Human specific anti-type IV collagen monoclonal antibodies, characterization and immunohistochemical application

This paper describes two new monoclonal antibodies reactive with human specific type IV collagen epitopes in frozen as well as routinely fixed and processed tissue sections. The antibodies (1042 and 1043) were raised against human placental type IV collagen and were shown by immunoblotting and ELISA tests to react exclusively with type IV collagen determinants. Extensive immunohistochemical survey studies on panels of tissues from various species, using unfixed cryostat sections, demonstrated that antibody 1042 reacted only with human type IV collagen whereas antibody 1043 in addition reacted with rabbit type IV collagen. All tissues showed homogeneous staining of the basement membrane, indicating that the detected epitopes did not show organ-specific distribution. Tissue processing protocols for using these monoclonal antibodies on routinely processed paraffin embedded tissues were developed. It was found that whereas polyclonal anti-type IV collage antisera required pepsin digestion, our monoclonal antibodies required pronase or papain digestion to restore type IV collagen immunoreactivity in paraffin sections. It is concluded that these monoclonal anti-type IV collagen antibodies detect species specific epitopes which can be detected in routinely processed paraffin embedded tissues after appropriate enzyme pretreatment.     

Basement membrane in differentiating mesonephric and paramesonephric ducts of male and female rat fetuses

The differentiation of male and female rat genital ducts and their basement membranes were studied by light- and electron-microscopic localization of type-IV and -V collagen, laminin, and heparan sulfate proteoglycan at the fetal ages of 15-21 days. At 15 days, the basement membrane of the mesonephric duct was continuous in both sexes, whereas on the medial side of the paramesonephric duct, it was incomplete. The male mesonephric duct remained enveloped by a continuous basement membrane. Increasing accumulation of basement-membrane material in the periductal mesenchyme was regarded as incipient epididymal differentiation. Local expansions and slow degradation of the basement membrane were noted in the regressing female mesonephric duct. The female paramesonephric duct had acquired a continuous basement membrane by the age of 16 days. At this age, the incomplete basement membrane in the medial side of the male paramesonephric duct disappeared, and breaks in the lateral portion appeared. The formation of epitheliomesenchymal contacts and basal cytoplasmic blebs in the epithelial cells of the regressing paramesonephric duct coincided with the disappearance of the basement-membrane material in the condensed periductal mesenchyme. The asymmetric regression of the male paramesonephric duct was initiated in the immature medial side. The changes in the periductal matrix are indications of basic differences in the regulation of the development and regression of the genital ducts in different sexes.     

Immunohistochemical studies on the tissue localization of collagen types I, III, IV, V and VI in schwannomas. Correlation with ultrastructural features of the extracellular matrix

The distinctive tissue localization of collagen types in typical schwannomas with Antoni type A and B areas was demonstrated immunohistochemically using affinity-purified antibodies against types I, III, IV, V and VI collagen and comparative ultrastructural studies were made on the extracellular matrix components. Antoni type A tissue, which was composed of tightly packed spindle cells with long cytoplasmic processes surrounded by a continuous basement membrane and a few fibrillar components of the extracellular matrix, was almost exclusively immunoreactive for type IV collagen, presumably representing the basement membrane. Verocay bodies, which are organoid structures of Antoni type A tissue, had a variety of more abundant extracellular fibrous components, such as banded collagen fibrils, fibrous long-spacing fibrils and microfibrils. These were positive for type I and III, as well as type IV collagen. In Antoni type B areas, where two types to tumor cells designated Schwann cell-like and fibroblast-like were scattered in large amounts of amorphous extracellular matrix containing microfibrils and thick banded collagen fibrils, type VI collagen as well as types I, III and IV collagen were consistently detected. Type V collagen was localized in dense fibrous tissue areas and around blood vessels. These findings indicate that the differently organized cellular patterns of schwannomas, identified as Antoni types A and B, are characterized not only by the ultrastructural features of the extracellular matrix, but also by the distinctive collagen types produced by neoplastic Schwann cells.     

