Introduction

Skin inflammation and remodeling are important pathophysiological features of chronic eczematous skin diseases such as allergic contact dermatitis and atopic dermatitis (AD). Skin lesions in such eczematous disorders are characterized by the recruitment of inflammatory cells, especially cutaneous lymphocyte-associated antigen-positive activated memory/effector T-lymphocytes, monocytes, and mast cells (Werfel et al., 1996a; Leung et al., 2003). Migration of inflammatory cells into the epidermis and mobilization of Langerhans cells (LCs) in and out of the epidermis involve the complex regulation of cell–cell and cell–matrix interaction by means of adhesion molecules, matrix metalloproteinases (MMPs), and cytokines (Wittmann and Werfel, 2006).

IL-13 has been shown to be a crucial mediator of Th-2-dominant immune responses (Wynn, 2003). In inflammatory skin conditions, IL-13 is produced by infiltrating CD4⁺ T cells and mast cells (Leung and Boguniewicz, 2003). In murine models of asthma and in monocytes and fibroblasts, IL-13 has been shown to modulate the expression of cytokines, chemokines, and MMPs by means of which cell migration is facilitated and tissue fibrosis may be induced (Zhu et al., 1999; Lee et al., 2001a). Recently, we have shown that IL-13-stimulated human primary keratinocytes (KCs) attract CD4⁺CCR4⁺ T cells by increased secretion of CCL22 (Purwar et al., 2006). A higher number of IL-13-expressing CD4⁺ T cells in peripheral blood and in the skin compartment have been observed in AD patients and correlated with the severity of AD in children (Hamid et al., 1996; Nakatani et al., 2001). However, very little is known about the modulation of cell–cell and cell–matrix interaction by IL-13 in the epidermal compartment.

MMPs are a family of Zn-dependent proteases with common functional and structural properties (Goetzl et al., 1996). MMP-9, also known as 92 kDa gelatinase/type IV collagenase, is one of the most important endopeptidases involved in the pathophysiology of tumor invasion and metastasis, in bullous skin diseases, contact hypersensitivity, toxic epidermal necrolysis, chronic inflammatory diseases, and in UVB-related dermatoses (Kobayashi, 1997; Liu et al., 1998; Wang et al., 1999; McCawley and Matrisian, 2001; Giannelli et al., 2002; Onoue et al., 2003; Gaultier et al., 2004; Ram et al., 2006). In the skin compartment, eosinophils, mast cells, LCs, and KCs express MMP-9 and MMP-3 (Kobayashi, 1997; Schwingshackl et al., 1999; Onoue et al., 2003; Kobayashi, 2005; Sawicki et al., 2005). MMP-9 cleaves fibronectin, elastin, collagen IV of the basement membrane, and degrades type VII collagen, the major structural component of the anchoring fibrils (Atkinson and Senior, 2003). MMP-9, like other MMPs, is secreted from cells as an inactive zymogen. Cell surface association of MMP-9 has been described for a number of cells including KCs (Makela et al., 1998; Fridman et al., 2003). MMP-3, known as stromelysin, is an effective activator of proMMP-9, but the broad substrate specificity of MMP-3 also includes laminin, collagen type IV, fibronectin, and proteoglycans (Ogata et al., 1992). Recently, it has been demonstrated that MMP-9 enables the emigration of LCs from the basal membrane to the lymph node and migration of inflammatory cells into the epidermis (Ratzinger et al., 2002). Thus MMPs play a critical role in the migration of immunocompetent cells into and out of the epidermal compartment.

In this study, we examined the hypothesis that IL-13 can influence epidermal tissue remodeling in human skin by its impact on KCs. Here we demonstrate that KCs respond to IL-13 with increased MMP-9 expression. Interestingly, we observed a coexpression of MMP-9 and IL-13 in biopsies taken from patients with acute eczema. On the basis of recent evidences, we conclude that IL-13 in KCs may contribute to the initiation and chronification of skin inflammation also by means of degradation of the basement membrane, and thus facilitating migration of inflammatory cells into the epidermis, which is a hallmark feature of eczema.

