Differential Expression of Basement Membrane Components in Lymphatic Tissues
Departments of Pathology (MM,AL,HA-H) and Biochemistry (SS), University of Oulu, Oulu, Finland; Clinic of Dermatology (KT), University Hospital of Oulu, Oulu, Finland; and Department of Dermatology, University of Freiburg, Freiburg, Germany (LB-T)
Correspondence to: Marko Määttä, MD, PhD, Dept. of Ophthalmology, University of Helsinki, PO Box 220, 00029 HUS, Helsinki, Finland. E-mail: mmaatta{at}mailcity.com
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Summary |
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Key Words: collagen endothelium laminin reticular fiber ring fiber
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Introduction |
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Previous immunohistochemical studies have shown the abundant presence of the main extracellular matrix (ECM) components laminin (Ln), type IV collagen, vitronectin, fibronectin, and types I and III collagen in the RFs of spleen, lymph nodes, and tonsil (Liakka et al. 1991; van den Berg et al. 1993
; Liakka 1994
; Jaspars et al. 1996
). During embryonal development, specific changes can be distinguished in the ECM composition (Liakka and Autio-Harmainen 1992
), and many in vitro studies have shown that the ECM components may affect lymphocyte migration, adhesion, and proliferation (Li and Cheung 1992
; Geberhiwot et al. 2001
).
The most typical molecules of basement membranes (BM) are Lns and type IV collagen. They are connected to each other via nidogen-1, to which other BM components are joined as well (Colognato and Yurchenco 2000). We currently know of 11 different Ln chains, and there is evidence of at least 15 Ln heterotrimers and 6
-chains of type IV collagen forming also several type IV collagen heterotrimers. Immunohistochemical (IHC) studies have shown that BMs may have heterogeneous compositions in different locations. In lymphoid tissues, especially, Ln and type IV collagen are abundant (Karttunen et al. 1989
; Liakka et al. 1991
; van den Berg et al. 1993
). In some of these tissues the BMs are highly discontinuous in areas where the cell traffic is high, e.g., the venous sinuses of the splenic red pulp (RP) and the tonsillar crypt epithelium. In addition to serving as adhesive matrix for the stationary cells, the RFs and BMs rich in Ln and type IV collagen can contribute to the function of non-stationary cell types through signal transduction via integrin-type receptors (Hemler 1990
; Ogata et al. 1996
; Madri and Graesser 2000
).
Many of the previous investigations have been performed by using polyclonal antibodies (PAbs) without knowledge of their chain specificity (Liakka et al. 1991; van den Berg et al. 1993
). Now when chain-specific antibodies are available, a more detailed description of the expression of the individual BM components in lymphoid tissues is possible. In this study we used IHC to evaluate the distribution of Ln
15, ß13, and
12 chains as well as collagen types IV, VII, XVII (previous bullous pemphigoid antigen 180; BP-180), and XVIII in adult human spleen, lymph node, and tonsil. The results showed that these molecules have their own characteristic expression patterns, which indicate their specific contribution to the function of the organs.
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Materials and Methods |
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Antibodies and Immunohistochemistry
Antibodies used are listed in Table 1. For IHC, 4-µm frozen sections were air-dried for 15 min, followed by fixation in cold acetone for 10 min, and then incubated with fetal calf serum (FCS) (Hyclone Laboratories; Logan, UT) 1:5 in PBS for 20 min to block nonspecific binding of IgG. The sections were then incubated with the primary antibody at 4C for 2 hr, followed by biotinylated anti-mouse or anti-rabbit IgG secondary antibody (DAKO; Glostrup, Denmark) for 30 min and avidinperoxidase complex for 30 min. The color was developed with diaminobenzidine tetrahydrochlorideH2O2 (DAB) (Sigma; St Louis, MO) in Tris buffer, pH 7.4. Finally, the sections were lightly counterstained with hematoxylin. As a negative control, the sections were treated as described above but PBS was used instead of the primary antibody.
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Positive Controls
Frozen skin sections were used as positive controls. All antibodies, except for Ln 1-,
2-, and ß2-chains, reacted with the epidermal BM. Collagen XVII showed a cell membrane-bound and partially intracytoplasmic immunoreactivity in basal keratinocytes. Ln ß2-chain reacted with the endothelial BMs of dermal vascular structures and Ln
2 was present in the stroma of dermis. Because of the negative staining reaction for the
1-chain in skin sections, we used the BM of pharyngeal mucous glands as a positive control for the Ln
1-chain stainings.
