FcγRIIb inhibits -induced VEGF-A production and intranodal lymphangiogenesis

Menna R. Clatworthya,1, Sarah K. Harfordb, Rebeccah J. Mathewsa, and Kenneth G. C. Smithb

aDepartment of Medicine, University of Cambridge Research Unit, Medical Research Council Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge CB2 0QH, United Kingdom; and bDepartment of Medicine, Cambridge Institute for Medical Research, University of Cambridge School of Clinical Medicine, Cambridge Biomedical Campus, Cambridge CB2 0XY, United Kingdom

Edited* by Jeffrey V. Ravetch, The Rockefeller University, New York, NY, and approved November 13, 2014 (received for review July 22, 2014) IgG immune complexes (ICs) are generated during immune the likelihood of such encounters (8). Lymphatic vessels responses to infection and self-antigen and have been implicated transport antigen and DCs from peripheral tissues and provide in the pathogenesis of autoimmune diseases such as systemic a distribution network within lymph nodes, providing access erythematosus (SLE). Their role, and that of the fragment lanes to the T-cell area (9). During tissue inflammation, there crystallizable (Fc) receptors that bind them, in driving local in- is an expansion of lymphatic vasculature (lymphangiogenesis) flammation is not fully understood. Low affinity-activating Fcγ within draining lymph nodes (10, 11). This increases the avail- receptors (FcγRs) that bind immune complexes are controlled by a single inhibitory receptor, FcγRIIb (CD32b). We investigated able conduits through which antigen or DCs may travel, en- whether FcγR cross-linking by IC might induce VEGF-A and lymph hancing transit to, and distribution within, draining lymph nodes. node lymphangiogenesis. Murine macrophages and dendritic cells Vascular endothelial growth factor A (VEGF-A) appears to be (DCs) stimulated with ICs produced VEGF-A, and this was inhibited particularly important in mediating this process, via its receptor by coligation of FcγRIIb. Similarly, IC-induced VEGF-A production VEGFR2 (10-13). Lymph node-resident B cells provide an im- by B cells was inhibited by FcγRIIb. In vivo, IC generation resulted portant source (10, 13) but macrophages and stromal cells can in VEGF-A–dependent intranodal lymphangiogenesis and increased also produce VEGF-A (11, 14, 15). A variety of stimuli result in DC number. We sought to determine the relevance of these findings VEGF-A production, including proinflammatory cytokines such to autoimmunity because elevated serum VEGF-A has been ob- as TNF-α (16), toll-like receptor (TLR) agonists, and in B cells, served in patients with SLE; we found that lymphangiogenesis B-cell receptor (BCR) cross-linking (10). We sought to deter- and VEGF-A were increased in the lymph nodes of mice with colla- γ gen-induced arthritis and SLE. In humans, a SLE-associated polymor- mine whether Fc R cross-linking with IgG immune complexes phism (rs1050501) results in a dysfunctional FcγRIIBT232 receptor. (ICs) would stimulate VEGF-A production in lymph node im- -derived macrophages from subjects with the FcγRIIBT/T232 mune cells, resulting in intranodal lymphangiogenesis. This genotype showed increased FcγR-mediated VEGF-A production, dem- would identify a novel effector function for IgG and an addi- onstrating a similar process is likely to occur in humans. Thus, ICs tional process, which might be negatively regulated by FcγRIIb. contribute to inflammation through VEGF-A–driven lymph node We demonstrate that ICs drive VEGF-A production by immune lymphangiogenesis, which is controlled by FcγRIIb. These findings cells, and hence intranodal lymphangiogenesis. This is controlled have implications for the pathogenesis, and perhaps future treat- by FcγRIIb in mice and also in humans, indicating that FcγRIIb ment, of autoimmune diseases. could potentially limit immunoreactivity via control of lym- phatics and suggests an additional therapeutic target in auto- Fc gamma receptors | autoimmunity | VEGF-A | lupus | lymphangiogenesis immune disease. ntibodies are important for defense against infection but Amay also be pathogenic in some autoimmune diseases. Significance Many effector functions of are mediated via fragment crystallizable