A Previously Unknown Dermal Blood Vessel Phenotype in Skin ... - Core

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ORIGINAL ARTICLE

A Previously Unknown Dermal Blood Vessel Phenotype in Skin Inflammation Marion Gro¨ger1, Heide Niederleithner1, Dontscho Kerjaschki2 and Peter Petzelbauer1 Podoplanin and lymphatic vascular endothelial hyaluronan receptor-1 (LYVE-1) are considered as lineage markers for lymphatic vessel (LV) endothelial cells (LECs). We have recently shown that IL-3 induces de novo expression of these genes in cultured blood vessel (BV) endothelial cells (BEC). To ask, if this is transdifferentiation or activation, we analyzed inflamed skin samples and cytokine-stimulated organ-cultured skin and found a subset of blood capillaries within the papillary dermis expressing low amounts of podoplanin and LYVE-1 as well as high amounts of cytokine-inducible adhesion molecules. In contrast, neighboring lymphatic capillaries express high amounts of podoplanin, LYVE-1 and low amounts of cytokine-inducible adhesion molecules. The different response patterns to inflammatory stimuli were reproducible in cell culture, when cytokine-stimulated BEC and LEC were analyzed. These findings signify that expression of ‘‘lymphatic proteins’’ on BEC corresponds to cell activation. Journal of Investigative Dermatology (2007) 127, 2893–2900; doi:10.1038/sj.jid.5701031; published online 20 September 2007

INTRODUCTION Blood endothelial cells (BEC) and lymphatic endothelial cells (LEC) differ by morphology and protein expression patterns. Blood capillaries are best characterized by the continuous basement membrane investing the lumen and by a surrounding layer of smooth muscle actin (SMA)-positive smooth muscle cells and pericytes (Braverman, 2000; Helmbold et al., 2001). In contrast, lymphatic capillaries possess a discontinuous basement membrane and no surrounding smooth muscle cells or pericytes (Sauter et al., 1998). At the protein level, a growing number of discriminative marker proteins have been identified. Among others, BEC express endothelial antigen PAL-E (Schlingemann et al., 1985), N-cadherin (Petrova et al., 2002; Podgrabinska et al., 2002; Hirakawa et al., 2003), and CXCR-4 (Petrova et al., 2002; Podgrabinska et al., 2002). LEC express prospero-related homeobox 1 (Prox-1) (Wigle et al., 2002), podoplanin (Breiteneder-Geleff et al., 1999; Kriehuber et al., 2001), VEGFR-3 (Kaipainen et al., 1995; Karkkainen et al., 2002), D6 (Nibbs et al., 2001), and lymphatic vascular

endothelial hyaluronan receptor-1 (LYVE-1) (Banerji et al., 1999). In normal tissues, immunostainings with the respective antibodies (Abs) results in a virtually exclusive reactivity with blood or lymphatic vessels (LVs) (Kriehuber et al., 2001; Petrova et al., 2002; Hirakawa et al., 2003). In contrast to the lineage-specific expression profile of unstimulated cells, stimulation with IL-3 (Gro¨ger et al., 2004), IL-7 (Al-Rawi et al., 2005), HHV8 infection (Carroll et al., 2004; Hong et al., 2004; Wang et al., 2004), or overexpression of Prox-1 (Hong et al., 2002; Petrova et al., 2002), has been shown to induce expression of podoplanin and LYVE-1 in cultured BEC. We therefore aimed to investigate, whether true blood capillaries in vivo may express lymphatic markers. Results were compared with cytokine-stimulated organ-cultured skin samples and cytokine-stimulated cultures of BEC and LEC. Here, we describe a previously unknown ‘‘inflammatory’’ phenotype of BEC, expressing low amounts of podoplanin and LYVE-1 and high amounts of E-selectin, ICAM-1, and VCAM-1. RESULTS