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Results |
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Faint immunoreactivity for type VII collagen was seen in capillaries of the WP, but other structures were negative.
The type XVII collagen immunoreacted with the sinusoidal ring fibers of the spleen (Figure 1G) but other structures were negative. It was accumulated in the wall of the venous sinuses and appeared under the endothelial cells in a dot-like manner.
Lymph Node
The RFs of the lymphatic follicles immunoreacted for all the Ln chains except the 1- (Figure 2A) and ß2-chains. Follicular capillaries showed a chain composition similar to that of follicular RFs (Figure 2B). In the paracortex, RFs and vascular BMs were stained for Ln
2-,
3- (Figure 2C),
4-,
5-, ß1- (Figure 2D), ß2-, and
1- (Figure 2E) chains. In addition, a faint staining for Ln
2 was seen in the paracortical vessels. Marginal sinuses immunoreacted for Ln
2-,
3-,
5-, ß1-, ß3-, and
1-chains. Capsular tissue showed staining reaction for Ln
2-,
5-, ß1-, ß2-, and
1-chains.
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Types VII and XVII collagen were not detected in the lymph node.
Tonsil
The RFs of the tonsillar lymphatic follicles immunoreacted for Ln 2-,
3-,
5-, ß1-, ß3-,
1-, and
2-chains (Figure 3A). Follicular capillaries showed otherwise similar composition, but they additionally contained Ln
4- and ß2-chain immunoreactivity, and the
2- chain staining was weak and sporadic. Interfollicular RFs immunoreacted with Ln
2-,
3- (Figure 3B),
5-, ß1- (Figure 3C), ß2-, ß3-, and
1-chains and occasionally also with
4-chain. Endothelial BMs of the interfollicular blood vessels (Figure 3D) contained all the other chains except for
1. The BMs of the surface and crypt epithelium showed a similar composition, containing Ln
3-,
4-,
5-, ß1-, ß3-,
1-, and
2-chains. BM staining of the crypt epithelium was, however, more disrupted and irregular deeper in the tonsillar bed.
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Type VII collagen antibody immunoreacted faintly with the follicular RFs and capillaries, but the staining reaction was much fainter and sporadic in the interfollicular area. BMs of the surface and crypt epithelia strongly immunoreacted for type VII collagen (Figure 3F), whereas other compartments were negative.
Immunoreactivity against type XVII collagen was observed in the cell membranes and cytoplasm of the basal cells of the surface and crypt epithelia (Figure 3G). An identical staining pattern was seen in the skin, which served as a positive control.
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Discussion |
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Ln 1, a component of Ln-1 (
1ß2
1) and Ln-3 (
1ß2
1), seemed to be the most restricted chain. It was not detected in the tonsil and lymph node and was seen only in the RFs of the splenic WP and, unexpectedly, in the capillary BMs of the follicular structures of the spleen. Ln
1-chain has been shown to be restricted mainly to the subepithelial glandular BMs and to be absent from the endothelial BMs (Virtanen et al. 2000
; Määttä et al. 2001
). In addition to the presence of Ln
1-chain, another unique feature of the follicular capillaries was the additional presence of chains of Ln 5 (
3ß3
2) in their BMs. Ln 5 is a well-known component of BMs of squamous epithelia, in which it forms a part of the hemidesmosomal complex and usually has been immunolocalized to the epithelium (Marinkovich et al. 1992
; Rousselle et al. 1997
). However, it has been previously reported in capillaries of the human thymus and follicular capillaries of the lymph node and tonsil (Jaspars et al. 1996
). This indicates that Ln 5 can rarely be detected in unusual locations, such as endothelial BMs, and the co-expression of Ln 1 and Ln 5 in the lymphatic tissues may also indicate a unique role for these Lns in the migration of lymphocytes into the follicle parenchyma.
One of the functions of ECM components is the modulation of leukocyte extravasation through the subendothelial Lns (Madri and Graesser 2000). It has been previously shown that especially Ln 8 (
4ß1
1) and Ln 10 have a strong binding capacity for monocytic cells (Pedraza et al. 2000
). Moreover, lymphoid progenitor cells actively synthesize and adhere to Ln 8 and Ln 10 (Gu et al. 2003
). Geberhiwot et al. (2001)
have shown in an in vitro study that lymphoid cells are also able to synthesize Ln 8. Laminin molecules on the cell membranes of lymphocytes may also contribute to the cellcell interactions between lymphocytes and antigen-presenting cells, e.g., dendritic reticular cells and macrophages, whereby Lns play a role in the immunological function of lymphatic organs. However, it must be remembered that because RFs and BMs of lymphatic tissues are covered by the cytoplasmic processes of fibroblastic reticular cells and the cytoplasm of endothelial cells (Chen and Weiss 1972
), the lymphocytes traveling within the lymphatic tissues are usually not in direct contact with them.