gamma receptors (FcγRs) that bind to the Fc Antibody (IgG) plays an important role in defense against in- portion of IgG. FcγRs may be activating (in mice FcγRI, III, and fection and in the pathogenesis of autoimmune diseases such IV) or inhibitory (FcγRIIb) and are found on most cells of the as systemic lupus erythematosus (SLE). Low affinity-activating γ immune system (1). Following stimulation with IgG-opsonized fragment crystallizable gamma receptors (Fc Rs) that bind IgG antigen, the inhibitory receptor FcγRIIb negatively regulates immune complexes (ICs) mediate many effector functions of γ B-cell activation, macrophage and proinflammatory antibody and are controlled by an inhibitory receptor, Fc RIIb. cytokine release, and antigen presentation by dendritic cells Here we show a previously unappreciated role for IC in driving (DCs). Mice deficient in FcγRIIb demonstrate hyperactive an expansion of lymphatic conduits within lymph nodes. This was dependent on macrophage VEGF-A production and in- immune responses and are susceptible to antibody-mediated hibited by FcγRIIb. Lymphangiogenesis and VEGF-A were in- autoimmune diseases (2). In humans, a single nucleotide poly- creased in the lymph nodes of mice with arthritis and SLE and morphism in FCGR2B (rs1050501) results in an amino acid sub- in macrophages obtained from people with a SLE-associated, stitution (a threonine for an isoleucine) within the transmembrane defunctioning polymorphism in FCGR2B. These findings have domain of the receptor. This substitution is associated with re-

implications for the pathogenesis and treatment of auto- INFLAMMATION ceptor dysfunction and confers susceptibility to the autoimmune immune diseases. IMMUNOLOGY AND disease systemic lupus erythematosus (SLE) (3–5) but may en- hance protective responses against some pathogens (6, 7). Author contributions: M.R.C. and K.G.C.S. designed research; M.R.C., S.K.H., and R.J.M. An adaptive immune response requires the anatomical colo- performed research; M.R.C., S.K.H., and R.J.M. analyzed data; and M.R.C. wrote the paper. calization of antigen or antigen-loaded antigen presenting cells The authors declare no conflict of interest. (APCs), such as DCs, with rare antigen-specific B and T cells. *This Direct Submission article had a prearranged editor. These interactions take place within secondary lymphoid organs 1To whom correspondence should be addressed. Email: [email protected]. (spleen and lymph nodes), in which the microanatomical ar- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. rangement of immune cells and stromal cell networks optimizes 1073/pnas.1413915111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1413915111 PNAS | December 16, 2014 | vol. 111 | no. 50 | 17971–17976 Downloaded by guest on September 29, 2021 Results and Discussion littermates (Fig. 1B). Similarly, in bone-marrow–derived DCs IgG Immune Complex-Induced VEGF-A Production by Macrophages (BMDCs), culture with IC induced VEGF-A production, which γ and DCs Is Inhibited by FcγRIIb. Because FcγRs mediate many ef- was regulated by Fc RIIb (Fig. 1C). The time course of immune- – fector functions of antibody, and VEGF-A is critical for driving complex induced VEGF-A production in macrophages was in- intranodal lymphatic expansion (10, 13), we sought to determine vestigated, demonstrating VEGF-A in culture supernatants whether FcγR cross-linking by IgG immune complexes could induce within 6 hours and its production increased to 36 h (Fig. S1A). IC-induced VEGF-A production by mouse and human macro- VEGF-A secretion by macrophages and DCs. Following incubation phages was significantly attenuated by blocking activating FcγRs of peritoneal macrophages with immune complexes [ovalbumin (Fig. 1D and Fig. S1B). VEGF-A production was also observed in opsonized with rabbit anti-OVA IgG (IC)] for 24 h, a significantly murine macrophages and DCs following stimulation with synge- higher concentration of VEGF-A was detectable within culture neic murine IgG IC (Fig. 1E and Fig. S2 A and B)andin supernatants compared with macrophages cultured with ovalbumin human monocyte-derived macrophages following stimulation γ alone (O) and this was more marked in Fc RIIb-deficient