Abstract
During inflammation, accumulation of immune cells in activated lymph nodes (LNs), coupled with a transient shutdown in lymphocyte exit, results in dramatic cellular expansion. Counter-regulatory measures to restrain LN expansion must exist and may include re-establishment of lymphocyte egress to steady-state levels. Indeed, we show in a murine model that egress of lymphocytes from LNs was returned to steady-state levels during prolonged inflammation following initial retention. This restoration in lymphocyte egress was supported by a preferential expansion of cortical and medullary sinuses during late inflammation. Cortical and medullary sinus remodeling during late inflammation was dependent on temporal and spatial changes in vascular endothelial growth factor-A distribution. Specifically, its expression was restricted to the subcapsular space of the LN during early inflammation, whereas its expression was concentrated in the paracortical and medullary regions of the LN at later stages. We next showed that this process was mostly driven by the synergistic cross-talk between fibroblastic reticular cells and interstitial flow. Our data shed new light on the biological significance of LN lymphangiogenesis during prolonged inflammation and further underscore the collaborative roles of stromal cells, immune cells, and interstitial flow in modulating LN plasticity and function.
One of the hallmarks of local immune responses and inflammation is alterations in the traffic of lymphocytes through stimulated lymph nodes (LNs). Specifically, lymphocyte accumulation in draining LNs (DLNs) is markedly increased, whereas lymphocyte exit into the efferent lymph is transiently blocked from few hours to days, depending on the nature of the Ag and inflammation (1–4). In contrast to the well-documented existence of mechanisms controlling lymphocyte retention within the inflamed LN following the initiation of an immune response (5–7), little information is available on the mechanisms that re-establish lymphocyte egress to steady-state levels.
Organization of the stromal elements of LNs is initiated during embryogenesis and was believed to be silenced soon after gestation. However, recent studies indicated that plasticity in LN structural organization continues to exist throughout life, particularly during inflammation and infection (8, 9). Reorganization of LN microanatomy during inflammation is characterized by the remodeling and expansion of key LN stromal cells, including lymphatic endothelial cells (LECs) (10), blood endothelial cells (BECs) (11, 12), and fibroblastic reticular cells (FRCs) (13, 14). Importantly, increasing evidence reveal the immunological significance of the remodeling of these stromal elements. For example, the expansion of BECs and LECS was reported to support a more robust accumulation of lymphocytes and dendritic cells (DCs), respectively, within stimulated LNs during the initiation of the immune response (12, 15).
LN remodeling is a dynamic process orchestrated by signals emanating, in part, from lymph and immune cells that are in constant transit through LNs and carry with them immune footprints of the peripheral sites from which they trafficked (1, 13). Conceivably, reciprocity in terms of interaction between LN stromal elements and these messengers may exist. Although these messengers may shape the LN microenvironment during inflammation (9, 16), stromal cells may also sense inflammatory signatures in lymph flow and immune cells and respond in a way to restore homeostasis within the LN.
In this study, we examined whether the remodeling of stromal elements within inflamed LNs might contribute to the re-establishment of lymphocyte egress rates after being temporarily decreased for few days following initiation of the immune response. Our findings reveal that lymphocyte egress from stimulated LNs can be restored to steady-state levels under prolonged inflammation by the expansion of cortical and medullary sinuses, the major gateways for lymphocyte exit. Such preferential growth of cortical and medullary sinuses during late phases of inflammation is driven by a distinctive spatial-temporal distribution of vascular endothelial growth factor (VEGF)-A within activated LNs, in a process fostered by interstitial flow and the FRC network.
Materials and Methods
Mice
CD45.2 and CD45.1 congenic female mice on a C57/BL6 background, between 8 and 12 wk of age, were obtained from The Jackson Laboratory (Bar Harbor, ME) and maintained under specific pathogen-free conditions within National University of Singapore’s satellite animal housing unit. Following sedation, 20 μl an emulsion containing equal volumes of CFA and sterile PBS containing keyhole limpet hemocyanin (KLH; final concentration of 2.5 mg/ml) was injected s.c. in the front and rear footpads of the mice. LNs draining the inflamed footpads (brachial, axillary, and popliteal) were harvested following mice sacrifice. All studies were performed under protocols approved by the National University of Singapore and Biological Resource Center Institutional Animal Care and Use Committee.
Adoptive cell transfers
For adoptive transfers, CD45.2 recipient mice were injected i.v. with 2 × 107 CD45.1 spleen and LN cells. In short-term homing experiments to assess lymphocyte entry into LNs, CD45.1 cells were adoptively transferred into control and immunized CD45.2 recipient mice and allowed to equilibrate for 2.5 h before sacrifice (Supplemental Fig. 1A). At this time point, entry from the peripheral blood into LNs directly determined the CD45.1 cell numbers within recipient LNs (17).
To assess lymphocyte egress from LNs, CD45.1 cells were adoptively transferred into control and immunized CD45.2 recipient mice and allowed to equilibrate for 24 h. Following equilibrium (T0), half of the mice were sacrificed, whereas the other half of the mice were treated with anti-CD62L Ab and sacrificed 20 h after administration (T20) (Supplemental Fig. 1B). Intraperitoneal administration of anti-CD62L Ab at a dose of 100 μg/mouse (clone Mel-14 hybridoma; American Type Culture Collection) blocked further entry of circulating lymphocytes into LNs without affecting lymphocyte egress from LNs (7, 18). Therefore, the population of CD45.1 lymphocytes remaining within LNs at T20 compared with T0 is a measure of lymphocyte egress from LNs. In some lymphocyte-egress assays, FTY720 (Cayman Chemicals) was administered i.p. together with anti-CD62L Ab, at a dose of 1 mg/kg (Supplemental Fig. 1C).
Treatment with VEGFR2 and VEGFR3 Abs
Signaling by VEGFR2 and VEGFR3 was inhibited using selective blocking rat IgG from clones DC101 and mF4-31C1 (ImClone Systems), respectively (15, 19). Abs were injected i.p., at a dose of 0.8 mg/mouse, into mice 1 d before and every alternate day thereafter for 14 d after CFA/KLH immunization.
Implantation of control and VEGF-A–containing Matrigel pellets
C57BL/6J mice were anesthetized before growth factor-reduced Matrigel (BD Biosciences) pellets (0.3 ml) containing PBS or 400 ng recombinant mouse VEGF-A16420), we ensured that our implants contained a dose of mouse VEGF-A164 that could elicit lymphangiogenesis in vivo (20). The pellets were left in situ for 5 d before the mice were sacrificed, and the DLNs were harvested.
Spleen and LN cell suspensions
Isolation of LECs, BECs, and FRCs from LNs were carried out adapting a previously described method (21). Briefly, LNs were digested in HBSS medium containing 4 mg/ml collagenase IV (Life Technologies) at 37°C for 45 min with gentle agitation. For isolation of DCs from LNs, LNs were digested with 1 mg/ml Collagenase D (Roche Diagnostics) at 37°C for 60 min. All other cell suspensions from LNs and spleens were prepared by mechanical disruption.
