Are the polarization colors of picrosirius red-stained collagen determined only by the diameter of the fibers?

Polarization colors of various purified collagens were studied in fibers of similar thickness. Three different soluble collagens of type I, insoluble collagen type I, lathyritic collagen type I, two p-N-collagens type I, pepsin extract collagen type II, two soluble collagens type III, p-N-collagen type III, and soluble collagen type V were submitted to a routine histopathologic procedure of fixation, preparation of 5-microns-thick sections, staining with Picrosirius red and examination under crossed polars. Polarization colors were determined for thin fibers (0.8 micron or less) an thick fibers, (1.6-2.4 microns). Most thin fibers of collagens and p-N-collagens showed green to yellowish-green polarization colors with no marked differences between the various samples. Thick fibers of all p-N-collagens, lathyritic and normal 0.15 M NaCl-soluble collagens showed green to greenish-yellow polarization colors, while in all other collagens, polarization colors of longer wavelengths (from yellowish-orange to red) were observed. These data suggested that fiber thickness was not the only factor involved in determining the polarization colors of Picrosirius red-stained collagens. Tightly packed and presumably, better aligned collagen molecules showed polarization colors of longer wavelengths. Thus, packing of collagen molecules and not only fiber thickness plays a role in the pattern of polarization colors of Picrosirius red-stained collagens.     

A comparison of the immunofluorescent localization of collagen types I,III, and V with the distribution of reticular fibers on the same liver sections of the snow monkey (Macaca fuscata)

Summary Localizations of collagen types I, III, and V in monkey liver, as determined by the indirect immunofluorescence method, were photographically superimposed on the fibers revealed by silver-staining in the same tissue sections. Immunofluorescence for type I collagen was found to correspond with the brown collagen fibers and with some of the coarse reticular fibers, while that for type III collagen was found to correspond with most, but not all, reticular fibers of the liver as well as with the brown collagen fibers. The distribution of type V collagen coincides not only with the collagen fibers in the stroma of portal triads and around the central veins, but also with the coarse and fine reticular fibers in the liver lobules. By immuno-electron microscopy, reaction products with anti-type III and V collagens antibodies were demonstrated on cross-striated collagen fibrils, about 45 nm in diameter, in the space of Disse. From these observations, it is concluded that: (1) the fine reticular fibers are mainly composed of type III and type V collagens, and (2) the collagen fibers and coarse reticular fibers in the periphery of liver lobules are composed of type I, type III and type V collagens.     

Collagen fibrils of an invertebrate (Sepia officinalis) are heterotypic: immunocytochemical demonstration

Collagen fibrils from the dermis of Sepia officinalis were processed for immunoelectron microscopy to reveal reactions to antibodies against mammalian types I, III, and V, teleost type I and cephalopod type I-like collagens, by single and double immunogold localization. The fibrils were observed: (a) in suspensions of prepared fibrils, (b) in ultrathin sections of embedded fibril preparations, and (c) in ultrathin sections of dermal tissue. Some samples were subjected to acetic acid or urea dissociation. It was found that collagen fibrils from Sepia dermis are heterotypic in that they are composed of type I-like and type V collagens. Type I-like collagen epitopes were present mainly at the periphery of the fibrils; type V collagen epitopes were present throughout the fibrils. This is the first demonstration that collagen fibrils from an invertebrate are heterotypic, suggesting that heterotypy may be an intrinsic characteristic of the fibrils of fibrillar collagens, independent of evolutionary or taxonomic status.     

Areas of asbestoid (amianthoid) fibers in human thyroid cartilage characterized by immunolocalization of collagen types I,II, IX,XI and X

The distribution of type I, II, IX, XI and X collagens in and close to areas of asbestoid (amianthoid) fibers in thyroid cartilages of various ages was investigated in this study. Asbestoid fibers were first detected in thyroid cartilage from a 3-year-old male child. Areas of asbestoid fibers functionally appear to serve as guide rails for vascularization of thyroid cartilage. Alcian blue staining in the presence of 0.3 M MgCl₂ revealed a loss of glycosaminoglycans in areas of asbestoid fibers. In addition, the fibers reacted positively with antibodies against collagen types II, IX and XI, but showed no staining with antibodies to collagen types I and X. Territorial matrix of adjacent chondrocytes showed the same staining pattern. In addition to staining for type II, IX and XI collagens, asbestoid fibers showed strong immunostaining for type I collagen after puberty but not for type X collagen. However, groups of chondrocytes within areas of asbestoid fibers reacted strongly with antibodies to type X collagen, suggesting that this collagen plays an important role in matrix of highly differentiated chondrocytes. The finding that these type X collagen-positive chondrocytes also revealed immunostaining for type I collagen confirms previous studies showing that hypertrophic chondrocytes can further differentiate into cells that are characterized by the synthesis of type X and I collagens.     

