Immuno-electron-microscopic localization of types III pN-collagen and IV collagen,laminin and tenascin in developing and adult human spleen

The distribution of the extracellular matrix proteins types III pN-collagen and IV collagen, laminin and tenascin was investigated in fetal, infant, and adult human spleens by using immuno-electron microscopy. The presence of type III pN-collagen was assessed by using an antibody against the aminoterminal propeptide of type III procollagen. All the proteins other than type III pN-collagen were found in reticular fibers throughout development. In the white pulp of the fetus aged 16 gestational weeks, only an occasional type III pN-collagen-containing fibril was present, although type III pN-collagen was abundant in the reticular fibers of the red pulp. Conversely, in adults, most of the reticular fibers of the white pulp, but not of the red pulp, were immunoreactive for type III pN-collagen. Ring fibers, the basement membranes of venous sinuses, were well developed in both infant and adult spleens. The first signs of their formation could be seen as a discontinuous basement membrane, which was immunoreactive for type IV collagen, laminin, and tenascin in the fetus aged 20 gestational weeks. Intracytoplasmic immunoreactivity for all the proteins studied was visible in the mesenchymal cells of the fetus aged 16 gestational weeks and in the reticular cells of the older fetuses, which also showed labeling for type IV collagen and laminin in the endothelial cells. The results suggest that proteins of the extracellular matrix are produced by these stationary cells.     

Distribution of laminin and types IV and III collagen in fetal,infant and adult human spleens

Summary The immunohistochemical distribution of the basement membrane (BM) proteins, laminin and type IV collagen, and interstitial type III collagen was investigated in 12 fetal spleens at the 15th–38th gestational weeks (g.w.) and in spleens of 8 infants from term to 4 years. The results were compared with the distribution of the same proteins in adult human spleen. BM proteins were found to be abundantly present in the red pulp of all spleens during the whole of development. The content of type III collagen gradually decreased with advancing age and, in adult spleen, there were only occasional positively staining fibers in Billroth's cords. This finding indicates that the composition of reticular fibers in the red pulp of spleen is different from the reticular fibers elsewhere in lymphoreticular tissue. Early signs of ring fiber formation in the walls of venous sinuses were detectable at the 15th–19th g.w., although their more complete development occurred relatively late from the 36th g.w. onwards. Ring fibers contained both laminin and type IV collagen in all the investigated spleens. They never stained for type III collagen. The developing white pulp was positive for BM proteins, but showed no staining for type III collagen at the 15th g.w. At later ages, the white pulp stained similarly for both BM proteins and type III collagen.     

An ultrastructural and immunohistochemical study of extracellular matrix in meningiomas

Extracellular matrix of meningiomas was studied by light and electron microscopy with the aid of immunohistochemical techniques. Special attention was paid to the distribution of type I, III, IV, V collagens and laminin with a comparison between meningothelial and fibroblastic types. Connective tissue fibers and basement membrane were not found among the tumor cells in the meningothelial type, but were found in the fibroblastic type. The immunolocalizations were consistently demonstrated extracellularly, but were not within the cytoplasm. Type I, III and V collagens were usually demonstrated in the fibrous septum in the meningothelial type, while they were localized among the tumor cells in the fibroblastic type. Furthermore, type IV collagen and laminin were demonstrated within the vascular walls or around the syncytium in the meningothelial type, while they were localized among the tumor cells in the fibroblastic type. In both types the expression of type IV collagen and laminin was closely related to the distribution of basement membrane. Although meningothelial and fibroblastic meningiomas showed quite different distribution of extracellular matrices, the profile of collagen types expressed by these two basic types was essentially the same. The cellular derivation of meningiomas was discussed with particular attention to the structure of human arachnoid villi and meninges.     

