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B Cell-Derived Vascular Endothelial Growth Factor A Promotes Lymphangiogenesis and High Endothelial Venule Expansion in Lymph Nodes This information is current as of September 26, 2021. Binita Shrestha, Teruto Hashiguchi, Takashi Ito, Naoki Miura, Kazunori Takenouchi, Yoko Oyama, Ko-ichi Kawahara, Salunya Tancharoen, Yuya Ki-i, Noboru Arimura, Narimasa Yoshinaga, Satoshi Noma, Chandan Shrestha, Takao Nitanda, Shinichi Kitajima, Kimiyoshi Arimura, Masahiro Sato, Taiji Sakamoto and Ikuro Downloaded from Maruyama J Immunol 2010; 184:4819-4826; Prepublished online 22 March 2010;

doi: 10.4049/jimmunol.0903063 http://www.jimmunol.org/ http://www.jimmunol.org/content/184/9/4819

Supplementary http://www.jimmunol.org/content/suppl/2010/03/22/jimmunol.090306 Material 3.DC1

References This article cites 38 articles, 15 of which you can access for free at: by guest on September 26, 2021 http://www.jimmunol.org/content/184/9/4819.full#ref-list-1

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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 © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

B Cell-Derived Vascular Endothelial Growth Factor A Promotes Lymphangiogenesis and High Endothelial Venule Expansion in Lymph Nodes

Binita Shrestha,*,1 Teruto Hashiguchi,*,1 Takashi Ito,*,1 Naoki Miura,†,1 Kazunori Takenouchi,* Yoko Oyama,* Ko-ichi Kawahara,* Salunya Tancharoen,* Yuya Ki-i,‡ Noboru Arimura,‡ Narimasa Yoshinaga,‡ Satoshi Noma,x Chandan Shrestha,* Takao Nitanda,{ Shinichi Kitajima,{ Kimiyoshi Arimura,|| Masahiro Sato,# Taiji Sakamoto,‡ and Ikuro Maruyama*

Vascular endothelial growth factor A (VEGF-A) is a prominent growth factor for both angiogenesis and lymphangiogenesis. Recent Downloaded from studies have shown the importance of VEGF-A in enhancing the growth of lymphatic endothelial cells in lymph nodes (LNs) and the migration of dendritic cells into LNs. VEGF-A is produced in inflamed tissues and/or in draining LNs, where B cells are a possible source of this growth factor. To study the effect of B cell-derived VEGF-A, we created transgenic mice (CD19Cre/hVEGF-Afl) that express human VEGF-A specifically in B cells. We found that the human VEGF-A produced by B cells not only induced lymphangiogenesis in LNs, but also induced the expansion of LNs and the development of high endothelial venules. Contrary

to our expectation, we observed a significant decrease in the Ag-specific Ab production postimmunization with OVA and in the http://www.jimmunol.org/ proinflammatory cytokine production postinoculation with LPS in these mice. Our findings suggest immunomodulatory effects of VEGF-A: B cell-derived VEGF-A promotes both lymphangiogenesis and angiogenesis within LNs, but then suppresses certain aspects of the ensuing immune responses. The Journal of Immunology, 2010, 184: 4819–4826.