Results

IL-13 induces MMP-9 in epidermal KCs

To study the effect of IL-13 on MMP-9 expression, KCs were subjected to IL-13 (50 ng/ml), IFN- (10 ng/ml), tumor necrosis factor- (TNF-) (10 ng/ml), IL-1 (10 ng/ml), and tissue growth factor-1 (TGF-1) (1 ng/ml) or combinations of these cytokines for 6 hours. The mRNA level of MMP-9 was then determined by quantitative real-time PCR (qRT-PCR). IL-13 upregulated the MMP-9 mRNA by two- to threefold (Figure 1a). We could confirm the induction of MMP-9 by TGF-1 as shown previously (Salo et al., 1991); however, we could not observe additive or synergistic effects of IL-13 and TGF-1 on MMP-9 induction (Figure 1a). Furthermore, IFN- and IL-1 alone or in combination with IL-13 did not induce MMP-9 in human KCs (data not shown).

Figure 1.

IL-13 induces MMP-9 in epidermal KCs and facilitates trans-Matrigel migration of lymphocytes. KCs were left unstimulated (ns) or stimulated with the indicated cytokines (IL-13 50 ng/ml, TGF-1 1 ng/ml) for 6 hours (RNA) and 24 hours (protein). MMP-9 induction was determined by (a) qRT-PCR or (b) ELISA-based assay (n=7). Data are shown as mean valueSEM. (c) For analysis of trans-basement membrane migration, purified CD4+ lymphocytes were added to the upper chamber of a Matrigel insert along with IL-13 (50 ng/ml)-stimulated or non-stimulated (ns) KCs. Chemokines were added to the lower chamber and cells migrated after overnight incubation were analyzed using Trucount tubes. Number of cells per 500 beads counted is depicted (meanSEM of n=12).

Full figure and legend (56K)

As MMP-9 is secreted from the cells as an inactive pro-form, we were interested in the functional significance of the observed mRNA induction. We investigated the bioactivity of MMP-9 in cell-free supernatants of KCs using an ELISA-based assay (BioTrak Activity Assay; Figure 1b). KCs were stimulated with IL-13 (50 ng/ml) or IL-4 (50 ng/ml) in combination with or without other cytokines for 24 hours. As shown in Figure 1b, we could confirm our mRNA data on the protein level. Both IL-13 and IL-4 induced a functionally active form of MMP-9 in KCs. Previously, TNF- and TGF-1 have been shown to induce MMP-9 in KCs (Salo et al., 1991; Han et al., 2001). To determine whether IL-13 modulates TGF-1- or TNF--induced MMP-9 in KCs, we stimulated KCs with IL-13 and TGF-1 or TNF-. We could not observe additive/synergistic effects of IL-13 and TNF- on MMP-9 induction in this setting (data not shown).

Dose kinetic studies revealed that 50 and 100 ng/ml of IL-13 were most effective in inducing the release of active MMP-9, with 50 ng/ml giving the most consistent results (standard error of mean (SEM) pg/ml for medium=102 (68), IL-13 1 ng/ml=206 (127), IL-13 10 ng/ml=87 (74), IL-13 50 ng/ml 232 (70), IL-13 100 ng/ml 271 (73), and TGF-1 529 (99)). A significant induction (P<0.05) of active MMP-9 secretion was observed when 50 or 100 ng/ml IL-13 or 1 ng/ml TGF-1 was incubated with KCs over a period of 24 hours. Longer incubation time (e.g., 48 or 72 hours) neither lead to higher MP-9 release nor to more MMP-9 intracellular accumulation as determined by flow cytometric analysis. Comparison between total and active form of MMP-9 showed that only about 5% total MMP-9 was endogenously active.