Extracellular matrix components synthesized by human amniotic epithelial cells in culture

Epithelial cells from human post-partal amniotic membrane in primary culture secreted two major matrix proteins, fibronectin and procollagen type III, and small amounts of laminin and basement membrane collagens (types IV and AB). Identified in the culture medium by immunoprecipitation, these components were located by immunofluorescence to a pericellular matrix beneath the cell monolayer. Deposition of fibronectin, laminin and procollagen type III occurred under freshly seeded spreading cells. In the matrix of confluent cultures, fibronectin and procollagen type III had a moss-like distribution. Matrix laminin had predominantly a punctate pattern and was sometimes superimposed on the fibronectin-procollagen type III matrix. In the human amniotic membrane in vivo, laminin, type IV collagen and fibronectin were located to a narrow basement membrane directly beneath the epithelial cells. Fibronectin and procollagen type III were detected in the underlying thick acellular compact layer. Fibronectin secreted by amniotic epithelial cells is a disulfide-bonded dimer of slightly higher apparent molecular weight (240 kilodaltons) than fibronectins isolated from human plasma or fibroblast cultures. Laminin was detected in small amounts in the culture medium. Laminin antibodies precipitated a polypeptide of about 400 kilodaltons, and two polypeptides with slightly faster mobility in electrophoresis under reducing conditions than fibronectin. Procollagen type III was by far the major collagenous protein whereas little or no production of procollagen type I could be observed. Basement membrane collagens were identified as minor components in the medium by immunoprecipitation (type IV) or chemical methods (αA and αB chains).     

Distribution of type IV collagen, laminin, nidogen and fibronectin in the haemodynamically stressed vascular wall

Changes in the extracellular matrix of haemodynamically stressed blood vessel walls were studied by immunofluorescence histochemistry in venous-pouch aneurysms fashioned on the site of the common carotid artery of nine sheep. Tissues from the thickened walls of the experimental aneurysms were examined from 11 to 98 months post-operatively for changes in the distribution of the basement membrane components type IV collagen, laminin, nidogen and fibronectin. In the younger aneurysms, there was an increase of the basement membrane components in the thickened area. Very little basement membrane was detected in older aneurysms. Diffuse staining for fibronectin was noted in aneurysms of all ages. Thick deposits of basement membrane material were observed in calcified tissues. The changes in the matrix proteins were similar to alterations occurring during the development of atherosclerosis in human vascular tissue.     

Immunocytochemical localization of collagen types I,III, IV,and fibronectin in the human dermis

Summary The distribution of collagen types I, III, IV, and of fibronectin has been studied in the human dermis by light and electron-microscopic immunocytochemistry, using affinity purified primary antibodies and tetramethylrhodamine isothiocyanate-conjugated secondary antibodies. Type I collagen was present in all collagen fibers of both papillary and reticular dermis, but collagen fibrils, which could be resolved as discrete entities, were labeled with different intensity. Type III collagen codistributed with type I in the collagen fibers, besides being concentrated around blood vessels and skin appendages. Coexistence of type I and type III collagens in the collagen fibrils of the whole dermis was confirmed by ultrastructural double-labelling experiments using colloidal immunogold as a probe. Type IV collagen was detected in all basement membranes. Fibronectin was distributed in patches among collagen fibers and was associated with all basement membranes, while a weaker positive reaction was observed in collagen fibers. Ageing caused the thinning of collagen fibers, chiefly in the recticular dermis. The labeling pattern of both type I and III collagens did not change in skin samples from patients of up to 79 years of age, but immunoreactivity for type III collagen increased in comparison to younger skins. A loss of fibronectin, likely related to the decreased morphogenetic activity of tissues, was observed with age.     

Immunogold localization of basement membrane molecules in rat retinal capillaries

Vascular basement membrane contains laminin, fibronectin, proteoglycan and collagens. These molecules have been identified in various tissues by immunolabeling methods and biochemical analyses. We have previously localized laminin, fibronectin and type IV collagen to the basement membrane of rat retinal vessels at the ultrastructural level using an immunoperoxidase method. In this study, we use an immunogold method to re-examine the distribution of these molecules and also to study the localization of heparan sulfate proteoglycan and types I, III and V collagen in the retinal capillary basement membrane. Gold labeling for laminin, type IV collagen and proteoglycan were found diffusely on the basement membrane of the endothelium and pericyte, while that for fibronectin and type V collagen was spotty and variable and that for types I and III collagen was negligible. The segment of basement membrane between the endothelial cell and pericyte appeared less reactive to anti-laminin and anti-type IV collagen than the membrane between the pericyte and perivascular neuroretina. The immunogold method may be useful in quantitative studies of thickened basement membranes under abnormal conditions.     