ost defense against infection requires the integrated which results in the upregulation of CCR7. Expression of CCR7 function of both the innate and the adaptive immune allows the DCs to enter draining lymphatic vessels that express the H systems. Innate immune responses, which represent the CCR7 ligands CCL21 and CCL19 (2). On reaching the draining front line of the immune system, are elicited by a variety of cell lymph nodes (LNs), the DCs interact with T and B cells, thus types, including granulocytes, macrophages, mast cells, NK cells, inducing adaptive immune responses. by guest on September 26, 2021 and dendritic cells (DCs). DCs are the professional APCs that form Lymphatic vessels are essential for transporting tissue fluid, the bridge between innate and adaptive immune responses (1). DCs extravasated plasma proteins, and cells back to the blood circu- process material from invading pathogens and damaged tissues, lation (3). Lymphatic vessels contribute to the immune surveil- lance of the body by transporting Ag-bearing DCs from peripheral tissues to the regional LNs, where they present Ags to lympho- *Department of Laboratory and Vascular Medicine, ‡Department of Ophthalmology, cytes. Congenital or acquired dysfunction of lymphatic vessels and ||Department of Neurology and Geriatrics, Graduate School of Medical and leads to chronic swelling, adipose degeneration, immune dys- Dental Sciences, †Laboratory of Diagnostic Imaging, Department of Veterinary Med- icine, Faculty of Agriculture, and #Section of Gene Expression Regulation, Frontier function, and susceptibility to infection (3). Science Research Center, Kagoshima University; and xDivision of Respiratory Med- Lymphatic vessels are not simply inert drainage ducts; rather, { icine, Respiratory and Stress Care Center and Division of Surgical Pathology, Ka- they are actively involved in many physiologic and pathologic goshima University Medical and Dental Hospital, Kagoshima, Japan 1 processes. For example, remodeling of lymphatic vessels by tumor- B.S., T.H., T.I., and N.M. contributed equally to this work. derived lymphangiogenic factors actively promotes cancer metas- Received for publication September 23, 2009. Accepted for publication February 17, tasis (4–6). Lymphatic vessels are also remodeled in various in- 2010. flammatory conditions (7), and these remodeled vessels promote This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (C: 17590888, C: 13670659, C: inflammation (8–10). Recent studies have revealed that lymphatic 15590901, and B: 19390156) and by a grant from Mitsubishi Pharma Research vessel growth (lymphangiogenesis) is regulated by vascular en- Foundation, Japan (to T.H.). dothelial growth factor (VEGF)-C and -D via their receptor, Address correspondence and reprint requests to Dr. Teruto Hashiguchi, Department of VEGFR-3 (10, 11). In addition, VEGF-A and its receptor, VEGFR-2, Laboratory and Vascular Medicine, Cardiovascular and Respiratory Disorders, Kagoshi- ma University Graduate School of Medical and Dental Sciences, 8-35-1, Sakuragaoka, also play an important role in lymphangiogenesis, especially in Kagoshima 890-8520, Japan. E-mail address: [email protected] the enlargement of lymphatic vessels (6, 12, 13). The online version of this article contains supplemental material. During inflammatory conditions, remodeling of lymphatic vessels Abbreviations used in this paper: CAG, CMV enhancer/chicken b-actin promoter; occurs not only in inflamed peripheral tissues, but also in the CAT, chloramphenicol acetyltransferase; DC, dendritic cell; EGFP, enhanced green regional LNs. Expansion of lymphatic vessels within LNs is impor- fluorescent protein; HEV, high endothelial venule; hVEGF-A, human vascular endothelial growth factor A; LN, lymph node; LYVE-1, lymphatic vessel endothelial tant because it enhances the mobilization of DCs to the draining hyaluronan receptor-1; mVEGF-A: mouse vascular endothelial growth factor A; LNs (14). Expansion of lymphatic vessels within LNs can be locally ND, not detected; p-hVEGF-A, plasmid of human vascular endothelial growth factor controlled by lymphangiogenic factors released within the LNs A; Tg, transgenic; VEGF-A, vascular endothelial growth factor A; WT, wild-type. (14, 15) or remotely controlled by factors released in the peripheral Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 tissues (16). In the former case, this process depends upon the www.jimmunol.org/cgi/doi/10.4049/jimmunol.0903063 4820 IMMUNOMODULATORY EFFECTS OF VEGF-A presence of B cells within the LNs (14, 15). B cells in inflamed LNs Immunohistochemical staining express VEGF-A and can be stimulated to secrete VEGF-A in vitro For immunohistochemistry, paraffin sections were heated in a microwave (14), suggesting the involvement of B cell-derived VEGF-A in oven for 20 min, dewaxed in xylene, and rehydrated through a graded series lymphangiogenesis and DC mobilization. However, the exact role of ethanol solutions. Endogenous peroxidase activity was blocked by of B cell-derived VEGF-A in vivo is still unknown. incubation with 0.3% hydrogen peroxide in absolute methanol for 15 min at In this study, we investigated the effect of B cell-derived VEGF- room temperature. Ag epitopes were heat-retrieved in Antigen Unmasking Cre fl Solution (Vector Laboratories, Burlingame, CA). Samples were then in- A in vivo using CD19 /hVEGF-A mice that express human cubated overnight at 4˚C with primary Abs: rabbit polyclonal anti- VEGF-A (hVEGF-A) specifically in B cells. We found that these lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) (1/500 mice had enlarged LNs, with expanded lymphatic vessels and dilution; Upstate Biotechnology, Temecula, CA), rabbit polyclonal anti- increased high endothelial venules (HEVs), even when they were mouse PECAM-1 (M-20) (1/500 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), rat monoclonal anti-mouse CD45R/B220 (clone RA3- not immunized. To the best of our knowledge, this is the first study 6B2, rat IgG2a,k, 1/50 dilution; BD Biosciences, San Jose, CA), and rat describing the effect of B cell-derived VEGF-A in vivo. monoclonal anti-CD3 (clone CD3-12, IgG1, 1/1000 dilution; Acris Anti- bodies, Hiddenhausen, Germany). Primary Abs were diluted using 1% BSA in PBS containing 0.01% Tween. The incubation with the secondary Materials and Methods Ab was carried out for 1 h using Histofine simple stain mouse MAX-PO Mice (rabbit) or Histofine simple stain mouse MAX-PO (rat) (Nichirei, Tokyo, 9 Mice were kept under environmentally controlled pathogen-free conditions Japan) at room temperature. Peroxidase activity was visualized using 3,3 - (light from 7:00 to 19:00; water, and standard, rodent diet ad libitum; 23˚C; diaminobenzidine (DakoCytomation, Carpinteria, CA), and the slides were lightly counterstained with Lillie-Meyer’s hematoxylin (Wako, Osaka, 55% humidity). Mice of C57BL/6N background were used to generate the

