Endothelial Cells Markers Prox-1 and Podoplanin in Human IL-3 Induces

IL-3 Induces Expression of Lymphatic Markers Prox-1 and Podoplanin in Human Endothelial Cells

This information is current as Marion Gröger, Robert Loewe, Wolfgang Holnthoner, of September 25, 2021. Robert Embacher, Manuela Pillinger, G. Scott Herron, Klaus Wolff and Peter Petzelbauer J Immunol 2004; 173:7161-7169; ; doi: 10.4049/jimmunol.173.12.7161

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

IL-3 Induces Expression of Lymphatic Markers Prox-1 and Podoplanin in Human Endothelial Cells1

Marion Gro¨ger,*† Robert Loewe,* Wolfgang Holnthoner,* Robert Embacher,*† Manuela Pillinger,*† G. Scott Herron,‡ Klaus Wolff,*† and Peter Petzelbauer2*†

Factors determining lymphatic differentiation in the adult organism are not yet well characterized. We have made the observation that mixed primary cultures of dermal blood endothelial cells (BEC) and lymphatic endothelial cells (LEC) grown under standard conditions change expression of markers during subculture: After passage 6, they uniformly express LEC-speciﬁc markers Prox-1 and podoplanin. Using sorted cells, we show that LEC but not BEC constitutively express IL-3, which regulates Prox-1 and podoplanin expression in LEC. The addition of IL-3 to the medium of BEC cultures induces Prox-1 and podoplanin. Blocking IL-3 activity in LEC cultures results in a loss of Prox-1 and podoplanin expression. In conclusion, endogenous IL-3 is required to maintain the LEC phenotype in culture, and the addition of IL-3 to BEC appears to induce transdifferentiation of BEC into Downloaded from LEC. The Journal of Immunology, 2004, 173: 7161–7169.

