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,§
,§
*
Department of Bacteriology and Immunology, Haartman Institute, University
of Helsinki, Helsinki, Finland.
Department of Pathology, Haartman Institute, University of Helsinki,
Helsinki, Finland.
Transplantation Laboratory, Haartman Institute, University of Helsinki,
Helsinki, Finland.
§
Helsinki University Central Hospital Laboratory Diagnostic, Helsinki,
Finland.
Correspondence to Dr. Risto Renkonen, Haartmaninkatu 3, The Haartman Institute, University of Helsinki, Department of Bacteriology and Immunology, P.O. Box 21, FIN-00014 Helsinki, Finland; Phone: +358-9-1912-6387; Fax: +358-9-1912-6382; E-mail: risto.renkonen{at}helsinki.fi
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The tethering and rolling phase of the lymphocyte extravasation is
suggested to be initiated by L-selectin
(13). This molecule recognizes
endothelial ligands, such as CD34, podocalyxin, MAdCAM-1, and sgp200, provided
that they are decorated with 
2,3sialylated, 1,3fucosylated, and
sulfated lactosamines (13). In
lymph node high endothelium, sulfated sialyl Lewis x (sLex) glycans, which are
necessary for L-selectin—mediated binding of lymphocytes to endothelium,
are constantly expressed. Concomitantly, flat endothelium, which are present
in most parenchymal organs under normal conditions, do not express these
proper glycoforms of L-selectin ligands
(13,14,15,16).
However, induction of sLex-decorated L-selectin ligands on the postcapillary
microvascular endothelium has been shown in rodent models of heart and kidney
allograft rejection
(17,18).
Moreover, our earlier studies suggest that enzymatically synthesized
multivalent sLex glycans can prevent selectin-dependent lymphocyte adhesion to
endothelium
(17,18).
Results obtained from animal models of inflammatory conditions cannot be directly extrapolated to the pathogenesis of human disease. Thus, we recently studied a large series (more than 600) of endomyocardial biopsies taken from human heart allografts and showed a significant correlation between the intensity of expression of endothelial-sulfated sLex-decorated L-selectin ligands on one hand and the histologic severity of the rejection episode on the other hand. Furthermore, this induced sulfo sLex expression in human heart allografts decreased rapidly as the rejection episode was successfully treated (19).
That kidney is the most common solid organ transplant in humans prompted us to investigate the oligosaccharide-dependent leukocyte extravasation in this transplant. Biopsies from kidneys with normal histology or with different grades of acute rejection were identified and investigated with regard to expression of functionally active L-selectin ligands. Although no expression of endothelial sLex (2F3) and sulfated epitopes (MECA-79) were present in control specimens, the expression of these glycans was significantly de novo induced at the onset and during acute allograft rejection. The more severe the rejection was, the more endothelial sLex glycans were detected on graft, and this expression decreased after the rejection resolved. These results suggest that expression of functionally active, properly decorated L-selectin ligands may participate in the initiation of acute rejection in human kidney allograft transplantation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Histopathologic Diagnosis of Rejection
The histologic intensity of acute rejection was graded by one transplant
pathologist according to the Banff classification
(20). No rejection indicates
that the histology did not present any signs of rejection. In the specimens
taken within 30 min after revascularization, occasional small focal leukocyte
infiltrations were seen regardless of otherwise normal histology. Grade 1,
"mild acute rejection," represents cases with significant
interstitial infiltration (>25% of parenchyma affected) and foci of
moderate tubulitis (>4 mononuclear cells/tubular cross section or group of
10 tubular cells). Grade 2, "moderate acute rejection,"
represented cases with (1) significant interstitial infiltration and
foci of severe tubulitis (>10 mononuclear cells/tubular cross section)
and/or (2) mild or moderate intimal arteritis. Grade 3, "severe
acute rejection," represents cases with severe intimal arteritis and/or
transmural arteritis with fibrinoid change and necrosis of medial smooth
muscle cells. Recent focal infarction and interstitial hemorrhage without
other obvious cause also are regarded as evidence for grade 3 rejection. We
also omitted from the study biopsies that represented "borderline
changes" according to the Banff classification.
Antibodies
The following monoclonal antibodies (mAb) against glycan epitopes on
L-selectin ligands were used (Table
1). Both 2F3 (mouse IgM, 5 µg/ml, kindly provided by R.
Kannagi, Aichi Cancer Center, Nagoya, Japan
(21,22))
and HECA-452 (rat IgM, 1:50 culture supernatant, kindly provided by S.