1

Division of General Dermatology, Department of Dermatology, Medical University of Vienna, Vienna, Austria and 2Institute of Pathology, Medical University of Vienna, Vienna, Austria Correspondence: Dr Peter Petzelbauer, Division of General Dermatology, Department of Dermatology, Medical University of Vienna, Waehringer Guertel 18-20, Vienna A-1090, Austria. E-mail: [email protected] Abbreviations: Ab, antibody; AEC, 3-amino, 9 ethyl-laisazole; BEC, blood endothelial cell; BV, blood vessel; EC, endothelial cell; LEC, lymphatic endothelial cell; LV, lymphatic vessel; LSM, laser scan microscope; LYVE-1, lymphatic vascular endothelial hyaluronan receptor-1; Prox-1, prosperorelated homeobox 1; SMA, smooth muscle actin; TRITC, tetramethyl ruodamine isothiocyanate

Received 13 February 2007; revised 5 June 2007; accepted 21 June 2007; published online 20 September 2007

& 2007 The Society for Investigative Dermatology

Podoplanin and LYVE-1 are expressed on blood capillaries of inflamed skin

Skin sections of formalin-fixed and paraffin-embedded biopsies were taken from normal skin, eczema, and psoriasis and analyzed for podoplanin expression. In normal skin—as expected—we found two types of vessels, the first expressing high levels of podoplanin representing LVs and the other being negative for podoplanin representing blood vessels (BVs, Figure 1a). In contrast, within the papillary dermis from eczema and psoriasis we found a third type of vessel expressing low amounts of podoplanin (Figure 1b–d). Some of them contained red blood cells within their lumina, suggesting that they belong to the BV tree (Figure 1b and c). www.jidonline.org 2893

M Gro¨ger et al. Blood Vessels Express Podoplanin and LYVE-1

Normal skin, podoplanin (mAb)

Eczema, podoplanin (mAb)

Psoriasis, podoplanin (mAb)

Psoriasis, podoplanin polyclonal

Podoplanin polyclonal

Podoplanin polyclonal +rec. podoplanin

Psoriasis, LYVE-1

Isotype control

Figure 1. Psoriasis and eczema skin samples contain a population of podoplaninlow þ and LYVE-1low þ vessels, some of which contain erythrocytes, suggesting that they belong to the BV tree. Formalin-fixed, paraffin-embedded skin samples were deparaffinized and subjected to immunohistochemistry. BV denotes blood vessel, LV denotes LV. (a) Normal skin sample showing a podoplanin BV and a podoplanin þ LV. (b, c) Eczema and psoriasis: BVs containing erythrocytes within their lumina are podoplaninlow þ . (d) Psoriasis: podoplaninlow þ vessels within the tips of the papillae. (e, f) Serial sections of eczema, in (e) stained with the polyclonal rabbit anti-podoplanin serum showing podoplanin þ vessels and some podoplanin þ inflammatory cells; (f) serial section; the anti-podoplanin Ab was preincubated with recombinant podoplanin protein, which abolished Ab reactivity. (g) Psoriasis: BVs with erythrocytes within lumina are LYVE-1low þ . (h) Isotype control. Bars: a–d, g ¼ 20 mm; e, f, and h ¼ 100 mm.

Moreover, in psoriatic skin, the tortuous vessels within the tips of the papillae also displayed this podoplaninlow þ phenotype (Figure 1d). Both, the mAb and the polyclonal anti-podoplanin Abs gave identical staining patterns (Figure 1c and d). Preincubation with the recombinant podoplanin protein resulted in a complete loss of staining (Figure 1e and f). Also the rabbit pre-immune serum gave negative results (Figure 1h). Similar to results seen with podoplanin, also stainings with anti-LYVE-1 Abs revealed two types of LYVE-1 þ vessels, one expressing high and the other low amounts of LYVE-1 2894 Journal of Investigative Dermatology (2007), Volume 127

(Figure 1g). In the reticular dermis expression of podoplanin or LYVE-1 was only seen in LV but not in BV (data not shown). The population of podoplaninlow þ vessels was further characterized in triple stained specimen; CD31 to determine the total number of vessels, anti-SMA stains to identify BVs, and anti-podoplanin (Figure 2). In the papillary dermis of normal skin, all CD31 þ /SMA þ BVs were podoplanin and all CD31 þ /SMA LVs were podoplaninhigh þ giving a clear distinction between both vascular beds. In contrast to normal skin, most BVs of eczema and psoriasis expressed low amounts of podoplanin (examples are provided in Figure 2; for summary see Table 1). As an additional control, urticarial skin lesions were analyzed, which gave virtually the same picture as normal skin (Table 1). Podoplanin and LYVE-1 are induced on BVs in organ culture

