VISUALIZATION OF COLLAGEN IN SOME HUMAN CONNECTIVE TISSUES BY IMMUNOFLUORESCENCE M. B. ENGEL and H. R. CATCHPOLE Department
of Histology, College of Dentistry, University of Illinois at the Medical Center, Chicago. IL 60680. U.S.A.
Summary-An antiserum to a urea-soluble fraction of human tendon collagen was raised in rabbits. The antiserum was used by the indirect method for immunofluorescent visualization of certain forms of collagen in several human connective tissues, including oral mucosa. Prominent reactive sites included basement membranes of small blood vessels, renal tubules, young collagen fibres, endomysium and elements of the lamina propria of oral mucosa and skin. In some loci, collagen did not react, presumably due to the state of aggregation or to interactions with other structural macromolecules. Similarities were noted between the altered staining of inflamed gingiva and collagenase-treated tissue.
INTRODUCTION
We have reported the visualization of certain forms of collagen in the rat by immunofluorescence microscopy (Engel and Catchpole, 1972; Mashouf and Engel, 1975). With similar techniques we developed an antiserum to human tendon collagen which we used to demonstrate collagen in several types of human connective tissue, including the lamina propria of oral tissues. Rothbard and Watson (1954, 1961, 1972) used skin as the source of collagen with different procedures of antigen preparation and tissue processing. Timpl (1976) used heterologous antigens consisting of types I-IV collagen, derived from bovine or rat skin, to generate antibodies which cross-reacted with components in human tissues. Nowack et al. (1976) proposed to use such antisera as probes for human collagen types although antibodies to types I -and III collagens show unexplained cross-reactions (Wick et al., 1976). We preferred to avoid the use of heterologous antisera. A feature of our earlier work was the recognition of different intensities of collagen reactivity, which was attributed to differences in the degree of collagen aggregation. This finding, which is also recognized in Cleaned ground
and washed in the frozen
human collagenous tissues, was made possible by methods of tissue preparation reported here. MATERIALS
Preparation
AND METHODS
of antigen
Adult human achilles tendon, which had a relatively low concentration of non-collagenous protein and is not highly cellular, was the source of the antigen and was obtained at surgery. Tissues were freed of fat and muscle and washed with distilled water to remove visible blood. Fibre bundles were teased apart and the tissue mass frozen in liquid nitrogen and dried in uucuo while frozen. The dried
tissue was ground in a Culatti mill kept thoroughly chilled with liquid nitrogen. The ground tissue was sequentially extracted with distilled water, 0.2 M NaCl and 0.2 M citrate buffer, pH 3.5. The residue thus exhausted of the usual soluble collagens was extracted with 8 M urea. The collagen solubilized by this step, designated collagen D, was separated, dialysed against distilled water and lyophilized, and was the collagen antigen used. A flow chart (Text Fig. I) summarizes the method of preparation. The
collagen state
fibres frozen in liquid nitrogen, to powder and lyophilized Extract
with 20 vok/w
I
dist.
H,O (x2)
I Residue
Combined supernatants, lyophilized collagen A, serum proteins
Extract with 20 vol 0.2 M NaCl (x2)
I
I
1
Residue
Combined supernatants,dialysed, lyophilized. Collagen B, serum proteins
I
I
Residue
Combined supernatants,dialysed, lyophilized.Collagen C, serum proteins
Fig. 1. Fractionation
Extract with 20 vol Na citrate-citric acid buffer, pH 3.5 (x2)
Solubilize in IO vol B M urea with stirring in cold (x2) Combine,dialyse, lyophilize Cal lagen D
of human achilles tendon 385
M. B. Engel and H. R. Catchpole
386
Table 1. Amino acid composition of human collagen-D antigen*
0 D
Collagen-D Hydroxylysine Lysine Histidine “Post-histidine”? Ammonia Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine A lanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Hydroxyproline
12 35 9.3 20 78 63 22 32 114 112 228 79 26 6.8 1.5 34 1.4 18.7 150
*Values given as residues per 1000 residues. tAppears in the same position as l-methyl dine.