Changes in the distribution of type IV collagen,laminin, proteoglycan,and fibronectin during mouse tooth development

The distribution of certain basement membrane (BM) components including type IV collagen, laminin, BM proteoglycan, and fibronectin was studied in developing mouse molar teeth, using antibodies or antisera specific for these substances in indirect immunofluorescence. At the onset of cuspal morphogenesis, type IV collagen, laminin, and BM proteoglycan were found to be present throughout the basement membranes of the tooth. Fibronectin was abundant under the inner enamel epithelium at the region of differentiating odontoblasts and also in the mesenchymal tissues. After the first layer of predentin had been secreted by the odontoblasts at the epithelial-mesenchymal interface, laminin remained in close association with the epithelial cells whereas type IV collagen, BM proteoglycan, and fibronectin were distributed uniformly throughout this area. Later when dentin had been produced and the epithelial cells had differentiated into ameloblasts, basement membrane components disappeared from the cuspal area. These matrix components were not detected in dentin while BM proteoglycan and fibronectin were present in predentin. The observed changes in the collagenous and noncollagenous glycoproteins and the proteoglycan appear to be closely associated with cell differentiation and matrix secretion in the developing tooth.     

Isolation and characterization of type IV procollagen, laminin, and heparan sulfate proteoglycan from the EHS sarcoma

We have studied the extractability of type IV collagen, laminin, and heparan sulfate proteoglycan from EHS tumor tissue growth in normal and lathyritic animals. Laminin and heparan sulfate proteoglycan were readily extracted with chaotropic solvents from both normal and lathyritic tissue. The collagenous component was only solubilized from lathyritic tissue in the presence of a reducing agent. These results indicate that lysine-derived cross-links and disulfide bonds stabilize the collagenous component in the matrix but not the laminin or the heparan sulfate proteoglycan. The majority of the collagen present in the extracts had a native triple helix based upon the pattern of peptides resistant to pepsin digestion and visualization in the electron microscope by the rotary shadow technique. This protein was composed of chains (Mr 185000 and 170000) identical in migration to the chains of newly synthesized type IV procollagen. This finding confirms earlier work that indicates that the biosynthetic form, type IV procollagen, is incorporated as such in the basement membrane matrix. Material with smaller chains (Mr 160000 and 140000) appeared on storage in acetic acid solutions. These results indicate that the lower molecular weight collagen in acid extracts of basement membrane arises artifactually due to an endogenous acid-active protease.     

Immunohistochemical localization of laminin, nidogen, and type IV collagen during the early development of human liver

 There is evidence that basement membrane components control differentiation of liver sinusoids and bile ducts. These processes occur in humans in the 9th gestational week (GW). Distribution of laminin, nidogen, and type IV collagen was studied during human liver development between the 6th and the 10th GW. Laminin and nidogen lined intrahepatic microvessels in the 6th and 7th GW decreasing in quantity at the beginning of the fetal period (9th–10th GW). Type IV collagen was detected in microvessels only from the 9th GW onward. In the early periportal matrix (9th–10th GW) laminin, nidogen, and type IV collagen were diffusely distributed. At these stages, basement membrane zones of larger portal vessels and of early bile ducts were also stained for all three glycoproteins. These results show that laminin and nidogen are localized in microvessels during early human liver development and decrease in concentration at the developmental stage during which microvessels become discontinuous. In contrast, type IV collagen is not present in early microvessels but occurs when laminin and nidogen disappear. The three glycoproteins occur together only in those areas of the developing liver in which, from the 9th GW onward, the differentiation of immature liver cells into biliary epithelium takes place. Accepted: 20 August 1998     

Connective tissue of rat lung. II: Ultrastructural localization of collagen types III, IV, and VI