Human specific anti-type IV collagen monoclonal antibodies,characterization and immunohistochemical application

Summary This paper describes two new monoclonal antibodies reactive with human specific type IV collagen epitopes in frozen as well as routinely fixed and processed tissue sections. The antibodies (1042 and 1043) were raised against human placental type IV collagen and were shown by immunoblotting and ELISA tests to react exclusively with type IV collagen determinants. Extensive immunohistochemical survey studies on panels of tissues from various species, using unfixed cryosat sections, demonstrated that antibody 1042 reacted only with human type IV collagen whereas antibody 1043 in addition reacted with rabbit type IV collagen. All tissues showed homogeneous staining of the basement membrane, indicating that the detected epitopes did not show organ-specific distribution.Tissue processing protocols for using these monoclonal antibodies on routinely processed paraffin embedded tissues were developed. It was found that whereas polyclonal antitype IV collagen antisera required pepsin digestion, our monoclonal antibodies required pronase or papain digestion to restore type IV collagen immunoreactivity in paraffin sections.It is concluded that these monoclonal anti-type IV collagen antibodies detect species specific epitopes which can be detected in routinely processed paraffin embedded tissues after appropriate enzyme pretreatment.     

The spatial organization of Descemet''s membrane-associated type IV collagen in the avian cornea

The organization of type IV collagen in the unconventional basement membrane of the corneal endothelium (Descemet's membrane) was investigated in developing chicken embryos using anti-collagen mAbs. Both immunofluorescence histochemistry and immunoelectron microscopy were performed. In mature embryos (greater than 15 d of development), the type IV collagen of Descemet's membrane was present as an array of discrete aggregates of amorphous material at the interface between Descemet's membrane and the posterior corneal stroma. Immunoreactivity for type IV collagen was also observed in the posterior corneal stroma as irregular plaques of material with a morphology similar to that of the Descemet's membrane-associated aggregates. This arrangement of Descemet's membrane-associated type IV collagen developed from a subendothelial mat of type IV collagen-containing material. This mat, in which type IV collagen-specific immunoreactivity was always discontinuous, first appeared at the time a confluent endothelium was established, well before the onset of Descemet's membrane formation. Immunoelectron microscopy of mature corneas revealed that the characteristic nodal matrix of Descemet's membrane itself was unreactive for type IV collagen, but was penetrated at intervals by projections of type IV collagen-containing material. These projections frequently appeared to contact cell processes from the underlying corneal endothelium. This spatial arrangement of type IV collagen suggests that it serves to suture the corneal endothelium/Descemet's membrane to the dense interfacial matrix of the posterior stroma.     

Formation of a supramolecular complex is involved in the reconstitution of basement membrane components

Basement membrane macromolecules, including type IV collagen, laminin, and heparan sulfate proteoglycan, do not aggregate when incubated alone. Rather, precipitation occurs in the presence of equimolar amounts of laminin and type IV collagen but variable amounts of heparan sulfate proteoglycan. This interaction requires native laminin and type IV collagen. Heparan sulfate proteoglycan increases the precipitation of laminin particularly in the presence of type IV collagen. Fibronectin does not cause type IV collagen to precipitate. These studies show that the components of basement membrane interact in a highly specific manner and suggest that such interactions may be involved in the deposition of basement membrane in situ.     

Basement membrane collagen requirements for attachment and growth of mammary epithelium.

In vivo mammary epithelial cells rest upon a basement membrane composed in part of type IV collagen which is synthesized by these cells. In this study, basement membrane collagen is shown to be selectively recognized by normal mammary ducts and alveoli for attachment and growth when compared to the types of collagen derived from stroma (types I or III) or cartilage (type II). Cell attachment and growth on type I collagen is inhibited by the proline analogue, cis-hydroxyproline, which blocks normal collagen production. These effects of cis-hydroxyproline are not apparent when a basement membrane collagen substratum is provided. Unlike normal mammary epithelium, mammary fibroblasts show little preference for the collagen to which they will attach. A requirement of type IV collagen synthesis for normal mammary epithelial cell attachment and growth on stromal collagen in vitro may have significance in vivo where a basement membrane scaffold may be necessary for normal mammary morphogenesis and growth.     