0 0
Fig. 2. lmmunodiffusion plate. Rabbit anti-collagen D (absorbed with human serum) in centre well. D, Collagen D; S, human serum. The antiserum reacts only with collagen
histi-
antigen was free of serum proteins when tested by immunodiffusion against goat anti-human serum protein antiserum. The amino acid composition for this form of insoluble tendon collagen is somewhat different from that reported for some other collagens, chiefly soluble collagens derived from skin (Table I). The number of glycine residues is lower and the glutamic acid and hydroxyproline values are somewhat higher. The chemistry of insoluble collagens has been little investigated. Preparation of tissues
Portions of oral mucosa, mainly free and attached gingiva, were obtained from patients undergoing minor surgical procedures. Usually the tissues were taken from the mandibular arch where block anaesthesia was feasible, thus avoiding tissue disruption by infiltrated anaesthetic solution. Blocks approximately 1-2 mm’ were placed on small metal cryostat holders and rapidly frozen in isopentane chilled to approximately - 150°C with liquid nitrogen. Other normal tissues, including tendon, skin, muscle and kidney. were obtained where possible as surgical specimens, or at autopsy. Preparation of anti-collagen antiserum
The collagen antigen was dissolved in 6 M urea at a concentration of lOmg/ml. This solution was diluted at the time of injection with an equal volume of aluminium hydroxide adjuvant prepared by the method of Tracy and Welker (1915). Two millilitres of this material were injected intramuscularly in equally-divided doses into young male albino rabbits every 2 weeks. This schedule was followed for 3-4
months, with test bleedings at intervals after the first month. When antibody titres appeared to be adequate as visualized by double diffusion in gel, the animals were exsanguinated 10-15 days after the last injection. By Ouchterlony plate analysis, some, but not all, antisera reacted weakly to human plasma proteins. TO remove this component from the antisera, all antisera were adsorbed with human plasma before use in immunofluorescence localization of collagen (Text Fig. 2). Preparation of tissue sections
Tissue sections were cut in a cryostat held at - 30°C to - 40°C. at a section thickness of 4-6 pm. The sections were dehydrated in vacua at -30°C and stored in a deep-freeze over Drierite until examined. Before staining, sections were affixed to slides coated with a thin layer of egg albumin. They were then incubated at room temperature with various specific and non-specific sera as follows. Immunofluorescence techniques
Sections were flooded with rabbit anticollagen serum and incubated for 30 min. The serum was decanted and the sections rinsed 5 times in phosphate-buffered saline (PBS), pH 7.4. The sections were then treated with the IgG fraction of fluorescein-conjugated goat anti-rabbit serum, at a dilution of 1:5 for 5 min. Excess fluid was decanted and the sections washed in 5 changes of PBS. They were then mounted in 5 per cent Gelvatol. The slides were examined with a Leitz Ortholux microscope using an HBO 200 mercury lamp, dark field condenser and a UGl excitation filter combined with a K490 barrier filter. Various controls were used to ensure specificity including: (I) Pretreatment of sections with Clostridium histolytica collagenase (Worthington, 1 mg/ml) at 37°C for 24 h. (2) Treatment with control, nonimmune rabbit serum followed by reaction with the conjugated goat anti-rabbit serum. (3) Treatment with conjugated goat anti-rabbit serum alone. (4) Blocking reaction, i.e. application of rabbit anti-collagen serum followed by unconjugated goat anti-rabbit serum, then application of conjugated goat anti-rabbit serum (each step separated by 5 washings with PBS). To compare the effects of staining due to serum proteins with specific collagen localization, several sections were treated with a potent rabbit anti-human
Collagen in some human connective tissues
serum antibody. Sites of serum were visualized by subsequent interaction with fluorescein conjugated goat anti-rabbit serum. RESULTS
Tendon (surgical)
The tibrils of young tendon were clearly delineated because they were moderately fluorescent, whereas the interfibrillar substance was unstained. Bundles of fibrils were demonstrated by the more intense reaction of the sheath material. Mature tendon, however, was only barely reactive, except in regions of the sheath. (Plate Figs. 3-5.) Kidney (autopsy)
The basement membranes of tubules and of contiguous renal vessels were outlined by fluorescence. Basement membranes of the glomerular vessels and of Bowman’s capsule also reacted. (Plate Fig. 6.) Musck
(autopsy)
Connective tissue surrounding muscle bundles and individual muscle cells, including perimysium, epimysium and endomysium, displayed fluorescence. Small arterioles and capillaries were revealed through the staining of their immediate surrounding connective tissue (Plate Fig. 7.) Skin (autopsy) Selected groups of dermal fibres reacted with the fluorescent antibody. The basement membranes of capillaries were prominently outlined. Substantial groups of fibres were unreactive. In general, the hypodermis showed fewer groups of reactive fibres than the dermis. (Plate Fig. 11.) Gingiua (surgical)