Flow cytometry analysis
Flow cytometric analysis of LN cell suspensions stained for CD45, podoplanin, and CD31 allowed differentiation and quantification of LEC (CD45−, CD31+, podoplanin+), BEC (CD45−, CD31+, podoplanin−) and FRC (CD45−, CD31−, podoplanin+) populations. Abs used included the following: rat anti-mouse CD31 (Serotec) detected with anti-rat–allophycocyanin and hamster anti-mouse podoplanin (clone 8.1.1; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) detected with anti-hamster PE and PerCPcy5.5-conjugated anti-mouse CD45.2 (BD Biosciences). For detection of VEGF-A expression in FRCs, surface Ag staining for FRCs were first performed, as detailed above. Intracellular staining for VEGF-A was then performed with the BD Fixed/Permeabilization Kit, using rabbit purified anti–VEGF-A (Santa Cruz) Ab and revealed with allophycocyanin-Cy7–conjugated anti-rabbit IgG (Santa Cruz).
FACS analysis was also used to quantify DC and T and B cell populations and congenic transferred lymphocytes. Abs used included the following: PE-conjugated anti-CD11c, FITC, PerCPCy5.5-, and Pacific Blue-conjugated anti-B220, allophycocyanin-conjugated anti-CD3e, PerCPCy5.5-conjugated anti-CD45.2, and biotin-conjugated anti-CD45.1 revealed with streptavidin-PE.
Live and dead cells were discriminated during flow cytometry using a LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Molecular Probes, Invitrogen). Cell counts were determined during flow cytometry using Count Bright Absolute Counting Beads (Molecular Probes, Invitrogen). FACS analysis was performed using a CyAn ADP Analyzer (Beckman Coulter), and data were analyzed with FlowJo software (TreeStar).
Immunohistochemistry
LNs were either freshly embedded in tissue-freezing medium or fixed overnight in 2% paraformaldehyde/30% sucrose solution at 4°C and embedded in tissue-freezing medium. Six to eight- and 25–30-μm-thick cryostat sections were cut for imaging by fluorescence and confocal microscopy, respectively.
Microscopy, image analysis, and area density measurements
The entire LNs were sectioned through in sequence, and immunofluorescence analysis was performed on every alternate slide in sequence. Low-magnification images of LN sections stained for LYVE-1 and B220 were first captured with a fluorescence microscope (Axio imager.Z1, Axiocam HRM camera; Carl Zeiss Micro Imaging, Jena, Germany) to anatomically locate the lymphatic sinuses in the subcapsular space, paracortex, and medulla. Next, high-magnification images of the same sections stained for CD11c, B220, and CD169 were captured with a confocal microscope (Leica TCS SP5; Leica Microsystems, Deerfield, IL). LYVE-1+ lymphatics were identified as subcapsular sinuses if they were found in the subcapsular space and contained both B cells and DCs. LYVE-1+ lymphatics were identified to be cortical or medullary sinuses if they were found in the LN paracortex or medulla and contained B cells but not DCs within their lumen. Total lymphatics area was quantified using ImageJ software (http://rsb.info.nih.gov/ij), as the total amount of LYVE-1+ sinuses found in the entire LN. To measure the area density of identified subcapsular sinuses in ImageJ, these LYVE-1+ lymphatics were outlined and selected as the region of interest. The area of LYVE-1+ lymphatics lying in this region of interest was determined and expressed relative to total lymphatic sinuses area. The same process was repeated to determine the area of subcapsular sinuses relative to total lymphatic sinuses area.
To quantify the proportion of VEGF-A residing in the different locations of the LNs, the subcapsular space, paracortex, and medulla were first outlined on low-magnification images. The area density of VEGF-A present in these regions of interest was then expressed relative to the total area in the LN occupied by VEGF-A. To assess VEGF-A association with subcapsular, cortical, and medullary sinuses and with ERTR-7+ reticular fibers lining these lymphatics, staining with VEGF-A, CD11c, and LYVE-1 or VEGF-A, CD11c, and ERTR-7 was performed on sequential sections.
Additional three-dimensional (3D) image analysis and volume rendering of confocal z-stacks images were processed using Imaris software (version 5.0.3; Bitplane). Voxels with fluorescence intensities above a certain threshold were assigned for each color channel and kept constant between different samples. Projections were rendered from these voxels to create 3D and volumetric reconstructions in which the spatial resolution was conserved. Images were processed using Adobe Photoshop CS4 (Adobe Systems, San Jose, CA) for adjustment of image brightness.
VEGF-A and VEGF-C ELISA
Footpads and LNs were harvested and homogenized in lysis buffer (RIPA buffer [Sigma Chemicals] with a protease inhibitor mixture [Roche Diagnostics]). Homogenates were centrifuged for 10 min at 4°C at 14,000 × g
Disruption of lymphatic flow through the LNs
Mice were sedated with i.p. injection of ketamine (100 mg/kg) and xylazine (10 mg/kg) for the duration of the surgery. Twenty microliters of 2000-kDa FITC-dextran (Molecular Probes) was injected into the hind footpads to allow visualization of lymphatic vessels (LVs). The size of the FITC–dextran complex excluded its access into FRC conduits. Afferent LVs draining the right popliteal LNs were cut (LV resection), whereas LVs on the contralateral side were left exposed in the same way but not cut (sham treated). The mice were treated with buprenophine and enrofloxacin after surgery and were sacrificed 1 d after surgery. Following sacrifice, 20 μl 2000-kDa FITC-dextran was injected into the footpads of each mouse to check lymph flow to the popliteal LNs in the sham-treated side and in the side with LV resection.
Quantitative RT-PCR
Total RNA from footpads and LNs was homogenized and extracted using TRIzol reagent (Invitrogen) and NucleoSpin RNA II kit (Macherey-Nagel). First-strand cDNA was synthesized using TaqMan Reverse Transcription Reagents (Applied Biosystems). Real-time PCR were performed using iTaq SYBR Green Supermix with ROX (Bio-Rad) on a 7500 Real-Time PCR System (Applied Biosystems). Expression of genes of interest was normalized to the expression of GAPDH. The following primers were used: VEGF-A forward, 5′-CAGAAGGAGAGCAGAAGTCC-3′ and reverse, 5′-CTCCAGGGCTTCATCGTTA-3′; VEGF-C forward, 5′-GTAAAAACAAACTTTTCCCTAATTC-3′ and reverse, 5′-TTTAAGGAAGCACTTCTGTGTGT-3′; VEGF-A120 forward, 5′-TGCAGGCTGCTGTAACGATG-3′ and reverse, 5′-CCTCGGCTTGTCACATTTTTCT-3′; VEGF-A164 forward, 5′-TGCAGGCTGCTGTAACGATG-3′ and reverse, 5′-GAACAAGGCTCACAGTGATTTTCT-3′; VEGF-A188 forward, 5′-TGCAGGCTGCTGTAACGATG-3′ and reverse, 5′-GAACAAGGCTCACAGTGATTTTCT-3′; and GAPDH forward, 5′-GACGGCCGCATCTTCTTGTG-3′ and reverse, 5′-CTTCCCATTCTCGGCCTTGACTGT-3′. The primers used for VEGF-A detect all alternatively spliced isoforms of VEGF-A.