Japan). Photographs were taken using a Zeiss Axiophot microscope with Downloaded from transgenic (Tg) mice. Mice heterozygous for Cre recombinase inserted into the CD19 locus (CD19Cre mice) (17) were kindly provided by an AxioCam MRc5 camera equipped with AxioVision Release 4.6 soft- Dr. Ursula Lichtenberg, Institute for Genetics, University of Cologne, ware (Carl Zeiss, Oberkochen, Germany). Cologne, Germany. Animal experiments were performed in accordance VEGF-A ELISA with the guidelines of the Frontier Science Research Center, Kagoshima University, Kagoshima, Japan. All efforts were taken to minimize the Human and mouse VEGF-A level was measured in spleen homogenates number of animals used and their suffering. and serum obtained from 13–19-wk-old CD19Cre/hVEGF-Aﬂ and Cre

CD19 mice using Human or Mouse VEGF Immunoassay (Quantikine; http://www.jimmunol.org/ Cre fl Establishment of CD19 /hVEGF-A mice R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions (n =6). The plasmid construct, containing human VEGF-A flanked by second loxP fl fl site (p-hVEGF-A ), is shown in Fig. 1Ai. To construct the p-hVEGF-A , the Measurement of inflammatory cytokines lacZ gene in pCETZ-17 (18) was replaced by the 576 bp cDNA encoding human VEGF-A. The resulting DNA construct (p-hVEGF-Afl) contains TNF-a, IFN-g, IL-2, IL-4, and IL-5 levels were determined using a mouse a CMV enhancer/chicken b-actin promoter (CAG), an enhanced green fluo- Th1/Th2 Cytokine Kit (BD Biosciences). IL-1b, IL-6, and IL-10 were rescent protein (EGFP)/chloramphenicol acetyltransferase (CAT) sand- determined using Mouse IL-1b, Mouse IL-6, and Mouse IL-10 ELISA wiched between two loxP sites and human VEGF-A flanked by loxP. kits, respectively (BioSource International, Camarillo, CA). IL-9 was de- The 5.4-kb SpeI fragment containing the hVEGF-Afl transgene was termined using a Mouse IL-9 ELISA Kit (RayBiotech, Norcross, GA). removed from the p-hVEGF-Afl vector and microinjected into the pro- by guest on September 26, 2021 nuclei of the fertilized eggs of C57BL/6N mice (18). The Tg founder (F0) Total RNA isolation and quantitative RT-PCR analysis fl mice (termed hVEGF-A mice) were identified by EGFP fluorescent blood Total RNAwas extracted from spleen, LNs, and isolated B220+ and B2202 cells using flow cytometry, as EGFP fluorescence is expressed ubiquitously cells using a total RNA isolation kit (RNAqueous, Ambion, Austin, TX). under the control of the CAG promoter system (19). Blood samples used Total RNA was quantified spectrophotometrically. Total RNA (2 mg) was for the analysis were obtained at the time of tail cut and immersed im- reverse-transcribed using the High-Capacity cDNA Reverse Transcription mediately into 1 ml 3.13% sodium citrate buffer. fl Kit (Applied Biosystems, Foster City, CA). Quantitative RT-PCR was The presence of the hVEGF-A transgene was confirmed by PCR. All performed using the TaqMan Gene Expression assay (Applied Bio- F0 Tg mice were then crossed onto wild-type (WT) C57BL/6N mice (aged Cre fl systems). Reactions were run in 96-well plates in an ABI Prism 7300 Se- 12–20 wk). CD19 mice were mated with