The interlacing collagen bundles of the gingiva were intensely stained. The basement membrane at the epithelium connective tissue junction was evident as a more intensely fluorescent structure. In general, the connective tissue of the rete pegs was somewhat more reactive than that of the deeper layers. In some preparations, the gingival epithelial cells (cytoplasm) or intercellular substance showed weak fluorescence. This was obviously a non-specific staining. In inflamed gingiva, the disruption and dissolution of collagen was indicated by the change in density of fluorescent fibres. The cpmpact network of fibres was interrupted in those regions around infiltrating lymphocytes and granulocytes. In dark field illumination. these areas were non-fluorescent (Plate Figs. 8- IO). Mucosa of cheek (surgical)
Strands of reactive fibres mingled with a larger mass of unstained collagen. The basement membranes surrounding small blood vessels and capillaries were prominently stained (Plate Fig. 12). GENERAL OBSERVATIONS
Intracellular forms of collagen were not seen. In the tissues which we chose to study, the reactive sites
387
usually included basement membranes and regions where young collagen and a high concentration of reticular fibres would be expected. When gingival sections were treated with collagenase, most of the collagen was removed and consequently did not fluoresce. However, there were aggregates of resistant material which continued to react. Some preparations were stained to show the distribution of plasma proteins for purposes of comparison. In general, the intensity of fluorescence was lower and more diffuse when compared to the reaction for collagen and the distribution was different. Material in blood vessels and on blood cells fluoresced. In almost all tissues studied the cells showed more or less autofluorescence. For this reason, tendon fibroblasts appeared prominently visible by contrast. The non-specific binding of elements of rabbit serum and subsequent interaction with conjugated goat antirabbit serum occasionally led to a degree of non-specific staining. This was sometimes the case in epitheha1 cells of the oral mucosa and gingiva. This effect could be differentiated from the specific one by comparing with the various controls. DISCUSSION
Collagen in tissue sections is generally identified by staining with various mixtures of aniline dyes as in the van Gieson or Mallory methods but the result in both colour value and intensity is empiric. Reticular fibres, which are carbohydrate-rich collagen (in some instances pre-collagen) elements, are stained black by silver methods. By developing an antibody to a collagen fraction, certain forms of human collagen are specifically visualized. The collagen antiserum was developed against a form of collagen antigen from which plasma proteins were proven absent. The antigen was made by exhaustively extracting native tendon collagen with solvents which removed both plasma proteins and readily soluble collagen fractions. All earlier fractions than the one used developed antibodies to both collagen and serum proteins when injected into rabbit recipients. The final product obtained by 8 M urea extraction was free of demonstrable serum proteins, It represents only a small part of the total insoluble collagen which has not been further investigated. Some antisera to this collagen fraction still showed a faint serum protein reaction which was removed by absorption before use. The collagen antiserum is highly reactive against certain forms of collagen, e.g. basement membrane, endomysium, lamina propria of gingiva and skin. It is thus probable that it is reacting to collagen types I, III and IV in the accepted nomenclature of collagen subunits. Human antigens of these types were not available for us to test this supposition. Collagen type may not be the sole or most important reason why a tissue stains with a given antiserum. Adult tendon, for example, was barely stained except at regions of the sheath, whereas young tendon fibres were more clearly delineated. In the lamina propria of the skin and cheek, only limited groups of fibres were visualized. All tested specimens were prepared by freezing and drying of cryostat sections, thereby avoiding denatu-
M. B. Engel and H. R. Catchpole
388
ration. The collagen can thus be considered to be the native form of the particular collagen moiety, a feature that cannot be claimed following air drying
Engel M. B. and Catchpole H. R. 1972. Collagen distribution in the rat demonstrated by a specific anticollagen antiserum. Am. J. Anat. 134, 23-29.
(Wick, Furthmayr and Timpl, 1975) or chemical fixation. Failure to react with the collagen antiserum would then lie in the nature of the binding or linkage of the collagen itself. The inference is that in adult native tendon, for example, the reactive sites are buried by collagen-collagen or collagen-ground substance interactions or cross linkages. Conversely, the immunofluorescence of fibres in young tendon, and particularly in the lamina propria of the gingiva, in basement membrane of renal tubules and glomeruli, and in sheaths of muscle bundles and other cells, suggests a less highly aggregated and therefore more reactive form of collagen. Possibly this material is being turned over metabolically at a greater rate. Page and Ammons (1974) presented evidence for this kind of interpretation for the insoluble collagen of gingiva of the marmoset, where the turnover of radio-proline is far greater than in palatal mucosa, skin or tendon. Sections of inflamed gingiva show a loss of both fibrous and ground substance components. Collagen dissolution is revealed by a low density of immunofluorescent substance, comparable to what occurs after collagenase. Indeed the aetiology of these conditions may include the release of collagenase by invading microorganisms or from affected cells in the tissue (Gersh and Catchpole, 1949; Engel. Orban and Ray, 1950; Fullmer and Gibson, 1966).
