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BASIC SCIENCE |
Departments of *Pathology, Internal Medicine III, and Surgery, Medical University of Vienna, Allgemeines Krankenhaus, Vienna, Austria; and Molecular/Cancer Biology Laboratory, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland
Correspondence to Dontscho Kerjaschki, Department of Pathology, University of Vienna-Allgemeines Krankenhaus, A-1090 Vienna, Austria. Phone: 43-1-40400 5176; Fax: 43-1-40400 5193; E-mail: dontscho.kerjaschki{at}akh-wien.ac.at
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Abstract |
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- and 
-chain-expressing plasmacytoid cells. Numerous chemokine receptor CCR7+ cells within the nodular infiltrates seemed to be attracted by secondary lymphatic chemokine (SLC/CCL21) that is produced and released by lymphatic endothelial cells in a complex with podoplanin. From these results, it is speculated that lymphatic neoangiogenesis not only contributes to the export of the rejection infiltrate but also is involved in the maintenance of a potentially detrimental alloreactive immune response in renal transplants and provides a novel therapeutic target. |
Introduction |
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Acute interstitial rejection is accurately defined as a special form of inflammation, with a mixed infiltrate of mononuclear inflammatory cells (CD4+ and CD8+ T lymphocytes, macrophages) that invade the cortical tubulointerstitial spaces (4). Despite many years of experience with transplant biopsies, little is known about the kinetics of infiltrate clearing after immunosuppressive therapy, and it is possible that nodular infiltrates of mononuclear cells in the cortical stroma are residues of the diffuse infiltrate found in acute rejection. Disappearance of the rejection infiltrate is in part due to apoptosis that occurs in the renal cortex at a rate similar to that in the thymus (5). However, even this relatively high apoptotic rate is obviously too low to account for the disappearance of inflammatory infiltrate cells after immunosuppressive treatment, thus demanding additional mechanisms of elimination. This has raised the possibility that, similar to other inflamed tissues, cortical lymphatic vessels could serve as exit routes (6), as suggested by previous studies in experimental renal transplant rejection in which lymphatic vessels drained large amounts of fluid and mononuclear cells (7). Therefore, we investigated here the distribution and local density of lymphatic vessels and their topographic relation to the rejection infiltrate in acute rejection, taking advantage of recently developed specific markers for lymphatic endothelial cells (8). We chose to examine biopsies and explants from human renal transplants rather than experimental models that only imperfectly mirror the course of rejection and its corresponding morphologic features in humans.
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Materials and Methods |
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Immunohistochemical Localization of Lymphatic Vessels
Localization of lymphatic vessels in paraffin or frozen sections was performed with purified rabbit anti-podoplanin IgG (5 µg/ml) or mouse monoclonal anti-podoplanin IgG (1 µg/ml), using a biotin–streptavidin–horseradish-peroxidase method, as described (10). The lymphatic vessel density was quantified as number of podoplanin-positive vascular profiles per medium-power field (MPF; median lymphatic vascular density [MLVD], 20x objective). Typically, 10 to 20 fields per biopsy were evaluated. As renal tissue cores are compressed by the biopsy procedure, no attempt was made to record the size and patency of lymphatic vessels. For confirming the identification of lymphatic vessels, indirect double immunofluorescence on acetone-fixed cryostat sections of renal explants was performed with primary antibodies specific for podoplanin and Prox-1 or with LYVE-1. Alexa 488– and Alexa 633–labeled secondary antibodies were used (Molecular Probes, Eugene, OR), and triple-channel confocal laser scanning microscopy was performed with a ZEISS LSM 510, as described (10).
Double and Triple Immunohistochemistry
These methods were performed on 2-µm sections of formalin-fixed, paraffin-embedded archival biopsies or renal explants that were classified for diagnostic purposes according to the Banff nomenclature, as described (10, 11). As immunohistochemical controls, the primary antibodies were omitted.
Immunoelectron Microscopy
Segments of biopsy cores were fixed in a mixture of 4% formaldehyde and 0.2% distilled glutaraldehyde in 20 mM cacodylate buffer (pH 7.2) for 6 to 12 h, the blocks were embedded in Lowicryl-K4M resin (Polysciences, Warrington, PA), and sections were immunolabeled with rabbit anti-podoplanin IgG and 10 nm of gold conjugated to sheep anti-rabbit IgG, as described (12). Double immunogold labeling was performed by incubation of the ultrathin sections in a first step with rabbit anti-podoplanin IgG and 10 nm of sheep-anti rabbit IgG gold conjugate and in a second layer with mouse monoclonal anti-SCL/CCL21 IgG, followed by 5 nm of gold sheep anti-mouse IgG conjugate. As positive controls, rabbit and monoclonal murine antibodies to podocalyxin were used. As negative controls, primary antibodies were omitted.