heterozygous hVEGF-A mice quence Detection System (Applied Biosystems). Data collection and to obtain bigenic (double Tg) offspring expressing Cre in a B cell-specific Cre fl analysis was performed using SDSv1.4 software (Applied Biosystems), manner (CD19 /hVEGF-A mice). after which data were exported and further analyzed. Data were normalized In vivo assay for Cre-mediated DNA recombination based on the expression levels of GAPDH. Absence of contaminating genomic DNA was confirmed by RT-PCR of the RNA samples. In the absence of Cre, hVEGF-A expression is prevented by the intervening + transcriptional EGFP/CAT sequence flanked by loxP sites. Cre-mediated Isolation of B220 B cells DNA recombination results in the removal of the EGFP/CAT sequence, fol- Single-cell suspensions were prepared from the spleens of 16–49-wk-old lowed by hVEGF-A expression (Fig. 1Ai, Supplemental Fig. 1A). Genomic CD19Cre/hVEGF-Afl and CD19Cre mice by dissociation of the isolated DNA was isolated and amplified using PCR primers Chi5’ (59-GGC GGG tissues with glass slides in MACS separation buffer (Miltenyi Biotec, GTT CGG CTT CTG GCG TGT GAC CGG-39)andVeg3’ (59-TCA CCG Bergisch Gladbach, Germany) and passage through a 30-mm nylon mesh. CCT CGG CTT GTC ACATCT GCA AGT-39), which recognize sequences B cells were enriched by positive selection using CD45R (B220) mi- of chicken b-actin promoter and hVEGF-A, respectively. In the case of fl crobeads and an AutoMACS Magnetic cell Sorter according to the man- hVEGF-A mice, a 3.2-kb fragment including the EGFP/CATand hVEGF-A ufacturer’s instructions (Miltenyi Biotec). was amplified, whereas Cre-mediated recombination in CD19Cre/hVEGF-Afl mice resulted in the amplification of a 788-bp PCR product (Fig. 1Aii). Flow cytometry LPS challenge Blood was collected from sevofrane-anesthetized mice by cardiac puncture and was anticoagulated with sodium citrate. Single-cell suspensions from Cre fl Cre CD19 /hVEGF-A and CD19 mice (13 to 14 wk, n = 5) were injected spleen and LNs were prepared as described above. Nonspecific binding i.p. with LPS (1 mg/kg, Escherichia coli 055:B5; Sigma-Aldrich, St. was blocked by incubation with an Fc-blocking Ab (10 mg/ml; BD Bio- Louis, MO) in sterile saline prior to cytokine analysis. Blood samples were sciences). Samples were then stained with mAbs to mouse CD4, CD8, collected 8 h later by cardiac puncture. Serum was isolated from the blood CD19 (Beckman Coulter, Marseille, France), CD11b, CD14, and 33D1 samples and stored at 280˚C until required. (BD Biosciences) conjugated to PE, for 15 min at room temperature. Following surface staining, RBCs were lysed with Optilyse C (Beckman Histopathological examination Coulter), and the remaining cells were fixed. Flow Count Beads (Beckman H&E staining. Spleen and LN specimens were fixed for 24 h in 10% Coulter, Fullerton, CA) were added to the samples for quantitation. Cells formaldehyde neutral buffer solution, embedded in paraffin wax, and were analyzed using an Epics XL flow cytometer (Beckman Coulter, sectioned (5–10 mm). Sections were stained with H&E. Miami, FL). The Journal of Immunology 4821