mac- with human IC (Fig. S2C). γ rophages (Fig. 1A). Conversely, Fc R ligation on macrophages To determine if macrophages within lymph nodes produce obtained from transgenic mice with macrophage-specific over- VEGF-A in response to FcγR cross-linking, we passively trans- expression of FcγRIIb (FcγRIIb-MTG) (17) resulted in signifi- ferred antiphycoerythrin (anti-PE) IgG to mice and immunized cantly lower VEGF-A production compared with nontransgenic them s.c. with PE 12 hours later to generate PE-immune com- plexes within draining lymph nodes, as described previously (18). Subcapsular sinus (SCS) macrophages are a specialized subset of macrophages specifically positioned at the periphery of lymph ABC nodes to filter incoming lymphatic fluid. They prevent viral dis- semination (19) and capture incoming ICs (18). At 24 h fol- lowing immunization, colocalization of VEGF-A staining was + noted with CD169 SCS macrophages in the draining lymph nodes of mice that received an anti-PE antibody (Fig. 1 F, Upper and G and Fig. S3 A and B) but not in animals that received isotype control antibody (Fig. 1F, Middle). VEGF-A was also − D E detectable in CD169 cells at the edge of the B-cell follicle (Fig. 1G). Less frequently, colocalization was observed with CD11c, indicating DC production of VEGF-A (Fig. 1H and Fig. S3 C– F). Thus, immune complexes induce VEGF-A production by macrophages and DCs, both in vitro and in vivo. Early immune complex-induced VEGF-A production is a di- G rect effect of FcγR signaling, but is indirectly enhanced by TNF- F α at later time points. VEGF-A expression has been shown to be increased by a number of inflammatory cytokines including TNF-α. Because activating FcγR cross-linking can induce significant TNF-α production in macrophages (6), we reasoned that the FcγR- mediated VEGF-A production we had observed might be a direct result of FcγR cross-linking or might occur indirectly, through the H effects of TNF-α. As noted above, VEGF-A was detectable in culture supernatants within 6 h of macrophage stimulation, in support of a direct effect by FcγR cross-linking (Fig. S1A). Fur- thermore, inhibition of extracellular signal-regulated kinase (Erk), a kinase located downstream of activating FcγRs (20), using the MEK1/2 inhibitor U0126 attenuated VEGF-A production to baseline at this early time point in both macrophages (Fig. 2A)and DCs (Fig. S4). Inhibition of other pathways, including TLR sig- Fig. 1. IgG immune complex-induced VEGF-A production by macrophages naling using an IRAK1/4 inhibitor, and calcineurin blockade had and DCs is inhibited by FcγRIIb. VEGF-A concentration in culture super- no impact on IC-induced VEGF-A production (Fig. S5). At 24 h, natants following 24-h incubation with media alone (−), ovalbumin (O), or TNF-α was detectable in macrophage culture supernatants fol- IgG-opsonized ovalbumin (IC) using (A) peritoneal macrophages from WT α −/− lowing immune complex stimulation (Fig. 2B) and TNF- and Fcgr2b mice, (B) peritoneal macrophages from nontransgenic (NTG) blockade partially abrogated IC-induced VEGF-A production and FcγRIIb-MTG mice, and (C) bone-marrow–derived DCs from WT and −/− (Fig. 2C). Similarly, there was significantly reduced VEGF-A Fcgr2b mice. (D) VEGF-A concentration in culture supernatants of murine γ macrophages stimulated with O, OVA-IC, and isotype (IC), or OVA-IC and production by TNFR1-deficient macrophages following Fc R anti-CD16 (FcγRIII) antibody (IC+αCD16). (E) VEGF-A production by unsti- cross-linking at 24 h (Fig. 2D). These data suggest that VEGF-A mulated (US) bone-marrow–derived macrophages or those stimulated with production occurs both as a direct result of immune complex

