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¶
From the Transplantation Laboratory*and Departments of Pathologyand Bacteriology and Immunology,¶Haartman Institute, Helsinki; the Rational Drug Design Program,Biomedicum, University of Helsinki, Helsinki; and Laboratory Diagnostics,Helsinki University Central Hospital, Helsinki, Finland
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Abstract |
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The tethering and rolling phases of lymphocyte extravasation have been proposed to be initiated by L-selectin. This molecule recognizes endothelial ligands, such as CD34, podocalyxin, MAdCAM-1, and sgp200, provided that these ligands are decorated with 
2,3-sialylated, 1,3-fucosylated, and sulfated lactosamines.1,5-7,10-12
These sulfated sialyl Lewis x (sLex) glycans are necessary for the L-selectin-mediated binding of lymphocytes to endothelium and are constantly expressed at least on the CD34 glycans of lymph node high endothelium.13
Under normal conditions, the flat endothelium in parenchymal organs does not express properly glycosylated modifications of L-selectin ligands14
and no extravasation occurs. However, the induction of sLex- or sulfo sLex-decorated L-selectin ligands onto the postcapillary microvascular endothelium occurs both in rodents and humans undergoing heart and kidney allograft rejection and also in the early specimens taken from bronchial asthma pa-tients.2-4,14
Moreover, the enzymatically synthesized multivalent sLex glycans can prevent the selectin-dependent lymphocyte adhesion to properly glycosylated endothelium ex vivo, pointing out a putative mean to inhibit the inflammatory reaction.14,15
Here we expand these previous observations and analyze the endothelial sLex or sulfo sLex expression patterns in various inflammatory diseases. For this purpose, a large series of histological biopsies (>400) from patients with ulcerative colitis, psoriasis, thyroiditis, myocarditis, and vasculitis and their corresponding noninflamed tissues were collected. The endothelium of control noninflamed tissues did not express essentially any sLex or sulfo sLex epitopes whereas on the endothelium of inflamed organs they were prominently expressed. Most importantly, the up-regulated sLex or sulfo sLex expression patterns of inflamed tissues were clearly different between every organ analyzed, and when combined with our previous data, it could be postulated that every inflamed organ expressed its own specific endothelial sLex or sulfo sLex zip code regulating lymphocyte traffic to a given organ. This thorough analysis of the endothelial sLex and sulfo sLex expression patterns in inflammatory diseases should warrant the more detailed analysis of the authentic glycosylations on the relevant endothelial glycoproteins acting as L-selectin ligands.13
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Materials and Methods |
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Formalin-fixed, paraffin-embedded archived blocks of normal and diseased human tissues and corresponding slides were obtained from the Department of Pathology, Helsinki University Central Hospital. The selection of human samples from archives was based on the primary pathological reports; all specimens were also re-evaluated independently to be representative for a given type of inflammation or serve as relevant controls of normal tissues by three observers (JR, OT, and TP). The ethical committee of the Helsinki University Central Hospital had approved the study plan.
Altogether, nearly 1000 histological specimens were identified from the records of the Department of Pathology and screened through for further study. One hundred twenty-two histological specimens with lymphocytic thyroiditis, 130 with ulcerative colitis, 126 with psoriasis, 108 skin biopsies with vasculitis, and 57 with myocarditis were screened, and 50 cases from each disease were chosen for the study. As control specimens, 131 samples with goiter, 150 samples with noninflammatory colon, 98 samples of normal skin, and 20 samples with myocardial hypertrophia or myofibrosis were screened, and 30 cases from each organ were selected.
Immunohistochemistry
The glycans on selectin ligands were identified by immunohistochemistry with three different monoclonal antibodies (mAbs) each recognizing specific glycosylation modifications (Table 1)
. mAb HECA-45216
(rat IgM) and mAb MECA-7917
(rat IgM) were kindly provided by Dr. S. Jalkanen (University of Turku, Turku, Finland). mAb 2F318
(IgM) was from Pharmingen (San Diego, CA). As a positive control for the detection of endothelial cells, anti-human CD34, class II (mIgG1) (1:25; DAKO, Glostrup, Denmark) and as negative controls mAbs 7C7 (mouse IgM) and TIB-146 (rat IgM), both kindly provided by Dr. S. Jalkanen, were used. For immunohistochemical analysis, previously described protocols was used.3,4
In short, tissue specimens originally embedded in paraffin were cut into 5-µm sections, deparaffinized, and rehydrated, followed by a microwave pretreatment and a serum blocking. Vector Vecstain IgG mouse and rat kits were used for immunoperoxidase stainings according to the manufacturer’s instructions (Vector Laboratories, Inc., Burlingame, CA).