Surface Plasmon-Resonance Analysis of SLC/CCL21 Binding to Podoplanin and Competition with Heparin
The binding of a podoplanin receptorglobuin, composed from a human Fc-domain and the glycosylated ectodomain of podoplanin and expressed in 293 human fibroblasts, was studied by surface plasmon-resonance analysis on a BiaCore 2000 instrument (BiaCore AB, Uppsala, Sweden), following the manufacturer’s protocol. Briefly, BiaCore sensor chips, type CM5, were activated with a 1:1 mixture of 0.2 M N-ethyl-N'-(3-dimethylamino-propyl)carbodiimide and 0.05 M N-hydroxysuccinimide in water. Podoplanin ectodomain fusion protein was purified by Protein-G affinity chromatography and yielded a single band by SDS-PAGE. The protein was immobilized at concentrations up to 50 ng/ml in 10 mM Na acetate (pH 4.5), and the remaining binding sites were blocked with 1 M ethanolamine (pH 8.5). The resulting densities were 1500 RU fusion protein/mm2. In some experiments, unglycosylated GSP-receptorglobulin was expressed in Escherichia coli, purified by affinity chromatography, and similarly immobilized. A control flow cell was made by performing the activation and blocking procedures only. Recombinant SCL/CCL21 was dissolved in sample running buffer (10 mM HEPES [pH 7.4], 150 mM NaCl, 2 mM CaCl2, and 0.005% Tween 20). In competition experiments, 1 to 3000 ng of heparin was included in the incubation buffer. In some experiments, the chips were coated with bacterially expressed unglycosylated podoplanin ectodomain-Fc fusion protein. Regeneration of sensor chips after each analysis was performed with 1.6 M glycine-HCl buffer (pH 3.0). The BiaCore response is expressed in relative response units, i.e., the difference in response between protein and control flow channel. Kinetic parameters were determined by use of BIAevaluation software.
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Results |
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The MLVD (expressed as number of podoplanin-positive vascular profiles per MPF) was very low in normal control kidney cortex (0.24 ± 0.1 vessels/MPF; Figure 1J). In the acute phases of rejection, identified by high Banff scores of acute rejection (Table 1) with diffuse mononuclear infiltrate and severe tubulitis, no significant proliferation of lymphatic vessels was observed, and their location and number resembled that of normal kidneys (Figure 1J). By contrast, significant amplification of the lymphatic microvasculature was observed in all 35 selected biopsies and explants that contained nodular infiltrates, with average density of 14.7 ± 8.6/MPF (Figure 1J), widely ranging from 41.6 to 3.7 vessels/MPF. Lymphatic vessels were found in the periarterial adventitia, as well as within the intertubular stroma (Figure 1A).
Numerous podoplanin-positive lymphatic endothelial cells expressed the nuclear proliferation marker Ki-67 (Figure 1, E through H). The identity of lymphatics was confirmed by double immunofluorescence with antibodies to LYVE-1 and Prox-1 (Figure 1, C and D). Glomeruli in the sections of the present study are by and large unlabeled because human podocytes express less podoplanin than lymphatic endothelial cells, in contrast to rat kidneys (Figure 1B).
Clinicopathologic Correlation
An attempt was made to correlate lymphatic proliferation and/or occurrence of nodular infiltrates with the clinical outcome criteria: graft loss, death of the patient with functioning graft, and functioning graft at present, taking also into consideration the time elapsed between transplantation, biopsy, and present end point of the study at the end of 2002. The patients were divided into two groups, one showing nodular infiltrates and lymphatic proliferation and the second without nodular infiltrates and few lymphatic vessels (Table 1).
Nine of ten cases of graft losses occurred in the first group and showed a long interval between transplantation and organ loss (average 334 wk). Thirteen patients in the same group had functioning grafts at the end of this study (average creatinine 2.1), with a similar average time distance to the transplantation of 358 wk. Two patients died with functioning grafts, and no information was available about the outcome of 10 patients.
The biopsies of patients without nodular infiltrates consistently showed a lower MLVD than those in the first group (Table 1). There was only one graft loss out of 12 patients, and for the remaining cases, the average time from transplantation to the end of the study was only 184 wk and the average creatinine amounted to 2.35. One patient of this group died with functioning graft, and no information was available on four patients.
VEGF-C Is Produced by Interstitial Macrophages
Within nodular infiltrates, VEGF-C was expressed by a small fraction of mononuclear cells that were identified as CD68+CD32+CD14+ macrophages coexpressing also VEGF-D and VEGFR-3 (Figure 2).
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) that were found on the periphery of nodules and sometimes also in their center (Figure 3, A and I). When the course of lymphatic vessels was followed, it became apparent that nodular infiltrates were built up only focally, leaving intervening stretches of "naked" lymphatic vessels in between (Figure 3C). No coincidence of Ki-67+ lymphatic endothelia and nodular infiltrates was obvious.