Jalkanen, University of Turku, Turku, Finland
(21,23))
are anti-sLex mAb, requiring the presence of both 2,3sialylation and
1,3fucosylation of lactosamine for recognition. HECA-452 also has been
reported to react with sialyl Lea
(24). MECA-79 (rat IgM, 1:50
culture supernatant, also from S. Jalkanen
(25,26))
recognizes 6 and or 6'-sulfation of lactosamine on a family of
endothelial proteins. QBEND10 (anti-CD34, mouse IgG1, 0.4 µg/ml, Dako,
Glostrup, Denmark) was used to localize the endothelial structures
(27). Isotype-matched mouse
IgG1 (3G6, 1:50 culture supernatant, a gift from Dr. O. Vainio, University of
Turku, Turku, Finland) as well as mouse -human, 7C7 (5 µg/ml), and
rat -mouse, TIB146 (10 µg/ml) (also gifts from S. Jalkanen) were
used as control reagents.
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Immunohistochemistry
Formalin-fixed, paraffin-embedded specimens were used for
immunohistochemistry. Five-µm sections were cut from the kidney core
biopsies and deparaffinized and rehydrated by rinsing with xylene (5 min
x 3), 100% ethanol (5 min x 2), 96% and 70% ethanol (5 min), and
aqua (5 min x 2). No pretreatment was used before antibody labeling. We
used a Histostain-plus kit (Zymed Laboratories Inc., San Francisco, CA) for
immunoperoxidase staining according to instructions. The intensity of
reactivity of mAb 2F3, HECA-452, MECA-79, and control CD34 was scored blinded
without knowledge of histopathologic diagnosis of the biopsy specimens. The
following semi-quantitative score was used: negative (0), mild (1), moderate
(2), and intense (3) staining. The analysis was performed separately from
vascular endothelium present in renal biopsies, i.e., vessels from
glomerulus, peritubular capillaries, major venules, and arteries. The
endothelial staining within a given anatomic localization was always very
homogenous, i.e., the great majority (>80%) of the vessels reacted
similarly within a given section. Fifty to 100 vessels were analyzed from each
specimen.
Statistical Analyses
We used the nonparametric Kruskal-Wallis test to determine the differences
in staining intensities between groups of different histologic diagnosis;
P < 0.05 was considered significant. Basically, this test is a
comparison of the medians of several unpaired groups and the null hypothesis
is that the medians all are equal. Unpaired t test adjusted with the
Bonferroni method was used to analyze the significance between the endothelial
staining intensity and different time groups during the progression of
rejection; P < 0.05 was considered significant. We also used the
nonparametric Spearman rank correlation test to determine whether the rank of
the staining intensity was correlated with the rank of the groups of
histologic diagnosis; P > 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Very low, if any, expression of endothelial sLex epitopes was detected with anti-sLex mAb 2F3 in biopsy specimens shortly after revascularization, i.e., during ischemia/reperfusion injury (mean staining intensity, 0.4 ± 0.13 ± SEM; Figures 1 and 2) or in specimens obtained from well-functioning transplants (mean staining intensity, 0.2 ± 0.1). The endothelia of glomerular capillaries and major vessels were always negative for sLex-decorated L-selectin ligands. The endothelial expression of sLex glycans detected by 2F3 was significantly induced de novo in peritubular capillaries and small venules of specimens taken at the onset of or during acute rejection episodes. The expression level of all rejection specimens was significantly different than in biopsies of control specimens (P < 0.0001) when comparing nonrejection specimens with rejection specimens by Kruskal-Wallis test. The rejection specimens were then divided into four categories according to the severity of rejection. Compared with nonrejection controls (staining intensity, 0.2 ± 0.1 SEM), endothelial reactivity of 2F3 was significantly elevated even in the biopsy specimens that represented "mild" grade 1 rejection (1.2 ± 0.4, P = 0.002 by unpaired t test adjusted with the Bonferroni method; Figures 1 and 2) and was even more pronounced in specimens with more severe rejection (grades 2A and 2B, intensity 1.6 ± 0.3 and 2.5 ± 0.4, respectively; P < 0.0001 by unpaired t test adjusted with the Bonferroni method). Of more than 500 renal allograft biopsies originally screened, only 3 represented rejection grade 3, with extensive hemorrhagia and necrotic tissue morphology. All of these specimens expressed sLex epitopes detected with 2F3, but the damaged tissue morphology did not allow quantitative analysis.