Immunohistochemical localization of type IV collagen in mouse tooth germ: an ultrastructural study

Localization of type IV collagen was analyzed at the ultrastructural level in mouse embryonic molars by using a preembedding technique. Cryostat sections were incubated with type IV collagen antibody and then treated with the peroxidase-antiperoxidase complex. This antibody was visualized at the epithelio-mesenchymal interface. Labeling was intense and uniformly distributed throughout the basement membrane. However, it was mainly restricted to the lamina densa. No immunostaining was detectable in the lamina lucida but it was crossed by fine filaments that appeared as projections from the lamina densa to the epithelial cell plasma membrane. At the mesenchymal aspect of the basement membrane, projections of labeled material extended from the lamina densa in the underlying dental mesenchyme. At the presecretory stage of odontoblasts, these projections were in close connection with mesenchymal cell processes.     

Synthesis of extracellular matrix glycoproteins by a differentiated thyroid epithelial cell line

We examined the synthesis of extracellular matrix macromolecules by the differentiated rat thyroid epithelial cell line FRTL-5. As shown by electron microscopy, the extracellular material produced by these cells is deposited at the basolateral surface and focally organized in the form of a basement membrane. Biochemical and biosynthetic studies demonstrated that laminin, type IV collagen, and fibronectin are synthesized and deposited in the culture monolayer. Secretion of fibronectin into the culture medium also occurred. By immunofluorescence we observed some peculiarities in the distribution patterns of the basement membrane glycoproteins; while fibronectin and laminin had an almost superimposable distribution, type IV collagen displayed a rather different pattern. Type IV collagen and laminin localization at sites where extracellular material was detected was confirmed by immuno electronmicroscopy using the protein A-colloidal gold technique. The results indicate that under appropriate culture conditions the differentiated thyroid epithelial cell line FRTL-5 synthesizes, secretes and organizes an extracellular matrix where some basement membrane glycoproteins are present.     

Developmental acquisition of basement membrane heterogeneity: type IV collagen in the avian lens capsule

To investigate potential heterogeneity and developmental changes in basement membranes during embryogenesis, we performed immunohistochemical analyses on lens capsules in chicken embryos of different ages using domain-specific monoclonal antibodies against type IV collagen. We found that the capsule of the newly formed lens stained uniformly with antibodies against this component of basement membranes, but with increasing age and differentiation of the lens cells the anterior lens capsule remained brightly fluorescent while staining of the posterior capsule became relatively much less intense. This antero- posterior gradient of anti-type IV collagen antibody reactivity demonstrated that developmentally-regulated changes can occur within a single, continuous basement membrane.     

Domain and basement membrane specificity of a monoclonal antibody against chicken type IV collagen

A monoclonal antibody, IV-IA8, generated against chicken type IV collagen has been characterized and shown to bind specifically to a conformational-dependent site within a major, triple helical domain of the type IV molecule. Immunohistochemical localization of the antigenic determinant with IV-IA8 revealed that the basement membranes of a variety of chick tissues were stained but that the basement membrane of the corneal epithelium showed little, if any, staining. Thus, basement membranes may differ in their content of type IV collagen, or in the way in which it is assembled. The specificity of the antibody was determined by inhibition ELISA using purified collagen types I-V and three purified molecular domains of chick type IV collagen ([F1]2F2, F3, and 7S) as inhibitors. Only unfractionated type IV collagen and the (F1)2F2 domain bound the antibody. Antibody binding was destroyed by thermal denaturation of the collagen, the loss occurring at a temperature similar to that at which previous optical rotatory dispersion studies had shown melting of the triple helical structure of (F1)2F2. Such domain-specific monoclonal antibodies should prove to be useful probes in studies involving immunological dissection of the type IV collagen molecule, its assembly within basement membranes, and changes in its distribution during normal development and in disease.     

Histochemical demonstration of type IV collagen in the renal glomerulus

Disulfide-groups are demonstrated in the basement membranes of glomerular capillaries, proximal convoluted tubules, and collecting ducts by means of a thiosulfathion/Alcian Blue +0.8 Mol MgCl2-staining sequence. It is suggested that the reaction shows type IV collagen of basal lamina material, which is characterized by a relatively high cystine content (8 half-cystine residues/1000).     