Organ culture allows cytokine stimulation and evaluation of protein expression of cells in situ. Moreover, by using cryostat sections, the affiliation podoplaninlow þ or LYVE-1low þ vessels can be further defined by the BV marker PAL-E (Schlingemann et al., 1985). In controls (unstimulated organcultured skin), all PAL-E þ vessels were surrounded by SMA þ cells, confirming that BVs of the papillary dermis can be defined by the surrounding of SMA þ cells. They were negative for podoplanin (Figure 3) and LYVE-1 (data not shown). All LVs were PAL-E and devoid of surrounding SMA þ cells, and expressed podoplanin (Figure 3) and LYVE1 (data not shown). Isotype controls were negative (Figure 3). Upon stimulation with IL-3 or more pronounced with a combination of TNFa, IFNg plus IL-3, most PAL-E þ /SMA þ BVs were found to express podoplanin and LYVE-1. Of note, not only BVs but also some LVs coexpressed E-selectin. Examples are shown in Figure 3, the summary of responding vessels is shown in Figure 4. Prox-1 was never detectable on PAL-E þ vessels (data not shown). Definition of cytokine response profiles in cultured BEC and LEC

To quantify the amount of protein expression following cytokine stimulation, we performed FACS analysis of cultured BEC and LEC. Following 24 hours of stimulation with TNFa, TNFa þ IFNg, or TNFa þ IFNg þ IL-3 (Figure 5a), BEC can be defined as podoplaninlow þ , E-selectinhigh þ , ICAM-1high þ , VCAM-1high þ , whereas LEC were podoplaninhigh þ , E-selectin þ /, ICAM-1low þ , VCAM-1 þ /. IL-3 induced low amounts of podoplanin in all BEC, which differs from the in vivo situation, where only a subset of BV expressed podoplanin (Figure S1). IL-3 induced low amounts of E-selectin in both BEC (Brizzi et al., 1993; Gro¨ger et al., 2004) and LEC, IFNg super-induced TNF-induced VCAM-1 expression in BEC (Lechleitner et al., 1998), but not in LEC. Of note, also after 6 hours of cytokine stimulation, E-selectin expression was significantly lower in LEC compared with BEC (comparable to 24 hours levels; not shown). Examples for individual FACS curves for podoplanin and the adhesion molecules following 24 hours cytokine stimulation are shown in Figures S1 and S2; results for 72 hours of cytokine


CD31

SMA

Podoplanin

Overlay

Normal skin

BV

LV

CD31

SMA

Podoplanin

Eczema

BV

LV

Figure 2. Podoplaninlow þ vessels are always surrounded by SMA-positive cells, suggesting that they belong to the BV tree. Formalin-fixed, paraffin embedded skin samples were deparaffinized and subjected to three-color immunofluorescence. CD31 in blue, SMA in red, podoplanin in green. Normal skin (first row): a BV positive for CD31, negative for podoplanin, and surrounded by SMA þ cells. Second row: an LV positive for CD31, positive for podoplanin, and not surrounded by SMA þ cells. Eczema skin (third row): a BV positive for CD31, low positive for podoplanin, and surrounded by SMA þ cells. Fourth row: an LV positive for CD31, positive for podoplanin, and not surrounded by SMA þ cells.

Table 1. In eczema and psoriasis skin, the majority of CD31+/SMA+ BVs express low amounts of podoplanin Normal skin Podoplanin

negative

blood vessel

1

Podoplaninlow+ blood vessel Podoplaninhigh+ lymphatic vessel3

65% (+7)

2

Urticaria

Eczema

Psoriasis

68% (+7)

8% (+8)*

9% (+11)*

0%

5% (+8)

61% (+12)*

69% (+14)*

35% (+8)

27% (+6)

31% (+6)

22% (+8)

100%4

100%

100%

100%

BV, blood vessel; SMA, smooth muscle actin. Numbers of vessels were quantified on triple stained laser scan images, as exemplified in Figure 2. 1 CD31+/SMA+ vessels were considered as blood vessels. 2 Mean7SD; n=9 in each condition, mean; *Po0.05 as compared with normal skin and urticaria. 3 CD31+/SMA vessels were considered as lymphatic vessels. 4 The total number of vessels was defined by CD31 reactivity and was set to 100%.

stimulation are shown in Figure S3. Results obtained by FACS were confirmed by real-time PCR, changes in cytokineinduced mRNA expression followed results obtained by FACS (Figure 5b and c). In summary, cytokine-induced phenotypic changes obtained in cultured BEC and LEC mirrored phenotypic changes seen on vessels in organ culture and in inflamed skin.