O, IC, anti-mouse F(ab′)2,(αFab2), or anti-mouse F(ab′)2/mouse IgG immune stimulation but can also be amplified by the indirect effects of +α complexes (mIgG Fab2). Graphs show the mean and SE of mean. P values the TNF-α induced by FcγR cross-linking. calculated using a Student’s t test. Graphs show representative experiments from three to five repeats. (F) Confocal micrograph of inguinal lymph node VEGF-A Production by B Cells Limited by Coligation of FcγRIIb. Angeli section obtained from WT mice following passive immunization with an et al. demonstrated that B cells produce VEGF-A following BCR anti-PE antibody or isotype control, followed by s.c. PE. Lymph nodes har- cross-linking (10), but it is not known whether this can be con- vested at 24 h and stained for CD169 (green), VEGF-A (red), and B220 γ (purple). (Lower) Secondary antibody alone for VEGF-A. Images represen- trolled by coligation of Fc RIIb. We confirmed VEGF-A secre- tion by B cells in response to BCR cross-linking with anti- tative of three lymph node sections. (G) High power image showing extent ′ of colocalization of CD169 and VEGF-A. (H) Confocal image of lymph node IgMF(ab )2 together with CD40 costimulation (Fig. 2 E and F). stained with CD11c antibody (blue) and VEGF-A (red). Colocalization is Coligation of the BCR and FcγRIIb with anti-IgM attenuated shown in white. VEGF-A production in WT B cells but not in FcγRIIb-deficient

17972 | www.pnas.org/cgi/doi/10.1073/pnas.1413915111 Clatworthy et al. Downloaded by guest on September 29, 2021 ABanti-IgM stimulation (Fig. 3H). Thus, BCR crosslinking directly induces Erk-dependent VEGF-A production that can be inhibited by coligating FcγRIIb.

Immune Complexes Induce Lymph Node Lymphangiogenesis in Vivo, Which Is Attenuated by FcγRIIb and Mediated via VEGFR2. Given our in vitro data demonstrating IC-induced VEGF-A production C D in macrophages, DCs, and B cells, including those in lymph nodes, we hypothesized that ICs may stimulate intranodal lym- phangiogenesis. To test this hypothesis, we immunized mice with ovalbumin in alum (OVA-Alum) intraperitoneally (leading to the production of anti-OVA IgG), followed 21 d later by a s.c. boost to generate immune-complexed antigen in immunized tissues and draining lymph nodes. Lymph nodes were harvested 3 d later and lymphatic vessels assessed both by immunohisto- chemistry (using an anti–Lyve-1 antibody to identify lymphatic endothelial cells, LECs) and by flow cytometry (CD45-negative, EF CD31/podoplanin double positive cells, as used previously to identify LECs) (11). In unimmunized mice, LECs were observed in the medulla and interlobar area of the inguinal lymph node. Following immunization and boost, an expansion of LECs was observed in draining lymph nodes, whereas only minimal lym- phatic expansion occurred in the absence of primary immunization (Fig. 3 A and B). IC-induced lymphangiogenesis was particularly

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Fig. 2. IC-induced VEGF-A production abrogated by Erk inhibition. VEGF-A concentration in culture supernatants following incubation with media alone (−), ovalbumin (OVA), IgG-opsonized ovalbumin (OVA-IC), or OVA-IC with the addition of the MEK1/2 inhibitor U0126 (OVA-ICi, hatched bars) using (A) − − peritoneal macrophages from WT and Fcgr2b / mice (B)TNF-α concentration G HI in culture supernatants following incubation of peritoneal macrophages from − − WT and Fcgr2b / mice with O or IC for 24 h. (C) VEGF-A concentration in culture supernatants following incubation of peritoneal macrophages from WT mice with OVA or OVA-IC for 6 h (white bars) and 24 h (hatched bars) with and without an anti–TNF-α blocking antibody at 0.5 or 1 μg/mL or an isotype control antibody. (D) VEGF-A concentration in culture supernatants following − − 24-h incubation of peritoneal macrophages from WT (white bars) and Tnfr1 / (striped bars) mice with O or IC. VEGF-A concentration in culture supernatants Fig. 3. Immune complexes induce lymph node lymphangiogenesis in vivo, ′ harvested 48 h following BCR cross-linking [with anti-IgM or anti-IgMF(ab )2] that is mediated via VEGFR2 and attenuated by macrophage expression of −/− together with CD40L of splenic B cells obtained from (E)WTandFcgr2b FcγRIIb. (A) Representative images of inguinal lymph nodes showing Lyve-1 γ γ mice and (F)FcRIIb B-cell transgenic mice (Fc RIIb-BTG, gray bars) and litter- positive lymphatic endothelial cells (LECs, green) and B cells (red) from mice −/− mate controls (NTG, white bars). (G)WTandFcgr2b B cells stimulated with immunized with OVA (OVA) s.c. in the flank (Left) or mice primed with OVA- ± −/− LPS or IgM MEK1/2 inhibitor U0126 (Erki). (H) WT (white bars) and Tnfr1 Alum IP to generate anti-OVA IgG, and boosted 3 wk later with s.c. OVA ± (striped bars) B cells stimulated with IgM or anti-IgM(fab)2 Erki. Graphs show (OVA-IC) (Right). (B) Quantification of inguinal lymph node LECs by flow the mean and SE of mean. Results are representative of two to four in- cytometry in unimmunized mice (Unimm) or mice immunized s.c. with OVA ≤ dependent experiments. P values were calculated using a Student t test. *P or OVA-IC, as decribed in A.