To compare in vitro data with ex vivo findings, we used 3 mm punch biopsies from healthy individuals and cultured them in KCs growth medium with or without IL-13 (50 ng/ml). After 24 hours of incubation, formalin-fixed and paraffin-embedded biopsies were stained for MMP-9 and collagen type IV. MMP-9 staining was more prominent in the basal epidermal layer in IL-13-stimulated biopsies as compared to unstimulated biopsies (Figure 2). The collagen type IV staining points to a degradation of this major basement membrane constituent in IL-13-stimulated biopsies as compared with unstimulated control (Figure 2).

Figure 2.

Immunohistochemistry of MMP-9 in epidermis. Punch biopsies (3 mm) from healthy individuals were incubated with IL-13 (50 ng/ml) for 24 hours at 37°C. Paraffin sections were stained for MMP-9 and collagen type IV as described in Materials and Methods. A representative set of MMP-9 (bar=40 m) and collagen type IV staining out of six experiments is depicted.

Full figure and legend (177K)

Intracellular staining of MMP-9 gave positive results only if cells were stimulated with PMA 2 hours before measurement (in addition to overnight stimulation with IL-13 or TGF-1). In this setting, both IL-13 and TGF-1 could increase intracellular MMP-9 expression (data not shown). IL-13 and TGF-1 also induced MMP-9 on the cell surface, as shown by flow cytometry (Figure 3a). Dose–response curves showed that 50 ng/ml IL-13 consistently induced MMP-9 surface expression. The level of MMP-9 induction observed was in the same range as observed for TGF-1. We performed a functional assay to confirm that IL-13-stimulated KC can enhance the trans-basement membrane migration of lymphocytes. For this purpose, we used a basement membrane equivalent (Matrigel™) and analyzed the number of migrated purified CD4+ lymphocytes in the presence of KC stimulated with IL-13 (50 ng/ml) or left unstimulated. Chemokines were added to the lower well to induce T-cell migration (Figure 1c). Experiments performed in the presence of the MMP inhibitor galardin reduced the migration of T cells in untreated cells by 52% and in IL-13-treated cells by 79% in one experiment and by 21% (untreated cells) and 47% (IL-13-treated cells) respectively in a second experiment. Previous studies have shown that IL-13 influences tissue remodeling by inducing TGF-1. Therefore, KCs were stimulated with IL-13 (50 ng/ml) for 24 hours and thereafter TGF-1 was measured in cell-free supernatant. Only a slight increase in the synthesis of TGF-1 was detected after IL-13 stimulation in KCs (unstimulated KCs: 42.268.62 pg/ml; IL-13-stimulated KCs: 58.659.98 pg/ml; n=8). These findings point to the fact that autocrine TGF-1 seems to be not critically involved in MMP-9 production in KCs. This notion is also supported by the MMP-9 mRNA induction (e.g., after 6 hours).

Figure 3.

Surface expression of MMP-9. KCs were left unstimulated (ns) or stimulated with different doses of IL-13 or 1 ng/ml TGF-1 as a control. MMP-9 surface staining was performed 16 hours later. (a) Mean values of cells positive for MMP-9SEM of 10 independent experiments are depicted. (b) A representative histogram out of these experiments is shown.

Full figure and legend (66K)

Besides MMP-9, we also investigated the effect of IL-13 on another gelatinase, MMP-2 in KCs. We detected neither MMP-2 mRNA in KCs nor the modulation of MMP-2 expression by IL-13 (data not shown). Furthermore, MMP-3, which can activate proMMP-9, was also studied. However, IL-13 stimulation of KCs (dose range 10–100 ng/ml) for 6 hours could not upregulate MMP-3 consistently (data not shown).

Coexpression of MMP-9 and IL-13 in eczema patients

As our data suggest that IL-13 upregulates MMP-9 in KCs, we were interested in investigating the expression of IL-13 and MMP-9 in biopsies taken from lesional eczematous skin. IL-13 and MMP-9 mRNA expressions were analyzed by qRT-PCR in 11 eczema patients. Out of 17 biopsies, 10 were taken from acute lesions and 7 from chronic lesions as assessed by clinical appearance (described in Materials and Methods). RNA was isolated from these biopsies and expression of MMP-9 and IL-13 was investigated using qRT-PCR (Figure 4). As expected, we observed a higher expression of IL-13 in acute as compared to chronic eczema. However, a higher expression of MMP-9 could also be detected in acutely inflamed skin as compared to chronic eczematous skin lesions. These sets of experiments demonstrate the co-expression of IL-13 and MMP-9 during the acute phase of skin inflammation.