DISCUSSION Here, we describe a previously unknown cytokine-induced phenotype of BEC within the papillary dermis of the skin. In addition to their well-recognized ability to express high levels of adhesion molecules E-selectin, ICAM-1, and VCAM-1 (Brizzi et al., 1993; Gerritsen et al., 1993; Lechleitner et al., 1998), BEC coexpressed low levels of podoplanin www.jidonline.org 2895


Control PAL-E+ SMA+ BV PAL-E– podoplanin+ LV

IL-3 stimulated PAL-E+ podoplanin+ CD31+ BV PAL-E– podoplanin+ CD31+ LV

E-selectin+ LYVE-1+ SMA+ BV E-selectin+ LYVE-1+ SMA– LV

TNF+IFN+IL-3 stimulated

PAL-E+ podoplanin+ SMA+ BV

PAL-E+ LYVE-1+ SMA+ BV PAL-E– LYVE-1+ SMA– LV

E-selectin+ LYVE-1+ SMA+ BV E-selectin+ LYVE-1+ SMA– LV

Figure 3. Blood vessels of cytokine-stimulated, organ-cultured skin express podoplanin and LYVE-1. Four millimeter punch biopsies from normal were placed in organ culture for 24 hours without (controls) or with the addition of the indicated cytokines. Thereafter, cryostat sections were subjected to three-color immunofluorescence. The respective Ab used is indicated in each image; the images on the right side show overlays of triple stains. Dotted arrows denote LVs; solid arrows denote BVs. (a) Controls (top row), PAL-E þ /SMA þ BVs are podoplanin. Isotype controls were negative (lower row). (b, c) IL-3 (b) and even more robust the combination of TNFa/IFNg/IL-3 (c) induced podoplanin, LYVE-1 and E-selectin on BVs and also E-selectin on LVs. Bar ¼ 20 mm.

2896 Journal of Investigative Dermatology (2007), Volume 127


100%

Blood vessels (PAL-E+/CD31+) E-selectin

Podoplanin

LYVE-1

60%

20%

Lymphatic vessels (PAL-E–/CD31+) 100%

60%

20% w/o culture

Medium control

IL-3

TNF/IFN/IL-3

Figure 4. Summary of E-selectin, podoplanin, and LYVE-1 expression profiles of BVs and LVs in organ-cultured skin. Randomly photographed, triple stained LSM images from the papillary dermis of organ-cultured skin were analyzed blinded to the condition (five images/sample; n ¼ 7 in each treatment condition, mean þ SD). Ab combinations were PAL-E and CD31, together with one of the following third Abs: E-selectin, podoplanin, or LYVE-1. Numbers of PAL-E þ /CD31 þ BVs or PAL-E/CD31 þ LVs expressing either E-selectin, podoplanin, or LYVE-1 were counted.

and LYVE-1. Clearly distinct from BEC, LEC express high levels of podoplanin and LYVE-1, but low levels of E-selectin, ICAM-1, and VCAM-1. The ability of LEC to express cytokineinduced adhesion molecules in culture has been described recently (Johnson et al., 2006), but was not compared with BEC from the same donor. More important and in contrast to previous studies, we have analyzed phenotypes of BEC and LEC in situ, which allows clear assignment of EC to the lymphatic or BV tree. We took advantage of the fact that BVs in situ are always surrounded by a layer of smooth muscle cells. Of note, SMA þ cells also surround collecting LVs, but the herein described podoplaninlow þ , LYVE-1low þ BVs were found within the superficial vascular plexus, at sites where these collectors are either absent or very few in number (Braverman, 2000; Karpanen and Makinen, 2006). Moreover, these podoplaninlow þ , LYVE-1low þ BVs also expressed PAL-E, an established BV marker (Schlingemann et al., 1985; Kriehuber et al., 2001). These findings strongly favor the concept that podoplanin and LYVE-1 expression on BEC is a sign of cell activation and not trans-differentiation of BEC into LEC. Of note, we were unable to induce Prox-1 on BV in situ, which contrasts the finding that Prox-1 was inducible in our cultured cells. The molecular basis of this phenomenon is unclear, as it is unknown whether expression of these three molecules is always synchronized. For example, following HHV8 infection Carroll et al. (2004) and Hong et al. (2004) detected expression of podoplanin, LYVE-1, and Prox-1 in BEC, whereas Wang et al. (2004) did not see Prox-1 expression in BEC under the same conditions. This raises the question on the function of these ‘‘lymphatic proteins’’ on BVs. In vivo, podoplaninlow þ and LYVE-1low þ vessels were mostly surrounded by a dense CD45RO þ infiltrate allegorizing them as ‘‘centers of activa-