(C) Percentage of cross-sectional area of inguinal ≤ ≤ ≤ 0.05, **P 0.01, ***P 0.001, ****P 0.0001. lymph node occupied by lymphatic endothelium and (D) number of LECs per lymph node and (E) number of myeloid DCs per lymph node (gating on live, CD11c positive, B220 negative cells) in unimmunized or OVA-IC immunized B cells (Fig. 2E). In keeping with these data, anti-IgM stimulation WT and Fcgr2b−/− (KO) mice. (F) Number of LECs per lymph node in OVA-IC INFLAMMATION −/− of B cells obtained from transgenic mice with supranormal ex- immunized WT and Fcgr2b (KO) mice following treatment with the IMMUNOLOGY AND pression of FcγRIIb on B cells (17) (FcγRIIb-BTG), resulted in VEGFR2 blocking antibody DC101 or an isotype control on the day of boost the abrogation of VEGF-A secretion (Fig. 2F). Of note, the and 48 h later. (G) Percentage of cross-sectional area of inguinal lymph node quantity of VEGF-A produced by B cells in vitro in response to occupied by lymphatic endothelium and (H) number of LECs per lymph node immune complexes was 100-fold less than that observed in mac- and (I) number of myeloid DCs per lymph node in unimmunized or OVA-IC immunized FcγRIIB macrophage transgenic (MTG) mice and littermate NTG rophages and DCs. Erk pathway inhibition reduced VEGF-A controls. In all cases, each point represents a single lymph node. Combined production to baseline (Fig. 2 G and H), whereas there was results are of three independent experiments. P values were calculated using equivalent secretion of VEGF-A by TNFR1-deficient B cells a Student t test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. ns, compared with WT B cells, both following anti-IgMF(ab′)2 and nonsignificant.

Clatworthy et al. PNAS | December 16, 2014 | vol. 111 | no. 50 | 17973 Downloaded by guest on September 29, 2021 prominent in subcapsular sinus, the medulla, interfollicular expansion in LECs in mice with disease (Fig. 4A). Lymph node − − regions, and in the cortical sinus (Fig. 3A, Right). IC-induced lymphangiogenesis was greater in Fcgr2b / mice compared lymphangiogenesis was also observed in a second model in which with WT mice (Fig. 4B), and in keeping with previous studies, − − mice passively immunized intraperitoneally with an anti-PE an- Fcgr2b / mice also had a more severe arthritis (data not shown). tibody were subsequently challenged with PE s.c. (Fig. S6A). SLE is a systemic autoimmune disease characterized by the γ Given that the inhibitory receptor Fc RIIb negatively regu- presence of circulating autoantibodies, in which the deposition of lates VEGF-A production in vitro in macrophages, DCs, and B ICs results in tissue inflammation. VEGF-A blockade has been – cells (Figs. 1 A D and 2 A and B), we hypothesized that intra- used in murine models of lupus nephritis, but any effect on renal nodal lymphangiogenesis would be increased in mice lacking the outcome was confounded by effects on podocyte integrity (23). inhibitory receptor. At baseline, both the cross-sectional area of Peritoneal macrophages stimulated with autoantibody-containing, lymph node composed of lymphatic vessels (determined by his- tological analysis) and the number of LECs (determined by flow cytometry) were similar in WT and FcγRIIb-deficient mice (Fig. 3 C and D). Following OVA immunization and boost, a modest A D increase in lymph node lymphatics was observed in WT mice, but this was significantly greater in Fcgr2b-knockout animals (Fig. 3 C and D). Previous reports demonstrated that inflammation- associated lymphangiogenesis leads to increased DC accumulation in lymph nodes (10). Similarly, we found a significantly higher − − number of DCs in Fcgr2b / mice following immunization and boost (Fig. 3E), in keeping with the increased expansion of lymph node lymphatic vessels observed in these mice.