Figure 4.

Expression of MMP-9 and IL-13 in eczematous patients. From patients suffering from acute or chronic phase of allergic eczematous skin disease, 2 mm punch biopsies were taken. RNA was isolated and subjected to qRT-PCR for MMP-9 and IL-13. Data are depicted as normalized ratio of MMP-9 or IL-13 versus glyceraldehyde-3-phosphate dehydrogenase. Median values are depicted.

Full figure and legend (11K)

Discussion

During inflammation, migration of cells is tightly regulated by the coordinated expression of proteinases, cytokines, and chemokines. There are evidences suggesting that MMPs are also important for the recruitment of cells at the site of inflammation (Lee et al., 2001a; McMillan et al., 2004). In this study, we have demonstrated that IL-13 increases the production of MMP-9 by human KCs. This may contribute to the migration of inflammatory cells into the epidermis through the basement membrane. This study further describes the coexpression of IL-13 and MMP-9 in acute lesions of eczema. CD4⁺ T cells are major sources of IL-13 in lesional skin and in the peripheral blood of AD (Hamid et al., 1996). During acute skin inflammation, T cells and T-cell-derived cytokines come in contact with KCs in the epidermal compartment.

Induction of MMP-9 by IL-13 might be relevant in several skin diseases. In MMP-9-deficient mice, no blister formation was described using the bullous pemphigoid model, suggesting an important role of MMP-9 in epidermal splitting at the level of basement membrane (Liu et al., 1998). MMP-9 has been implicated in the pathophysiology of lung diseases (Atkinson and Senior, 2003). In accordance with our data, it seems mainly expressed during the acute phase or upon allergen challenge (Kelly et al., 2000; Lee et al., 2001b, 2006; Belleguic et al., 2002; Mattos et al., 2002; Cataldo et al., 2002a; Han et al., 2003; Wenzel et al., 2003). However, there is still some controversy about the net effect of the action of MMP-9 in this disease state. In some murine models of asthma, the presence of MMP-2 and MMP-9 seems to be not required for the development of the allergic and obstructive lung phenotype (Corry et al., 2002; Corry et al., 2004; McMillan et al., 2004), whereas others emphasize the functional importance of MMP-9 for disease progression/development (Lee et al., 2001c, 2004; Cataldo et al., 2002b; Nakashima et al., 2006). However, most authors agree that MMP-9 is more than just a marker for asthma severity. Conflicting data about the role of MMP-9 in different disease models may also result from the broad range and complex way of action of MMP-9. Most members of the MMP family of enzymes have been shown to play a significant role in both the development and the resolution of inflammatory processes. Obviously a higher cellular infiltrate can be detected in challenged lungs of MMP-9 knockout mice (McMillan et al., 2004; Greenlee et al., 2006). However, this might be due to reduced emigration of cells out of the lung tissue (Vermaelen et al., 2003; Corry et al., 2004). MMPs can affect chemotactic molecules through multiple mechanisms. MMPs may inactivate a protein through cleavage or they may activate a protein by releasing an active fragment. Greenlee et al. (2006) have recently identified novel substrates for MMP-2 and MMP-9, which, in addition to chemokines known to be modified by MMPs (McQuibban et al., 2000, 2002; Van den Steen et al., 2000, 2003), may account for the decrease in the concentration of chemokines in the BAL in the absence of MMP-2 and MMP-9 (Corry et al., 2002, 2004). In the lung, the chemokine-regulating functions of MMP-9 may predominate over the extracellular matrix-degradation effect with regard to the outcome of some mouse asthma models. As in the skin compartment, the situation is slightly different – cells do not leave the epidermis by emigration to the surface – skin inflammation might not be deteriorated but alleviated in MMP-9 knockout mice. Resolution of skin inflammation by MMP-9 inhibition has been shown (Kobayashi and Shinkai, 2005). Furthermore, granulocytes, which are a relevant cell type in asthmatic inflammation, are not found in eczematous epidermal inflammation. It should also be noted that skin epithelial cells behave quite different from lung epithelial cells. Furthermore, our study points to a role of MMP-9 in the second step of cell migration (dermis to the epidermis); however, in lung inflammation, the role of MMP-9 has been studied in cell migration from endothelium to the lung tissue (McMillan et al., 2004). So far no study has shown that MMP-9 could regulate the migration of lymphocytes across the subepithelial basement membrane of the skin.