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To analyze further the correlation between the intensity of endothelial L-selectin ligand expression, detected by 2F3, with the severity of the histologic grade of the rejection, we performed the Spearman rank correlation test. The result obtained indicated a strong and significant positive correlation between the staining intensity with endothelial sLex expression, as detected with 2F3, on one hand and the histologic severity of rejection on the other hand (P < 0.0001, by Spearman rank correlation test; Figure 3).
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Effect of Rejection Treatment on L-Selectin Ligand Expression
To investigate the regulation of endothelial L-selectin ligand expression
on kidney allografts with regard to the rejection status and antirejection
treatment with intensified immunosuppressive therapy, we investigated in
detail the follow-up biopsies from four cases when baseline, acute rejection,
and postrejection biopsies were available. The specimens from well-functioning
kidneys without signs of acute rejection did not express any amount of 2F3
reactive epitopes. At the onset of rejection, the expression of functionally
active L-selectin ligands was strongly induced (mean, 2.5 ± 0.5;
P = 0.032 to rejection in unpaired t test adjusted with the
Bonferroni method; Figure 4).
As the rejection grade decreased to normal or borderline levels after
successful antirejection therapy, the intensity of endothelial staining with
2F3 decreased in the same individuals almost to control levels (mean, 0.5
± 0.3; P = 0.032 in unpaired t test adjusted with the
Bonferroni method compared with specimens with signs of rejection;
Figure 4).
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Endothelial sLex Expression Detected by HECA-452
Contrary to results with 2F3, the endothelial sLex expression detected with
another anti-sLex mAb HECA-452 was strongly positive in all of the 15 biopsies
taken right after revascularization, i.e., during
ischemia/reperfusion injury (mean staining intensity, 1.6 ± 0.2;
Figure 2). This endothelial
HECA-452 reactivity was no longer detectable in specimens from
well-functioning kidney allografts with subsequent normal histology (0.1
± 0.1; P < 0.0001 by unpaired t test adjusted with
the Bonferroni method). However, at the onset of acute rejection episodes, the
endothelial sLex expression was strongly and significantly upregulated again
on peritubular capillaries and venules (mean, 1.9 ± 0.2; P
< 0.0001 by unpaired t test adjusted with the Bonferroni method;
Figures 1 and
2). With HECA-452, no
correlation between the intensity of staining and the severity of the
rejection episode was found. However, as with 2F3, the HECA-452 epitope was
always absent from glomerular capillaries and major vessels. Although both 2F3
and HECA-452 detect sLex, the latter has been suggested to react with sialyl
Lea (24).
Expression of Sulfated L-Selectin Ligands on Graft Endothelium during
Rejection
Another glycomodification that contributes to L-selectin—mediated
rolling of lymphocytes on endothelium is the 6 and/or 6' sulfation of
sLex glycans
(25,26).
Therefore, the expression of these sulfated lactosamine epitopes was
investigated with mAb MECA-79. In specimens taken from kidneys after
revascularization as well as in specimens from well-functioning transplants,
no endothelial MECA-79 reactivity was seen in any vessels. On the contrary, in
specimens that showed signs of acute rejection, a clear and significantly
elevated endothelial MECA-79 reactivity was observed compared with the two
control groups (mean, 1.2 ± 0.2; P < 0.0001 by unpaired
t test adjusted with the Bonferroni method compared with both control
groups to the rejection groups; Figure
2). Here again, in contrast to results obtained with anti-sLex
antibody 2F3, the endothelial MECA-79 staining intensity did not correlate
with the grade of rejection, i.e., the severity of the lesion. The
MECA-79 epitope was absent in glomerular capillaries and major vessels of the
kidney in all biopsies.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The strong correlation between the intensity of endothelial cell expression of sulfated and/or sLex-decorated ligands and the presence of acute rejection suggests that endothelial cells upregulate the expression of these inflammation-promoting glycoforms of L-selectin ligands at the onset of rejection, thus leading to increased lymphocytic traffic into the human kidney allografts. These observations are well in line with our previous results with both rodent and human allografts (17,18,19,29). In rat, the kidney and heart allograft venous endothelium expresses de novo sLex glycans at the onset of rejection, thus presumably promoting the lymphocyte extravasation to the tissue particularly at the sites of lymphocyte extravasation. This has been shown with the anti-sLex antibodies and the L-selectin-IgG fusion protein and in the ex vivo Stamper-Woodruff lymphocyte-binding assay in other models in rodents and in humans (17,18,29). Furthermore, we recently synthesized multivalent sLex polylactosamine glycans that have organ specificity. Some extended sdiLex glycans inhibited the lymphocyte binding to heart endothelium with 10-fold lower IC50 values compared with lymphocyte binding to lymph node endothelium in the same animals (18,29,30,31). The regulation of sLex ligands thus would provide means to site-specific immunosuppression by inhibiting lymphocyte traffic only to the transplanted organ but not interfering with normal host immunity in lymph nodes.