DC-migration assay
To study peripheral DC migration, 25 μl FITC solution (FITC dissolved in a 1:1 volume of acetone and dibutyl phthalate [all from Sigma] to give a final concentration of 8 mg/ml) was applied to the shaved back skin draining the brachial and axillary LNs on each side of the mouse 36 h before sacrifice (22). Following sacrifice, the brachial and axillary LNs were removed and processed, as described above (Supplemental Fig. 3A). The migration of DCs induced by FITC application, expressed as the total number of FITC+ DCs/LN, was calculated by multiplying the percentage of FITC+CD11c+ cells by the total number of LN cells. To distinguish subcapsular from cortical and medullary sinuses, migration of skin DCs was induced by sensitizing mice on the back skin with the contact sensitizer solution of acetone and dibutyl phthalate.
Statistical analysis
Statistical analysis was performed with Prism 5 (GraphPad Software). Statistical significances were determined using the unpaired two-tailed t test. Whenever more than two groups were compared, the one-way ANOVA test with the Bonferroni post test was applied. For all tests, a p value < 0.05 was considered significant.
Results
Lymphocyte egress from stimulated LNs is altered during prolonged inflammation
Consistent with our previous report (15), footpad immunization with CFA/KLH induced a marked cellular expansion in DLNs at day 4. In this study, we showed that this cellular expansion peaked at day 7, with a considerable decrease from day 14 postimmunization but sustained beyond day 30 (Fig. 1A). The decrease in LN cellularity between days 7 and 14 may be attributed to a programmed contraction of the lymphocyte compartment following the priming of adequate numbers of effector lymphocytes in the LN. However, this was an unlikely explanation, because formation of germinal centers, the hallmark of the peak of an immune response, occurred ≥14 d after immunization in our model of inflammation (data not shown).
T lymphocyte egress from stimulated LNs is increased during prolonged inflammation. (A) LN cellularity was evaluated in mice injected with PBS (control) or CFA/KLH (immunized). (B) Short-term homing experiments were performed in mice at baseline (control) or days 4 and 14 postimmunization to assess lymphocyte entry into LNs. (C) Using lymphocyte adoptive-transfer assay, T lymphocyte egress from LNs was assessed at baseline (control) or days 4 and 14 postimmunization. (D) Results were expressed as mean fraction of egressed T cells from LNs at baseline (control) or days 4 and 14 postimmunization. (E) The effect of treatment with FTY720 on T lymphocyte egress was analyzed in mice at baseline (control) or day 14 postimmunization. (F) Results were expressed as mean fraction of egressed T cells from inflamed LNs of FT720-treated mice compared with untreated mice. Results from short-term homing experiments and lymphocyte-egress experiments are pooled from three independent experiments with five or six mice/group in each experiment. Results from FTY720 experiment are pooled from two independent experiments with five mice/group in each experiment. Error bars represent SD. *p < 0.05, **p < 0.01. D, Day.
We hypothesized that the decrease in LN cellularity at day 14 resulted from either decreased lymphocyte entry or increased lymphocyte egress from the activated LNs. Because LN cellularity peaked at day 7 postimmunization, and changes in LN cellularity are influenced by events occurring just before and after day 7, we chose to focus our studies on days 4 and 14.
We first assessed lymphocyte entry into LNs in short-term homing assays (Supplemental Fig. 1A). At 2.5 h after cell transfer, entry of CD45.1-transferred lymphocytes into LNs from CD45.2-immunized mice was significantly higher compared with controls (Fig. 1B). Both T and B cell entry into LNs was significantly increased at day 14 postimmunization compared with day 4 (Fig. 1B), indicating that the decreased LN cellularity observed at day 14 could not have resulted from decreased lymphocyte entry into LNs.
In assays to assess lymphocyte egress from LNs (Supplemental Fig. 1B), exit of T cells from control LNs was reflected by a considerable decrease in the CD45.1 T cell numbers at T20 compared with T0 (Fig. 1C). The number of CD45.1 T cells in LNs decreased even more strikingly between T0 and T20 at day 14 postimmunization, whereas no significant decrease was detected at day 4 (Fig. 1C). We also expressed the data as mean fraction of egressed cells, which was calculated by dividing the mean number of CD45.1 T cells that exited by the mean population of CD45.1 T cells present at baseline (T0). Analysis revealed that T cell egress from LNs at day 4 postimmunization was decreased (22%) compared with nonimmunized mice (55%), but it returned to steady-state levels at 14 d postimmunization (50%). This phenomenon was not restricted to the T cell compartment, because similar observations were made for B cells (Supplemental Fig. 2A, 2B).
We considered the possibility that the greater decrease in transferred T cells in DLNs at day 14 compared with day 4 postimmunization might have resulted from increased death and not increased LN egress, even though this scenario is quite unlikely because it would imply that ∼50% of the transferred T lymphocytes died within 20 h. To clarify whether more T lymphocytes truly exited from LNs at day 14 postimmunization, we used the immunosuppressive drug FTY720 to block lymphocyte egress from LNs (23, 24) (Supplemental Fig. 1C). At T20, the number of T cells remaining in LNs from FTY720-treated immunized mice was significantly greater compared with untreated immunized mice (Fig. 1E). Specifically, FTY720 treatment decreased the mean fraction of egressed T cell from 0.49 to 0.14, a 3.5-fold decrease (Fig. 1F). FTY720 treatment also attenuated enhanced egress of B cells (Supplemental Fig. 2C) and total lymphocytes (Supplemental Fig. 2D). These findings demonstrated that the greater decrease in transferred T cells in DLNs at day 14 compared with day 4 postimmunization resulted from increased LN egress. Together, this indicated that restoration of lymphocyte egress to steady-state levels after an initial retention phase may represent homeostatic measures to restrain LN expansion during prolonged inflammation.
Immunization induces lymphangiogenesis in DLNs
Because lymphatics are critical routes for lymphocyte egress from LNs, we examined LN lymphangiogenesis in this model of long-lasting inflammation. We used flow cytometry to quantify LECs in LNs (Fig. 2A). Immunization stimulated significant, but similar, increases in LEC numbers over control at 4 and 14 d postimmunization (Fig. 2B, 2C). The increase in LEC population was sustained even at 30 d after immunization (2.5-fold over control) (Fig. 2B, 2C). This marked and prolonged expansion of lymphatic network in inflamed DLNs was also apparent in LN sections immunostained for LYVE-1, a marker for lymphatics (Fig. 2C). Immunostaining of LN sections for Ki67, a marker for cell division, revealed that this lymphatic expansion resulted from proliferation of pre-existing lymphatics (Fig. 2D).
Immunization induces lymphangiogenesis in DLNs. (A) LN cells were stained for CD45, podoplanin, and CD31 to identify LECs, BECs, and FRCs by FACS analysis. The number of LECs was enumerated in LNs following immunization (B) and expressed as fold change over baseline (C). Data are pooled from three independent experiments with four or five mice/group in each experiment. Error bars represent SD. (D) Control and immunized LN sections were immunostained for LYVE+. Scale bar, 200 μm. (E) LYVE-1+ vessels were analyzed for coexpression of the proliferative marker Ki67. Arrows indicate colocalization of lymphatics with Ki67. Ki67bright dividing lymphocytes were also observed. Scale bar, 50 μm. Images are representative of three or four independent experiments (n = 3–4). *p < 0.05, immunized versus control LNs.