Previously, other cells involved in epidermal/dermal inflammation, such as mast cells and LCs, have been described to secrete active MMP-9. In skin inflammation, mast cells have been observed mostly in the upper dermis and in the epidermis of AD patients (Groneberg et al., 2005). Treatment of human cultured mast cells with PMA led to conversion of pro-MMP-9 to an active form, suggesting that human mast cells not only produce MMP-9 but also activate its pro-form (Kanbe et al., 1999). LCs produce MMP-9, and intradermal injection of anti-MMP-9 mAb before hapten (rhodamine B) painting has been shown to markedly inhibit the contact allergen-induced decrease in LC number in the epidermis (Kobayashi et al., 1999). Thus, our study adds KCs as another source of active MMP-9 in skin pathology. Since KCs are high in number in the epidermis as compared to LCs and mast cells, this cellular source of MMP-9 might also play a very important role in vivo.

Previously, TGF-1 has been shown to induce MMP-9 in human primary KCs (Salo et al., 1991). TGF-1 has been shown to be an important mediator of fibrosis and healing processes (Kaviratne et al., 2004). We could confirm the induction of MMP-9 in KCs by TGF-1. However, we could not observe an additive/synergistic effect of IL-13 and TGF-1 on MMP-9 expression. It has recently been suggested that IL-13 mediates its profibrotic effect by regulating the production and activation of TGF-1 (Lee et al., 2001a). Using the Clara cell 10 kDa protein promoter to express IL-13 selectively in the lung, Zhu et al. (1999) showed that IL-13 induces MMP-9 and TGF-1 expression and that the activation of TGF-1 is mediated by MMP-9-dependent mechanisms. These studies indicate a direct functional link between IL-13 and TGF-1. From the data of our study, it appears unlikely that the induction of MMP-9 by IL-13 in KCs is dependent on autocrine TGF-1 as we were unable to detect relevant amounts of TGF-1 production by KCs (IL-13 50 ng/ml). MMP-9 mRNA induction seen within 6 hours also points to a direct effect of IL-13 on MMP-9. In Schistosoma mansoni infection, complete abrogation of fibrosis was seen despite the continued production of TGF-1 in IL-13 knockout mice; and in TGF-1 knockout mice, upregulation of MMPs expression by IL-13 could be detected, demonstrating that IL-13 also activates the fibrogenic machinery directly and independent of TGF-1 (Kaviratne et al., 2004). So the hypothesis that tissue remodeling/fibrosis and action of MMP-9 are TGF-1 dependent (IL-13–TGF-1 fibrosis) does not hold true for all cases (Lee et al., 2001a).

Our data suggest that IL-13 induces MMP-9 in KCs, which might facilitate trans-basement membrane migration of leukocytes and mobilization of LCs, which are characteristic features of eczematous skin diseases. Thus our results suggest the pathophysiological implication of IL-13 mainly in the acute phase of skin inflammation.