Enzyme-antibody histochemistry. A method for detection of collagens collectively

Different types of distinct molecular forms of collagen are components of the extracellular matrix in most tissues. The common types can usually be detected by immunohistochemical methods but others may escape detection for lack of specific antisera. However, all these collagens are substrates for the collagenase of Clostridium histolyticum. In this report we describe a method that allows the visualization of collagens, collectively, in a tissue preparation. The method is based on the affinity between clostridial collagenase and collagen on one hand, and collagenase and its antibody on the other. Under the conditions of low temperature used in the procedure, collagenase binds to collagen, but digestion does not occur. Subsequent reaction of the bound collagenase with the specific collagenase antibody is followed by reaction with a tagged anti-IgG reagent. This allows the visualization of the enzyme-substrate complex. The procedure is illustrated in sections of the heart and the aorta, as well as in the isolated cardiomyocytes and the collagen distribution is verified using collagens type I and IV specific antibodies. In all instances the collagenase staining pattern includes all structural features seen individually with the type specific anticollagen antibodies.     

Localization of types I, II, and III collagen mRNAs in developing human skeletal tissues by in situ hybridization

Paraffin sections of human skeletal tissues were studied in order to identify cells responsible for production of types I, II, and III collagens by in situ hybridization. Northern hybridization and sequence information were used to select restriction fragments of cDNA clones for the corresponding mRNAs to obtain probes with a minimum of cross-hybridization. The specificity of the probes was proven in hybridizations to sections of developing fingers: osteoblasts and chondrocytes, known to produce only one type of fibrillar collagen each (I and II, respectively) were only recognized by the corresponding cDNA probes. Smooth connective tissues exhibited variable hybridization intensities with types I and III collagen cDNA probes. The technique was used to localize the activity of type II collagen production in the different zones of cartilage during the growth of long bones. Visual inspection and grain counting revealed the highest levels of pro alpha 1(II) collagen mRNAs in chondrocytes of the lower proliferative and upper hypertrophic zones of the growth plate cartilage. This finding was confirmed by Northern blotting of RNAs isolated from epiphyseal (resting) cartilage and from growth zone cartilage. Analysis of the osseochondral junction revealed virtually no overlap between hybridization patterns obtained with probes specific for type I and type II collagen mRNAs. Only a fraction of the chondrocytes in the degenerative zone were recognized by the pro alpha 1(II) collagen cDNA probe, and none by the type I collagen cDNA probe. In the mineralizing zone virtually all cells were recognized by the type I collagen cDNA probe, but only very few scattered cells appeared to contain type II collagen mRNA. These data indicate that in situ hybridization is a valuable tool for identification of connective tissue cells which are actively producing different types of collagens at the various stages of development, differentiation, and growth.     

Enzyme-antibody histochemistry

Summary Different types of distinct molecular forms of collagen are components of the extracellular matrix in most tissues. The common types can usually be detected by immunohistochemical methods but others may escape detection for lack of specific antisera. However, all these collagens are substrates for the collagenase of Clostridium histolyticum. In this report we describe a method that allows the visualization of collagens, collectively, in a tissue preparation. The method is based on the affinity between clostridial collagenase and collagen on one hand, and collagenase and its antibody on the other. Under the conditions of low temperature used in the procedure, collagenase binds to collagen, but digestion does not occur. Subequent reaction of the bound collagenase with the specific collagenase antibody is followed by reaction with a tagged anti-IgG reagent. This allows the visualization of the enzyme-substrate complex.The procedure is illustrated in sections of the heart and the aorta, as well as in the isolated cardiomyocytes and the collagen distribution is verified using collagens type I and IV specific antibodies. In all instances the collagenase staining pattern includes all structural features seen individually with the type specific anticollagen antibodies.Abbreviations BSA Bovine serum albumin - PBS phosphate buffored saline     

Immunohistochemical localization of procollagens. I. Light microscopic distribution of procollagen I, III and IV antigenicity in the rat incisor tooth by the indirect peroxidase-anti-peroxidase method.