OVA sensitization and challenge followed by determination of (CD19Cre) (Fig. 1Ai). Prerecombination, the loxP flanked EGFP/ serum Ab concentration CAT hybrid sequence was expressed under the control of the CAG For sensitization, 10–12-wk-old CD19Cre/hVEGF-Afl and CD19Cre mice promoter, whereas the hVEGF-A gene was silent. Cre-mediated re- were injected s.c. on day 0 with 50 mg OVA (grade V; Sigma-Aldrich) combination resulted in the deletion of the EGFP/CAT sequence adsorbed on 100 mg (100 ml) of CFA (Sigma-Aldrich) and again on day 10 and subsequent expression of the hVEGF-A gene. This resulted in with 25 mg adsorbed on 100 ml incomplete Freund’s adjuvant. Mice were CD19Cre/hVEGF-Afl Tg mice. The loxP sequence, followed by challenged intranasally with 25 mg OVA in PBS on days 21, 22, and 23. VEGF-A sequence (Supplemental Fig. 1A) and the appearance of Mice were killed on day 25. Serum and spleen homogenates were obtained + Cre for measurement of OVA-specific IgG1 and IgE Abs by ELISA using a 788-bp PCR product in the DNA from B220 cells of CD19 / fl a mouse OVA-IgG1 and IgE kit (Shibayagi, Gunma, Japan) according to hVEGF-A mice (Fig. 1Aii), confirmed successful recombination. the manufacturer’s instructions. Because the EGFP gene is deleted from the B cells of hVEGF- Afl mice during recombination to create CD19Cre/hVEGF-Afl mice, Statistical analysis we measured the EGFP expression in CD19+ B cells using flow Statistical analysis was performed using the Student t test, Mann-Whitney’s cytometry (Fig. 1Bi). In CD19Cre/hVEGF-Afl mice, ∼80% of the U test, or Tukey-Kramer test through all of the experimental procedures. CD19+ B cells were EGFP negative. This percentage was similar in p values of pp , 0.05 and ppp , 0.01 were considered significant. CD19+ B cells from spleen, LN, and blood samples (Fig. 1Bii). Flow cytometry using Abs against CD19, CD8, and CD4 showed Results that EGFP had been selectively deleted from the CD19+ B cells in Generation of Tg mice expressing hVEGF-A specifically in B cells the CD19Cre/hVEGF-Afl mice (Supplemental Fig. 1B).

We generated mice overexpressing hVEGF-A in B cells by crossing The expression of hVEGF-A mRNA was observed in spleen and Downloaded from loxP-flanked (floxed) EGFP mice (hVEGF-Afl) onto mice express- LN samples from CD19Cre/hVEGF-Afl mice, but not in samples ing Cre under the control of the B cell-specific CD19 promoter from of CD19Cre mice (Fig. 1C). The expression of mouse VEGF-A

FIGURE 1. DNA constructs and Cre- mediated recombination event. Ai,Sche- http://www.jimmunol.org/ matic of the target plasmid (p-hVEGF-Afl) and its recombinated form are shown. LoxP sites are shown as triangles. The small arrows (Chi5’ and Veg3’) indicate the positions and directions of the primers used for PCR. Aii, Identification of Cre- mediated DNA recombination product by PCR amplification of DNA isolated from fl 2 spleen of hVEGF-A mice (lane 1), B220 by guest on September 26, 2021 cells of CD19Cre/hVEGF-Afl mice (lane 2), and B220+ cells of CD19Cre/hVEGF-Afl mice (lane 3). The 788-bp fragment rep- resents the successfully recombinated DNA. M indicates PCR marker. Bi, Flow cytometry analysis of B cells from the spleen of 19–22-wk-old hVEGF-Afl, CD19Cre, and CD19Cre/hVEGF-Afl mice. Cells were stained with Abs to CD19. Percentages refer to EGFP-positive and -negative cells among CD19+ cell population. Bii, Recombination ratio of hVEGF-A in the CD19+ cells from spleen, LN, and blood samples. The ratio was defined as the percentage of EGFP- negative cells in the total CD19+ cell population. C, hVEGF-A mRNA expression in spleen of CD19Cre/hVEGF-Afl and CD19Cre mice analyzed by quantitative RT-PCR (n = 6). D, hVEGF-A mRNA expression in B220+ and B2202 cells of CD19Cre/hVEGF-Afl (n = 4) and CD19Cre (n = 5) mice analyzed by quantitative RT-PCR. E, hVEGF-A protein expression in spleen homogenates and serum from CD19Cre/hVEGF-Afl and CD19Cre mice (n = 6). F, mVEGF-A protein expression in spleen homogenates from CD19Cre/hVEGF-Afl and CD19Cre mice. Data in A–F are representative of at least two independent experiments. ppp , 0.01. ND, not detected. 4822 IMMUNOMODULATORY EFFECTS OF VEGF-A