Restoration of lymphocyte egress to steady-state levels during later phases of inflammation is supported by LN lymphangiogenesis
Thus far, our findings suggested that the restored egress of lymphocytes at day 14 postimmunization is likely to be accounted for by LN lymphangiogenesis. To address this possibility, we next evaluated the effect of blocking lymphangiogenesis on lymphocyte egress from inflamed LNs. Because both VEGFR2- and VEGFR3-signaling pathways have been implicated in inflammatory LN lymphangiogenesis (15, 21, 25, 26), we treated animals with neutralizing mAbs against VEGFR2 and VEGFR3 to inhibit lymphangiogenesis. Consistent with previous reports (15), blocking VEGFR2 and VEGFR3 signaling had no effect on pre-existing lymphatic and blood vessels in resting LNs (data not shown). Compared with control rat IgG-treated immunized mice, blockade of VEGFR2 and VEGFR3 signaling markedly reduced, but did not completely abolish, inflammation-induced lymphangiogenesis and angiogenesis (Fig. 3A, 3B).
Blocking LN lymphangiogenesis reduces lymphocyte egress. (A) Immunized mice were treated with a combination of anti-VEGFR2 and anti-VEGFR3 Abs or control rat IgG. The number of LECs, BECs, and FRCs was determined in LN suspensions by flow cytometry and expressed as fold change over baseline. Results are pooled from two independent experiments with four mice/group in each experiment. (B) Immunoreactivity for LYVE-1+ was examined in LN sections from mice treated with anti-VEGFR2 and VEGFR3 Abs or control rat IgG. Images are representative of three independent experiments (n = 3). Scale bar, 200 μm. Effect of blocking VEGFR2 and VEGFR3 signaling on the egress of T lymphocytes (C, D) and B lymphocytes (E, F) from immunized LNs was determined. Results in (D) and (F) are expressed as mean fraction of egressed lymphocytes. Results in (C)–(F) are pooled from two independent experiments, with five mice/group in each experiment. Error bars represent SD. *p < 0.05.
Analysis indicated that abrogating lymphangiogenesis markedly diminished T cell egress at day 14 postimmunization (Fig. 3C). Because fewer transferred T cells entered the inflamed LNs of mice treated with anti-VEGFR2 and anti-VEGFR3 mAbs due to inhibition of angiogenesis, evaluating the mean fraction of egressed cells may provide a more meaningful measure. We observed a 3-fold decrease in the mean fraction of egressed T cells in mice treated with anti-VEGFR2 and anti-VEGFR3 mAbs compared with control rat IgG-treated mice (Fig. 3D). Similar observations were made for B cell egress following treatment of mice with anti-VEGFR2 and anti-VEGFR3 mAbs (Fig. 3E, 3F). These results demonstrated that the restoration of lymphocyte egress observed at day 14 postimmunization is supported by LN lymphangiogenesis.
LN lymphangiogenesis is initiated and sustained by lymphangiogenic factors derived from the inflamed peripheral site and LNs
We examined whether VEGF-A and VEGF-C, essential growth factors for inflammation-induced lymphangiogenesis (15, 21, 25, 26), may be involved in the prolonged LN lymphangiogenesis. ELISA analysis revealed that, compared with control LNs, VEGF-A and VEGF-C protein levels were highly elevated in homogenates of inflamed LNs at as early as 2 d postimmunization and remained elevated even at day 30 (Fig. 4A, 4B). Interestingly, the increase in VEGF-A and VEGF-C proteins in inflamed LNs did not coincide with an increased expression of their respective mRNA (Fig. 4C, 4D) or the mRNA expression of the major isoforms of VEGF-A (VEGF-A120, VEGF-A164, and VEGF-A188) (data not shown). In contrast, both protein (Fig. 4E, 4F) and mRNA (Fig. 4G, 4H) levels of VEGF-A and VEGF-C in inflamed footpad homogenates were highly elevated compared with control. Expression of mRNA of various VEGF-A isoforms in inflamed footpads was correspondingly upregulated compared with control (data not shown). Consistent with a previous report in a delayed-type hypersensitivity-induced LN lymphangiogenesis model (21), our results suggest that VEGF-A or VEGF-C within inflamed LNs may primarily originate from the peripheral inflamed site and subsequently be transported to DLNs via afferent LVs. Alternatively, it is possible that LN-resident cells, including B cells (15) or FRCs (27), are cellular sources of lymphangiogenic factors. However, the influx and proliferation of other nonlymphangiogenic factor-producing cells in the inflamed LNs could have artificially decreased whole-LN mRNA expression of the various lymphangiogenic factors.
LN lymphangiogenesis is initiated and sustained by lymphangiogenic factors derived from the inflamed peripheral site. VEGF-A (A) and VEGF-C (B) protein content were analyzed in homogenates of control and inflamed DLNs by ELISA. Quantitative RT-PCR analysis was performed on whole-LN RNA to examine expression of VEGF-A (C) and VEGF-C (D) in control and inflamed DLNs. VEGF-A (E) and VEGF-C (F) protein content in homogenates of control and inflamed footpads was examined by ELISA. Quantitative RT-PCR analysis was performed on whole-footpad RNA to examine expression of VEGF-A (G) and VEGF-C (H) in control and inflamed footpads. For ELISA and Q-PCR results, data are representative of two independent experiments with three or four mice/group. Error bars represent SD. *p < 0.05, **p < 0.01, immunized versus nonimmunized LNs. Imm, postimmunization; D, Day.
Interestingly, we noted that LN stimulation induced an increase in FRC numbers that was sustained for 30 d (Fig. 5A). Strikingly, the expansion and persistence of LEC and FRC populations appeared closely linked in both kinetics and magnitude, in contrast to the BEC population (Fig. 5B), and suggested a possible relationship between lymphatics and the FRC network within inflamed LNs. Because the 3D conduits ensheathed by FRCs can be identified by their reactivity with the anti–ER-TR7 mAb, we performed this staining in combination with anti–LYVE-1 Ab to probe for a possible relationship between lymphatics and FRCs. Indeed, we found that lymphatic channels were closely associated with ER-TR7+ reticular fibers (Fig. 5Ci), and this association was further confirmed when we performed volume rendering of confocal image stacks and image rotation (Fig. 5Cii, 5Ciii). This mirrored observations in an earlier study describing the endothelial walls of lymphatics as being continuously lined by reticular fibroblasts (28).
FRCs can produce VEGF-A during inflammation to drive LN lymphangiogenesis. (A) The number of FRCs in LNs was evaluated following immunization. (B) LEC, BEC, and FRC populations were examined and enumerated by flow cytometry, and results are expressed as fold change over baseline. Data in (A) and (B) were pooled from three independent experiments with four or five mice/group in each experiment, Error bars represent SD. (C) LN sections from immunized mice at 14 d postimmunization were immunostained for LYVE-1 and ER-TR7. Confocal microscopic images (i) were processed for volume rendering analysis (ii), and these images were rotated on the axis denoted by the blue line (iii). The thickness of the sections is indicated on the left. Images are representative of three independent experiments (n = 3). Scale bar, 50 μm. (D) Intracellular expression of VEGF-A by CD45−gp38+CD31− FRCs was evaluated by flow cytometry in LNs from control and immunized mice at 7 d postimmunization. Data are representative of three independent experiments (n = 3). *p < 0.05.