Inhibition of VEGF-A in Vivo Eliminates Differences in Lymphangiogenesis Between WT and FcγRIIb-Deficient Mice. We hypothesized that IC- induced intranodal lymphangiogenesis is mediated via VEGF-A and that the heightened lymphangiogenesis observed in FcγRIIb- deficient mice occurred due to an increase in VEGF-A in these mice. B To test this hypothesis, we used a VEGFR2 antibody that prevents VEGF-A binding to its receptor. Treatment of animals with a VEGFR2 blocking antibody reduced IC-induced lymph node lymphangiogenesis in WT and FcγRIIb-deficient mice, compared with those treated with isotype control antibody. This reduction was − − more marked in the Fcgr2b / mice, and indeed the number of lymphatic endothelial cells observed in anti–VEGFR2-treated − − Fcgr2b / mice was no different from that observed in WT mice C (Fig. 3F).

FcγRIIb Expression on Macrophages Rather than B Cells Moderates Lymph Node Lymphangiogenesis in Vivo. Previous reports have E shown that B cells play a critical role in inflammation-induced intranodal lymphangiogenesis (10, 13). Because our data showed that FcγRIIb modulates IC-mediated BCR activation in B cells and VEGF-A production in vitro (Fig. 2), we used the FcγRIIb- BTG mice to test the role of B cells in lymphatic expansion in vivo. Following immunization and boost, less lymphatic expan- γ Fig. 4. Lymphangiogenesis and VEGF-A production occurs in autoimmune sion was observed in the draining lymph nodes of Fc RIIb-BTG lymph nodes and immune complex-induced VEGF-A is increased in individuals mice compared with littermate controls (Fig. S6 B and C), but with the lupus-associated FcγRIIBT/T232 polymorphism. (A) Representative con- this did not reach statistical significance and there was a non- focal micrographs of inguinal lymph node sections obtained from WT and − − significant reduction in DC numbers in FcγRIIb-BTG mice (Fig. Fcgr2b / mice 31 d following induction of collagen-induced arthritis (CIA). S6D). In contrast, in mice overexpressing FcγRIIb on macro- Lymph node sections were stained for Lyve-1 (green) and B220 (red). (B)Per- phages (FcγRIIb-MTG), there was a significant reduction in centage of cross-sectional area of inguinal lymph node occupied by lymphatic −/− lymph node lymphangiogenesis (Fig. 3 G and H) and DC number endothelium in WT and Fcgr2b mice. Each point represents one lymph node (Fig. 3I) following immunization and boost. These data suggest (inguinal, axillary, or brachial) at days 31 and 40 following induction of CIA. (C) γ Representative flow cytometric histogram (Upper Left) and quantification that Fc RIIb attenuates IC-induced intranodal lymphangio- (Upper Right) of intracellular VEGF-A staining of murine peritoneal macro- genesis principally by its effects on macrophage activation. phages following stimulation for 24 h with heat-inactivated serum obtained from NZM2410 mice with lupus nephritis (dark green) or from control mice Increased Lymphangiogenesis and VEGF-A in Autoimmunity. We next without disease ( green). (Lower) Quantification of VEGF-A in culture sought to determine whether these observations were relevant to supernatants obtained from the same experiments. Graphs shown are mean autoimmune diseases in which autoantibodies are known to be and SE of mean of triplicates (SEM). (D) Confocal micrograph of inguinal lymph pathogenic. Collagen-induced arthritis (CIA) is a model of in- node section obtained from NZM2410 mice with and without autoimmune flammatory arthritis with some similarities to human rheumatoid diseases (lupus nephritis). Lymph node sections were stained for CD169 arthritis. Autoantibodies against type II collagen play an im- (green), VEGF-A (red), and LYVE-1 (cyan). High power images are shown γ (Lower panels 1 and 2). (E) VEGF-A concentration in culture supernatants portant role