Collagen types VII and XVII in the spleen and tonsil, as described in this study, have not been previously reported. They are both mainly expressed by the squamous epithelium and in agreement with this were both localized in this study to the tonsillar crypt epithelium, which is a direct extension of the surface squamous epithelium. Surprisingly, type VII collagen was also detected in the follicular capillaries and RFs of the tonsil and in capillaries of the splenic WP. All the evidence obtained from this study indicates that the capillaries in lymphoid tissues seem to be specified to possess a more "epithelium-like" BM composition than in other tissues. Of the hemidesmosomal components, the integrin 6ß4 has also been detected in human lymph node and tonsil (Liakka 1994
; Jaspars et al. 1996
). As far as we know, however, it is not known whether there are true hemidesmosomes in those locations of lymphatic tissues where hemidesmosomal proteins are found, and this issue needs future investigation.
An interesting finding of this study was that type XVII collagen was specifically present in ring fibers of the spleen. This raises the question of its possible stabilizing role for the sinusoidal structures. Ring fibers are unique BMs of the venous sinuses that surround the endothelial cells like hoops around a barrel. Earlier electron microscopic and IHC studies (Chen and Weiss 1972; Drenckhahn and Wagner 1986
) have shown that within the endothelial cells of the splenic venous sinuses there are basally located fine filamentous bands arranged longitudinal to the cell axis. They run from one ring fiber to the other, and appear to be inserted into the plasma membrane on the ring fiber and to connect the endothelial cells to the ring fiber. In fact, in this study the type XVII collagen staining reaction pattern of the ring fibers differs clearly from that of types IV and XVIII collagen and was seen as dots in the walls of the venous sinuses. We suggest that these dots represent the insertion site of the filamentous bands on the ring fiber matrix. In addition to our finding of type XVII collagen in the ring fibers, the integrin subunit
6 has been found previously in sinus endothelial cells (Liakka 1994
; van den Berg et al. 1993
). This subunit associates with the ß4 subunit to form one of the components of epithelial hemidesmosomes. However, according to our results ring fibers lack Ln 5 and type VII collagen, which are normal constituents of epithelial hemidesmosomes. This indicates that the contact of the sinus endothelial cells with the ring fibers is not mediated via conventional hemidesmosomes. But because endothelial cells must resist marked shear forces caused by the fluid and blood cells that travel through the interendothelial slits from the red pulp cords into the sinuses, our findings suggest that this resistance is maintained through the formation of a firm hemidesmosome-like contact site and that the type XVII collagen and the integrin subunit
6 are involved in the connection between the ring fiber matrix and the intermediate filaments of the sinusoidal endothelial cells.
The type XVIII collagen, a member of the non-fibril-forming collagens, is a widely distributed BM component in many tissues (Halfter et al. 1998; Saarela et al. 1998
; Erickson and Couchman 2000
). A particular interest is concentrated on its 20-kD proteolytic C-terminal peptide fragment, called endostatin (O'Reilly et al. 1997
). This fragment has a strong ability to inhibit endothelial cell activity, angiogenesis, and tumor growth. Thus far, type XVIII collagen distribution in lymphatic organs has not been published. We noted here that type XVIII collagen and type IV collagen showed a similar distribution pattern and were expressed in all the tissue compartments analyzed. The wide presence of type XVIII collagen/endostatin indicates its role not only as an important structural protein but also in controlling endothelial cell proliferation in the lymphatic organs.
In conclusion, our results demonstrate that BM-associated proteins show characteristic expression patterns in lymphatic tissues. The distributions of the various Ln chains, as well as types VII and XVII collagen, differ from those of BM proteins in non-lymphoid tissues and suggest a specific role for these proteins in the maintenance of tissue architecture and in the migration and immunological function of lymphocytes.
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Acknowledgments |
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Ln 1,
4, ß1 and
1 antibodies were a kind gift from professor Ismo Virtanen, University of Helsinki, Finland, and collagen type XVIII antibodies were provided by Prof Taina Pihlajaniemi, Universty of Oulu, Finland. The antibody to ß2 Ln chain purified by Dr Dale Hunter and Dr Joshua Sanes was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA.
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Footnotes |
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