Fullmer H. M. and Gibson W. 1966. Collagenolytic tivity in gingiva of man. Nature 209, 728-729.
.&knowLdgemmrs-We thank Mrs. Liuda Butikas and Mrs. Natalie Iwaniw for devoted and skilful technical assistance and Mrs. Isabele Stoncius for aid with the illustrations. This work was supported in part by grants DE 0236 and GRSG No. 120 from the U.S.P.H.S. REFERENCES Engel M. B.. Ray H. and Orban B. The pathogenesis of desquamative gingivitis. A disturbance of the connective tissue ground substance. J. dent Rex 29, 41&418.
ac-
Gersh 1. and Catchpole H. R. 1949. The organization of ground substance and basement membrane and its significance in tissue injury. disease and growth. Am. J. Anal. 85, 457-521 Mashouf K. and Engel M. B. 1975. Maturation of periodontal connective tissue in new-born rat incisor. Archs oral Biol. 20, 161-166. Nowack H., Gay S., Wick G., Becker U. and Timpl R. 1976. Preparation and use in immunohistology of antibodies specific for type I and type III collagen and procollagen. J. immunol. Meth. 12, 117-124. Page R. C. and Ammons W. F. 1974. Collagen turnover in the gingiva and other mature connective tissues of the marmoset, Saquinus Oedipus. Archs oral Viol. 19, 651-658. Rothbard S. and Watson R. F. 1954. The antigenicity of rat collagen. J. PX~. Med. 99, 535-549. Rothbard S. and Watson R. F. 1961. Demonstration of antibody to rat collagen in the renal glomuli of rats by Huorescence microscopy. J. exp. Med. 125. 595605. Rothbard S. and Watson R. F. 1972. Demonstration of collagen in human tissues by immunofluorescence. Lab. Invest. 27. 76-84. Timpl R. 1976. Immunological studies on collagen. In Biochrmisrry of Collagen. (Edited by Ramachandran G. M. and Reddi A. H.) pp. 319-375. Plenum Press, New York. 1976. Tracy G. and Welker W. H. 1975. The use of aluminum hydroxide cream for the removal of albumin in nitrogen partition in urinary analysis. J. hiol. Chem. 22. 5557. Wick G., Furthmayr H. and Timpl R. 1975. Purified antibodies to collagen: An immunofluorescence study of their reaction with tissue collagen. In?. Archs Allergy appl. Immun. 48, 664679. Wick G.. Nowack H., Hahn E., Timpl R. and Miller E. J. 1976. Visualization of type 1 & 11 collagens in tissue sections by immunohistologic techniques. Immunology I 17. 298-303.
Collagen in some human connective tissues
Plate 1. Sections were cut in a cryostat at a thickness of 4 pm and then freeze-dried. Immunofluorescence ’ 400. was elicited by the direct method. Original magnification was approximately Fig. 3. Tendon (autopsy) from newborn child showing reactive fibres mingled with unstained bundles. Fig. 4. Tendon (autopsy) from newborn treated with control rabbit serum followed by goat antirabbit conjugate. Weak non-specific staining and some autofluorescent structures are shown. Fig. 5. Adult tendon (surgical). Fibres of the tendon sheath show strong fluorescence. The rest of the tissue gives a weak general reaction. Fig. 6. Kidney (autopsy). Basement membranes of tubules and capillaries are demonstrated. Fig. 7. Skeletal muscle (surgical). Endomysium and capillary basement membranes are reactive.
389
390
M. B. Engel and H. R. Catchpole
Plate 2. Fig. 8. Normal gingiva (surgical). Most of the fibres of the lamina propria react and some non-specific staining persists in the inter-cellular substance of the epithelial layer. Fig. 9. Normal gingiva (surgical) treated for 24 h with bacterial collagenase. papillary fibres persist.
Reactive remnants
of
Fig. 10. Inflamed gingival tissue (surgical). Much of the collagen is dissolved but reactive material remains, especially around blood vessels (compare with Fig. 9). Fig. 11. Skin (surgical). Fluorescent and unreactive fibres mingle. Many small vessels are outlined by reaction. Fig. 12. Mucosa of cheek (surgical). Fluorescent and unreactive fibre groups are shown. Small vessels at e.
of Histology, College of Dentistry, University of Illinois at the Medical Center, Chicago. IL 60680. U.S.A.