Frozen sections of the growing end of the rat incisor tooth were exposed to antisera or affinity prepared antibodies against partially purified type I, II, or IV procollagen in the hope of detecting the location of the corresponding antigens by the peroxidase-anti-peroxidase technique. The distribution of immunostaining was similar with antisera as with purified antibodies of a given type, but differed for each type; that is, predentin, odontoblasts, pulp and periodontal tissue were the sites of type I; blood vessel walls, pulp and periodontal tissue, of type III; and basement membranes, of type IV antigenicity. It was demonstrated, at least in cases of type I and III, that immunostaining detected the corresponding procollagens and related substances, but not the corresponding collagens. The interpretation of these observations is that: 1) odontoblasts elaborate procollagen I for release to predentin and subsequent transformation to dentinal collagen I; 2) pulp and periodontal cells produce procollagens I and III which presumably become collagens I and III respectively, while the adventitial cells of blood vessels give rise to collagen III; and 3) procollagen IV is associated with basement membranes and, occasionally, adjacent cells.     

Immunohistochemical study of collagens of the extracellular matrix in cartilage of Sepia officinalis

We used various anti-collagen antibodies to perform indirect immunofluorescent staining of cartilage sections from cuttlefish (S. officinalis). On ultrathin sections and collagen fibril preparations from the same tissue, we performed immunostaining with colloidal gold. The extracellular matrix (ECM) of S. officinalis cartilage reacted intensely and homogeneously with an antibody directed against type I-like collagen isolated from the cartilage of cuttlefish and with anti-rat type V collagen antibody. A weak reaction was observed with anti-fish and anti-chicken type I collagen antibodies, while no reaction was observed with anti-rat type I and anti calf type II collagen antibodies. Anti-chicken type II, anti calf type IX and type XI collagen antibodies reacted weakly with ECM, while stained cell bodies and cell processes reacted more intensely. A similar pattern of reaction was observed on cartilage section and isolated collagen fibrils prepared for electron microscopy. These findings suggest that ECM of cuttlefish cartilage may be composed of molecules similar to the type I, type V, type IX and type XI collagen molecules of vertebrates. Cephalopods have evolved a cartilage of structure and macromolecular organisation similar to that of vertebrate cartilage. However, the main molecular components of S. officinalis cartilage--type I-like and type V collagens--differ from those of vertebrate cartilage. We suggest that this type I-like collagen can be considered an initial step toward the evolution of type II collagen typical of vertebrates.     

Involvement of HMGB1 and HMGB2 proteins in exogenous DNA integration reaction into the genome of HeLa S3 cells

Electrophoretic and Western blot studies were conducted on collagen fractions extracted from Sepia officinalis (cuttlefish) cartilage using a modified salt precipitation method developed for the isolation of vertebrate collagens. The antibodies used had been raised in rabbit against the following types of collagen: Sepia I-like; fish I; human I; chicken I, II, and IX; rat V; and calf IX and XI. The main finding was that various types of collagen are present in Sepia cartilage, as they are in vertebrate hyaline cartilage. However, the main component of Sepia cartilage is a heterochain collagen similar to vertebrate type I, and this is associated with minor forms similar to type V/XI and type IX. The cephalopod type I-like heterochain collagen can be considered a first step toward the evolutionary development of a collagen analogous to the typical collagen of vertebrate cartilage (type II homochain). The type V/XI collagen present in molluscs, and indeed all phyla from the Porifera upwards, may represent an ancestral collagen molecule conserved relatively unchanged throughout evolution. Type IX-like collagen seems to be essential for the formation of cartilaginous 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.     

Immunocytochemical study of collagen in epidermal growth factor (EGF)-treated osteoblastic cells

The alteration of collagen components in clone MC3T3-E1 cells by epidermal growth factor (EGF) was investigated immunocytochemically, using antibodies to type I and type III collagens. EGF transformed those cells that had become more slender than those of control cultures. Type I and type III collagens were observed in the same cells in both EGF-treated and control cultures. Type I collagen was decreased by EGF, whereas type III collagen appeared to be increased. However, no cells with only type III collagen were observed, suggesting that EGF influences collagen metabolism in clone MC3T3-E1 cells.     

Are the polarization colors of Picrosirius red-stained collagen determined only by the diameter of the fibers?