Materials and Methods

Sample material and cytokines

Biopsies (2–4 mm punch biopsies) were taken from patients suffering from extrinsic type of AD (n=8) or allergic contact dermatitis (n=9). Lesions were classified as acute (vesicles, oozing, erythema; spongiosis as determined by histological analysis) or chronic (erythema, scaling, lichenification) according to the appearance of clinical symptoms. Biopsies were taken from lesions that according to clinical appearance were clearly distinguishable as acute or chronic. None of the patients received any systemic immunosuppression or antihistamine treatment. The study was approved by the medical ethical committee of the Hannover Medical School and was conducted according to the Declaration of Helsinki Principles. All patients gave their written informed consent. After the biopsies were taken, any remaining blood was washed from the biopsies (with sterile NaCl) and transferred to RNA stabilizing solution (RNA later from Ambion, Frankfurt, Germany) and then stored at -80°C until RNA isolation.

All cytokines were used as purified recombinant human preparations. IL-13 and TGF-1 were purchased from PromoCell GmbH (Heidelberg, Germany), IFN- from ImmunoTools (Friesoythe, Germany) and IL-4 from R&D Systems (Wiesbaden, Germany). MMP inhibitor galardin (GM 6001) was purchased from Merck (Darmstadt, Germany) and used at a concentration of 10 M in the assays. This concentration was not toxic for keratinocytes as determined by 7AAD staining.

Cell isolation and culture

Primary cultures of normal human KCs were prepared from foreskin as described previously (Wittmann et al., 2005). In brief, the single-cell suspension of KCs was cultured in serum-free growth medium (Keratinocyte Growth Medium 2 Kit; PromoCell GmbH, Heidelberg, Germany). KCs (passages 2–5) cultured in hydrocortisone-free medium were used for all experiments.

Flow cytometric analysis of intracellular and membrane molecules

Expression of surface antigens was assessed as described previously (Wittmann et al., 1999). Before flow cytometric analysis, cells were detached by using 0.025% EDTA (10 minutes; PAN Biotech GmbH, Aidenbach, Germany) and HyQTase (10 minutes; HyClone, South Logan, UT). The cells were stained with FITC-labeled mAb: MMP-9 (clone 56129, R&D Systems). Stained cells were measured by flow cytometry (FACS Calibur) and analyzed using Cell QuestPro™ software (BD Biosciences, Heidelberg, Germany). Intracellular detection of MMP-9 was performed using the same mAb in fixed and permeabilized cells (Cytofix/Cytoperm kit, BD Biosciences). PMA was added to the cells 2 hours before measurement.

mRNA isolation, reverse transcription, and qRT-PCR

KCs were stimulated with an appropriate cytokine for 6 hours and were lysed in the well (no prior detachment or enzyme treatment). RNA isolation and reverse transcription were performed as previously described (Wittmann et al., 2004) by using the "High Pure mRNA Isolation Kit" (Roche Molecular Biochemicals, Mannheim, Germany) and "First Strand cDNA Synthesis Kit" (MBI Fermentas, St Leon-Rot, Germany). The qRT-PCR was performed on a LightCycler (Roche Molecular Biochemicals) by using "LightCycler^® FastStart DNA Master^PLUSSYBR Green I" (Roche Molecular Biochemicals). MMP-2, MMP-3, MMP-9, E-cadherin, IL-13, and glyceraldehyde-3-phosphate dehydrogenase were run in a touchdown PCR program (target temperature, 65°C; secondary target temperature, 55°C; step size, 0.5; step delay, 1). The following primers were used for PCR amplification: MMP-9 sense, 5'-GCC AAT CCT ACT CCG C-3'; MMP-9 antisense, 5'-TGA GGA ATG ATC TAA GCC C-3'; glyceraldehyde-3-phosphate dehydrogenase sense, 5'-CCA CAT CGC TCA GAC ACC AT-3'; glyceraldehyde-3-phosphate dehydrogenase antisense, 5'-GGC AAC AAT ATC CAC TTT ACC AGA GT-3'; IL-13 sense, 5'-CTC ATG GCG CTT TTG T-3'; IL-13 antisense, 5'-TGT AAG AGC AGG TCC TTT-3'. For MMP-2 and MMP-3, we used "Quantitect Primers" (Qiagen, Hilden, Germany). For quantitative analysis, standard curves were created and targets were quantified using the "Relative Quantification Software" (Roche Molecular Biochemicals).

