterized by numerous activated Th1 cells, focal intra-epidermal collections of neutrophils, and dermal accumulation of macrophages and dendritic cells. IFN-γ-producing Th1 cells dominate psoriatic lesions and are primarily responsible for epidermal changes (3, 5). Recently, the pathogenesis of psoriasis has evolved from a classical type 1 (Th1) disease activated by IFN-γ to include a new T-cell subset, Th17 cells, as well (3).
Recent studies demonstrated that infiltration of inflammatory cells into tissues is regulated by chemokines. Two main subfamilies, recently renamed chemokine (C-C motif) ligand (CCL) and chemokine (C-X-C motif) ligand (CXCL) chemokines, have been distinguished (6). Several studies have suggested a crucial involvement of CCL chemokines in allergic inflammation because they potently attract eosinophils, basophils, monocytes and T-cell subsets (6). By contrast, CXCL chemokines attract primarily Th1 lymphocytes and neutrophils. Chemokines and their receptors are implicated in the development of symptoms of AD and psoriasis (6–8). A subset of chemokines including CCL1, CCL11, CCL13, CCL17, CCL18, CCL22 and CCL27 are highly expressed in AD (6, 7), whereas a distinct chemokine network such as CCL2, CCL3, CCL4, CCL5, CCL17, CCL19, CCL20, CCL27, CXCL7, CXCL8, CXCL9 and CXCL10 plays a role in pathogenesis of psoriasis (8, 9). Patients with AD and psoriasis are frequently colonized with Staphylococcus aureus (10, 11).
A distinct percentage of S. aureus strains are able to produce α-toxin, a potent 33-kDa cytolysin which does not belong to the group of enterotoxins (superantigens) (12). In a former study, we found 30%α-toxin-producing S. aureus strains isolated from the skin of untreated patients with AD (13). More recently, we investigated 127 patients with AD who were undergoing standard anti-inflammatory and antiseptic treatment, and found a skin colonization of 63% with α-toxin-producing S. aureus in these patients (14). In psoriasis, Marples et al. (15) detected S. aureus in nonlesional skin of 27% of patients and in lesional skin of 46% of patients. Tomi et al. (11) investigated 25 patients with psoriasis: the skin of 60% of patients was positive for S. aureus, and isolated S. aureus strains were toxigenic for staphylococcal enterotoxin A-D in 36%. It has been shown that staphylococcal toxigenic strains isolated from both lesional skin and nares of patients with psoriasis were significantly higher than those of healthy controls. A significant relationship between PASI scores and toxin production was also demonstrated (11, 16). Macrophage infiltration is a hallmark of inflammation, especially in the skin where they can mediate chronic inflammation by producing cytokines and chemokines. This is seen in several inflammatory dermatoses including psoriasis and AD (17).
The effects of α-toxin on human macrophages in general and in chronic skin inflammation in particular still remain unclear. We show here for the first time that sublytic α-toxin induces the Th1-related chemokine CXCL10 in human macrophages. Moreover, we studied the effects of α-toxin on Th1- and Th2-related chemokines in macrophages from patients with AD and psoriasis where the intrinsic abnormal and different chemokines production profile is well defined.
Materials and methods
Peripheral blood samples were taken from healthy donors and patients with AD or psoriasis after obtaining informed consent, respectively, presented to our outpatient and inpatient department, as previously described (18). Atopic dermatitis was determined by the diagnostic criteria described by Hanifin & Rajka (19). Further information is provided in Online Repository (OR).
Cell isolation and culture
Peripheral blood mononuclear cells (PBMCs) were isolated by Lymphoprep density-gradient centrifugation from healthy donors and from patients with AD and psoriasis as previously described in OR. To culture macrophages, CD14+ cells were purified by negative selection according to the manufacturers’ instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were cultured in ‘macrophage medium’. Further information is provided in OR.
Stimulation of macrophages
Macrophages were either unstimulated or stimulated for various periods of time with lyophilized staphylococcal α-toxin (Sigma-Aldrich, Deisenhofen, Germany) in sublytic concentrations, recombinant human IFN-γ (10 ng/ml; ImmunoTools, Friesoythe, Germany) or TNF-α (20 ng/ml; ImmunoTools). Lipopolysaccharide (LPS) was not detected in any reagent, as determined by the Limulus amebocyte assay (Haemochrom Diagnostika, Essen, Germany).