Enhanced synthesis of type IV collagen in cultured arterial smooth muscle cells associated with phenotypic modulation by dimethyl sulfoxide

The effect of dimethyl sulfoxide (DMSO) on synthesis of basement membrane collagen in cultured smooth muscle cells was evaluated. DMSO promoted phenotypic modulation of cells from the synthetic state to the contractile state accompanied by formation of basement membranes. By immunofluorescence using monospecific antibody against type IV collagen, type IV collagen was identified not only in the cell cytoplasms but intensely along the cell surfaces in the cultures treated with DMSO for 7 days, as compared with untreated cultures. Electron microscopic immunohistochemistry revealed the presence of type IV collagen both in the basement membrane region and in the rough endoplasmic reticulum of DMSO-treated cells. Such an enhancement of type IV collagen synthesis appears to be expressed as a result of the phenotypic changes of smooth muscle cells to the contractile state modulated by DMSO.     

Basement membrane components of bronchial epithelium in humans suffering from chronic nonspecific lung diseases

Collagen IV and laminin are important constituents of the basement membrane (BM). By use of immunocytochemistry we examined the occurrence and distribution of these two components in the BM beneath normal, mucoid and metaplastic epithelium of large bronchi in 22 adults suffering from chronic nonspecific lung diseases. Both collagen IV and laminin were expressed as a thin and continuous layer beneath the epithelium in most tissue specimens with normal epithelium. In a few specimens the layer showed interruptions with a patchy distribution of the immunoreactivity. Three patterns of distribution of BM components were found under the metaplastic epithelium. Total absence of immunoreactive collagen IV and laminin was the most common variant. Weak and scarce staining for both proteins in the BM characterized the second pattern. The third variant showed strong collagen IV immunoreactivity but lack of laminin. The BM beneath the mucoid epithelium was characterized by irregular distribution of collagen IV and laminin. We suggest that the occurrence and distributional pattern of the BM components are related to the type of overlying epithelium and connected with an altered synthesis of these components.     

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     

Abnormal deposition of basement membrane and connective tissue components in dimethylbenzanthracene-induced rat mammary tumours: an immunocytochemical and ultrastructural study

Summary Dimethylbenzanthracene-induced rat mammary tumours consist of lobules of tumours cells surrounded by connective tissue. The interstitial connective tissue proteins, collagen types I, III and V, fibronectin and elastin are largely restricted to the interlobular connective tissue. The tumour lobules are surrounded by a basement membrane that stains with antiserum to laminin. Electron microscopy reveals a greatly thickened basement membrane to which striated interstitial collagen fibres are closely juxtaposed. The lumina within the tumour lobules are of two types. In the first type, the luminal surface is characterized by the presence of microvilli and tight junctions are reacts with antiserum to rat milk fat globule membrane. In the second type, the luminal surface is flattened and lined by a thickened basement membrane that stains with antiserum to laminin and type IV collagen. These abnormal patterns of growth and differentiation may be partly a consequence of the disorganization of extracellular matrix components at the interface between the tumour epithelial cells and the surrounding stroma.     

Immunohistochemical and biochemical studies on the collagenous proteins of human osteosarcomas

The distribution of type I, II, III, IV, V and VI collagens in 20 cases of osteosarcoma was demonstrated immunohistochemically using monospecific antibodies to different collagen types. In addition, biochemical analysis was made on collagenous proteins synthesized by tumor cells in short-term cultures obtained from seven representative cases and compared with dermal fibroblasts. In osteoblastic areas, most of the tumor osteoid consisted exclusively of type I collagen. Type V collagen was associated in some of them. Type III and type VI collagens were mainly localized in the perivascular fibrous stroma. Cultured tumor cells from osteoblastic osteosarcomas produced type I collagen exclusively and small amount of type V collagen constantly, while the synthetic activity of type III collagen was extremely low. In contrast, fibroblastic areas were characterized by the codistribution of type I, III, VI collagens and chondroblastic areas by type I, V, VI collagens as well as type II. Furthermore, type IV collagen was demonstrated in the stroma, other than the basement membrane region of blood vessels, in fibroblastic, intramedullary well-differentiated and telangiectatic osteosarcomas. In vitro, the production of variable amounts of type IV collagen, which was not detected in cultured dermal fibroblasts, was also recognized in the osteoblastic, fibroblastic, undifferentiated and intramedullary well-differentiated osteosarcomas examined. These findings suggest that the immunohistochemical approach using monospecific antibodies to different collagen types is useful not only in identifying some specific organoid components, such as tumor osteoid, but also in disclosing the biological properties of osteosarcoma cells with diverse differentiation.     