in CIA pathogenesis via Fc R ligation, and de- γ γ following Fc R cross-linking in primary human monocyte-derived macrophages ficiency of Fc RIIb on myeloid cells results in increased disease from individuals with differing FCGR2B genotypes (FcγRIIBI/I232,FcγRIIBI/T232, severity (21). Of note, VEGF-A inhibition ameliorates disease and FcγRIIBT/T232) after 48-h culture. Graphs show the mean and SEM. Com- (22). We examined the draining lymph nodes (inguinal, brachial, bined results are of three independent experiments. All P values were calcu- and axillary) of mice with active arthritis and observed a marked lated using a Student t test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

17974 | www.pnas.org/cgi/doi/10.1073/pnas.1413915111 Clatworthy et al. Downloaded by guest on September 29, 2021 heat-inactivated serum obtained from New Zealand mixed (NZM) produced in-house (17). NZM2410 mice were purchased from Jax and TNFR1- 2410 mice with lupus nephritis demonstrated significantly increased deficient mice were obtained from Jean Langhorne, National Institute for VEGF-A production compared with macrophages stimulated with Medical Research, Mill Hill, London. All procedures were conducted in ac- serum obtained from healthy control mice without disease (Fig. cordance with the United Kingdom Animals (Scientific Procedures) Act 1986. 4C). In addition, we investigated lymphangiogenesis and VEGF-A μ production in lymph nodes in diseased NZM2410 mice. We ob- Immunization. Mice were immunized with 200 g ovalbumin (Invitrogen) in + μ μ served significantly increased numbers of LYVE-1 LECs and 200 L alum (Imject) intraperitoneally and subsequently boosted with 100 g + ovalbumin in 100 μL alum s.c. 3–4 wk later. Alternatively, mice were passively VEGF-A cells in the lymph nodes of NZM mice with lupus immunized intraperitoneally with antiphycoerythrin antibody (Rockland) compared with age-matched controls without disease (Fig. 4D). + followed by s.c. administration of 10 μg of phycoerythrin (Invitrogen Molec- Of note, VEGF-A staining was observed in CD169 SCS macro- ular Probes) as described previously (18). phages in diseased NZM mice, but not in those without disease. Collagen-Induced Arthritis. The protocol followed to induce CIA in C57BL/6 Immune Complex-Induced VEGF-A Production by Human Monocyte- mice was reported previously (17). Derived Macrophages is Increased in Individuals with the Lupus- T/T232 Associated FcγRIIb Genotype. Elevated VEGF-A has been In Vivo Inhibition of VEGF-A. VEGF-A was inhibited using a VEGFR-2 rat noted in patients with rheumatoid arthritis and SLE and correlates , clone DC101 (ImClone Systems) (10). with disease activity (24, 25). In humans, a nonsynonymous SNP in FCGR2B (rs1050501) (isoleucine to threonine at position 232) Immunohistochemistry. Immunohistochemistry methodology is described in leads to receptor dysfunction and confers susceptibility to SLE (5, SI Materials and Methods. 26). We hypothesized that rs1050501 would affect VEGF-A pro- duction in individuals with the SLE-associated FcγRIIBT232 re- Histological Quantification of Lymphatics. The proportion of the total lymph ceptor. To test this assertion, peripheral blood from node cross-section occupied by LYVE-1 positive cells was quantified using individuals with FcγRIIBI/I232 (the wild-type receptor), FcγRIIBI/T232, Velocity Software (Perkin-Elmer). Colocalization analysis was performed and FcγRIIBT/T232 were treated with macrophage colony- using Imaris software. stimulating factor (M-CSF) to generate macrophages and were Flow Cytometric Quantification of Lymphatic Endothelium. Inguinal lymph subsequently stimulated with ICs. IC-induced VEGF-A pro- γ T/T232 nodes were harvested, weighed, counted, and stained. Details of the anti- duction was significantly higher in individuals