We localized collagen types III, IV, and VI in normal rat lung by light and electron immunohistochemistry. Type IV collagen was present in every basement membrane examined and was absent from all other structures. Although types III and VI had a similar distribution, being present in the interstitium of major airways, blood vessels, and alveolar septa, as in other organs, they had different morphologies. Type III collagen formed beaded fibers, 15-20 nm in diameter, whereas type VI collagen formed fine filaments, 5-10 nm in diameter. Both collagen types were found exclusively in the interstitium, often associated with thick (30-35 nm) cross-banded type I collagen fibers. Occasionally, type III fibers and type VI filaments could be found bridging from the interstitium to the adventitial aspect of some basement membranes. Furthermore, the association of collagen type VI with types I and III and basement membranes suggests that type VI may contribute to integration of the various components of the pulmonary extracellular matrix into a functional unit.     

Immunohistochemical localization of type IV collagen fibronectin and laminin in the juxtaglomerular apparatus of the rat kidney

The juxtaglomerular apparatus (JGA) is a complex structure containing several components: the vessels, the extraglomerular mesangium and the distal tubule. These structures include cellular elements and an extracellular matrix (ECM). Collagenous (type IV collagen) and noncollagenous components of the basement membranes were studied. The localization of type IV collagen and of two extracellular glycoproteins (laminin and fibronectin) was investigated using immunofluorescent and immunoperoxidase labelled antibodies. Type IV collagen and laminin have the same localization on the JGA basement membranes. On the other hand, fibronectin is limited to the entrance of the glomerular stalk. On electron microscopy, type IV collagen is found in the basement membrane while fibronectin is restricted to certain areas of the extracellular matrix. These findings confirm data concerning the distribution of these three components in basement membranes and allow a better understanding of the histoarchitecture of the juxtaglomerular apparatus.     

Assay for radiolabeled type IV collagen in the presence of other proteins using a specific collagenase

An enzymatic assay is described which quantitates radiolabeled type IV basement membrane collagen in the presence of large amounts of other proteins. A partially purified neutral protease is used which cleaves type IV collagen into fragments at 37°C which are not precipitated at 1.3% (final concentration) trichloroacetic acid-tannic acid. The kinetics of type IV collagen digestion by this enzyme are not significantly altered by the presence of a 10-fold excess of type III collagen. [¹⁴C]Tryptophan-labeled control proteins prepared from fibroblast cultures are not degraded significantly by this protease in the presence of 2.5 mmN-ethylmaleimide. The proportion of type IV collagen in a mixture of labeled placenta collagenous proteins was calculated after separate digestions with the type IV collagenolytic activity and bacterial collagenase: this value compared favorably with the proportion of type IV collagen estimated by gel electrophoresis.     

Influence of the subendothelial basement membrane components on fibrin assembly. Evidence for a fibrin binding site on type IV collagen

Effective repair of a vascular injury depends on establishment of a stable fibrin patch at the injury site. Data presented in this study demonstrate that structural modification of fibrin occurs as a result of fibrin interaction with naturally occurring components of the vascular basement membrane and subendothelial structures. Of the basement membrane components, type IV collagen produces the greatest structural modification, generating thick fibrin fibers; a 3-fold increase in the fiber mass/length ratio occurs when type IV collagen is increased from 0 to 100 ng/ml. Laminin and dermatan sulfate decrease the fibrin fiber mass/length ratio resulting in thinner fibers. However, the overall effect of the basement membrane on fibrin is to increase the fibrin fiber diameter. Electrophoretic light scattering and the binding of type IV collagen by fibrinogen-Sepharose further establish the interaction between type IV collagen and fibrinogen. Incorporation of laminin with type IV collagen onto coated surfaces decreases the ability of type IV collagen to bind fibrinogen. These studies emphasize that the final fibrin structure is influenced by the milieu in which the clot is assembled.     