A large number of mononuclear cells within nodular infiltrates expressed the nuclear proliferation marker Ki-67 (Figure 3). The nodular infiltrates were composed of variable proportions of T and B lymphocytes that were frequently segregated into different regions (Figure 3C), and occasionally clusters of - and -chain–producing plasmacytoid cells (Figure 3E) and arborized S-100+ dendritic cells were observed in proximity to lymphatic vessels (Figure 3H). CD4+CD25+ cells were absent, and CD45RA+ and CD53+ memory B cells and CD21+ follicular dendritic cells were rare (data not shown). Triple labeling revealed that cells expressing the proliferation marker Ki-67 were mostly of T lymphocyte lineage (approximately 75%) or B lymphocytes (25%).
CCR7+ Cells Cluster around Lymphatic Vessels
Double immunofluorescence showed the colocalization of podoplanin-positive lymphatic vessels and numerous CCR7+ cells within nodular infiltrates (Figure 4). CCR7+ cells frequently showed on their surfaces granular deposits of podoplanin (Figure 3D, inserts). RANKL-receptor, IL-7, lymphotoxin 
and 
receptors, ELK/CCL19, CXCL13, and CXCR5 were not detected (data not shown).
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) that co-localized with podoplanin. These complexes were found along the basolateral membrane of lymphatic endothelial cells and sequestered into the perivascular stroma as seen by double-labeling immunoelectron microscopy (Figure 5, B and C).
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Discussion |
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Critical for investigations in the nascent field of lymphangiogenesis is the reliability of the immunocytochemical markers used (13). Currently, four markers distinguish lymphatic from blood vessel endothelial cells: (1) the membrane mucoprotein, called by us podoplanin, that qualifies as highly specific for lymphatic endothelial cells (12) and was instrumental for the first isolation of pure lymphatic endothelial cell lineages from human dermis (14, 15); (2) VEGFR-3 that is also expressed in endothelial cells of newly formed blood vessels (16); (3) LYVE-1, a CD44-related hyaluronate receptor (17); and (4) Prox-1, a transcription factor that controls the lymphatic phenotype of endothelial cells (18). In this study, the immunohistochemical results were obtained primarily with antibodies to podoplanin, and identical results were obtained by double labeling with antibodies against LYVE-1 and Prox-1.
In the cortex of normal human kidney, podoplanin/LYVE-1/Prox-1–expressing lymphatic vessels were confined to the adventitia of large- and middle-sized arteries, as described previously (19). This pattern of distribution persisted in acute phases of transplant rejection with intense interstitial mononuclear infiltration. However, in biopsies containing nodular infiltrates, there was approximately 50-fold amplification of the lymphatic vessel density over controls, with lymphatic microvessels reaching deep into the tubulointerstitial space. These lymphatic vessels were formed by lymphangiogenesis, as many of them expressed the nuclear proliferation marker Ki-67 and presumably sprouted from preexistent perivascular lymphatics. In the lumina of the newly formed lymphatics, CD2+ and fewer CD2- cells were frequently encountered, indicating that the lymphatic vessels contribute to the clearing of the rejection infiltrate from the renal cortex. Moreover, previous results of experimental renal transplantation indicated that the hilar lymphatics of the graft drained large amounts of lymphatic fluid and mononuclear cells, thus supporting the functionality of these vessels (7). Lymphatic neoangiogenesis apparently involved the tubulointerstitial infiltration of CD68+CD23+ macrophages that produce VEGF-C and VEGF-D, similar to a recently discovered subset of tumor-associated macrophages that were related to peritumoral lymphatic vessel proliferation (10) and to tubulointerstitial mononuclear cells in the rat remnant kidney model of cortical fibrosis (20). These data provide further support for the hypothesis that VEGF-C–producing macrophages contribute to regionalized lymphatic neoangiogenesis.
A potentially important observation in all biopsies with nodular infiltrates is the co-localization of lymphatic vessels with the nodular mononuclear infiltrates that presumably are immunologically highly active organoid structures, with sometimes massive Ki67+ T and B lymphocyte activation. Immunohistochemical typing of the nodular mononuclear infiltrates revealed clusters of CD4+ and CD8+ cells, as well as CD20+ B lymphocytes and and chain-expressing plasmacytoid cells. S-100+ dendritic cells were observed in association with lymphatic vessels. These results provide evidence that within the perilymphovascular nodular infiltrates, activation and maturation of T lymphocytes occur by antigen presentation by dendritic cells and that CD-20+ B cells mature to Ig-producing plasmacytoid cells. Thus, nodular infiltrates resemble "tertiary" lymphatic organs in autoimmune diseases that locally perpetuate autoimmune reactions and support autoantigenic epitope spreading (21). As a consequence, it is possible that in transplants, a cellular and/or humoral alloantigenic response could be contained within the grafted organ and thus difficult to detect in the circulation. In contrast to lymphatic organs developing, for example, in lymphoid organogenesis or in autoimmune diseases, nodular infiltrates in transplants were not associated with high endothelial venules (data not shown), suggesting that their constituent lymphocytes were recruited locally and not from the circulation.