(mVEGF-A) mRNA was similar in both the CD19Cre/hVEGF-Afl temic circulation, as mice expressing systemic VEGF-A develop and CD19Cre mice (Supplemental Fig. 2A). The expression of widespread tissue edema and die within days (20). mVEGF-D mRNA was also similar in both (Supplemental Fig. Increased lymphangiogenesis and angiogenesis in LNs of 2C), but mVEGF-C mRNA expression was significantly increased Cre fl in CD19Cre/hVEGF-Afl mice (Supplementary Fig. 2B), which is in CD19 /hVEGF-A mice agreement with previous studies (6). At around 14 wk, the LNs of CD19Cre/hVEGF-Afl mice were sig- To examine whether hVEGF-A mRNA was specifically ex- nificantly larger than those of CD19Cre mice of the same age (Fig. pressed by the B cells, we isolated B220+ cells from the spleens of 2Ai, Supplemental Fig. 3A). Quantitative FACS analysis of LN cell CD19Cre/hVEGF-Afl and CD19Cre mice. B220+ cells, but not suspensions revealed significantly increased numbers of B and B2202 cells, expressed mCD19 mRNA, indicating the successful T cells in the LNs of CD19Cre/hVEGF-Afl mice (Supplemental isolation of B cells (data not shown). In this experimental con- Fig. 3B). The LNs from CD19Cre/hVEGF-Afl mice had an apparent dition, B220+ cells, but not B2202 cells, expressed hVEGF-A reddish color, which may be due to the increased vascularization mRNA (Fig. 1D), confirming the specific expression of hVEGF-A around LNs (Fig. 2Aii). Immunohistochemical analysis of the mRNA in B cells. The expression of mVEGF-A mRNA was not LNs showed an increase in both lymphangiogenesis and angiogen- significantly different in either the B220+ or the B2202 esis. LYVE-1+ lymphatic vessels and PECAM-1+ blood vessels were cells of both CD19Cre/hVEGF-Afl and CD19Cre mice (data not increased in the LNs of CD19Cre/hVEGF-Afl mice compared with shown). CD19Cre mice (Fig. 2B). Higher magnification showed an increase Next, we examined hVEGF-A protein levels in the CD19Cre/ in the number of HEVs within the LNs of the CD19Cre/hVEGF-Afl hVEGF-Afl mice. hVEGF-A was detected in spleen homogenates mice that were stained for PECAM-1 (Fig. 3A,3B). These findings Downloaded from of CD19Cre/hVEGF-Afl mice, but was not present in their serum suggest that B cell-derived VEGF-A promotes LN hypertrophy, (Fig. 1E), suggesting that hVEGF-A is localized to lymphoid lymphangiogenesis, and HEV expansion. organs and does not spread through the systemic circulation in Cre fl Enlargement and disorganization of spleens in CD19 /hVEGF-A mice. No hVEGF-A was detected in either Cre fl the spleen homogenates or the serum from the CD19Cre mice. CD19 /hVEGF-A mice

mVEGF-A protein levels in spleen homogenates were similar On gross anatomical examination, we observed splenomegaly in http://www.jimmunol.org/ in CD19Cre/hVEGF-Afl and CD19Cre mice (Fig. 1F), and the the CD19Cre/hVEGF-Afl mice (Fig. 4A), which developed from the total amount of VEGF-A (hVEGF-A plus mVEGF-A) in spleen age of 14 wk. Histological analysis of the spleens from CD19Cre/ homogenates was 3- to 4-fold higher in CD19Cre/hVEGF-Afl hVEGF-Afl mice revealed a severe distortion of the microscopic mice. structure, even in mice that were younger than 14 wk old, and this The CD19Cre/hVEGF-Afl mice appeared grossly normal in distortion was seen in both the red and white pulp areas (Fig. 4B). In weight and life span, and peripheral blood cell levels were com- addition, sinusoidal dilatations were observed in the CD19Cre/ parable to that of CD19Cre mice. CD19Cre/hVEGF-Afl, CD19Cre, hVEGF-Afl mice. The spleens from CD19Cre mice showed a normal hVEGF-Afl, and WT mice were born at the expected Mendelian structure. The distribution of T and B cells was similar in both ratio, but we noticed a slight reduction in the Mendelian distribution CD19Cre/hVEGF-Afl mice and CD19Cre mice (Supplemental Fig. 4) by guest on September 26, 2021 of hVEGF-Afl mice. The ratio of CD19Cre/hVEGF-Afl:CD19Cre: despite the distortion of splenic structure present in the CD19Cre/ hVEGF-Afl:WT mice was 32.41%:24.83%:19.31%:23.45%, re- hVEGF-Afl mice. The number of CD8+ T cells was significantly spectively, from a total of 145 mice. These findings further support decreased in the spleens of the CD19Cre/hVEGF-Afl mice, whereas the idea that hVEGF-A protein in CD19Cre/hVEGF-Afl mice is lo- that of CD19+ B cells was similar in CD19Cre/hVEGF-Afl and calized to lymphoid organs and does not spread through the sys- CD19Cre mice (Supplemental Fig. 5).