nterleukin-3 is a broadly acting regulatory protein of hemo- tively expressed markers are N-cadherin (22–24), pathologische poietic cell differentiation. It stimulates proliferation, sur- anatomie Leiden-endothelium Ag (PAL-E) (for microvessels) (16, I vival, and differentiation of pluripotent hemopoietic stem 25), or CXCR4 (22, 23). During embryogenesis, factors governing cells, and promotes expansion of cells that differentiate into mature lymphatic differentiation are well described. The transcription fac- http://www.jimmunol.org/ cell lineages. Interestingly, IL-3 also has the capacity to divert tor Prox-1 is the earliest detectable marker of lymphatic differen- differentiation of already committed cells. In the presence of IL-3, tiation and is essential for differentiation of lymphatic EC (LEC) cells of the osteoclast lineage differentiate into cells of the mac- from the anterior cardinal vein endothelium (14, 26, 27). Lym- rophage lineage (1). IL-3 signals to cells via the IL-3R, which is phangiogenesis is regulated by growth factors VEGF-C and composed of an IL-3-specific ␣-chain and a common ␤-chain VEGF-D and their receptor VEGFR-3 (20, 28–30). The trans- shared with the GM-CSFR ␣-chain and the IL-5R ␣-chain (2, 3). membrane mucoprotein podoplanin was shown to be important for Endothelial cells (EC)3 express IL-3R ␣- and ␤-chains, and ex- the correct lymphatic network formation (26). In the adult organ- pression can be increased by stimulation with TNF-␣ and/or IFN-␥ ism, the situation is more complex and less defined. For example, (4). EC respond to IL-3 by expressing IL-8 and E-selectin (5, 6). in some tumors and in inflammation, the lymphatic growth factor by guest on September 25, 2021 Moreover, IL-3 promotes endothelial tube formation and direc- receptor VEGFR-3 is expressed on proliferating lymphatic and tional migration (7). Cultured EC have been identified as a source blood vessels, resulting in VEGF-C-induced lymph and blood ves- of IL-3 (8). Whether EC-derived IL-3 has autocrine functions is sel formation (31–34). This raises the question whether additional unknown, as is the question of a role for IL-3 in EC differentiation. factors exist, which determine differentiation of LEC within the EC invest two distinct vascular trees, one building the transport adult organism. In this study, we demonstrate that IL-3 regulates system for blood and the other for lymph. They differ by morphol- expression of LEC-specific markers Prox-1 and podoplanin. ogy and phenotype as reviewed recently by several authors (9–13). Established markers for human lymphatic cells are, for example, Materials and Methods Prox-1 (14), podoplanin (15, 16) (also called T1␣ (17) or gp36 Cells (18)), vascular endothelial growth factor (VEGF)R-3 (19, 20), and lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) HUVEC were isolated as described (35) and cultured in IMDM with 20% (21). Also for blood EC (BEC), lineage-specific genes have been FCS, glutamine (2 mM; Invitrogen Life Technologies, Carlsbad, CA), pen- icillin (100 U/ml), streptomycin (100 ␮g/ml; both Invitrogen Life Tech- identified by transcriptional profiling (22–24), among constitu- nologies), and heparin/ECGS (PromoCell, Heidelberg, Germany). Foreskin-derived microvascular EC were isolated and cultured as described (36). Briefly, foreskins were treated with dispase (Invitrogen Life *Department of Dermatology, Division of General Dermatology, Medical University of Vienna, and †Ludwig Boltzmann Institute for Angiogenesis, Microcirculation and Technologies) for 20 min at 37°C, followed by mechanically scraping EC Inflammation, Vienna, Austria; and ‡Department Dermatology, Palo Alto Medical with a cell scraper. Cells were then seeded into fibronectin-coated wells Clinic, Palo Alto, CA 94301 and cultured in EC growth medium (MV/ECGS; PromoCell). At passage 1, BEC and LEC were separated by magnetic sorting with an anti-podoplanin Received for publication May 26, 2004. Accepted for publication October 4, 2004. serum (15, 16). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Antibodies 1 This work was supported by a research grant from the Stavros Niarchos Foundation. First step Abs were as follows: anti-Prox-1 (RDI, Flanders, NJ), anti- 2 Address correspondence and reprint requests to Dr. Peter Petzelbauer, Depart- macrophage mannose receptor (BD Pharmingen, San Jose, CA), PAL-E ment of Dermatology, Division of General Dermatology, Medical University of (RDI), anti-N-cadherin (BD Pharmingen), FITC-labeled CD31 (Immuno- Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria. E-mail address: tech, Marseille, France), anti-IL-3R ␣-chain Ab for FACS (clone 9F5), and [email protected] blocking anti-IL-3R ␣-chain Ab (clone 7G3; both BD Pharmingen). As a 3 Abbreviations used in this paper: EC, endothelial cell; BEC, blood EC; LEC, lymphatic specificity control for the anti-podoplanin serum, we raised a mouse mono- EC; VEGF, vascular endothelial growth factor; LYVE, lymphatic vessel endothelial clonal anti-human podoplanin Ab using peptide GASTGQPEDDTETT hyaluronan receptor; PAL-E, pathologische anatomie Leiden-endothelium Ag. GLEGG (aa 22–40) as the Ag; a rabbit anti-human podoplanin serum was