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Statistical Analysis
Fifty cases from inflamed specimens and 30 cases from specimens with normal histology entered the statistical analysis. The results were first analyzed by the Kruskal-Wallis one-way analysis of variance by ranks. This test is basically a comparison of medians of several unpaired groups and the null hypothesis is that the medians are equal; P values <0.05 were considered as significant. The Kruskal-Wallis test indicated that we could use the nonparametric Mann-Whitney U-test for the multiple comparisons of medians in control and inflammation groups. The null hypothesis is that the medians are equal. In the Mann-Whitney U-test, differences were considered to be significant at P < 0.05.
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Results |
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, whereas the vascularity in noninflamed skin biopsies was only 34 ± 1.8/mm2. The numbers of anti-CD34+ vessels were modified only marginally in the inflammatory tissues when compared to their control noninflamed tissue samples (107 ± 2.0/mm2 in thyroiditis, 99 ± 1.8/mm2 in myocarditis, 87 ± 2.9/mm2 in colitis, 47 ± 1.9/mm2 in psoriasis, 49 ± 2.6/mm2 in vasculitis).
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. The original antigens in the immunization yielding mAbs 2F3 and HECA-452 sLex decorated glycolipids and stromal components of human lymph nodes, respectively.16,18
mAbs 2F3 and HECA-452 detect 2,3-sialylation and 1,3-fucosylation of lactosamine, but they do not distinguish between sulfated and nonsulfated epitopes. MECA-79 was originally defined to recognize an antigen-entitled peripheral lymph node adressin (PNAd).17,19
This is actually a family of glycoproteins, most of which are able to bind L-selectin. In humans only CD34 and podocalyxin have been identified at the molecular level among the glycoprotein species reacting with MECA-79.20
Later the recognizing epitope of MECA-79 was shown to recognize an extended sulfated core 1 lactosamine structure. This antibody does not recognize 2,3-sialylation or 1,3-fucosylation, but is however able to block in vitro and in vivo lymphocyte homing to murine lymph nodes.17,19 The sLex moiety and its sulfated forms are of importance in the extravasation process leading to leukocyte invasion into different human tissues. To assess whether distinct inflammatory diseases differ from each other in the relation to their endothelial glycosylation, the profiles of 50 specimens representing inflammation and 30 control specimens from five different noninflammatory conditions were studied by immunohistochemistry using two different sLex mAbs and one sulfation-dependent mAb with relevant controls.
Thyroid Gland
In the thyroid gland (Figure 2
; A to F), the anti-sLex antibody mAb 2F3 did not react with the endothelium in control goiter specimens with noninflammatory histology. As noninflamed controls, both the diffuse and multinodular types of thyroid hyperplasias but not adenomas were included. In thyroiditis specimens, the percentage of mAb 2F3-reactive vessels was significantly increased to 26.3 ± 2.4% (mean ± SEM) (Figure 3A)
when compared to control specimens (P < 0.001, Mann-Whitney U-test). It should be noted that only capillaries and venules expressed sulfo sLex-decorated L-selectin ligands whereas arteries or arterioles did not.
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, and Figure 3A
). To analyze the sulfated variants of functionally active L-selectin ligands on the endothelium, the antibody recognizing the extended core 1 polylactosamine structure, mAb MECA-79, was used. Although only 0.6 ± 0.3% of the vessels reacted positively with mAb MECA-79 in specimens with noninflamed histology, a strong endothelial reactivity was observed in thyroiditis specimens (37.5 ± 2.3%) (P < 0.001, Mann-Whitney U-test).
Heart
In myocarditis, a similar analysis of the endothelial expression of various glycosylation variants was performed (Figure 2
; G to L). Although specimens with noninflamed myocardium remained essentially negative with all of the three antibodies used (Figure 3B)
, myocarditis lesions showed some slight, but yet significant endothelial up-regulated expressions of sLex epitopes, detected with mAb HECA-452, rising from 0.0 ± 0.0% to 2.5 ± 0.9% (mean ± SEM, P = 0.017).