tion’’. Indirect evidence exists that both molecules are involved in leukocyte adhesion. LYVE-1 contains a hyaluronan-binding domain termed ‘‘Link’’ module (Banerji et al., 1999). Based on sequence similarities with CD44, another hyaluronan-binding molecule, LYVE-1 could support leukocyte adhesion (Jackson, 2004). With regard to podoplanin, it induces platelet aggregation by interacting with platelet surfaces in a sialylated O-glycan-dependent manner (Kato et al., 2003; Kaneko et al., 2004). Sialylated O-glycans serve pro- and antiadhesive functions and mediate leukocyte rolling on endothelial surfaces (Fukuda, 2002). Anyway, a direct role for podoplanin and LYVE-1 in leukocyte rolling or adhesion to BVs has yet to remain hypothetical. The observation that podoplaninlow þ , LYVE-1low þ BVs are found in chronic skin lesions like eczema or psoriasis and not in acute urticarial rashes deserves additional comments. Inflammatory cells in eczema or psoriasis are mainly lymphocytes (Gaspari, 2006), whereas in urticarial rashes, the infiltrate mainly consists of neutrophils and eosinophils. It is therefore possible that expression of podoplanin and LYVE-1 on BVs is related to changed leukocyte recruitment patterns in acute versus chronic lesions. It is known that adhesion molecule expression patterns change during time. In the early phase of skin inflammation, EC express high levels of E-selectin, P-selectin, some VCAM-1, and are negative for sialyted, fucosylated, and/or sulfated carbohydrate moieties. In chronic lesions, EC express some E-selectin, robust VCAM-1 (Petzelbauer et al., 1993, 1994), and express fucosylated and sulfated carbohydrates (Lechleitner et al., 1999; Renkonen et al., 2002). We thus suggest that podoplanin and LYVE-1 expression by BV endothelium extents the list of molecules displayed on BECs in chronic skin inflammation. In conclusion, we describe a previously unknown cytokine-induced BV phenotype in chronic skin inflammation. They express low amounts of podoplanin and LYVE-1. This clearly distinguishes them from LVs, which constitutively express high amounts of these molecules. Thus, podoplanin and LYVE-1 are not only lineage markers for LEC, but have to be considered as activation markers for BEC. MATERIALS AND METHODS Cells Foreskin-derived microvascular ECs were isolated as described (Petzelbauer et al., 1995). Briefly, foreskins were treated with dispase (Invitrogen/GIBCO, Carlsbad, CA) for 20 minutes at 371C, followed by mechanically scraping ECs with a cell scraper. Cells were seeded into fibronectin-coated wells and cultured in EC growth medium (MV/ECGS; PromoCell, Heidelberg, Germany). Fibroblasts were removed by magnetic sorting using CD31 mAbs. BEC and LEC were separated by sorting with an anti-podoplanin serum. Phenotypes of BEC and LEC cultures have been described recently (Kriehuber et al., 2001; Gro¨ger et al., 2004); BEC were PAL-E, N-cadherin, CXCR4 positive; LEC were podoplanin, LYVE-1, Prox-1 positive.