Flow cytometric analysis was performed as described previously (18). Further information is provided in OR.
Cell viability assay
Approximately 3 × 105 macrophages were prestimulated with α-toxin in a dose-dependent manner. Cell viability was assessed by flow cytometry by staining with 7-amino-actinomycin D (7-AAD) (BD Biosciences, Heidelberg, Germany) after 10–15 min to determine the percentage of viable cells that were treated with α-toxin in a dose-dependent manner. 7-amino-actinomycin D intercalates into double-stranded nucleic acids. It is excluded by viable cells but can penetrate cell membranes of dying or dead cells. Viability of cells with <1 μg/ml α-toxin stimulation was not affected in macrophages. Therefore, we chose <1 μg/ml for our investigations. Figure S1 shows one representative of five independent experiments.
mRNA isolation, reverse transcription, quantitative RT–PCR and microarray experiments
RNA isolation, reverse transcription and quantitative real-time PCR were performed as previously described (18). Further information is provided in OR.
Microarray experiments were performed with the human inflammation microarray (Ocimum Biosolutions, Corporate Office Europe, Ijsselstein, The Netherlands), which contains 136 gene probes for inflammatory genes and 19 gene probes for housekeeping genes (13, 20, 21).
Cytokine assessment by ELISA
Cell-free culture supernatants were harvested and analysed for CXCL10 or CCL22 (each Duo Set; R&D-Systems, Minneapolis, MN, USA), using a commercially available enzyme-linked immunosorbent assay (ELISA) system following the manufacturer’s instructions.
Immunohistochemistry was analysed as described previously (21). Further information is provided in OR.
The chemotactic activity of supernatants from α-toxin-stimulated macrophages on lymphocytes (CD4+ T cells) was determined using a Boyden chamber technique as previously described (21). Further information is provided in OR.
A total of 106 macrophages were prestimulated with α-toxin (100 ng/ml), IFN-γ (10 ng/ml), TNF-α (10 ng/ml) or LPS, which was derived from Escherichia coli serotype 055:B5 (500 ng/ml; Sigma-Aldrich) for various periods of time (5, 15, 30, 60 min or 2, 4, 6, 24 h). Cell extracts were subjected to Western blot analysis as described previously (21). Further information is provided in OR.
Data were analysed with either the Student’s t-test (normality test passed) or the Mann–Whitney rank sum test (normality test failed). P-values below 0.05 were regarded as significant. P < 0.05 is depicted with *, P < 0.01 with ** and P < 0.001 with ***. The program GraphPad prism version 3.02 (GraphPad Software Inc., San Diego, CA, USA) and the software Sigma stat for Windows (Systat Software, San Jose, USA) were used for statistical analysis.
Induction of IP-10/CXCL10 by staphylococcal α-toxin in human macrophages
To screen for the potential effects of sublytic α-toxin concentrations that did not induce cell death (Fig. S1), we investigated the expression of a selected set of 136 genes with known relevance to inflammation (13, 20, 21), such as cytokines, chemokines and matrix metalloproteinases in purified macrophages derived from healthy controls which had been treated with α-toxin (100 ng/ml) for 24 h. Microarray analysis revealed that CXCL10 was the most strongly up-regulated gene in macrophages (215.1-fold induction) (Table S1). In the same experimental set-up, the mean ratio for CXCL9, CCL8 and CCL15 were 11.6, 8.8 and 3.8, respectively. An increased expression was not observed for other chemokines (including CCL2, CCL3, CCL4, CCL5, CCL7, CCL11, CCL20, CCL27, CXCL3, CXCL5 and CXCL8). The mean ratio for all these chemokines was between 0.2 and 2 when comparing not stimulated with α-toxin-stimulated macrophages (Table S1). CXCL10 expression and secretion following α-toxin stimulation were investigated at the mRNA level as determined by quantitative RT–PCR (Fig. 1) as well as at the protein level as determined by ELISA (Fig. 2). As shown in Fig. 1A, an efficiency-controlled quantitative real-time PCR for CXCL10 confirmed the microarray results. A marked up-regulation of CXCL10 was observed both after 6 and 24 h following α-toxin stimulation at the mRNA level in macrophages derived from healthy controls (Fig. 1). Low concentrations of α-toxin (10 ng/ml) also up-regulated CXCL10 in macrophages derived from healthy controls (data not shown). Higher concentrations of α-toxin (10, 20 μg/ml) led to reduction in the expression of CXCL10 