Surprisingly little is known about the occurrence and function in graft rejection of nodular infiltrates so far, and it is not clear yet whether they indicate a distinct clinicopathologic entity. The current study was aimed primarily at establishing the connection of lymphovascular neoangiogenesis and the occurrence of peri- and paralymphovascular lymphoid organ-like infiltrates. The clinical correlation with the pathomorphologic data suggests that nodular infiltrates are found in approximately 10 to 15% (35 of <350) of transplant biopsies. Also, some correlation was observed between the occurrence of nodular infiltrates with the chronicity indices of the Banff classification. The occurrence of nodular infiltrates and lymphatic proliferation could be associated with an increased chance of graft loss, as all graft losses but one clustered within this group of patients (Table 1). However, the significant differences in the time periods between transplantation, taking of the biopsy, and end point of this study, between the patients with nodular infiltrates and robust lymphatic proliferation and those without nodular infiltrates and lower MLVD, impairs this speculation. Thus, because of the relatively small number of samples and the inhomogeneity of the patient groups, no relevant information can be deduced from this preliminary study, and therefore the precise chronology of nodular infiltrates and their relation to lymphatic neoangiogenesis remain to be determined in a systematic large-scale analysis of protocol biopsies, which is currently under way (Stuhte and Kerjaschki, unpublished observations).
The close association of nodular infiltrates with lymphatic vessels raises the possibility that the lymphatic endothelial cells actively recruit lymphocytes. A good candidate chemokine for this purpose is SLC/CCL21, which organizes lymphatic follicles when expressed ectopically in cells of mouse pancreas (22) and attracts mononuclear cells in inflammatory diseases (23), whereas mice with deletion of the SLC/CCL21 gene fail to develop lymph nodes in certain regions (24). We previously discovered that SLC/CCL21 is produced and polarly secreted by isolated lymphatic endothelial cells (14). Here we show that lymphatic vessels in transplants produce SLC/CCL21 in situ and are surrounded by CCR7+ cells, providing direct evidence for SLC/CCL21-mediated lymphocyte and presumably also dendritic cell chemoattraction by lymphatic vessels. These findings assign a novel, active role to lymphatic vessels in the organization of the perilymphovascular nodular infiltrates in renal transplants and perhaps in lymphoid organogenesis in general.
In tissues, SLC/CCL21 gradients are required for the directed migration of CCR7+ cells (25) and are established by charge interactions with proteoglycans in basement membranes (26), which, however, are not produced by lymphatic vessels. Here we provide evidence by double-labeling immunoelectron microscopy that podoplanin contributes to the perilymphovascular SLC/CCL21 gradient formation, as SLC/CCL21-podoplanin complexes appear on the basal cell membrane of lymphatic endothelia and are shed into the perivascular stroma. This process is presumably facilitated by a reorientation of the expression of podoplanin from the luminal membrane of lymphatic endothelia in normal tissue (12, 27 ) toward the abluminal membrane in the setting of transplant rejection shown here. As ultimate proof for podoplanin-SCL/CCL21 complex formation, surface plasmon resonance binding indicated a high-affinity charge and carbohydrate side chain–dependent binding of podoplanin to SLC/CCL21. Its ligation with other chemokines remains to be determined. Collectively, these data provide evidence that SLC/CCL21-podoplanin complexes contribute to the perivascular gradient formation and assign a novel function to podoplanin as a binder of chemokines in lymphatic endothelial cells.
In conclusion, the results of this study put newly formed lymphatic vessels and peri- and paralymphovascular infiltrates center stage as foci of immunologic activity in human renal transplants. By virtue of their cellular composition, nodular infiltrates have the potential to launch and perpetuate specific immune responses to graft alloantigens and thus could contribute to recurrent episodes of acute rejection, support humoral rejection (28), pave the way for chronic rejection and eventual loss of transplant function, and thus could provide a novel therapeutic target. Although the observations obtained in this study on human biopsy material are necessarily primarily descriptive, they offer several functional hypotheses that now can be tested in experimental systems.
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Acknowledgments |
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We thank Dr. Agnes Fogo for critical reading of this manuscript.
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References |
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/podoplanin deficiency disrupts normal lymphatic vasculature formation and causes lymphedema. EMBO J 22: 3546–3556, 2003[CrossRef][Medline]
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J. Am. Soc. Nephrol. 2004 15: A24-A26.
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