a gift from Dr. D. Kerjaschki (Department of Pathology, Medical Univer- cells. As a positive control, we used CD31 Abs, which reacted sity of Vienna). Isotype controls were purchased from Sigma-Aldrich (St. equally with LEC and BEC (Fig. 1B). Louis, MO). Second step Abs were as follows: tetramethylrhodamine iso- thiocyanate-labeled goat anti-rabbit F(ab)2 (Jackson ImmunoResearch IL-3 is constitutively expressed in LEC Laboratories, West Grove, PA). PE-labeled goat anti-rabbit F(ab)2 (BD Pharmingen), Alexa 488-labeled anti-rabbit IgG (Molecular Probes, Lei- The molecular characterization of sorted LEC and BEC is shown den, The Netherlands), FITC-labeled anti-mouse-IgG (Sigma-Aldrich), and in Fig. 1C. LEC are characterized by the expression of podoplanin, HRP-labeled anti-mouse or anti-rabbit IgG (Bio-Rad, Hercules, CA). Flt-4, LYVE-1, Prox-1, and mannose receptor, and BEC by PAL-E FACS and laser scan analysis and N-cadherin expression. To determine factors inducing podoplanin expression of mixed (nonsorted) LEC/BEC in culture, we For FACS, cells were detached from culture dishes using trypsin/EDTA. screened cell culture supernatants of sorted LEC and BEC for cy- For laser scan imaging, cells were directly fixed on culture dishes with acetone/methanol (1:1) at Ϫ20°C for 10 min. Samples were then incubated tokines. We found IL-3 consistently present in LEC and absent in with indicated first-step Abs followed by the appropriate fluorescence-la- BEC supernatants (Fig. 2A). HUVEC were analyzed for compar- beled second-step Abs. Isotype-matched control Abs were used in parallel. ison and also found negative for IL-3 (see also Nilsen et al. (8)). Bound fluorescence was analyzed by FACScan (BD Biosciences, Moun- Primary cultures, which, due to the isolation procedure, consisted tain View, CA) or laser scan microscope (LSM 520; Zeiss, Oberkochen, of a mixture of LEC and BEC (mixed LEC/BEC), had slightly Germany). higher IL-3 release than in sorted LEC (by Student’s t test, the RT-PCR difference compared with LEC was NS). IL-3 expression could be ␣ ␥ Cells were lysed in lysis buffer (10 mM NaOH-HEPES (pH 7.8), 1.5 mM induced following stimulation with TNF- /IFN- in LEC and in MgCl, 10 mM KCl, 1 mM DTT, 0.1% Nonidet P-40, and1UofRNase BEC, but IL-3 levels were ϳ10 times higher in stimulated LEC Downloaded from inhibitor (Roche, Basel, Switzerland)), and mRNA was isolated by using than in stimulated BEC (Fig. 2A). To confirm results, IL-3 mRNA oligo(dT) beads (Dynal, Oslo, Norway). RNA was reverse transcribed with was analyzed by real-time PCR: IL-3 mRNA was detectable in Superscript Reverse Transcriptase (Invitrogen Life Technologies) and hex- unstimulated LEC after a mean of 37 cycles, and was absent in amer primers (Roche) for 90 min at 42°C. One microliter of cDNA was then used for PCR with primers for podoplanin (forward, 5Ј-GAA GGT unstimulated BEC even after 45 cycles (GAPDH was detectable GTC AGC TCT GCT CT-3Ј; reverse, 5Ј-ACG TTG GCA GGG CGT after 21 cycles). Fig. 2B gives an example of a representative semi- Ј Ј AA-3 ; 35 cycles), Prox-1 (forward, 5 -AAG ACA GAG CCT CTC CTG quantitative RT-PCR for IL-3. http://www.jimmunol.org/ AAT C-3Ј; reverse, 5Ј-TTG CAC TTC CCG AAT AAG GTG AT-3Ј;30 It was previously shown that HUVEC express IL-3R ␣- and cycles), Lyve-1 (forward, 5Ј-GTG CTT CAG CCT GGT GTT G-3Ј; re- ␤ ␣ ␥ verse, 5Ј-GCT TGG ACT CTT GGA CTC TTC-3Ј; 30 cycles), flt-4 (for- -chain, and that expression is induced by TNF- /IFN- (4). In ward, 5Ј-AGC TCT CAG AGC TCA GAA GAG-3Ј; reverse, 5Ј-TTC TCT this study, we show that also LEC and BEC express IL-3R CTC TCT GCT TCA GCT-3Ј; 30 cycles). Primers for GAPDH were pur- (slightly higher in LEC than in BEC), and that expression was chased from BD Clontech (Palo Alto, CA). As control for genomic DNA enhanced by TNF-␣/IFN-␥. Fig. 2C shows results for the ␣-chain; contamination, PCR was performed without reverse transcription. PCR