dramatically, which was because of cell death (data not shown). IFN-γ and TNF-α, which have been shown previously to induce CXCL10 in human monocytes (22), were used as positive controls. α-Toxin was a stronger inducer of CXCL10 compared with TNF-α but not with IFN-γ at mRNA level (Fig. 1). As shown in Fig. 2, our mRNA data could also be reproduced by ELISA at the protein level: A higher amount of CXCL10 was already observable after 24 h stimulation and with α-toxin, which was stable after 48 and 120 h (Fig. S2A). This induction was observable in a dose-dependent manner (10, 50, 100 ng/ml, 1 μg/ml) in macrophages from healthy controls (Fig. S2B). α-Toxin induced CXCL10 secretion in a similar manner to IFN-γ, whereas TNF-α was ineffective in induction of CXCL10 at the protein level (Fig. 2). To compare in vitro data with ex vivo findings, we used 3-mm punch biopsies from healthy individuals and cultured them in keratinocyte growth medium with or without α-toxin (100 ng/ml) or IFN-γ (100 ng/ml). After 24 h of incubation, formalin-fixed and 5-μm paraffin-embedded biopsy sections were stained for CXCL10 as well as CD68. CXCL10 staining was more prominent in the dermis in α-toxin-stimulated biopsies as compared to unstimulated biopsies. IFN-γ was used as a positive control for CXCL10 staining. CD 68 staining points to infiltration of macrophages in the dermis (Fig. 3).
α-Toxin-stimulated macrophages could induce the migration of human CD4+ T cells via CXCR3
The biological activity of CXCL10 induced by staphylococcal α-toxin was examined by means of a chemotaxis assay (Boyden chamber). As shown in Fig. 4A, supernatants of α-toxin-stimulated macrophages induced significantly the migration of CD4+ T cells. CD4+ T lymphocytes were pretreated with a blocking anti-CXCR3 antibody known to neutralize the biological activity of surface CXCR3, or IFN-γ-induced protein of 10-kDa (IP-10) secretion was directly blocked using an anti-IP-10 antibody. Both inhibitors abrogated the migration induced by α-toxin-treated macrophage supernatants (supernatants from unstimulated macrophages attracted a mean of 267 000 T cells, and supernatants from α-toxin [100 ng/ml]-stimulated macrophages attracted a mean of 360 000 cells compared with 193 000 or 237 000 cells when pretreated with neutralizing CXCR3 or IP-10 antibodies, respectively) (Fig. 4B). α-Toxin in cell culture medium without macrophages did not directly induce the migration of human CD4+ lymphocytes. Medium free of cells was used as a negative control (Fig. 4B). IL-27-stimulated keratinocytes that were shown to induce CXCL10 were used as a positive control (data not shown) (21). Isotype controls of neutralizing antibodies did not abrogate the migration induced by α-toxin-treated macrophages (data not shown).
α-Toxin could up-regulate HLA-DR (MHC class II) expression in macrophages
α-Toxin induced a moderate up-regulation of MHC class II on the cell surface of macrophages (Fig. S3). This effect was visible on cells from healthy donors and from patients with AD or psoriasis (data not shown). No effect was detected on CD14+, CD64+, CD80+, and CD86+ expression (data not shown).
Low effect of α-toxin on CXCL10 induction (Th1-related chemokine) in macrophages from patients with AD
Macrophages from patients with AD and psoriasis as well as from healthy controls were either left unstimulated or were stimulated with 10, 50, 100 ng/ml or 1 μg/ml α-toxin, respectively, for the indicated time points. To compare α-toxin effects in CXCL10 production with other previously described inducers, cells were stimulated with IFN-γ or TNF-α as well.
CXCL10 expression and secretion following α-toxin stimulation were investigated at the mRNA level as determined by quantitative RT–PCR as well as at the protein level as determined by ELISA. α-Toxin increased CXCL10 expression as well as secretion in macrophages from patients with AD and psoriasis and from healthy controls compared with the appropriate medium control both at the mRNA (Figs 5A and S4) and at the protein levels (Fig. 5B–D). However, α-toxin induced significantly lower levels of CXCL10 expression (Figs 5A and S4) or secretion (Fig. 5B–D) in patients with AD compared with healthy controls as well as psoriasis at all time points and doses tested. There was no significant difference in CXCL10 induction between psoriasis and healthy controls following α-toxin stimulation (Fig. 5A–D). In contrast to α-toxin, IFN-γ strongly enhanced CXCL10 secretion in AD as well (Fig. 5C and D).