Antibodies First step Abs were as follows: PAL-E (RDI, Flanders, NJ), antipodoplanin rabbit serum (Kriehuber et al., 2001; Gro¨ger et al., www.jidonline.org 2897


MFI

1,000

400

Podoplanin

10

* w/o

2,000

IFN

*

*

TNF

IL-3

ICAM-1

MFI

1,000

200

*

*

*

0

TNF TNF +IFN +IFN +IL-3

w/o

120 100 80 60 40 20 0

*

*

500

C t value (×106)

w/o

IFN

TNF

IL-3

TNF +IFN

w/o

3 2

1

1

1

C t value (×106 )

BEC 15

E-selectin

10

10

5

5

LEC

BEC

IL-3

TNF

TNF +IFN

TNF +IFN +IL-3

LYVE-1

2

15

TNF +IFN +IL-3

12

3

LEC

TNF +IFN

*

IFN

2

BEC

IL-3

*

3

LEC

TNF

*

Prox-1

Podoplanin

IFN

VCAM-1

TNF +IFN +IL-3

12

12

*

*

100

*

1,500

0

E-selectin

300

100

LEC

BEC

VCAM-1

LEC

BEC

Figure 5. Cultured BEC and LEC differ in their cytokine-induced phenotype. Foreskin-derived BEC and LEC were cultured in the absence or presence of the indicated cytokines for 24 hours. (a) Protein expression was determined by FACS using the indicated Abs (mean þ SD; three independent experiments). The y-axis shows mean fluorescence intensity (MFI). (*) denotes significant differences in antigen expression between unstimulated and cytokine-stimulated BEC, as well as between BEC and LEC (Po0.05). BEC ’, LEC . (b) Real-time PCR for mRNA expression of the indicated lymphatic markers (one of three experiments shown, each performed in triplicates). (c) Real-time PCR for mRNA expression of the indicated adhesion molecules (one of three experiments shown, each 6 performed in triplicates). The y-axis represents DDCt values ( 10 ). The Ct value represents the cycle number at which the fluorescence passes a fixed threshold value. Ct values were normalized to glyceraldehyde-3-phosphate dehydrogenase and calculated relative to the water control using the formula 2DDCt. For (b) and (c): control, TNFa/IFNg, and ’ TNFa/IFNg/IL-3.

2004), mouse monoclonal anti-human podoplanin (Kerjaschki et al., 2006), anti-LYVE-1 (RDI), anti-E-selectin (R&D Systems Inc., MN), CD45RO (Dako, Glostrup, DK), FITC-labeled anti-E-selectin (RDI), FITC-labeled anti-ICAM-1 (R&D Systems Inc.), and FITC-labeled anti-VCAM-1 (R&D Systems Inc.), FITC-labeled CD31 (Immunotech, Marseille-Cedex, France), and Cy3-labeled anti-smooth muscle alpha actin (clone1A4, Sigma). As negative controls, the respective isotype controls were used (Sigma-Aldrich, St Louis, MO). As an additional negative control, the anti-podoplanin serum was preincubated with a recombinant Fc-podoplanin fusion protein (Breiteneder-Geleff et al., 1999). Second step Abs were: Alexa488-labeled anti-mouse IgG (Invitrogen/Molecular Probes, Carlsbad, CA), tetramethyl ruodamine isothiocyanate (TRITC) or Alexa488-labeled anti-rabbit IgG (Invitrogen/Molecular Probes). The following second step Abs were F(ab)2 fragments; Cy5-labeled goat anti-mouse IgG (Jackson Laboratories, West Grove, PA), phycoerythrin-labeled goat anti-rabbit IgG (BD Biosciences Pharmingen, NJ), Cy5-labeled goat anti-rabbit IgG (Jackson Laboratories). 2898 Journal of Investigative Dermatology (2007), Volume 127

Inflamed skin samples and organ-cultured skin Inflamed skin sections were obtained from formalin-fixed, paraffinembedded material from patients that underwent a diagnostic biopsy for medical reasons; only biopsies from trunks or upper limbs were included into this evaluation. All patients had signed an informed consent before the diagnostic biopsy stating that material that is not needed for the diagnostic procedures can be used for scientific purposes under the premise that enough material remains in case additional diagnostic procedures will become necessary thereafter. This was approved by the Institutional Review Board of the Medical University of Vienna and conducted according to declarations of Helsinki Principles. The patients’ diagnosis was established by clinical and histological means. From each biopsy, 10 sections (5 mm each) were taken for these experiments and deparaffinized according to standard procedures. For organ cultures, skin derived from plastic surgery specimen according to the protocol described above was incubated at 371C, 5% CO2 in a humidified incubator for 24 hours in EC growth medium (MV w/o ECGS; PromoCell), without or with the addition of