These observations and the fact that FRCs were described to produce VEGF-A during inflammation to support angiogenesis (27) prompted us to examine the expression of VEGF-A by FRCs in our model of inflammation using flow cytometry. Basal levels of VEGF-A were detected in FRCs isolated from resting LNs, which were further upregulated upon LN stimulation (Fig. 5D). Thus, the growth of lymphatic network induced in our model of prolonged inflammation may also be supported by VEGF-A produced locally by FRCs in the LNs.
Preferential expansion of cortical and medullary sinuses occurs at later phases of inflammation
We hypothesized that the return of lymphocyte egress to baseline levels at day 14 postimmunization compared with day 4 may occur through an expansion of cortical and medullary sinuses, because they are the major exit paths for lymphocytes (29–31). To investigate this possible scenario, we developed a strategy to distinguish cortical and medullary sinuses from subcapsular sinuses given that specific markers to differentiate them are currently lacking. First, we differentiated subcapsular sinuses from cortical and medullary sinuses according to their anatomical location within the LNs after staining with LYVE-1 and B220. Consistent with earlier studies (32, 33), we identified LYVE-1+ subcapsular sinuses in the B cell-rich capsular region of the LN, whereas cortical sinuses were in the paracortical region (the boundary of the B and the T cell zones), and medullary sinuses were in the medulla of the LN (Fig. 6Ai). To verify the identity of the sinuses, and because subcapsular sinuses have been described to extend into medullary sinuses (34), we used a second criteria to distinguish subcapsular sinuses from cortical and medullary sinuses. Unlike cortical and medullary sinuses, subcapsular sinuses support the trafficking of different immune cell types. Therefore, we used colocalization of LYVE-1+ channels with immune cells as another approach to distinguish these channels. Indeed, subcapsular sinuses contained T cells (data not shown), B cells, and DCs (Fig. 6Aii), because these cells can enter LNs via such subcapsular sinuses (10, 35, 36). In contrast, cortical and medullary sinuses contained T cells (data not shown) and B cells but not DCs (Fig. 6Aiii, 6Av), because very few DCs egress from LNs compared with lymphocytes (7, 10, 15, 37, 38). Because few DCs from noninflamed skin migrate to the LNs, we induced DC migration by epicutaneous application of a contact sensitizer. This method allowed us to detect more DCs within the LNs and, thus, to identify the subcapsular sinuses more accurately. Furthermore, migration assay revealed that DC migration into inflamed LNs was clearly increased at days 4 and 14 postimmunization compared with controls, although the increase was more dramatic at day 4 (Supplemental Fig. 3B). Because we could detect abundant incoming DCs in sinuses innervating the capsule of control LNs after sensitization (Supplemental Fig. 3C), differences in the number of migrating DCs did not bias the ability to detect subcapsular sinuses at days 4 and 14 after immunization. As reported by other investigators, we observed that the LYVE-1+ medullary lymphatic plexus colocalized with CD169+ macrophages (Fig. 6Av) (39, 40), whereas the cortical sinuses did not (Fig. 6Aiv) (30).
Using this method based on the anatomical location of LYVE-1+ sinuses in the LN and their colocalization with different immune cells, the lymphatic network was noted to be largely confined to the subcapsular regions of the activated LN at day 4 postimmunization. In contrast, LYVE-1+ lymphatic channels extended into the LN paracortical and medullary regions at day 14 postimmunization (Fig. 6B). In addition, we observed more dilated and sinusoidal cortical sinuses on day 14 compared with day 4 postimmunization (Fig. 6C). Quantitative image analyses to assess the area density of LN covered by subcapsular, cortical, and medullary sinuses revealed that subcapsular sinuses constituted the majority of total lymphatics (∼ 91%) at day 4 postimmunization (Fig. 6D). In contrast, a greater proportion of cortical and medullary sinuses was present at day 14 (∼43%) compared with day 4 postimmunization (∼9%) (Fig. 6D).
Cortical and medullary sinuses undergo preferential expansion at later phases of inflammation. (Ai) LN sections were immunostained for LYVE-1 (green) and B220 (red) to determine the anatomical location of LYVE-1+ subcapsular, medullary, and cortical sinuses. (Aii–v) To specifically distinguish subcapsular from cortical and medullary sinuses, LN sections were stained for LYVE-1, B220, and CD11c or LYVE-1, B220, and CD169 and analyzed by confocal microscopy. LYVE-1+ subcapsular sinuses contain B220+ B cells and CD11c+ DCs (ii), whereas both cortical (iii, iv) and medullary sinuses contain B220+ B cells but not CD11c+ DCs. LYVE-1+ sinuses in the medulla colocalize with CD169+ macrophages (v), but the cortical sinuses did not (iv). White arrows indicate the presence of DCs within subcapsular sinuses. Scale bars, 200 and 50 μm (low- and high-magnification images, respectively). (B) LN sections from mice at baseline and days 4 and 14 were stained for LYVE-1, B220, and CD11c. Images show only LYVE-1 and B220 staining. Dotted white lines demarcate LYVE-1+ subcapsular sinuses identified by their anatomical location in the LN and their colocalization with lymphocytes and DCs. Scale bar, 200 μm. (C) LNs from mice at baseline and days 4 and 14 were stained for LYVE-1, B220, and CD11c. Note more dilated and sinusoidal cortical sinuses on day 14 compared with day 4 postimmunization. Scale bar, 50 μm. (D) The area density of LN covered by subcapsular, cortical, and medullary sinuses was assessed in LN sections from days 4 and 14 postimmunization by quantitative image analyses. Images in (B) and (C) are representative of six independent experiments (n = 6). Quantitative analysis (D) is pooled from six independent experiments (n = 6). Error bars represent SD. **p < 0.01. L, Lumen of sinuses.
Immunostaining of LN sections from lymphocyte-egress experiments confirmed that at T20, numerous CD45.1 T cells transversed the abundant cortical and medullary sinuses present at day 14 postimmunization (Supplemental Fig. 4A). In contrast, such LYVE-1+ sinuses in the LNs from FTY720-treated mice were conspicuously devoid of transferred T cells (Supplemental Fig. 4B). These observations complemented our flow cytometry data (Fig. 1C–F), further supporting our hypothesis that the remodeling of cortical and medullary sinuses supported the restoration of lymphocyte egress to steady-state levels.
Overall, these data demonstrate that early inflammation following immunization (e.g., day 4 postimmunization) is accompanied by the expansion of subcapsular sinuses, a transient retention of lymphocytes, and the accumulation of DCs within the inflamed LNs (Supplemental Fig. 3B). It is only during protracted phases of inflammation (e.g., day 14 postimmunization) that sinuses preferentially expand in the cortical and medullary regions of the inflamed LNs. The remodeling of such cortical and medullary sinuses is associated with an increased egress of lymphocytes, with the ultimate aim of restoring lymphocyte egress to baseline rates.