IFN-γ that was used as a positive control stimulus was a stronger inducer of CXCL10 in macrophages from patients with psoriasis compared to healthy controls and patients with AD by trend (Fig. 5C and D). No significant effect of TNF-α on CXCL10 production was found in macrophages from all three groups (Fig. 5C and D).
Effect of α-toxin on MDC production (Th2-related chemokine) in macrophages from patients with chronic inflammatory skin diseases
Based on the effect of α-toxin on Th1-related chemokine (IP10/CXCL10) production in macrophages, we investigated the Th2-related chemokine (macrophage-derived chemokine, MDC/CCL22) production.
We chose CCL22 that plays a crucial role in pathogenesis of AD. We recently showed that IL-13-stimulated human primary keratinocytes (HPKs) express CCL22, which preferentially attract CD4+ CCR4+ T cells. Furthermore, CD4+ CCR4+ T cells from patients with AD showed higher chemotactic indices towards IL-13-stimulated HPKs than those from healthy individuals (23).
Macrophages from patients with AD as well as patients with psoriasis showed an intrinsically higher CCL22 production compared to healthy controls by trend. On the whole, α-toxin did not regulate CCL22 secretion. A slight increase of 17% of CCL22 was observed in patients with AD and psoriasis, while there was a slight decrease (significant) in CCL22 secretion in cells from HC (Fig. 6).
There was no significant difference in CCL22 production following α-toxin stimulation between patients with AD and psoriasis (Fig. 6).
Atopic dermatitis and psoriasis are the most common immune-mediated chronic inflammatory skin diseases with frequent colonization with S. aureus which correlates with disease severity (10, 11). More recently, we described a skin colonization of 63% with α-toxin-producing S. aureus in patients with AD undergoing anti-inflammatory and antiseptic treatment (14). In higher concentrations, α-toxin may lead to necrosis in a wide range of human cells by forming small pores (1–2 nm diameter) into cell membranes (13). Besides this well-described mechanism of α-toxin lysis (24), subcytocidal concentrations of α-toxin seem to induce pro-inflammatory (IL-1β and TNF-α production) effects in a variety of cells involved in inflammatory processes, for example monocytes (25), polymorphonuclear cells (26) and endothelial cells (27), presumably because of the alteration in the cellular ion balance following pore formation, specifically because of calcium influx (28). Previously, we showed that sublytic concentrations of α-toxin were able to effectively activate T lymphocytes, which led to proliferation and induction of IFN-γ (13).
In this study, we demonstrate for the first time an inflammatory effect of α-toxin on induction of the Th1-related chemokine CXCL10 both in vitro and ex vivo and up-regulation of HLA-DR expression in human macrophages. The biological activity of α-toxin-induced CXCL10 which was measured by means of a chemotactic assay and blocking experiments was confirmed by resulting in the attraction of CXCR3+ Th1 cells as well. Moreover, the lack of up-regulation of the Th2-related chemokine CCL22 following α-toxin stimulation is in favour of a role of α-toxin in human Th1 responses. This indicates that besides pore-forming abilities, sublytic concentrations of α-toxin have immunological properties and therefore may serve as a pathogenic factor in chronic inflammatory skin diseases.
CXCL10 is secreted by activated T cells, monocytes, endothelial cells, keratinocytes and fibroblasts (22, 29). It belongs to the CXC chemokine subfamily and acts through CXCR3 receptors that attract T lymphocytes (22, 29, 30).
Very few studies have investigated the production pattern of CXCL10 in skin-homing cells other than keratinocytes (29, 30), such as macrophages, or their role in chronic inflammatory skin diseases such as AD and psoriasis.
One study investigated the mechanisms regulating CXCL10 production in the human monocytic cell line (THP-1 cells) following treatment with IFN-γ and TNF-α alone or in combination (22). They showed that TNF-α has more potential than IFN-γ to induce CXCL10 production in human THP-1 monocytes. In addition, IFN-γ synergistically enhances TNF-α-induced production of CXCL10 by activating signal transducer and activator of transcription factor (STAT)-1 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) through Janus-activated kinases (JAKs) pathways (22).