Immunofluorescent localization of collagens,fibronectin, and laminin during terminal differentiation of odontoblasts

The localization of collagens types I, II, III, IV, laminin, and fibronectin was analyzed in mouse embryonic molars by indirect immunofluorescence. Using affinity-purified antibodies, all these antigens except collagen type II were detected in tooth germs and particularly at the epithelio-mesenchymal junction. Collagens type I, type IV, and laminin were localized at the junction before, during, and after odontoblasts terminal differentiation. The staining patterns corresponding to type III collagen and fibronectin were modified during the polarization of odontoblasts. Collagen type III present at the epithelio-mesenchymal junction could no longer be detected in this region when odontoblasts were polarized. Fibronectin, surrounding preodontoblasts, was confined to the epithelio-mesenchymal junction when odontoblasts were fully polarized. Previous studies had shown that the presence of a basement membrane and associated material was a prerequisite for the polarization of odontoblasts. Therefore, the redistribution of collagen type III and fibronectin was discussed in terms of fibronectin-collagen interactions and transmembranous control of the cytoskeleton activity in the differentiating odontoblasts.     

Production and characterization of a monoclonal antibody to human type III collagen

Human type III collagen from placenta was isolated and purified for use as an immunogen. A monoclonal antibody was produced which specifically recognizes epitopes unique to type III collagen. The specificity of the antibody was determined by inhibition ELISA, an immunoblot assay, and by immunoprecipitation. Results indicated that the monoclonal antibody recognized only the alpha 1(III) polypeptide chains and did not crossreact with type I, IV, or V collagen. The monoclonal antibody was also used for immunohistochemical localization of type III collagen in tissue sections of human placenta, bovine spleen, and lymph node. In placenta, both large and small blood vessels showed pronounced staining of the tunica media, which contains largely smooth muscle cells, known to synthesize type III collagen. In contrast, the intimal areas and endothelial cells showed no staining with the antibody. In the placental villi, staining was limited to the villous core, where fine fibrillar structures showed strong staining. In lymph nodes, the capsule and pericapsular adipose cells were surrounded by a covering of type III collagen. Within the parenchyma of the node, staining was localized to a branching, reticular array of fine fibers. In the spleen, staining was pronounced in the capsule, splenic trabeculae, and white pulp, where blood vessel staining was especially prominent. The red pulp and splenic sinuses contain little or no type III collagen. The fine network-like or reticular staining pattern found in the lymph node parenchyma is consistent with the staining pattern of the protein reticulin, and suggests that type III collagen may be closely associated with reticulin in certain tissues. Since the role of type III in tissues is unclear, this reagent will be useful in providing new information in this regard.     

Migratory interaction of amphibian epidermal cells with components of the basement membrane

In adult newts, basal epidermal cells adjacent to a fresh wound move toward the damaged area by migrating over the epidermal basement membrane. In an attempt to determine which basement membrane components mediate this migration, small pieces of glass coated with various natural matrices, purified proteins, or fragments of proteins were implanted into skin wounds such that epidermal cells attempting to form a wound epithelium would encounter the implants. Laminin derived from a cell line (M1536-B3) that produces no type IV collagen was inactive as a migration substrate. Migration on recombinant entactin was somewhat better than on laminin but was still only ～ 14% of that on type I collagen. M15 matrix, a laminin and entactin-containing product of M1536-B3 cells, was no better than entactin alone. Type IV collagen was an excellent substrate, producing slightly more migration than corresponding concentrations of type I collagen at nearly all concentrations tested. Migration on type IV lacking the NC1 domain was at least as good as on intact type IV. All the activity in type IV was present in a 95 kD fragment (al (IV)95) from the carboxy terminal two-thirds of the α1 chain. Approximately 60% of the activity on β1(IV)95 was obtained on implants coated with a 110 amino acid fragment of the α1 chain derived from the carboxy terminal half of α1(IV)95. Adding the synthetic peptide, arg-gly-asp-ser (RGDS) to the medium, biocked migration on fibronectin-coated implants but had no effect on implants coated with type IV, suggesting that migration on type IV involves different cell surface receptors than those mediating migration over fibronectin. Matrigel, a commercial product containing most basement membrane components, was a poor migration substrate. Thus if type IV mediates basal cell migration toward a wound in vivo, there may have to be some alterations in basement membrane structure to allow epidermal receptors to access type IV active site(s). © 1994 Wiley-Liss, Inc.     