Histochemical Demonstration of type IV collagen in the renal glomerulus

Summary Disulfide-groups are demonstrated in the basement membranes of glomerular capillaries, proximal convoluted tubules, and collecting ducts by means of a thiosulfation/Alcian Blue +0.8 Mol MgCl₂-staining sequence. It is suggested that the reaction shows type IV collagen of basal lamina material, which is characterized by a relatively high cystine content (8 half-cystine residues/1000).     

Immunohistochemical distribution of type VI collagen in developing human kidney

Summary The distribution of type VI collagen was investigated immunohistochemically in the developing human kidney from 15 to 32 weeks gestational age and it was compared with that observed in the normal infantile and adult human kidney. In fetal kidney, type VI collagen was widely distributed as a fibrillar network in the subcapsularly undifferentiated mesenchyme and intertubular interstitium, and as a basement membrane-like structure around the ureteral bud branches, tubules, and collecting ducts. During nephrogenesis, type VI collagen disappeared from the induced mesenchyme close to the tips of ureteral branches, while it formed a distinct basement membrane-like structure around the early stages of nephron differentiation (comma-shaped and S-shaped bodies) and later along Bowman's capsule of capillary loop and maturing glomeruli. A strong immureactivity for type VI collagen was also found in the glomerular basement membrane and mesangial areas of capillary loop and maturing glomeruli. In infantile kidney, type VI collagen showed a distribution pattern similar to that observed during the fetal period. In adult human kidney, glomerular basement membrane showed a weak positivity for type VI collagen and the basement membrane-like staining around Bowman's capsule, tubules, and collecting ducts was less evident than in fetal and infantle kidney. Our immunohistochemical findings suggest that type VI collagen is a normal component of the glomerular and extraglomerular extracellular matrix of developing human kidney and that it undergoes changes in the expression during maturation.     

Synthesis of basement membrane components by differentiated thyroid cells

Morphological studies indicate that basement membrane formation or maintenance can be achieved in cultures of thyroid cells. In the present investigation we have studied the biosynthesis of this extracellular matrix by differentiated porcine thyroid cells in culture. They were prepared by two procedures: (1) thyroid cells isolated by dispase digestion of the thyroid gland were maintained in serum-free medium on poly(L-lysine) coated dishes; (2) thyroid follicles released by collagenase treatment of the gland were isolated by differential filtration and cultured in suspension on agarose-coated dishes. In both cases, functional follicular-like structures were obtained as shown by their ability to organify Na125I and to respond to thyrotropin stimulation (250 microU/ml). After incubating the cells with radiolabeled proline or methionine, collagen synthesis was observed with the two types of culture, as shown by the formation of radioactive hydroxyproline and by the synthesis of peptides with electrophoretic properties identical to those of authentic collagen molecules and susceptible to collagenase. Besides variable amounts of type I and type III collagen-like peptides, significant proportions of labeled peptides migrated with type IV collagen chains and were precipitated by anti-type IV collagen antibody; thyrotropin had no significant effect either on the total collagen synthesis or on the relative amounts of the different collagen peptides. When thyroid cells were incubated with [35S]sulfate, a labeled glycosaminoglycan with chromatographic properties analogous to that of heparan sulfate could be obtained in both culture conditions; here again, no effect of thyrotropin was observed. The ability of differentiated porcine thyroid cells to synthesize basement membrane was suggested by their production of type IV collagen and heparan sulfate, two of its potential components. Thyrotropin, which drastically enhanced the functional property of the cells, did not seem to regulate this synthesis.