Skin
The control noninflamed skin specimens expressed a small proportion of positively stained sulfo sLex vessels (Figure 2
, mol/L-R), especially when detected with mAb HECA-452 (9.2 ± 1.3%, mean ± SEM) (Figure 3C)
. In the psoriasis specimens with active skin lesions, the endothelial reactivity with mAb HECA-452 (24.6 ± 1.3%) was significantly elevated (P < 0.001, Mann-Whitney U-test). Concomitantly, the endothelial reactivity with mAb 2F3 remained totally negative.
In the skin biopsy specimens with vasculitis, the mAb 2F3 and MECA-79 reactivities remained essentially at a background level, and the staining with mAb HECA-452 was practically not altered either (9.2 ± 1.3% to 11.8 ± 1.2%).
Colon
In the colon, the endothelial sulfo sLex expression was already present in control specimens with noninflamed histology (Figure 2, S–X
). However, the endothelial expression levels analyzed with all three mAbs were even more enhanced in colitis specimens. The percentage of mAb 2F3-expressing vessels increased from 2.6 ± 0.5% to 12.8 ± 1.4%, the mAb HECA-452-reactive vessels from 7.5 ± 1.4% to 15.8 ± 1.7%, and mAb MECA-79 from 1.0 ± 0.4% to 11.0 ± 1.1% (mean ± SEM) (Figure 3D)
. All these elevations in the expression of endothelial sulfo sLex variants were statistically significant between the noninflamed controls and the inflammatory specimens (P < 0.001 for each pair, Mann-Whitney U-test).
Putative Endothelial Zip Codes
In addition to the de novo-induced or -enhanced endothelial sLex or sulfo sLex expression shown in this study, we propose that each organ also possessed its own sLex- or sulfo sLex-specific zip code. A zip code could be determined by combining the percentages of reactive vessels for each of the three mAbs used, as shown in Table 2
. Thus the glycosylation zip code for endothelium in thyroid gland would be 00-02-00 during noninflamed conditions and 26-33-38 during thyroiditis. Similarly the sulfo sLex-specific zip code for heart endothelium is normally 00-00-00, 00-003-00 during mild myocarditis and 38-25-45 during severe allograft rejections (Table 2)
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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These observations are well in line with previously published data and extend further the earlier results with both rodent and human inflammatory lesions, such as allograft rejection.2,4,14,19,21 In rat kidney and heart allograft studies, the venous endothelium expressed de novo sLex glycans at the onset of rejection. This phenomenon did presumably promote the lymphocyte extravasation into tissue parenchyma as shown with anti-sLex antibodies, the L-selectin-IgG fusion protein, as well as with the ex vivo Stamper-Woodruff lymphocyte-binding assay.14,15,22 In addition, after synthesizing multivalent sLex polylactosamine glycans with different organ specificities, we were able to show that these extended sialyl diLex glycans were able to inhibit lymphocyte binding to heart endothelium with 10-fold lower IC50 values when compared to the lymphocyte binding to lymph node endothelium in the same animals.23 Thus, the manipulation of the regulation of sLex ligand expression could provide means to a site-specific immunosuppression, by inhibiting lymphocyte traffic only to a given organ, but not interfering the common normal host immunity in lymph nodes.
For this study, we selected five different inflammatory diseases and compared the endothelial expression of sLex- and sulfo sLex-decorated L-selectin ligands in inflamed tissues to their noninflamed counterparts. In lymphocytic thyroiditis, there is a marked and intense infiltrate of lymphocytes and plasma cells in thyroid gland that finally and virtually ends up in replacing the actual thyroid parenchyma and, thus, the histology of a late-stage disease may resemble the histology of lymphatic tissue. Myocarditis is defined as an inflammatory involvement of the heart muscle characterized by a leukocyte infiltrate and the necrosis or degeneration of myocytes. In psoriasis, the most typical lesions are well-demarcated pink plaques, which have a characteristic marked epidermal thickening and a superficial lymphocytic infiltrate, but the most diagnostic sign is the great number of neutrophils forming small aggregates. The term vasculitis is applied to any blood vessel inflammation. The classic histological feature is an aggregation of neutrophils within and about venules, which can in severe cases lead to a lymphocyte infiltration of venule walls. Ulcerative colitis is a common recurrent disease expressing both active inflammatory ulceration areas and healing areas, both showing a microscopic evidence of inflammation. Histologically, the active phase is characterized by crypt abscesses and ulcerations extending down to the muscular layer and surrounded by a prominent mucosal infiltrate of inflammatory cells.24
Recently, a novel sulfotransferase (HEC-GlcNAc6ST or LSST) responsible for synthesizing the 6 sulfation of GlcNAc has been cloned.25,26 The gene deletion of this high endothelial venule-specific enzyme results in a significantly hampered lymphocyte traffic to lymph nodes in vivo.27 Because such sulfations could be of a crucial importance for the recruitment of lymphocytes into the site of inflammation, the presence and the role of such enzymes warrant study in future. The endothelial expressions of MECA-79 epitopes, sulfated extended core 1 lactosamine structures,28 have been shown earlier to be induced during inflammation at the sites of acute transplant rejection, bronchial asthma, as well as inflamed muscle or skin lesions.3,29,30 Here we expand these observations and show that endothelial mAb MECA-79 reactivity was present in many sites of inflammations, such as thyroiditis, psoriasis, ulcerative colitis, asthma, and organ transplant rejections.