with Fc RIIB bodies used are found in SI Materials and Methods. compared with FcγRIIBI/I232 and FcγRIIBI/T232 (Fig. 4E), raising γ the possibility that reduced Fc RIIb function may have a similar Macrophage, Dendritic Cell, and B-Cell in Vitro Assays. Peritoneal macro- effect on human lymph node lymphatics, potentially propagating phages, BMDCs, and splenic B cells were harvested (details in SI Materials and autoimmunity. We did not assess the effect of immune complex Methods) and stimulated with OVA (Invitrogen) or OVA opsonized with size and glycosylation state on VEGF-A production, but these rabbit polyclonal anti-ovalbumin antibody (Sigma). A total of 10 μM1, factors are known to alter FcγR binding (27) and may well impact 4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene (U0126; Sigma) on IC-induced VEGF-A production. was used to inhibit ERK. Anti-TNFα antibody (polyclonal goat anti-mouse In summary, previous studies have shown that macrophages antibody; R&D Systems) was used at 0.5 and 1 μg/mL. B cells were stimulated μ – ′ produce VEGF-A in response to innate stimuli such as TLR with goat anti-mouse IgM -chain specific F(ab )2 or intact IgG (Jackson agonists and proinflammatory cytokines. Here we find that ImmunoResearch Laboratories) at 10 μg/mL and CD40L (Peprotech), or 10 VEGF-A is also secreted in an adaptive immune response via μg/mL of LPS (Salmonella typhimurium; Sigma). engagement of FcγR by IgG ICs and that this is limited by FcγRIIb. Our study demonstrates a previously unappreciated Primary Human Cells. Primary human monocyte-derived macrophages were obtained from peripheral blood obtained from healthy volunteers who immune-enhancing effect of IgG in increasing VEGF-A–induced γ had given informed consent, via the Cambridge BioResource (www. intranodal lymphangiogenesis. Because Fc RIIb inhibits IC- cambridgebioresource.org.uk). Macrophages were generated by cultur- induced VEGF-A production, it indirectly controls immune re- ing monocytes for 7 d with M-CSF (400 ng/mL; Peprotech). sponses by limiting the available lymphatic channels in lymph nodes through which antigen and APCs travel. This may have Cytokine and VEGF-A Measurement. VEGF-A and TNF-α concentrations in additional implications, because intranodal LECs also provide culture supernatants were measured by ELISA (Duokit; R&D Systems) ac- a source of sphingosine1-phosphate (S1P), enabling lymphocyte cording to the manufacturer’s instructions. egress from lymph nodes (28). These observations may also be relevant to vaccine design, in that FcγRIIb blockade during boost Statistics. Statistical comparisons were made using GraphPad PRISM software. might enhance lymphangiogenesis and promote DC and antigen A two-tailed Student t test was applied, unless otherwise indicated. Results delivery to the relevant area of the lymph nodes. Our data also are expressed as means and SE of mean. All experiments were subject to at support the concept that VEGF-A inhibition may be a potential least three replicates per experimental parameter. treatment target for antibody-mediated autoimmune diseases. ACKNOWLEDGMENTS. We thank Jean Langhorne for providing bone marrow −/− Materials and Methods from TNFR1 mice and Cambridge BioResource for assistance. This work was supported by a Wellcome Trust Intermediate Fellowship (WT081020) to M.R.C., γ Mice. Fc RIIb-deficient BALB/c and C57BL/6 mice were kindly provided by Jeff a Wellcome Trust Programme Grant (083650/Z/07/Z), a Lister Prize Fellowship Ravetch and Silvia Bolland, The Rockefeller University, New York. FcγRIIb– (to K.G.C.S.), and the National Institute for Health Research Cambridge Bio- B-cell and macrophage transgenic mice and nontransgenic controls were medical Research Centre. INFLAMMATION

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