MMP-9 bioactivity assay

In all experiments, KCs were grown in a 24-well plate (Nunc, Wiesbaden, Germany) to a 60–90% cell density in 0.5 ml of KCs medium. MMP-9 activity in cell-free supernatants was determined by the Biotrak MMP-9 activity assay system (Amersham Biosciences/GE Healthcare Europe GmbH, Munich, Germany). The test was performed according to the manufacturer's instructions. In brief, standards and samples were incubated overnight at 4°C in microtiter 96 wells precoated with anti-MMP-9 antibodies. Concentration of MMP-9 was detected through the activation of a modified urokinase proenzyme and the subsequent cleavage of its chromogenic peptide substrate. The absorbance was read at 405 nm, and the concentration of active MMP-9 was determined by interpolation from the standard curve.

Immunohistochemistry

Immunohistochemistry was performed as described previously (Werfel et al., 1996b) with few modifications. Immediately after surgery, 3 mm punch biopsies from non-eczematous and non-atopic patients with clinically unaffected skin were obtained. The ex vivo punch biopsies were incubated overnight in KC culture medium alone or were stimulated with IL-13 (50 ng/ml). The next day, biopsies were embedded in paraffin. The fixed paraffin sections (5 m thick) were overlaid with undiluted anti-MMP-9 mAb (clone RB-9234-R7, Neomarkers, Fremont, CA). Corresponding concentrations of appropriate isotype antibodies were used in the same buffer. After 1 hour incubation, the sections were overlaid with 1:200 horse anti-mouse biotin (Vector Laboratories Inc.) for 30 minutes at room temperature and stained as described previously (Werfel et al., 1996b). For collagen type IV staining, the mouse–anti-human mAb (clone CIV22, Dako, Hamburg, Germany) was used at a 1:50 dilution. For collagen type IV staining, paraffin sections were incubated at 70°C for 24 hours and treated with proteinase K (Dako Real™, Dako) according to the manufacturer's instructions. The Dako Real Detection System, Alkaline Phosphatase/Red, Mouse (Dako) was used with a Dako-automated immunostaining instrument according to the manufacturer's instructions.

Trans-basement membrane migration assay

Human CD4+ lymphocytes were purified as described previously (Purwar et al., 2006) using a negative selection kit (Miltenyi Biotech, Bergisch Gladbach, Germany). To study the migration of CD4+ lymphocytes through basement membrane components, Matrigel (BD Biosciences)-coated cell culture inserts were used according to the manufacturer's instructions. KCs were added to the upper chamber of the Matrigel insert and stimulated overnight with IL-13 (50 ng/ml). Purified CD4⁺ lymphocytes were added the next day (2 10⁶ cells/well) and chemokines were added to the lower chamber (recombinant human CCL22 5 ng/ml, CCL2 5 ng/ml, and CXCL3 5 ng/ml were purchased from Peprotech, Rocky Hill, NJ). Plates were incubated overnight at 37°C in 5% CO₂. Migrated lymphocytes were counted by use of Trucount™ tubes (BD Biosciences) as described previously (Purwar et al., 2006). Donor-to-donor variation in this assay was quite high. Only cells from healthy individuals were used.

Statistical analysis

The software used to perform the statistical analysis was Prism 3.03. Data were depicted as mean valueSEM. Unpaired t-test (Figures 1a, b and 3), paired t-test (Figure 1c), or Mann–Whitney U-test (Figure 4) was performed. In the figures ^* stands for a P-value <0.05, ^** for P<0.02, and ^*** for P<0.01.

Conflict of Interest

The authors state no conflict of interest.

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Acknowledgments

We thank Christina Hartmann and Melanie Drenker for excellent technical assistance. We also thank Bernward Völker for helping with the immunohistochemical analysis. This study was supported by DFG Grant SFB 566, A6.