expression of the ␤-chain followed the pattern of the ␣-chain (data conditions were 94°C for 30 s, 56°C for 40 s, and 72°C for 45 s. IL-3 mRNA was analyzed by real-time PCR using primers from assay not shown) (6, 38, 39). As a negative control, dermal fibroblasts 4327037T (Applied Biosystems, Foster City, CA) according to the instruc- were analyzed in parallel (Fig. 2C). tions of the manufacturer. by guest on September 25, 2021 IL-3 up-regulates podoplanin and Prox-1 expression ELISA Cell surface expression of podoplanin was analyzed by FACS. Cell culture supernatants were collected, protease inhibitors added (PMSF, Following IL-3 stimulation, podoplanin expression was enhanced leupeptin, and aprotinin), and analyzed using an IL-3 ELISA kit from R&D in LEC (3-fold). In BEC and HUVEC, IL-3 induced de novo ex- Systems (Minneapolis, MN) according to the instructions of the manufacturer. OD was measured. pression of podoplanin, albeit to a weaker extent (2-fold above 450 baseline; Fig. 3). As a negative control, IL-3 was heat inactivated, Western blot which did not up-regulate podoplanin expression (Fig. 3). Also ␣ ␥ Western blotting was performed as described previously (37). Briefly, fol- TNF- /IFN- induced podoplanin expression in LEC, BEC, and lowing cell lysis with Tris lysis buffer (containing 10 mM Tris-HCl (pH HUVEC. The addition of 1 ␮g/ml anti-IL-3R ␣-chain Ab (clone ␮ 7.5), 150 mM NaCl, 2 mM CaCl2, 1 mM PMSF, 10 g/ml aprotinin, 15 7G3, blocks signal transduction of the IL-3R (40)) inhibits TNF- ␮g/ml leupeptin, 1% Nonidet P-40, and 1% Triton X-100), proteins were ␣/IFN-␥-induced podoplanin expression (Fig. 3), which indicates separated on SDS polyacrylamide gels and blotted on polyvinylidene di- that a TNF-␣/IFN-␥-induced IL-3 release is responsible for the fluoride membranes. Blots were then incubated with indicated first-step Abs, followed by incubation with appropriate HRP-labeled second-step observed podoplanin up-regulation. Following prestimulation with Abs. Signals were visualized by chemiluminescence (Super ECL System; TNF-␣/IFN-␥, subsequent IL-3 stimulation further enhanced po- Amersham Biosciences, Buckinghamshire, U.K.) and documented on Hy- doplanin expression in LEC (4-fold). Also in BEC and HUVEC, perfilm MP (Amersham Biosciences). TNF-␣/IFN-␥ prestimulation followed by IL-3 was additive; podoplanin expression was further induced (Fig. 3). Dermal fibro- Results blasts analyzed as a negative control neither expressed podoplanin Skin-derived EC acquire a lymphatic phenotype during at baseline nor following TNF-␣/IFN-␥ and IL-3 stimulation subculture (Fig. 3). Normal human skin contains approximately equal numbers of po- FACS data were confirmed by podoplanin Western blots using doplanin-positive lymphatic vessels and podoplanin-negative whole-cell lysates of LEC and BEC (Fig. 4A). Prox-1 expression blood vessels (an example of an immunofluorescence staining of was analyzed by immunofluorescence. In unstimulated LEC, human skin is shown in Fig. 1A). As a consequence, mechanically Prox-1 was found in the nucleus in 100% of cells. In contrast, in scraped EC consisted of LEC and BEC at a ratio of ϳ1:1 (ranging unstimulated BEC, Prox-1 was undetectable, but was inducible from 2:1 to 1:2). A FACS analysis using anti-podoplanin Abs of upon stimulation with IL-3 (Fig. 4B). Podoplanin and Prox-1 primary isolated cells (passage 0 cells) is shown in Fig. 1B. During mRNA expression was analyzed by semiquantitative RT-PCR and subculture, the numbers of podoplanin-negative cells continuously were readily detectable in LEC at baseline and induced following decreased, and numbers of podoplanin-positive cells increased IL-3 stimulation (Fig. 4C). In contrast, unstimulated BEC and (Fig. 1B). Passage 6 cells consisted of 100% podoplanin-positive HUVEC were negative for podoplanin and Prox-1 mRNA, but was The Journal of Immunology 7163 Downloaded from