FIGURE 2. Lymphangiogenesis and angiogenesis in CD19Cre/hVEGF-Afl mice. Ai, Weight of LNs taken from 19–22-wk-old CD19Cre/hVEGF-Afl (n =9)and CD19Cre (n = 8) mice. Both individual and mean weights are presented. Data obtained from five independent experiments. Aii, The gross macroscopic structure of the ileocolic LNs of CD19Cre/hVEGF-Afl and CD19Cre mice. Scale bar, 2.5 mm. Arrowheads indicate the LNs. Rep- resentative images from five experiments are shown. B, Immunohistochemical analysis of expression of LYVE-1 and PECAM-1 in the ileocolic LNs of age- and sex-matched CD19Cre/hVEGF-Afl and CD19Cre mice. Serial sections were taken and stained for LYVE-1 and PECAM-1 as indicated in the figure. Data are representative of three independent experiments. Scale bar, 500 mm. Original magnification 325. pp , 0.05. The Journal of Immunology 4823

FIGURE 3. Increased numbers of HEVs in the LNs of CD19Cre/hVEGF-Afl. A, PECAM-1 staining of serial sections of ileocolic LNS from CD19Cre/hVEGF-Afl and CD19Cre mice. Ai, Low-power view of HEVs. Scale bar, 200 mm. Original magnification 350. Aii, High-power view of the inset shown in Ai showing the characteristics of high endothelial cell structure. Data are representative of three independent experiments. Scale bar, 20 mm. Original magnification 3400. B, Number of HEVs in the LNs. Individual data and values are presented. ppp , 0.01.

Decreased adaptive immune responses in CD19Cre/hVEGF-Afl significantly lower in CD19Cre/hVEGF-Afl mice. IL-1b, IL-2, IL-4, mice and IL-10 levels were similar in CD19Cre/hVEGF-Afl and CD19Cre mice. The cytokine levels in mice injected with saline alone were Next, we examined whether B cell-derived VEGF-A accelerated or Cre fl

Cre fl very low and were not significantly different in CD19 /hVEGF-A Downloaded from suppressed the immune response in CD19 /hVEGF-A mice. To Cre Cre and CD19 mice. These findings indicate that B cell-derived examine the adaptive immune response, we challenged CD19 / Cre fl hVEGF-Afl and CD19Cre mice with OVA and then measured OVA- VEGF-A induces LPS tolerance in CD19 /hVEGF-A mice. specific IgG1 levels. OVA-specific IgG1 levels were significantly lower in the serum of CD19Cre/hVEGF-Afl mice compared with Discussion CD19Cre mice (Fig. 5A). The OVA-specific IgG1 levels were also Besides being an angiogenic factor, VEGF-A has recently been Cre fl identified as a pivotal mediator of inflammation-induced LN lower in spleen homogenates from CD19 /hVEGF-A mice http://www.jimmunol.org/ (Fig. 5B), whereas the spleens were significantly larger in these lymphangiogenesis (14, 16). However, the precise role of VEGF- mice (Fig. 5C). The OVA-specific IgE levels were not significantly A–induced inflammatory lymphangiogenesis in the modulation of different (data not shown). These findings indicate that B cell- immune function remains unclear. Hosts utilize various compo- derived VEGF-A promotes splenomegaly, but can suppress the Ab nents of the immune system to carefully maintain the delicate production. balance between promoting a proper immune response to invading pathogens and preventing an excessive immune response that can LPS tolerance in CD19Cre/hVEGF-Afl mice We examined cytokine levels in CD19Cre/hVEGF-Afl and CD19Cre mice. As shown in Fig. 6, LPS challenge resulted in the induction by guest on September 26, 2021 of TNF-a,IFN-g, IL-5, and IL-6 production in both CD19Cre/ hVEGF-Afl and CD19Cre mice, but the levels of these cytokines were