Summary Polarization colors of various purified collagens were studied in fibers of similar thickness. Three different soluble collagens of type I, insoluble collagen type I, lathyritic collagen type I, two p-N-collagens type I, pepsin extract collagen type II, two soluble collagens type III, p-N-collagen type III, and soluble collagen type V were submitted to a routine histopathologic procedure of fixation, preparation of 5-m-thick sections, staining with Picrosirius red and examination under crossed polars. Polarization colors were determined for thin fibers (0.8 m or less) and thick fibers, (1.6–2.4 m). Most thin fibers of collagens and p-N-collagens showed green to yellowish-green polarization collors with no marked differences between the various samples. Thick fibers of all p-N-collagens, lathyritic and normal 0.15 M NaCl-soluble collagens showed green to greenish-yellow polarization colors, while in all other collagens, polarization colors of longer wavelengths (from yellowish-orange to red) were observed. These data suggested that fiber thickness was not the only factor involved in determining the polarization colors of Picrosirius red-stained collagens. Tightly packed and presumably, better aligned collagen molecules showed polarization colors of longer wavelengths. Thus, packing of collagen molecules and not only fiber thickness plays a role in the pattern of polarization colors of Picrosirius red-stained collagens.     

Immunohistochemical localization of collagens and fibronectin in human breast neoplasms

Forty four specimens from neoplastic, hyperplastic and normal human breast tissues were studied for localization of collagens and fibronectin. Affinity purified antihuman type I, III and IV collagens and antifibronectins were utilized by the indirect immunoperoxidase technique on fixed and paraffin-embedded sections. 86% of the cell cytoplasm of infiltrating ductal and 83% of the lobular cancers were positively stained for collagen type I and III. Collagen type IV, however, was detected in 100% of infiltrating ductal and 83% of lobular carcinomas. Focal cytoplasmic staining is a predominant feature for all antigens in the intraduct carcinoma while a diffuse pattern is encountered in the infiltrating types. Intact basement membranes in various lesions always stained for type IV collagen and showed variable staining for type III collagen and fibronectin. Epithelia of normal, benign, hyperplastic breast and most medullary carcinoma were negative for the three collagen types. Our results are in favour of the view that infiltrating breast carcinoma cells produce inappropriately the majority of collagens and inconsistently other proteins such as fibronectin.     

Inhibition of collagen-induced platelet aggregation by antibodies to distinct types of collagens.

Aggregation of platelets by fibrils formed from collagens type I, II and III could be inhibited by coating the fibrils with anti-collagen antibodies or Fab fragments. Similar results were obtained in a clot-retraction assay. Inhibition was achieved with stoichiometric amounts of antibodies and was specific for each type of collagen. Aggregation caused by a mixture of type-I and -III collagens could only be inhibited by a mixture of antibodies against both collagens. The data show that each interstitial collagen is capable of interacting with platelets and do not support the concept of an outstanding activity of type-III collagen.     

Collagens,collagen-binding heat shock protein 47 and transforming growth factor-beta 1 are induced in cicatricial pemphigoid: possible role(s) in dermal fibrosis

Cicatricial pemphigoid (CP) is an autoimmune mucocutaneous blistering disease associated with scarring. Heat shock protein 47 (HSP47) is thought to play an important role in fibrogenesis, but its role in skin lesions of cicatricial pemphigoid is not yet known. In the present study, we examined the role of HSP47 in dermal fibrosis in cutaneous lesions of a CP patient. Skin biopsies from a patient with CP, and from normal subjects were studied for the expression of HSP47, and interstitial collagens (type I and type III collagens) by immunohistochemistry. Dermal fibroblasts isolated from skin of normal individuals and from fibrotic skin of a CP patient were used to study the expression of HSP47, transforming growth factor beta 1 (TGF-beta 1), type I and type III collagens. Compared to the control skin sections, an increased expression of HSP47 was associated with an increased deposition of interstitial collagens in the fibrotic skin section of the CP patient. Similarly, in contrast to control dermal fibroblasts, the fibroblasts isolated and cultured from fibrotic skin of the CP patient, and grown in vitro, exhibited increased expression of HSP47, type I and type III collagens. Furthermore, compared to the normal control fibroblasts, an increased expression of TGF-beta 1 was detected in the dermal fibroblasts isolated from fibrotic skin of the CP patient. When dermal fibroblasts were treated with various concentrations of TGF-beta 1 (6.25, 12.5, 25, 50 and 100 ng/ml for 24 h), it induced the expression of both type I collagen and HSP47, as determined by quantitative real-time PCR. In conclusion, the expression of TGF-beta 1, HSP47, type I collagen and type III collagen was up-regulated in the fibrotic skin of CP patient, and a complex interaction of these molecules may initiate and propagate the fibrotic cascade in the skin of CP patients.     