FIGURE 1. A, Immunoﬂuores- cence double-staining of normal human skin samples with anti- podoplanin serum and CD31 Abs. Double-positive vessels represent

LEC and are marked by arrowheads. http://www.jimmunol.org/ The two right panels show staining with the corresponding negative controls. B, FACS analysis for podoplanin and CD31 expression of freshly isolated EC cultures (p0), passage 1 cells (p1), and passage 6 cells (p6) stained with anti-podoplanin serum (left panel), CD31 (middle panel), and controls (right panels). C, by guest on September 25, 2021 Marker proﬁle of sorted BEC and LEC (passage 4 cells). FACS histo- grams are shown at the left side, Western blots at the right side, and RT-PCR at the bottom. Podoplanin was analyzed with two Abs, an anti- podoplanin serum or a mouse mAb (marked by asterisks). Note that immunoblots with anti-podoplanin serum reveal two bands of 38 and 28 kDa (18), whereas the mouse mAb detects a 38-kDa band only. 7164 IL-3 INDUCES Prox-1 AND PODOPLANIN

FIGURE 2. A, IL-3 secretion into the culture medium of HUVEC, sorted BEC and LEC (passage 4 cells), and primary cultures of freshly isolated skin-derived EC consisting of a mixture of BEC and LEC (mixed BEC and LEC) as determined by ELISA. Basal IL-3 release under standard conditions is shown in the left panel. The right panels show IL-3 release following stimu- Downloaded from lation with TNF-␣/IFN-␥ (10 ng/ml each) for 24 h (mean Ϯ SEM of three independent experiments performed each in triplicate; the difference between BEC and LEC is signiﬁcant, p Ͻ 0.01; the difference between mixed cultures and LEC is NS). B, Semiquantitative RT-PCR for IL-3 mRNA expression in BEC and LEC is http://www.jimmunol.org/ shown (35 cycles). As a control, GAPDH mRNA expression is shown (20 cycles). C, IL-3R ␣-chain expression as determined by FACS is shown. The left panels show IL-3R ␣-chain expression of indicated cells grown under standard conditions. The right panels show IL-3R ␣-chain expression following stimulation with TNF-␣/IFN-␥ (10 ng/ml each) for 12 h.

Thin lines are isotype controls; bold lines are by guest on September 25, 2021 staining with the anti-IL-3R␣ Ab.