Summary-An antiserum to a urea-soluble fraction of human tendon collagen was raised in rabbits. The antiserum was used by the indirect method for immunofluorescent visualization of certain forms of collagen in several human connective tissues, including oral mucosa. Prominent reactive sites included basement membranes of small blood vessels, renal tubules, young collagen fibres, endomysium and elements of the lamina propria of oral mucosa and skin. In some loci, collagen did not react, presumably due to the state of aggregation or to interactions with other structural macromolecules. Similarities were noted between the altered staining of inflamed gingiva and collagenase-treated tissue.
INTRODUCTION
We have reported the visualization of certain forms of collagen in the rat by immunofluorescence microscopy (Engel and Catchpole, 1972; Mashouf and Engel, 1975). With similar techniques we developed an antiserum to human tendon collagen which we used to demonstrate collagen in several types of human connective tissue, including the lamina propria of oral tissues. Rothbard and Watson (1954, 1961, 1972) used skin as the source of collagen with different procedures of antigen preparation and tissue processing. Timpl (1976) used heterologous antigens consisting of types I-IV collagen, derived from bovine or rat skin, to generate antibodies which cross-reacted with components in human tissues. Nowack et al. (1976) proposed to use such antisera as probes for human collagen types although antibodies to types I -and III collagens show unexplained cross-reactions (Wick et al., 1976). We preferred to avoid the use of heterologous antisera. A feature of our earlier work was the recognition of different intensities of collagen reactivity, which was attributed to differences in the degree of collagen aggregation. This finding, which is also recognized in Cleaned ground
and washed in the frozen
human collagenous tissues, was made possible by methods of tissue preparation reported here. MATERIALS
Preparation
AND METHODS
of antigen
Adult human achilles tendon, which had a relatively low concentration of non-collagenous protein and is not highly cellular, was the source of the antigen and was obtained at surgery. Tissues were freed of fat and muscle and washed with distilled water to remove visible blood. Fibre bundles were teased apart and the tissue mass frozen in liquid nitrogen and dried in uucuo while frozen. The dried
tissue was ground in a Culatti mill kept thoroughly chilled with liquid nitrogen. The ground tissue was sequentially extracted with distilled water, 0.2 M NaCl and 0.2 M citrate buffer, pH 3.5. The residue thus exhausted of the usual soluble collagens was extracted with 8 M urea. The collagen solubilized by this step, designated collagen D, was separated, dialysed against distilled water and lyophilized, and was the collagen antigen used. A flow chart (Text Fig. I) summarizes the method of preparation. The
collagen state
fibres frozen in liquid nitrogen, to powder and lyophilized Extract
with 20 vok/w
I
dist.
H,O (x2)
I Residue
Combined supernatants, lyophilized collagen A, serum proteins
Extract with 20 vol 0.2 M NaCl (x2)
I
I
1
Residue
Combined supernatants,dialysed, lyophilized. Collagen B, serum proteins
I
I
Residue
Combined supernatants,dialysed, lyophilized.Collagen C, serum proteins
Fig. 1. Fractionation
Extract with 20 vol Na citrate-citric acid buffer, pH 3.5 (x2)
Solubilize in IO vol B M urea with stirring in cold (x2) Combine,dialyse, lyophilize Cal lagen D
of human achilles tendon 385
M. B. Engel and H. R. Catchpole
386
Table 1. Amino acid composition of human collagen-D antigen*
0 D
Collagen-D Hydroxylysine Lysine Histidine “Post-histidine”? Ammonia Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine A lanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Hydroxyproline
12 35 9.3 20 78 63 22 32 114 112 228 79 26 6.8 1.5 34 1.4 18.7 150
*Values given as residues per 1000 residues. tAppears in the same position as l-methyl dine.