the indicated cytokines, as described previously (Petzelbauer et al., 1993). Then, samples were snap frozen, cut into 4 mm sections, and fixed in acetone. For immunohistochemistry, sections were incubated with the indicated first step reagents followed by appropriate second step Abs. Bound Abs were visualized by incubation with a streptavidin-HRP conjugate (Dako) and 3-amino, 9 ethyl-laisazole (AEC), resulting in a red-colored reaction product. Sections were counterstained with hematoxylin. For immunofluorescence triple stainings, the indicated first step Abs were either directly labeled with a fluorescent dye or bound Abs were visualized by fluorescence-labeled second step Abs. In all experiments, the respective isotype controls were run in parallel. Sections were analyzed by a confocal laser scan microscope (LSM 510, Zeiss, Oberkochen, Germany). The pinhole of the LSM was set to an airy unit of 1, resulting in a section thickness of 0.8 mm.

Quantification of immunofluorescence The following procedure was followed for evaluating paraffin sections: for each Ab used, at least two sections from each biopsy were stained by triple immunofluorescence technique and five images from the papillary dermis were randomly obtained. Each LSM image was stored as an electronic file and then analyzed by of three independent observers blinded to the conditions. Numbers of SMA/podoplaninhigh þ , SMA þ /podoplaninlow þ , and SMA þ / podoplanin were counted were set in relation to the total numbers of CD31 þ vessels. For evaluation of cryostat sections, following was the procedure: Ab combinations were PAL-E and CD31, together with one of the following third Abs, E-selectin, podoplanin, or LYVE-1. Five randomly selected LSM images of the papillary dermis were analyzed blinded to the conditions. Numbers of PAL-E þ /CD31 þ BVs or numbers of PAL-E/CD31 þ LVs expressing either E-selectin, podoplanin, or LYVE-1 were counted.

FACS Cells were detached from culture dishes using trypsin/EDTA (Invitrogen/GIBCO). Samples were incubated with the indicated first step fluorescence labeled Abs. Isotype-matched control Abs were used in parallel. Bound fluorescence was analyzed by FACScan (Becton Dickinson, BD Sciences, San Jose, CA).

Real-time PCR BEC and LEC were cultivated in the absence or presence of the indicated cytokines. Then, RNA was isolated and reverse transcribed as described (Gro¨ger et al., 2004). Real-time PCR was carried out with a TaqMAN Universal PCR Master Mix kit (Applied Biosystems, Foster City, CA), according to the manufacturer’s instructions. Primer kits for glyceraldehyde-3-phosphate dehydrogenase (assay-IDs Hs9999990 m1), podoplanin (Hs00366764 m1), Prox-1 (Hs00896293 m1), LYVE1 (Hs00272659 m1), E-Selectin (Hs00174057 m1), and VCAM-1 (Hs00174239 m1) were purchased from Applied Biosystems. PCR was performed on an ABI Prism 7700 Sequence Detector using the following cycling conditions: 951C for 10 minutes followed by 40 cycles of 951C (15 seconds) and 601C (1 minute). The Ct value represents the cycle number at which the fluorescence passes a fixed threshold value. Ct values were normalized to glyceraldehyde-3phosphate dehydrogenase and calculated relative to the water control using the formula 2DDC t .

Statistical analysis Results are presented in a primary descriptive manner (mean7SD). Significance among the groups was calculated by Student’s t-test and considered significant when Po0.05. CONFLICT OF INTEREST The authors state no conflict of interest.

ACKNOWLEDGMENTS This work was supported in part by a research grant from the Austrian Science Foundation (FWF S9402-BII).

SUPPLEMENTARY MATERIAL Figure S1. Examples of individual FACS curves for podoplanin surface expression. Figure S2. Examples of individual FACS curves for E-selectin, ICAM-1, and VCAM-1 expression. Figure S3. Adhesion molecule expression after 72 hours of cytokine stimulation.

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2900 Journal of Investigative Dermatology (2007), Volume 127