inducible following IL-3 stimulation (Fig. 4C). As a negative con- podoplanin (Fig. 5A, first panel). Results were confirmed by FACS trol, dermal fibroblasts were analyzed and found negative for po- analysis. Fig. 5B gives the cumulative result of three independent doplanin and Prox-1 mRNA (Fig. 4C). experiments. After 168 h of anti-IL-3R␣ Ab treatment, ϳ90% of cells were negative for podoplanin expression, whereas an isotype- ␣ Anti-IL-3R -chain Abs inhibit Prox-1 and podoplanin matched control Ab had no effect (Fig. 5B). After 168 h, the anti- expression IL-3R␣ Ab was removed by repeated washing and replaced by To analyze the role of endogenous IL-3, sorted LEC were cultured fresh medium. As shown in Fig. 5C, cells remained negative for in the presence of an anti-IL-3R ␣-chain Ab (1 ␮g/ml) (40). In the podoplanin even after withdrawal of the Ab. Results from immu- presence of this blocking Ab, podoplanin expression in LEC de- nofluorescence and FACS were confirmed by RT-PCR; podopla- creased (as analyzed by immunofluorescence LSM imaging; Fig. nin and Prox-1 mRNA were undetectable following 72-h treatment 5A). After 24 h of treatment of LEC, islands of cells emerged, with anti-IL-3R␣ Ab (Fig. 5B, inset). which had lost podoplanin expression, whereas other islands Anti-IL-3R␣ Ab treatment also altered cell morphology of cul- showed persisting podoplanin expression. Islands of podoplanin- tured LEC. A representative example of transmission light micros- positive cells became continuously smaller, and after 168 h, po- copy images of LEC (cultured with isotype Ab), BEC (cultured doplanin expression was almost completely lost in all LEC (Fig. with isotype Ab), and LEC cultured with anti-IL-3R␣ Ab is shown 5A). In the presence of a control Ab, cells remained positive for in Fig. 5D. LEC appeared as flattened cells, whereas BEC had a The Journal of Immunology 7165 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 3. IL-3 induces podoplanin as determined by FACS analysis. Sorted LEC, sorted BEC, HUVEC (passage 4 cells), or dermal fibroblasts were cultured for 24 h in medium alone or in medium supplemented with IL-3 (25 ng/ml; 24 h), heat-inactivated IL-3, TNF-␣/IFN-␥ (10 ng/ml each; 24 h), TNF-␣/IFN-␥ plus anti-IL-3R␣ Ab 7G3 (1 ␮g/ml), or TNF-␣/IFN-␥ (12 h) followed by IL-3 (24 h). Staining was performed with the control serum (row at the top) or with anti-podoplanin serum (all others). Geometric mean fluorescence values are inserted into each panel and represent the mean of three independent experiments. more spindled morphology. In the presence of anti-IL-3R␣, a pop- markers Prox-1 and podoplanin. Blocking IL-3 effects leads to a ulation of spindled cells emerged comparable to those seen in pure loss of expression of these molecules in cultured LEC. BEC cultures. These islands correspond to the podoplanin-nega- It should be noted that, during embryogenesis, IL-3 does not tive cells shown in Fig. 5A. appear to be the critical factor for the formation of the lymphatic tree, because IL-3-deficient animals have no severe lymphatic dys- Discussion function. However, current reports have not specifically searched Prox-1 is the earliest lymphatic marker during embryonic devel- for subtle changes in lymphatic function (42, 43); thus, the role of opment (14, 26) and is thought to be the critical gene programming IL-3 during development has to remain open. the lymphatic phenotype (14, 23, 27, 41). Podoplanin expression Another issue concerns the question of why the expression of occurs later and appears to be important for the lymphatic network IL-3 has not yet been identified as a LEC-specific gene in previous formation (23, 26, 41). Both molecules are constitutively ex- gene expression arrays (22–24). On the one hand, this might be pressed on lymphatic endothelium in the adult organism, but fac- due to the reported instability of IL-3 mRNA, which complicates tors that induce or maintain their expression are currently un- detection of IL-3 mRNA (44, 45). In contrast, this seeming dis- known. In this study, we identified for the first time a soluble crepancy might be due to the very low IL-3 mRNA levels in un- factor, IL-3, which is expressed in cultured LEC but not in BEC, stimulated LEC cultures, which may have escaped detection in and which is required to maintain expression of LEC-specific arrays. However, constitutive IL-3 expression levels are generally 7166 IL-3 INDUCES Prox-1 AND PODOPLANIN