0 0
Fig. 2. lmmunodiffusion plate. Rabbit anti-collagen D (absorbed with human serum) in centre well. D, Collagen D; S, human serum. The antiserum reacts only with collagen
histi-
antigen was free of serum proteins when tested by immunodiffusion against goat anti-human serum protein antiserum. The amino acid composition for this form of insoluble tendon collagen is somewhat different from that reported for some other collagens, chiefly soluble collagens derived from skin (Table I). The number of glycine residues is lower and the glutamic acid and hydroxyproline values are somewhat higher. The chemistry of insoluble collagens has been little investigated. Preparation of tissues
Portions of oral mucosa, mainly free and attached gingiva, were obtained from patients undergoing minor surgical procedures. Usually the tissues were taken from the mandibular arch where block anaesthesia was feasible, thus avoiding tissue disruption by infiltrated anaesthetic solution. Blocks approximately 1-2 mm’ were placed on small metal cryostat holders and rapidly frozen in isopentane chilled to approximately - 150°C with liquid nitrogen. Other normal tissues, including tendon, skin, muscle and kidney. were obtained where possible as surgical specimens, or at autopsy. Preparation of anti-collagen antiserum
The collagen antigen was dissolved in 6 M urea at a concentration of lOmg/ml. This solution was diluted at the time of injection with an equal volume of aluminium hydroxide adjuvant prepared by the method of Tracy and Welker (1915). Two millilitres of this material were injected intramuscularly in equally-divided doses into young male albino rabbits every 2 weeks. This schedule was followed for 3-4
months, with test bleedings at intervals after the first month. When antibody titres appeared to be adequate as visualized by double diffusion in gel, the animals were exsanguinated 10-15 days after the last injection. By Ouchterlony plate analysis, some, but not all, antisera reacted weakly to human plasma proteins. TO remove this component from the antisera, all antisera were adsorbed with human plasma before use in immunofluorescence localization of collagen (Text Fig. 2). Preparation of tissue sections
Tissue sections were cut in a cryostat held at - 30°C to - 40°C. at a section thickness of 4-6 pm. The sections were dehydrated in vacua at -30°C and stored in a deep-freeze over Drierite until examined. Before staining, sections were affixed to slides coated with a thin layer of egg albumin. They were then incubated at room temperature with various specific and non-specific sera as follows. Immunofluorescence techniques
Sections were flooded with rabbit anticollagen serum and incubated for 30 min. The serum was decanted and the sections rinsed 5 times in phosphate-buffered saline (PBS), pH 7.4. The sections were then treated with the IgG fraction of fluorescein-conjugated goat anti-rabbit serum, at a dilution of 1:5 for 5 min. Excess fluid was decanted and the sections washed in 5 changes of PBS. They were then mounted in 5 per cent Gelvatol. The slides were examined with a Leitz Ortholux microscope using an HBO 200 mercury lamp, dark field condenser and a UGl excitation filter combined with a K490 barrier filter. Various controls were used to ensure specificity including: (I) Pretreatment of sections with Clostridium histolytica collagenase (Worthington, 1 mg/ml) at 37°C for 24 h. (2) Treatment with control, nonimmune rabbit serum followed by reaction with the conjugated goat anti-rabbit serum. (3) Treatment with conjugated goat anti-rabbit serum alone. (4) Blocking reaction, i.e. application of rabbit anti-collagen serum followed by unconjugated goat anti-rabbit serum, then application of conjugated goat anti-rabbit serum (each step separated by 5 washings with PBS). To compare the effects of staining due to serum proteins with specific collagen localization, several sections were treated with a potent rabbit anti-human
Collagen in some human connective tissues
serum antibody. Sites of serum were visualized by subsequent interaction with fluorescein conjugated goat anti-rabbit serum. RESULTS
Tendon (surgical)
The tibrils of young tendon were clearly delineated because they were moderately fluorescent, whereas the interfibrillar substance was unstained. Bundles of fibrils were demonstrated by the more intense reaction of the sheath material. Mature tendon, however, was only barely reactive, except in regions of the sheath. (Plate Figs. 3-5.) Kidney (autopsy)
The basement membranes of tubules and of contiguous renal vessels were outlined by fluorescence. Basement membranes of the glomerular vessels and of Bowman’s capsule also reacted. (Plate Fig. 6.) Musck
(autopsy)
Connective tissue surrounding muscle bundles and individual muscle cells, including perimysium, epimysium and endomysium, displayed fluorescence. Small arterioles and capillaries were revealed through the staining of their immediate surrounding connective tissue (Plate Fig. 7.) Skin (autopsy) Selected groups of dermal fibres reacted with the fluorescent antibody. The basement membranes of capillaries were prominently outlined. Substantial groups of fibres were unreactive. In general, the hypodermis showed fewer groups of reactive fibres than the dermis. (Plate Fig. 11.) Gingiua (surgical)