FIGURE 4. Enlargement and disorganization of spleens in CD19Cre/ hVEGF-Afl mice. Ai, Gross macroscopic comparison of the spleens from age- and sex-matched CD19Cre/hVEGF-Afl and CD19Cre mice. Scale bar, 1 cm. Representative images from 10 experiments are shown. Aii, Spleen weights of 16–75-wk-old CD19/h-VEGF (n = 27) and CD19Cre (n = 16) mice. Individual data and mean values are presented. Data obtained from FIGURE 5. Decreased adaptive immune responses in CD19Cre/hVEGF- 10 independent experiments. B, H&E-stained sections of spleen from Afl mice. OVA specific IgG1 levels in the serum (A) and the spleen (B) CD19Cre/hVEGF-Afl and CD19Cre mice. Representative images from 10 homogenates of 10–12-wk-old CD19Cre/hVEGF-Afl (n = 4) and CD19Cre experiments are shown. Scale bar, 200 mm. Original magnification 325. (n = 4) mice postimmunization with OVA. C, Spleen weight (n = 4). Data pp , 0.05. are representative of two independent experiments. pp , 0.05; ppp , 0.01. 4824 IMMUNOMODULATORY EFFECTS OF VEGF-A

FIGURE 6. LPS tolerance in CD19Cre/hVEGF-Aﬂ mice. Cytokine levels in the serum of 13- to 14-wk- Cre ﬂ

old CD19 /hVEGF-A (n = 5) and Downloaded from CD19Cre (n = 5) mice after i.p. challenge of saline or LPS (1 mg/ kg). Data are representative of two independent experiments. pp , 0.05; ppp , 0.01. http://www.jimmunol.org/ by guest on September 26, 2021

lead to immunopathology (21). In this study, we have shown that (but not of VEGF-D mRNA) were increased in CD19Cre/hVEGF-Afl B cell-derived VEGF-A might play a role in maintaining the mice (Supplemental Fig. 2B,2C), which is in agreement with a pre- balanced immune responses by orchestrating many aspects of the vious report showing that VEGF-A treatment upregulates VEGF-C immune responses, including the expansion of lymphatic networks expression in cultured endothelial cells (23). It is suggested that and the suppression of Ab production. Although our study does VEGF-A promotes lymphangiogenesis in CD19Cre/hVEGF-Afl mice not reveal endogenous roles of B cell-derived VEGF-A or roles of either directly or via the upregulation of VEGF-C. other cell-derived VEGF-A, it provides an indication of the likely The cellular mechanisms of de novo lymphangiogenesis remain role of B cell-derived VEGF-A. poorly defined and may involve the division of local pre-existing endothelial cells (24) or the incorporation of lymphatic endothelial Lymphangiogenic roles of VEGF-A progenitor cells of myeloid origin (25–27). We observed the ac- A recent study suggested the involvement of B cell-derived VEGF- cumulation of a CD11b+ cell population in the LNs of our A in lymphangiogenesis and DC mobilization (14). In this study, CD19Cre/hVEGF-Afl mice. CD11b+ cells might play an important we examined the role of B cell-derived VEGF-A in vivo using role in lymphangiogenesis either by secreting VEGF-C, which a Tg mouse model in which the B cells express hVEGF-A. We stimulates the division of pre-existing local lymphatic endothelial found that these mice had enlarged LNs, with expanded lymphatic cells, (7) or by transdifferentiating and directly incorporating into vessels and increased HEVs, even when they were not immunized. the endothelial layer (25, 28). These findings suggest that B cell-derived VEGF-A promotes lymphangiogenesis as well as angiogenesis in vivo. Immunosuppressive roles of VEGF-A VEGF-A induces lymphangiogenesis either directly or via upreg- Previous studies have indicated that lymphangiogenic responses ulation of the lymphangiogenic factors VEGF-C and VEGF-D. lead to the increased migration of APCs to draining LNs, thereby Wirzenius et al. (13) reported that VEGF-A can directly promote boosting immuneresponses(14,15,29).Other studies have suggested lymphatic vessel enlargement via VEGFR-2 signaling. Crusiefen that the growth of HEVs is associated with increased lymphocyte et al. (22) reported that inflammatory macrophages, in response to entry into the LNs, which, again, boosts the immune responses stimulation with VEGF-A, release VEGF-C/-D that contributes to (30, 31). These findings led us to speculate that the increase in lym- lymphangiogenesis. We observed that the levels of VEGF-C mRNA phangiogenesis and the growth of HEVs within LNs might stimulate The Journal of Immunology 4825 immune responses in CD19Cre/hVEGF-Afl mice. However, VEGF-A 6. Hirakawa, S., S. Kodama, R. Kunstfeld, K. Kajiya, L. F. Brown, and M. Detmar. 2005. VEGF-A induces tumor and sentinel lymph node lymphangiogenesis and is known to suppress both the development of T cells and the matu- promotes lymphatic metastasis. J. Exp. 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