Codistribution of collagen types IV and AB2 in basement membranes and mesangium of the kidney. an immunoferritin study of ultrathin frozen sections

Affinity-purified rabbit antibodies specific for collagen types I, III, AB2 and for a partially characterized type IV collagen derived from a murine tumor were used to study the distribution of collagens in the normal mouse kidney. Immunofluorescence staining of conventional frozen sections demonstrated that types I and III were present in bundles around large vessels and in fibers surrounding glomeruli and tubules, whereas types IV and AB2 were distributed in a linear fashion along basement membranes of tubules, glomeruli, and Bowman's capsule and in the mesangial stalk. The distribution of types IV nd AB2 was examined at the ultrastructural level by staining of 600- to 800-A thick frozen sections with a three-stage procedure employing specific collagen antibodies, biotinyl sheep antirabbit IgG, and avidin-ferritin conjugates. Labeling by this procedure demonstrated codistribution of types AB2 and the putative type IV in all three basement membranes. In addition, mesangial matrix was shown to contain both of these collagen types. These results support recent biochemical evidence of collagen heterogeneity in basement membranes, and also support the concept of a structural relationship between mesangial matrix and glomerular basement membranes.     

The localization and synthesis of some collagen types in developing mouse embryos.

The location of type IV (basement membrane)collagen in early post-implantation mouse embryos was examined by immunoperoxidase reactions using a specific immunoglobulin raised against mouse lens capsule collagen. Reaction was positive in the earliest embryos studied--on the fifth day of gestation (the day of detection of the copulation plug is the first day). It was found only in the primitive endoderm adjacent to the blastocoelic cavity. Subsequently in development, strong staining reactions were found in the parietal endoderm, Reichert's membrane and an acellular layer which separates the visceral endoderm of the egg cylinder from the ectoderm. In tenth to eighteenth day visceral yolk sacs, the mesodermal portion was stained, which is consistent with the presence of basement membranes around blood vessels. The endodermal portion of the visceral yolk sac did not react, while small amounts were found in the amnion. By incubation of various embryonic tissues with tritiated amino acids, purification of the biosynthesized secreted collagens and their partial characterization, the differential expression of several collagen genes was detected. Identification of collagen types was made by: reaction with specific antibodies to type I and IV collagens; electrophoretic mobility; sensitivity to reduction and to collagenase; analysis of the proportions of 3-hydroxyproline, 4-hydroxyproline and hydroxylysine; and CNBr peptides. In agreement with the data of Minor et al. (1976a) for the rat, mouse parietal endoderm synthesizes large amounts of type IV collagen. In contrast to their findings, however, the 165,000 molecular weight polypeptide is not converted to one of 100,000 after reduction, alkylation and repepsinization (Dehm and Kefalides, 1978). The endoderm of the visceral yolk sac was shown to be synthesizing primarily type I collagen, while the mesoderm layer of this membrane synthesized both type I and IV collagens. Little or no type IV collagen synthesis was detected in the endoderm of the visceral yolk sac. If it is correct that the visceral endoderm of the early embryo makes a major contribution to the formation of the endoderm portion of the visceral yolk sac, then it is clear that a switch in collagen gene expression must occur as it does so.     

Collagen type I and type V are present in the same fibril in the avian corneal stroma

The distribution, supramolecular form, and arrangement of collagen types I and V in the chicken embryo corneal stroma were studied using electron microscopy, collagen type-specific monoclonal antibodies, and a preembedding immunogold method. Double-label immunoelectron microscopy with colloidal gold-tagged monoclonal antibodies was used to simultaneously localize collagen type I and type V within the chick corneal stroma. The results definitively demonstrate, for the first time, that both collagens are codistributed within the same fibril. Type I collagen was localized to striated fibrils throughout the corneal stroma homogeneously. Type V collagen could be localized only after pretreatment of the tissue to partially disrupt collagen fibril structure. After such pretreatments the type V collagen was found in regions where fibrils were partially dissociated and not in regions where fibril structure was intact. When pretreated tissues were double labeled with antibodies against types I and V collagen coupled to different size gold particles, the two collagens colocalized in areas where fibril structure was partially disrupted. Antibodies against type IV collagen were used as a control and were nonreactive with fibrils. These results indicate that collagen types I and V are assembled together within single fibrils in the corneal stroma such that the interaction of these collagen types within heterotypic fibrils masks the epitopes on the type V collagen molecule. One consequence of the formation of such heterotypic fibrils may be the regulation of corneal fibril diameter, a condition essential for corneal transparency.