FIGURE 4. A, Western blotting for IL-3-induced podoplanin expression. IL-3 (25 ng/ml) was added to the medium for 24 h. Cells were then lysed, and immunoblots were performed with the polyclonal rabbit

anti-podoplanin serum, which detects two different Downloaded from gylcosylation forms, 28 and 38 kDa, respectively. As a loading control, immunoblots for CD31 expression are shown. B, Immunoﬂuorescence laser scan images for Prox-1 expression. Prox-1 is seen in a nuclear local- ization (red) in untreated LEC, and immunoﬂuores- cence is enhanced following IL-3 stimulation. In contrast, Prox-1 is absent in unstimulated BEC and http://www.jimmunol.org/ induced following IL-3 stimulation. To visualize cell margins, cells were double-stained with CD31 Abs in green. C, Semiquantitative RT-PCR for podoplanin and Prox-1 expression in unstimulated cells or following IL-3-stimulation for indicated times is shown. Fi- broblasts were used as a negative control. by guest on September 25, 2021

low in most cell types previously analyzed (46), and even such low lation was additive to subsequent IL-3-induced Prox-1 and podo- concentrations are biologically active in settings of an autocrine planin expression. This additive effect is most likely based on IL-3 release. IL-3 supplemented to the medium requires much TNF-␣/IFN-␥-induced IL-3R expression. Such a mechanism has higher concentrations to achieve biological activity (46). been previously described for IL-3-induced MHC class II expres- In our assays, committed cells were used, which were isolated sion in HUVEC, where TNF-␣/IFN-␥ prestimulation increased and sorted from human foreskins and thus already differentiated IL-3R expression and subsequently MHC class II expression in- into either a BEC or a LEC phenotype. Even in such differentiated duced by IL-3 (4). cells, IL-3 was able to induce Prox-1 and podoplanin expression. To date, IL-3 is the first soluble factor identified that is able to Although IL-3-induced expression levels of Prox-1 and podopla- induce Prox-1 and podoplanin expression in differentiated EC. nin were much lower in BEC than in LEC, it involved the whole This IL-3 effect was seen in LEC and in BEC, which raises the cell population, excluding growth selection as the basis of this intriguing question whether IL-3-treated BEC transdifferentiate phenomenon. Moreover, phenotypic changes appeared within into LEC. Based on current knowledge, the answer is complex, 12–24 h, and population doubling times in EC are 36–48 h, which because (first) cultured LEC have a certain heterogeneous expres- also argues against a growth selection phenomenon. We also show sion of lymphatic markers (e.g., LYVE-1 or VEGFR-3 may be that first-line inflammatory cytokines TNF-␣/IFN-␥ induce podo- absent). This phenomenon was reviewed by Saharinen et al. (47) planin expression, and this also depended on IL-3 (because it is and interpreted as the appearance of different LEC subpopulations. inhibited by anti-IL-3R␣ Abs). Moreover, TNF-␣/IFN-␥ prestimu- Second, the situation is more complicated in experiments where a The Journal of Immunology 7167 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 5. Anti-IL-3R␣ Abs inhibit podoplanin and Prox-1 expression. A, LEC were cultured in the presence of anti-IL-3R␣ Ab 7G3 (1 ␮g/ml) for indicated times, and then double-stained with anti-podoplanin (red) and CD31 (green) Abs and analyzed by laser scan microscopy. The left image shows persisting podoplanin expression in the presence of a control Ab. After 24-h treatment with anti-IL-3R␣ Abs, islands of cells emerged, which had lost podoplanin expression, whereas surrounding cells showed persisting podoplanin expression. After 168 h, podoplanin expression was almost completely lost in all LEC (right panel). B, FACS analysis for podoplanin expression in LEC treated with anti-IL-3R␣ or isotype control Abs. Geometric mean