In addition to the de novo-induced or -enhanced endothelial sLex or sulfo sLex expression shown in this study, we propose that each organ could also possess its own sLex- or sulfo sLex-specific zip code. When expressed as a percentage of reactive vessels for each of the three mAbs used, we could designate these zip codes as shown in Table 2
. These induced expression levels of glycans that fulfill the criteria of L-selectin ligands suggest that these glycans could be crucial in the initiation of leukocyte extravasation into sites of inflammations. To test this hypothesis either an indirect Stamper-Woodruff type of ex vivo lymphocyte-binding assay could be performed in each organ, but this would require the use of fresh and not formalin-fixed tissues. Another more direct approach could be to isolate the endothelial glycoproteins, such as CD34, known to act as L-selectin ligands and directly analyze with mass spectrometry the O-glycans on both normal and inflamed tissues and testing their ability to support L-selectin-dependent leukocyte rolling. We have already began the latter approach and characterized the glycans on CD34 of tonsillar high endothelial venules.13
Furthermore, we were able to observe differences even between the inflammations of the same organ, such as between psoriasis and vasculitis in the skin. Nonetheless, whether this observation was caused by the severity of the inflammatory incidence or by the difference in the putative pathogenesis of lymphocytic infiltration remains to be defined. However, our observations about inducible organ- and inflammation-specific zip codes could be understood as an extension to the previously reported organ-specific peptides and their ligands.31-34 Both the glycan and peptide specificities of various organs could serve as the means of targeted interventions for re-circulating cells, whether or not these cells are leukocytes or malignant tumor cells during hematogenous metastasis.
The down-regulation of the endothelial expression of sulfo sLex-bearing glycoforms is taking place concomitantly with the inflammation resolving.2
As previously shown, of six or more human 1,3-fucosyltransferases, FucTVII is the one capable of synthesizing sLex glycans at the level of endothelial expression and, thus, linked to the induction of heart allograft rejections.23,35,36
The studies with L-selectin-deficient mice as well as with FucTVII-null or FucTIV and FucTVII double-null animals have demonstrated and supported the importance of L-selectin in the extravasation of lymphocytes into lymph nodes and inflamed tissues.37-39
Taken together, we show here that the de novo induction of endothelial sLex or sulfo sLex glycans is a common event in all organs analyzed here and, thus, suggests that these glycans play a crucial role in the early-state induction of tissue inflammation. In addition, because each organ was shown to carry its own zip code, ie, a specific modification of sLex or sulfo sLex glycans, it suggests that this phenomenon could putatively provide a mean for the organ-selective inhibition of leukocyte trafficking.
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Acknowledgements |
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Footnotes |
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Supported in part by research grants from the Academy of Finland and the Technology Development Center, Helsinki; the Sigrid Juselius Foundation; grants from the Helsinki University Central Hospital Fund; and a grant from the Heart Association, Helsinki, Finland.
Accepted for publication May 2, 2002.
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References |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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3'sialylated and multiply 3fucosylated biantennary polylactosamines. A bivalent sialyl diLex-saccharide inhibited lymphocyte endothelial adhesion organ selectively. Eur J Biochem 1999, 261:208-215[Medline]
(1,3)fucosyltransferase Fuc-TVII controls leukocyte trafficking through an essential role in L-, E-, and P-selectin ligand biosynthesis. Cell 1996, 86:643-653[Medline]
(1,3)fucosyltransferase FucT-IV and FucT-VII exert collaborative control over selectin-dependent leukocyte recruitment and lymphocyte homing. Immunity 2001, 15:115-126[Medline]Related articles in Am J Pathol:
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