Spatial differences in VEGF-A distribution accompany the differential remodeling of lymphatic sinuses during prolonged inflammation
We next investigated the possible mechanisms underlying the preferential remodeling of cortical and medullary sinuses at later stages of inflammation. Because similar amounts of lymphangiogenic factors were detected at days 2 and 10 postimmunization in the inflamed LNs (Fig. 4A), we evaluated whether the differential remodeling of cortical and medullary sinuses may be related to changes in the distribution of these factors in the inflamed LNs. Consistent with our previous study (15), we could not detect VEGF-C in LNs by immunostaining (data not shown). In contrast, VEGF-A expression in immunized LNs was markedly increased compared with control LNs (Fig. 7A). Clear differences in VEGF-A distribution could be discerned between inflamed LNs at days 4 and 14 postimmunization. Although VEGF-A expression was mainly detected in the subcapsular region of the activated LNs at day 4, VEGF-A could also be found within the LN paracortex and medulla at day 14 postimmunization (Fig. 7A). Quantification of VEGF-A distribution confirmed that the bulk of it within DLNs at day 4 after immunization was found within the subcapsular regions. Conversely, VEGF-A was present in the subcapsular, paracortical, and medullary regions of DLNs at day 14 after immunization (Fig. 7B). VEGF-A expression in the paracortical and medullary regions was significantly greater at day 14 (∼45%) compared with day 4 (∼18%) postimmunization, pointing toward differences in VEGF-A distribution as inflammation persists. This distribution of VEGF-A within the LN was reminiscent of the pattern of the lymphatic network during different phases of inflammation. On closer examination, we observed that, although VEGF-A colocalized with LYVE-1+ subcapsular sinuses on both days 4 and 14 after immunization, VEGF-A was associated with cortical and medullary sinuses only at day 14 postimmunization (Fig. 7C).
Spatial differences in VEGF-A distribution accompany the differential remodeling of lymphatic sinuses during prolonged inflammation. (A) Immunoreactivity for TCR-β, ERTR7, and VEGF-A or rabbit IgG (isotype control) was evaluated in sections from LNs at baseline and days 4 and 14 postimmunization. Scale bar, 200 μm. (B) Sections were costained for VEGF-A, CD11c, and LYVE-1+, to examine by confocal microscopy, VEGF-A colocalized with subcapsular, cortical, and medullary sinuses at days 4 and 14 after immunization. (C) VEGF-A distribution within DLNs at days 4 and 14 after immunization was quantified. Data are pooled from three to five independent experiments (n = 3–5). Error bars represent SD. (D) VEGF-A colocalization with FRCs lining subcapsular, cortical, and medullary sinuses at days 4 and 14 after immunization was examined by confocal microscopy. Dotted white lines demarcate lymphatics. (E) Orthogonal plane view of how VEGF-A is aligned on the FRCs lining cortical sinuses. Enlarged image of confocal image stack of boxed region is shown (right panel). VEGF-A can be found on the surface and interior of FRCs. (F) Interaction between cortical and medullary sinuses, FRCs, and VEGF-A was evaluated by confocal microscopy at day 14 postimmunization on LN sections stained for ERTR7, VEGFR3, and VEGF-A. All images are representative of three to five independent experiments (n = 3–5). Scale bar (C–F), 50 μm. **p < 0.01. L, Lumen.
Given the intricate relationship between FRC network and lymphatic channels (Fig. 5C), we also examined VEGF-A distribution with respect to the ER-TR7+ reticular fibers associated with lymphatics. VEGF-A colocalized with the reticular network that lined subcapsular sinuses network on both days 4 and 14 postimmunization (Fig. 7D). Interestingly, although VEGF-A was largely absent from the ER-TR7+ network associated with cortical and medullary sinuses on day 4 postimmunization, there was an obvious association of VEGF-A with the ER-TR7+ FRC conduit system that lined these sinuses at day 14 (Fig. 7D). Association between FRCs lining cortical and medullary sinuses and VEGF-A was ascertained to occur at two interfaces: VEGF-A was present at the surface and inside reticular fibers (Fig. 7E). Although the presence of VEGF-A within and on the reticular fibers is suggestive that it is produced by FRCs and secreted into the LN parenchyma, we cannot exclude the possibility that extranodal VEGF-A is transported within reticular conduits and subsequently displayed on FRCs. To further explore the relationship between lymphatics and FRCs, we used VEGFR3 as another marker for lymphatics. We observed a similar close spatial association between lymphatics and FRCs (Fig. 7F). Closer examination also revealed that cortical sinuses, FRCs, and VEGF-A in LNs from day 14 postimmunization engaged in a tripartite interaction, whereas such an interaction was not observed at day 4 (Fig. 7F).
Altogether, these data suggest that as the inflammation evolves, spatial differences in the distribution of VEGF-A within LNs may mediate the remodeling of cortical and medullary sinuses.
Supplementing VEGF-A during early inflammation accelerates remodeling of cortical and medullary sinuses in LNs and restores lymphocyte egress
Based on our earlier observations, we speculated that the presence of VEGF-A in LN paracortical and medullary areas during late inflammation may be a key factor driving cortical and medullary sinus expansion. Thus, we investigated whether we could promote cortical and medullary sinus remodeling during early inflammation by providing exogenous VEGF-A to the paracortical and medullary areas of LNs. To this end, we s.c. implanted Matrigel pellets containing mouse VEGF164 or PBS into the back skin of mice draining to immunized brachial and axillary LNs. Analysis of LN sections at day 5 after immunization revealed increased VEGF-A accumulation in mice that received Matrigel pellets impregnated with VEGF-A compared with control PBS (Fig. 8A). Furthermore, delivery of exogenous VEGF-A resulted in the preferential distribution of VEGF-A in the paracortex and medulla of the DLNs and was accompanied by a remarkable expansion of cortical and medullary sinuses, observations not typically seen at this time point (5 d) postimmunization (Fig. 8B, 8C). More significantly, such augmented remodeling of cortical and medullary sinuses at an early stage of inflammation induced by VEGF-A supplementation functionally restored egress of T and B lymphocytes from the inflamed LNs (Fig. 8D, 8E). These findings support our hypothesis that, in the absence of intervention, the distribution of VEGF-A in the LN paracortex and medulla during late inflammation is, in part, a key event leading to the expansion of cortical and medullary sinuses.
Supplementing VEGF-A during early inflammation accelerates remodeling of cortical and medullary sinuses in LNs. (A) VEGF-A expression in LNs was examined 5 d after implantation of Matrigel pellets containing VEGF-A or PBS by immunostaining for TCR-β and VEGF-A. (B) The effect of Matrigel pellets containing PBS or VEGF-A on LYVE-1+ lymphatic vessel network in the LNs was evaluated by immunostaining. (C) Area density of subcapsular, cortical, and medullary sinuses relative to total lymphatics area in LNs was determined as described in Fig 6. Egress of T (D) and B (E) lymphocytes from LNs was examined after lymphocyte adoptive transfer in immunized mice that received Matrigel pellets impregnated with VEGF-A or PBS. For (A) and (B), images are representative of data from six mice in each group (n = 6). Scale bar, 200 μm. Data in (C) are pooled from six independent experiments (n = 6). Lymphocyte egress experiments in (D) and (E) consist of data from six mice in each group (n = 6). Error bars represent SD. *p < 0.05, **p < 0.01.