The interlacing collagen bundles of the gingiva were intensely stained. The basement membrane at the epithelium connective tissue junction was evident as a more intensely fluorescent structure. In general, the connective tissue of the rete pegs was somewhat more reactive than that of the deeper layers. In some preparations, the gingival epithelial cells (cytoplasm) or intercellular substance showed weak fluorescence. This was obviously a non-specific staining. In inflamed gingiva, the disruption and dissolution of collagen was indicated by the change in density of fluorescent fibres. The cpmpact network of fibres was interrupted in those regions around infiltrating lymphocytes and granulocytes. In dark field illumination. these areas were non-fluorescent (Plate Figs. 8- IO). Mucosa of cheek (surgical)
Strands of reactive fibres mingled with a larger mass of unstained collagen. The basement membranes surrounding small blood vessels and capillaries were prominently stained (Plate Fig. 12). GENERAL OBSERVATIONS
Intracellular forms of collagen were not seen. In the tissues which we chose to study, the reactive sites
387
usually included basement membranes and regions where young collagen and a high concentration of reticular fibres would be expected. When gingival sections were treated with collagenase, most of the collagen was removed and consequently did not fluoresce. However, there were aggregates of resistant material which continued to react. Some preparations were stained to show the distribution of plasma proteins for purposes of comparison. In general, the intensity of fluorescence was lower and more diffuse when compared to the reaction for collagen and the distribution was different. Material in blood vessels and on blood cells fluoresced. In almost all tissues studied the cells showed more or less autofluorescence. For this reason, tendon fibroblasts appeared prominently visible by contrast. The non-specific binding of elements of rabbit serum and subsequent interaction with conjugated goat antirabbit serum occasionally led to a degree of non-specific staining. This was sometimes the case in epitheha1 cells of the oral mucosa and gingiva. This effect could be differentiated from the specific one by comparing with the various controls. DISCUSSION
Collagen in tissue sections is generally identified by staining with various mixtures of aniline dyes as in the van Gieson or Mallory methods but the result in both colour value and intensity is empiric. Reticular fibres, which are carbohydrate-rich collagen (in some instances pre-collagen) elements, are stained black by silver methods. By developing an antibody to a collagen fraction, certain forms of human collagen are specifically visualized. The collagen antiserum was developed against a form of collagen antigen from which plasma proteins were proven absent. The antigen was made by exhaustively extracting native tendon collagen with solvents which removed both plasma proteins and readily soluble collagen fractions. All earlier fractions than the one used developed antibodies to both collagen and serum proteins when injected into rabbit recipients. The final product obtained by 8 M urea extraction was free of demonstrable serum proteins, It represents only a small part of the total insoluble collagen which has not been further investigated. Some antisera to this collagen fraction still showed a faint serum protein reaction which was removed by absorption before use. The collagen antiserum is highly reactive against certain forms of collagen, e.g. basement membrane, endomysium, lamina propria of gingiva and skin. It is thus probable that it is reacting to collagen types I, III and IV in the accepted nomenclature of collagen subunits. Human antigens of these types were not available for us to test this supposition. Collagen type may not be the sole or most important reason why a tissue stains with a given antiserum. Adult tendon, for example, was barely stained except at regions of the sheath, whereas young tendon fibres were more clearly delineated. In the lamina propria of the skin and cheek, only limited groups of fibres were visualized. All tested specimens were prepared by freezing and drying of cryostat sections, thereby avoiding denatu-
M. B. Engel and H. R. Catchpole
388
ration. The collagen can thus be considered to be the native form of the particular collagen moiety, a feature that cannot be claimed following air drying
Engel M. B. and Catchpole H. R. 1972. Collagen distribution in the rat demonstrated by a specific anticollagen antiserum. Am. J. Anat. 134, 23-29.
(Wick, Furthmayr and Timpl, 1975) or chemical fixation. Failure to react with the collagen antiserum would then lie in the nature of the binding or linkage of the collagen itself. The inference is that in adult native tendon, for example, the reactive sites are buried by collagen-collagen or collagen-ground substance interactions or cross linkages. Conversely, the immunofluorescence of fibres in young tendon, and particularly in the lamina propria of the gingiva, in basement membrane of renal tubules and glomeruli, and in sheaths of muscle bundles and other cells, suggests a less highly aggregated and therefore more reactive form of collagen. Possibly this material is being turned over metabolically at a greater rate. Page and Ammons (1974) presented evidence for this kind of interpretation for the insoluble collagen of gingiva of the marmoset, where the turnover of radio-proline is far greater than in palatal mucosa, skin or tendon. Sections of inflamed gingiva show a loss of both fibrous and ground substance components. Collagen dissolution is revealed by a low density of immunofluorescent substance, comparable to what occurs after collagenase. Indeed the aetiology of these conditions may include the release of collagenase by invading microorganisms or from affected cells in the tissue (Gersh and Catchpole, 1949; Engel. Orban and Ray, 1950; Fullmer and Gibson, 1966).