Expression and adhesive properties of basement membrane proteins in cerebral capillary endothelial cell cultures

Previous studies have indicated the importance of basement membrane components both for cellular differentiation in general and for the barrier properties of cerebral microvascular endothelial cells in particular. Therefore, we have examined the expression of basement membrane proteins in primary capillary endothelial cell cultures from adult porcine brain. By indirect immunofluorescence, we could detect type IV collagen, fibronectin, and laminin both in vivo (basal lamina of cerebral capillaries) and in vitro (primary culture of cerebral capillary endothelial cells). In culture, these proteins were secreted at the subcellular matrix. Moreover, the interaction between basement membrane constituents and cerebral capillary endothelial cells was studied in adhesion assays. Type IV collagen, fibronectin, and laminin proved to be good adhesive substrata for these cells. Although the number of adherent cells did not differ significantly between the individual proteins, spreading on fibronectin was more pronounced than on type IV collagen or laminin. Our results suggest that type IV collagen, fibronectin, and laminin are not only major components of the cerebral microvascular basal lamina, but also assemble into a protein network, which resembles basement membrane, in cerebral capillary endothelial cell cultures.     

Collagen-binding proteins secreted by type II pneumocytes in culture

The alveolar epithelial basement membrane is believed to play important roles in lung development, in maintaining normal alveolar architecture, and in guiding repair following lung injury. However, little is known about the formation of this structure, or of the mechanisms which mediate interactions between the epithelium and specific matrix macromolecules. Since type IV collagen is a major structural component of basement membranes, we investigated the production of type IV collagen-binding proteins by primary cultures of rat lung type II pneumocytes. Cultures were labeled for up to 24 h with 3H-labeled amino acids or [3H]mannose. Soluble collagen-binding proteins which accumulated in the culture medium were isolated by chromatography on collagen-Sepharose and examined by SDS-polyacrylamide gel electrophoresis. The major type IV collagen-binding protein (CBP1) was identified as fibronectin. We also identified a novel disulfide-bonded collagen-binding glycoprotein (CBP2; Mr = 45,000, reduced). This protein was not recognized by polyclonal antibodies to fibronectin, and showed no detectable binding to denatured type I collagen. The protein was resolved from fibronectin and partially purified by sequential chromatography on gelatin and type IV collagen-Sepharose. We suggest that type II pneumocyte-derived collagen-binding proteins contribute to the formation of the epithelial basement membrane and/or mediate the attachment of these cells to collagenous components of the extracellular matrix.     

Participation of hepatocytes in the production of basement membrane components in human and rat liver during the perinatal period

Little is known about the role of the extracellular matrix in cellular growth, migration and differentiation in the developing liver. The distribution and origin of the main constituents of the hepatic extracellular matrix have never been studied during liver differentiation. We have investigated the extracellular and intracellular distribution of fibronectin, laminin and types I, III and IV collagen in both rat and human liver during the perinatal period by light and electron microscopy, using the indirect immunoperoxidase method. All these components were demonstrated extracellularly, located mainly in portal spaces and, to a lesser extent, surrounding central veins. In perisinusoidal spaces, variations in distribution were observed depending on the matrix protein, the age of the donor and the species. In fetal rat liver, fibronectin formed a continuous layer around hepatocyte clusters while laminin and type III procollagen were present in small amounts. Collagens and laminin were visualized more easily in newborn rat liver. Fetal and newborn human liver contained higher amounts of matrix components than their rat counterparts. Fibronectin also reacted strongly in the sinusoid, and laminin and collagens formed discontinuous deposits. The source of this extracellular matrix was demonstrated to be of mixed origin. The major finding was the presence of immunoreactive laminin in the rough endoplasmic reticulum of hepatocytes irrespective of the age or species. In addition, hepatocytes contained large amounts of fibronectin and little of type I collagen. Another basement membrane component, type IV collagen, was also found in hepatocytes from all groups except fetal rat. Perisinusoidal cells also contained various matrix components including laminin, type III procollagen and, again with the exception of fetal rat liver, type IV collagen. The greater amounts of basement membrane components in the sinusoids of developing liver than in adult tissue and the participation of immature hepatocytes in the production of laminin and to a lesser degree of type IV collagen suggest that these matrix proteins play a critical role during liver differentiation.