fluorescence levels from three independent experiments are shown and expressed as the percentage of inhibition of podoplanin expression compared with untreated cells (mean Ϯ SD). The corresponding RT-PCR for podoplanin and Prox-1 mRNA expression is shown as an inset in B. C, Representative FACS histogram for podoplanin expression in untreated LEC (first panel) and anti-IL-3R␣ Ab-treated LEC (168 h; second panel). Thereafter, the Ab was washed out, and cells were cultured in medium alone for another 48 and 168 h, respectively. Podoplanin expression remained negative even after withdrawal of the Ab (third and fourth panels). D, Anti-IL-3R␣ Ab treatment changes cell morphology. Transmission microscopic images of LEC (first image) and BEC (third image) treated with a control Ab. The image in the middle shows LEC treated with 1 ␮g/ml anti-IL-3R␣ Ab for 48 h. The asterisk denotes flattened cells resembling LEC, and the circles denote more spindled cells resembling BEC. phenotypic switch from BEC into LEC was induced by, for ex- induced in all experiments (48, 49). Thus the definition of a true ample, overexpressing Prox-1 (23, 41) or by infection with Kar- LEC or BEC phenotype still floats with accumulating data on these posi sarcoma-associated herpes virus (HHV8) (48, 49). Prox-1 cells (reviewed by Saharinen et al. (47)). In other words, we think overexpression in BEC induced only ϳ20% of lymphatic genes that the question whether IL-3 induces a true BEC-to-LEC con- and repressed only ϳ40% of blood vessel genes. This is also true version or a hybrid form cannot be answered at present. for HHV8 infection of blood vessel endothelium, where some, but With regard to in vivo effects of IL-3, others have shown new not all, lymphatic markers were up-regulated, and some blood- vessel formation in mice injected with IL-3-containing Matrigel specific markers disappeared. Surprisingly, even Prox-1 was not plugs, but at the time of this report, phenotypic markers for LEC 7168 IL-3 INDUCES Prox-1 AND PODOPLANIN were yet not well characterized, and thus distinctions between LEC 16. Kriehuber, E., S. Breiteneder-Geleff, M. Groeger, A. Soleiman, and BEC were not made (7, 50). Moreover, this type of experiment S. F. Schoppmann, G. Stingl, D. Kerjaschki, and D. Maurer. 2001. Isolation and characterization of dermal lymphatic and blood endothelial cells reveal stable and would not differentiate between lymphangiogenesis from pre-ex- functionally specialized cell lineages. J. Exp. Med. 194:797. isting lymph vessels and lymph vessel formation through transdif- 17. Ma, T., B. Yang, M. A. Matthay, and A. S. Verkman. 1998. Evidence against a role of mouse, rat, and two cloned human T1␣ isoforms as a water channel or a ferentiation or a combination thereof. Anyway, the possibility for regulator of aquaporin-type water channels. Am. J. Respir. Cell Mol. Biol. IL-3 to induce lymphangiogenesis raises the question whether in- 19:143. creased IL-3 activity in tumors is associated with a high metastatic 18. Zimmer, G., F. Oeffner, V. Von Messling, T. Tschernig, H. J. Groness, H. D. Klenk, and G. Herrler. 1999. Cloning and characterization of gp36, a potential. This was shown for rat mammary adenocarcinoma cell human mucin-type glycoprotein preferentially expressed in vascular endothe- clones, which were highly metastatic when they produced high lium. Biochem. J. 341:277. levels of IL-3, but were poorly metastatic when they had no IL-3 19. Kaipainen, A., J. Korhonen, T. Mustonen, V. W. van Hinsbergh, G. H. Fang, D. Dumont, M. Breitman, and K. Alitalo. 1995. Expression of the fms-like ty- activity (51). As a natural source of IL-3, tumor-infiltrating lym- rosine kinase 4 gene becomes restricted to lymphatic endothelium during devel- phocytes have to be considered (50, 52). Finally, the effects of the opment. Proc. Natl. Acad. Sci. 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