In this article, we show for the first time that endothelial sLex
expression increases in the graft endothelium during reperfusion injury within
15 to 30 min postrevascularization presumably induced by the cold-ischemia and
reperfusion injury. This rapid upregulation of sLex glycans on the peritubular
capillaries and small venules of the kidney allograft may contribute to the
selectin-mediated extravasation of granulocytes into the tissue, thus
contributing to the reperfusion injury that leads to acute tubular necrosis
and anuria (32). Although the
sLex induction in well-functioning grafts is decreased to background levels,
at the onset of acute rejection episodes, both the endothelial sulfation and
the sLex expression are once again de novo induced. This must be due
to increased synthesis and/or decreased degradation of these glycans in
endothelial cells. We have preliminary evidence for the enhanced synthesis of
sLex glycans during acute rejections, as the presence of the
1,3-fucosyltransferase VII is elevated in heart allografts during
rejection (19). Work with
L-selectin—deficient mice as wells as with Fuc TVII null animals
(absence of endothelial sLex) demonstrated the importance of L-selectin in
extravasation of lymphocytes to other inflamed tissues
(33,34).
However, the importance of L-selectin and/or selectin ligands in transplant
rejection of visceral organ transplants has not been shown in gene-deficient
mice, although it has been documented with skin transplants in L-selectin
knockout mice (35).
Recently, several sulfotransferases that are capable of synthesizing 6 and/or 6' sulfations have been cloned. Their presence and role in the inflammation-induced endothelial MECA-79 reactivity at the sites of acute transplant rejection, as well as other lymphocyte mediated inflammations such as bronchial asthma, muscle (36), or skin (37), have yet to be studied. It is noteworthy that there are also newly described mAb that recognize the whole epitope of sulfated sLex (13,21). However, these mAb do not work in paraffin sections; therefore, we could not use them on our large archive of clinically and histologically graded kidney transplant specimens.
Comparison of the endothelial L-selectin ligand expression of kidney allografts with our recent heart allograft study reveals a similar observation (19). A few interesting differences can be noted, however. Whereas the 2F3 reactivity correlated significantly to the severity of both the heart and the kidney rejections, the endothelial HECA-452 and MECA-79 reactivities were strongly correlated to the severity of only heart allograft rejection. This suggests that the postcapillary venous endothelia are different in these two organs. Furthermore, acute reperfusion injury was linked to endothelial expression of HECA-452 but not 2F3, a pattern that might be due to local induction of sLea, also know to act as a L-selectin ligand (14,24).
The heterogeneity in endothelial L-selectin ligands within different vascular beds is potentially an interesting observation. This may be due to different expression, subcellular localization, and/or activity of transferases responsible for the synthesis of sulfated sLex glycans (38). Staining with the indicated mAb is strongly predictive for L-selectin ligand activity, but direct proof of this remains to be obtained, e.g., transplantation experiments with relevant transferase knockout animals, which are not yet available. Expression of several other adhesion molecules, in addition to L-selectin, have been described to be induced on peritubular capillaries and venules during teh acute kidney transplant rejection (5,9,39,40), but the whole picture of the multistep process of lymphocyte recruitment into the allograft at the onset of rejection is still beyond our understanding (2,3).
These observations broadened our previous studies in experimental and human allografts and demonstrate a strong induction of properly decorated endothelial ligands for L-seletin in acute rejection. Taken together, selectin-dependent leukocyte extravasation is a crucial step in the initiation of lymphocytic inflammatory reaction, such as acute allograft rejection. Our data suggest that the inducible endothelial glycoforms of L-selectin ligands that participate in the lymphocyte traffic into the sites of inflammation, thus providing a new target to glycan-based immunomodulatory interventions. It may also provide a new, sensitive tool for histopathologic analysis and grading of acute allograft rejection.
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Acknowledgments |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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