Interstitial flow is required for the differential distribution of VEGF-A in LNs during inflammation
Interstitial flow acting in concert with lymphangiogenic factors was reported to be a key driving force of lymphangiogenesis (41, 42). Moreover, alterations in interstitial flow were shown to affect the expression of chemokines by FRCs (43). Therefore, we considered the possibility that interstitial flow through the LNs during inflammation might influence the spatial-temporal distribution of VEGF-A and, thereby, support differential lymphatic remodeling. To address this, we designed a surgical strategy to perturb LN interstitial flow by cutting the afferent lymphatics draining the popliteal LNs at day 10 postimmunization. Mice that received sham operations served as controls (Fig. 9A). Mice were sacrificed 1 d after surgery, a time point at which the structure of the LN was observed to be intact (Fig. 9B). Patency or obstruction of lymphatic flow to the popliteal LNs was verified (Fig. 9B). Although VEGF-A expression was preserved in the sham-treated LNs, perturbation of interstitial flow dramatically decreased expression of VEGF-A in LNs (Fig. 9C). In addition, perturbation of interstitial flow altered VEGF-A distribution in LNs, such that it was confined to the superficial cortex. This indicated that during late inflammation, interstitial flow within LNs governed distribution of VEGF-A into the paracortex and medulla. To further support this, disruption of interstitial flow was noted to obliterate association of VEGF-A with cortical and medullary sinuses compared with sham-treated LNs (Fig. 9D). This implies that lymph flow through the LN could influence the distribution of VEGF-A during the course of inflammation and, as a consequence, modulate the remodeling of cortical and medullary sinuses.
Interstitial flow is required for the differential distribution of VEGF-A in LNs during inflammation. (A and B) Afferent lymphatic vessels were cut on one side (LV resection), whereas a sham operation was performed on LVs on the contralateral side (Sham). One day later, FITC-dextran was injected into the rear footpads, and its transport to the popliteal LNs was examined. The effect of LV resection and resultant perturbation of interstitial flow on VEGF-A distribution (C) and association with cortical and medullary sinuses (D) and FRCs lining these sinuses (E) in DLNs were determined by immunostaining. Dotted white lines demarcate lymphatics. Scale bars in (A) and (C), 200 μm; in (B) and (D), 50 μm. Images are representative of four independent experiments (n = 4). L, Lumen.
Because other groups described that interstitial flow can modulate FRC organization and function (43) and, in our model, extranodal VEGF-A may be transported within the FRC conduit system and subsequently displayed (Fig. 7D) and/or produced by FRCs (Figs. 5D, 7D), we next investigated the repercussions of disrupting interstitial flow on VEGF-A interaction with the fibroblastic reticular network. In contrast to sham-treated LNs, perturbation of interstitial flow through LNs ablated VEGF-A colocalization with ER-TR7+ reticular fibers (Fig. 9E). This suggests that LN interstitial flow is an important regulator of VEGF-A production and/or presentation by FRC network.
Blocking angiogenesis and lymphangiogenesis during inflammation affects distribution of VEGF-A in LNs
Because blocking VEGFR2 and VEGFR3 signaling during inflammation abrogated angiogenesis and lymphangiogenesis, we determined whether this may affect VEGF-A distribution in inflamed LNs. Compared with mice receiving control rat IgG, VEGF-A expression in DLNs from mice treated with anti-VEGFR2– and anti-VEGFR3–blocking Abs was markedly diminished at day 14 postimmunization (Fig. 10A). In addition, distribution of VEGF-A in DLNs from mice that received anti-VEGFR2– and anti-VEGFR3–blocking Abs was generally restricted to the subcapsular space and B cell follicles, in contrast to a broader VEGF-A distribution in mice that received control rat IgG (Fig. 10A).
Blocking angiogenesis and lymphangiogenesis during inflammation affects distribution of VEGF-A in LNs. (A) Distribution of VEGF-A was evaluated in LN sections from immunized mice receiving a combination of VEGFR2- and VEGFR3-blocking Abs (right panel) or control rat IgG (left panel). Scale bar, 200 μm. To measure the effect of blocking VEGFR2 and VEGFR3 signaling on VEGF-A association with cortical and medullary sinuses (B) and with FRCs lining these sinuses (C), LN sections from control or anti-VEGFR2– and anti-VEGFR3–immunized mice were immunostained for VEGFR3, CD11c, and VEGF-A (B) or podoplanin and VEGF-A (C). Scale bar, 50 μm. Images are representative of five independent experiments (n = 5).
In the presence of rat anti-VEGFR2– and anti-VEGFR3–blocking Abs, the use of anti-rat secondary IgG to reveal ER-TR7 staining resulted in cross-reactive staining. Therefore, we used podoplanin (gp38) as an alternate marker to identify the FRC conduit system by immunofluorescence (13, 43). Blocking VEGFR2 and VEGFR3 signaling abolished VEGF-A association with cortical and medullary sinuses (Fig. 10B), as well as with the podoplanin+ reticular fibers lining these sinuses (Fig. 10C). Collectively, these data indicate that disruption of angiogenesis and lymphangiogenesis resulted in the alterations of the distribution of VEGF-A within inflamed LNs, likely precipitated by perturbed interstitial flow through the LNs as a consequence of VEGFR2 and VEGFR3 blockade.
Discussion
In recent years, there has been growing appreciation that LN lymphangiogenesis can be a significant determinant of the immune response during inflammation (15, 25, 26). However, all of these studies focused on lymphangiogenesis occurring at early phases of inflammation and did not evaluate the exact location of the expanded lymphatic network within the LN. To our knowledge, our study provides the first evidence that inflammation, as it evolves from early to late phases, can induce a biphasic remodeling of lymphatics, with the subcapsular sinuses being expanded first, followed by the cortical and medullary sinuses, and that this differential remodeling is biologically and functionally important.
Notably, we demonstrated that the expansion of cortical and medullary sinuses can regulate lymphocyte egress from DLNs during prolonged inflammation. By providing routes for lymphocyte exit during inflammation, remodeling of cortical and medullary sinuses serves as a compensatory measure to re-establish steady-state egress of lymphocytes from inflamed LNs. In the steady state, as much as 50% of recirculating lymphocytes passing through an LN exit through the efferent lymph (5). During inflammation, LN activation triggers remodeling of high endothelial venules (HEVs) to increase lymphocyte entry by manyfold (11, 12). Undesirable situations, including LN hypertrophy and hyperactivation, which are often associated with autoimmune and chronic inflammatory diseases, may arise if these lymphocytes fail to leave the inflamed LNs in a timely manner. Indeed, we have some evidence that the LN hypertrophy described in dyslipidemic mice (44) mainly results from the complete block of lymphocyte egress from the inflamed LNs (M.H. Tay and V. Angeli, unpublished observations). Thus, an increase in the recruitment of lymphocytes to LNs during inflammation has to be accompanied by a proportional increase in lymphocyte output in the efferent lymph. Indeed, studies suggested decades ago that increased blood flow in the stimulated DLNs was associated with increased cell output in the efferent lymph. However, the dynamics of these events, especially in the setting of protracted inflammation, have never been fully characterized (2, 45). In this study, we showed that increased lymphocyte entry into LNs during inflammation was paralleled by a transient decreased lymphocyte output, followed by a return to steady-state lymphocyte output that consisted of ∼50% of T cells and ∼40% of B cells that entered the LNs. Re-establishment of steady-state egress from inflamed LNs may offer an elegant solution to keep pace with increased lymphocyte entry into LNs when inflammation persists. This will prevent these LNs from becoming a sink that may impede proper lymphocyte recirculation and the timely development of an appropriate immune response (16, 46). Although it is possible that enhanced lymphocyte egress at day 14 may be modulated by decreased lymphocyte retention signals arising from downregulated expression of chemokines, such as CCL21 and CXCL13 (47), our experiments inhibiting lymphangiogenesis seemed to indicate otherwise. Blocking VEGFR2 and VEGFR3 signaling had remarkably little effect on the expansion of FRCs (Fig. 3A) and presumably minimal alterations in the expression of CCL21 and CXCL12, which are chiefly produced by FRCs (48–50). However, arrested lymphangiogenesis arising from a blockade of VEGFR2 and VEGFR3 signaling abrogated restoration of lymphocyte egress and underpins that lymphangiogenesis, specifically that of cortical and medullary sinuses, plays an important part in the re-establishment of lymphocyte egress during protracted inflammation.