Fullmer H. M. and Gibson W. 1966. Collagenolytic tivity in gingiva of man. Nature 209, 728-729.
.&knowLdgemmrs-We thank Mrs. Liuda Butikas and Mrs. Natalie Iwaniw for devoted and skilful technical assistance and Mrs. Isabele Stoncius for aid with the illustrations. This work was supported in part by grants DE 0236 and GRSG No. 120 from the U.S.P.H.S. REFERENCES Engel M. B.. Ray H. and Orban B. The pathogenesis of desquamative gingivitis. A disturbance of the connective tissue ground substance. J. dent Rex 29, 41&418.
ac-
Gersh 1. and Catchpole H. R. 1949. The organization of ground substance and basement membrane and its significance in tissue injury. disease and growth. Am. J. Anal. 85, 457-521 Mashouf K. and Engel M. B. 1975. Maturation of periodontal connective tissue in new-born rat incisor. Archs oral Biol. 20, 161-166. Nowack H., Gay S., Wick G., Becker U. and Timpl R. 1976. Preparation and use in immunohistology of antibodies specific for type I and type III collagen and procollagen. J. immunol. Meth. 12, 117-124. Page R. C. and Ammons W. F. 1974. Collagen turnover in the gingiva and other mature connective tissues of the marmoset, Saquinus Oedipus. Archs oral Viol. 19, 651-658. Rothbard S. and Watson R. F. 1954. The antigenicity of rat collagen. J. PX~. Med. 99, 535-549. Rothbard S. and Watson R. F. 1961. Demonstration of antibody to rat collagen in the renal glomuli of rats by Huorescence microscopy. J. exp. Med. 125. 595605. Rothbard S. and Watson R. F. 1972. Demonstration of collagen in human tissues by immunofluorescence. Lab. Invest. 27. 76-84. Timpl R. 1976. Immunological studies on collagen. In Biochrmisrry of Collagen. (Edited by Ramachandran G. M. and Reddi A. H.) pp. 319-375. Plenum Press, New York. 1976. Tracy G. and Welker W. H. 1975. The use of aluminum hydroxide cream for the removal of albumin in nitrogen partition in urinary analysis. J. hiol. Chem. 22. 5557. Wick G., Furthmayr H. and Timpl R. 1975. Purified antibodies to collagen: An immunofluorescence study of their reaction with tissue collagen. In?. Archs Allergy appl. Immun. 48, 664679. Wick G.. Nowack H., Hahn E., Timpl R. and Miller E. J. 1976. Visualization of type 1 & 11 collagens in tissue sections by immunohistologic techniques. Immunology I 17. 298-303.
Collagen in some human connective tissues
Plate 1. Sections were cut in a cryostat at a thickness of 4 pm and then freeze-dried. Immunofluorescence ’ 400. was elicited by the direct method. Original magnification was approximately Fig. 3. Tendon (autopsy) from newborn child showing reactive fibres mingled with unstained bundles. Fig. 4. Tendon (autopsy) from newborn treated with control rabbit serum followed by goat antirabbit conjugate. Weak non-specific staining and some autofluorescent structures are shown. Fig. 5. Adult tendon (surgical). Fibres of the tendon sheath show strong fluorescence. The rest of the tissue gives a weak general reaction. Fig. 6. Kidney (autopsy). Basement membranes of tubules and capillaries are demonstrated. Fig. 7. Skeletal muscle (surgical). Endomysium and capillary basement membranes are reactive.
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Plate 2. Fig. 8. Normal gingiva (surgical). Most of the fibres of the lamina propria react and some non-specific staining persists in the inter-cellular substance of the epithelial layer. Fig. 9. Normal gingiva (surgical) treated for 24 h with bacterial collagenase. papillary fibres persist.
Reactive remnants
of
Fig. 10. Inflamed gingival tissue (surgical). Much of the collagen is dissolved but reactive material remains, especially around blood vessels (compare with Fig. 9). Fig. 11. Skin (surgical). Fluorescent and unreactive fibres mingle. Many small vessels are outlined by reaction. Fig. 12. Mucosa of cheek (surgical). Fluorescent and unreactive fibre groups are shown. Small vessels at e.