In contrast, we showed that IFN-γ and α-toxin are indeed stronger inducers of CXCL10 production compared to TNF-α both at the mRNA and at the protein levels in human macrophages. Based on the strong induction of CXCL10 following α-toxin stimulation, we investigated possible signalling pathways that are involved in CXCL10 production including STAT-1, extracellular signal-regulated kinase (ERK) and NFκB (22). However, we failed to detect activation of these molecules following α-toxin stimulation compared with not treated macrophages or using positive controls such as IFN-γ, TNF-α or LPS.
α-Toxin could not activate STAT-1, ERK 1/2 or NF-κB compared with unstimulated macrophages, whereas IFN-γ that was used as a positive control for STAT-1, and TNF-α or LPS which was used as a positive control for ERK1/2 or NF-κB activated clearly ERK1/2 and NF-κB, respectively (Fig S5A–C). Several previous studies have shown that TNF-α induced phosphorylation and degradation of IκB-α, thereby induced phosphorylation and nuclear translocation of NF-κB depending on the cell type (22). However, we failed to detect α-toxin-induced phosphorylation and degradation of IκB-α in macrophages compared with unstimulated or TNF-α-treated cells as well (data not shown). Moreover, the c-JUN–JNK complex has been implicated in the regulation of CXCL10 gene expression (31). We also investigated the possible involvement of c-Jun N-terminal kinases/stress-activated protein kinase (JNK/SAPK) signalling pathway in CXCL10 induction following α-toxin stimulation. In our experiments, staphylococcal α-toxin did not activate c-JUN as well as JNK phosphorylation in human macrophages (data not shown). The intracellular mechanism of cell activation by sublytic doses of proteins forming small pores such as α-toxin is not clear yet. Both direct actions on toll-like receptors on cell membranes and intracellular activities such as the activation of caspase-1 through NLRP-3 inflammasomes have been discussed (32, 33).
Chemokines and their receptors are implicated in the pathogenesis and maintenance of AD. The imbalance in serum concentration of Th1- and Th2-derived chemokines is one of the factors involved in pathogenesis of AD.
However, discrepancies obtained in many studies regarding the imbalance in chemokine serum levels in patients with AD and relatively small number of the patients included did not allow drawing equivocal conclusions (29, 34, 35). Narbutt et al. (34) found significant lower serum concentration of CXCL9, CXCL10 and CCL17 and higher concentrations of CXCL12, CCL22 and CCL27 in patients with AD compared to healthy controls.
These findings are in line with our observations that show the alteration of the Th1 axis in AD following α-toxin stimulation and might support Th2 responses.
An interesting observation of this study is that macrophages from patients with AD produced similar amounts of CXCL10 in response to IFN-γ but reduced amounts of CXCL10 in response to α-toxin when compared with macrophages from healthy controls or from patients with psoriasis. In contrast, macrophages from patients with psoriasis responded to the same stimuli with an exaggerated expression of CXCL10 production. These results may indicate that in addition to the general existence of genetically determined defects in the constitutive and induced chemokine production in patients with AD and psoriasis, this abnormal production appeared under a stimulus-specific control rather than complex chemokine-associated. Giustizieri et al. showed that CXCL10 was markedly expressed in the epidermis of patients with psoriasis vulgaris but only weakly and limited to some areas in AD lesions. They concluded that the higher expression of CXCL10 in the epidermis of patients with psoriasis compared with that of patients with AD likely reflects the presence of more numerous Th1 cells in the former, because IFN-γ is the most potent inducer of CXCL10 (30). In addition to this explanation, our data indicate that different responses to the staphylococcal exotoxin α-toxin in macrophages could be another factor that may play a role in the distinct pathogenesis of AD and psoriasis in CXCL10 induction.
Staphylococcal α-toxin contributes to Th1 polarization by induction of CXCL10 in macrophages. Thereby, it provides pathways linking adaptive and innate immune functions. In conclusion, our data support the hypothesis that the contribution of macrophages in the pathogenesis of AD and psoriasis is linked to the presence of distinct alterations in their capacity to respond to the staphylococcal exotoxin α-toxin and that these abnormalities can modulate the amplification and persistence of chronic skin inflammation. Further studies should be performed to clarify the mechanisms of macrophage activation by staphylococcal α-toxin.
This study was supported by grants of the Deutsche Forschungsgemeinschaft (Research Training Group (GRK) 1441/1) and SFB 566, A6). We would like to thank Gabriele Begemann, Kathrin Baumert and Manuela Gehring for their excellent technical assistance.
Conflict of interest
The authors state no conflict of interest.