On the basis of our findings, we propose a model whereby spatial-temporal distribution of VEGF-A, LN interstitial flow, and the fibroblastic reticular network formed coalescent forces to coordinate cortical and medullary sinus remodeling during late inflammation. During early inflammation, VEGF-A may be transported from its primary site of production (i.e., the inflamed peripheral tissue) to the DLNs (21) and/or produced by activated B cells residing in the cortex of the DLNs (15). At this stage, VEGF-A expression was mainly confined to the subcapsular region of the LN. The molecular mass of the predominant VEGF-A isoform, VEGF164 (46 kDa), would allow ready access into the LN parenchyma via the FRC conduit system (51). However, the heparin-binding domain of VEGF164, which anchors the molecule to the extracellular matrix, may limit its free diffusion within the LN via the reticular network (52). In contrast, at later phases of inflammation, VEGF-A expression was not restricted to the subcapsular region of the LN; it also extended into the paracortical and medullary regions, where it drives cortical and medullary sinus expansion. In support of our proposed model, we accelerated remodeling of functional cortical and medullary sinuses during the early days of inflammation by delivering exogenous VEGF-A to the paracortical and medullary areas of LNs. This supports our hypothesis that localization of VEGF-A to the LN paracortex and medulla is instrumental to cortical and medullary sinus remodeling. In addition, this differential distribution of VEGF-A within the activated LNs is dependent on interstitial flow, as shown by our lymphatic vessel-resection and VEGFR2- and VEGFR3-blocking experiments.
Interestingly, organization and function of FRCs were shown to be uniquely sensitive and responsive to changes in lymph flow (43) and contact with activated lymphocytes during inflammation (13). We demonstrated that FRCs in the inflamed LNs expanded with a kinetics and magnitude similar to LECs. It is plausible that, during late inflammation, increased interstitial flow may support the production of VEGF-A by FRCs, and/or combined with FRC expansion, increase delivery of VEGF-A to the LN paracortex and medulla. Whatever its site of production, VEGF-A may then be captured and displayed by sinus-associated reticular fibers to drive the remodeling of cortical and medullary sinuses. Given the physical relationship between lymphatics and FRCs, it is tempting to speculate that FRC expansion in the T cell zone may organize a structural framework to provide the scaffolding required for cortical and medullary sinus remodeling. Altogether, our data suggest that FRCs are essential to regulate HEVs (27), as well as cortical and medullary sinuses, and raise the possibility that LECs and FRCs cooperate during inflammation to modulate LN plasticity and function. Further studies directed at evaluating the significance of this cooperation will be required.
Most research on lymphocyte egress from DLNs has been carried out in homeostatic conditions and focused either on mechanisms that lymphocytes use to reach efferent lymphatic vessels, including regulation of CCR7 (7) and sphingosine 1-phosphate receptor-1 (S1P1) expression (18, 29, 30, 53, 54), or on mechanisms operating at the level of lymphatic endothelial barriers (31, 55). We showed that the expansion of cortical and medullary sinuses in the late phase of inflammation functionally supports the egress of naive lymphocytes from LNs. In one study, the investigators showed that S1P1 upregulation, paralleled by decreased CCR7-mediated retention signals, occurs in activated T cells to facilitate their egress (7). It would be intriguing to explore whether the expansion of cortical and medullary sinuses during inflammation, by providing more available exit routes, acts in conjunction with modulation of lymphocyte CCR7 and S1P1 expression to support egress of activated lymphocytes from DLNs.
Studying subcapsular versus cortical and medullary sinus expansion during inflammation has been constrained by the lack of specific markers to distinguish these structures. To overcome this limitation, we developed a microscopic-imaging method to identify and quantify specifically these three types of lymphatic channels following LN stimulation. Although we found that the increase in LEC population following inflammation was paralleled by proliferation of lymphatics, we cannot rule out the possibility that the apparent expansion of cortical and medullary sinuses may also result from dilation of the sinuses induced by increased flux of cells and/or fluid in response to inflammation. It is imperative to further identify markers that will allow us to discern subcapsular versus cortical and medullary sinuses by means other than imaging. This will allow subcapsular, cortical, and medullary sinuses to be identified and isolated by the use of techniques such as flow cytometry and pave the way for gene-profiling studies of these structures.
By delineating the remodeling of cortical and medullary sinuses and demonstrating how this expansion regulates lymphocyte egress from persistently inflamed DLNs, the current study reveals a novel biological function of LN lymphangiogenesis that is critical for optimal immune response and resolution of inflammation.
Disclosures
The authors have no financial conflicts of interest.
Acknowledgments
We thank Gwendalyn Randolph (Mount Sinai School of Medicine) for discussions and critical reading of the manuscript, Cornelia Halin (Institute of Pharmaceutical Sciences, Eidgenössische Technische Hochschule Zürich) for technical advice and discussions, Benson Chua (National University of Singapore) for breeding of the OTII transgenic mice, and Lew Fei Chuin and Paul Edward Hutchison (Flow Cytometry Lab, National University of Singapore) for sharing expertise. We are grateful to Laurent Renia (Singapore Immunology Network) for the gift of the anti-CD62L Ab and Bronislaw Pytowski (ImClone Systems) for the gift of anti-VEGFR3 and VEGFR2 Abs.
Footnotes
This work was supported by a Singapore Ministry of Education Tier 1 grant and a National Research Foundation grant (to V.A.).
The online version of this article contains supplemental material.
Abbreviations used in this article:
- BEC
- blood endothelial cell
- 3D
- three-dimensional
- DC
- dendritic cell
- DLN
- draining lymph node
- FRC
- fibroblastic reticular cell
- HEV
- high endothelial venule
- KLH
- keyhole limpet hemocyanin
- LEC
- lymphatic endothelial cell
- LN
- lymph node
- LV
- lymphatic vessel
- S1P1
- sphingosine 1-phosphate receptor-1
- VEGF
- vascular endothelial growth factor.
- Received June 23, 2011.
- Accepted February 16, 2012.
- Copyright © 2012 by The American Association of Immunologists, Inc.
References
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