LOCALIZATION OF CHONDROMUCOPROTEIN IN CARTILAGE · greatly increased by prior hyaluronidase treatment ofCMP;this is presumablydueto degradation and removal of polysaccharide and freeing

Ann. rheum. Dis. (1965), 24, 528.

LOCALIZATION OF CHONDROMUCOPROTEININ CARTILAGE

BY

G. LOEWIM.R.C. Rheumatism Research Unit, Canadian Red Cross Memorial Hospital, Taplow, Maidenhead, Berks

The histochemical localization of acid poly-saccharides involves difficulties of fixation andusually reactions of uncertain specificity. Thus,the aqueous phase used in most fixatives is liable toelute at least part of the material, while moststaining reactions lack specificity. The latter prob-lem has lately been successfully attacked by the useof the salt-controlled alcian blue technique (Scottand Dorling, 1965). The present paper describesthe application of an immuno-histochemical tech-nique to this problem. Chondromucoprotein(CMP) in cartilage was detected by the applicationof antibody directed against this substance. Theimmunological characteristics of the reaction andnature of antigen and antibody have been fullydescribed elsewhere (Loewi and Muir, 1965; Muir,1958). Antiserum to CMP is predominantlydirected against the protein part; for this reasonparallel studies have been carried out with the alcianblue technique to demonstrate the acid polysac-charide part of the CMP complex.

Material and MethodsCartilage was taken from fully-grown pigs immediately

after death in the slaughter-house, including specimensof costal cartilage, of tracheal cartilage, and of articularcartilage from the ankle joint. Part of each specimenwas snap-frozen in a mixture of solid CO2 and ethanol,and another part was fixed in 95 per cent. ethanolat 40 C. Some specimens were fixed in a mixture of 1part of 40 per cent. formaldehyde and 9 parts ofabsolute ethanol at -20° C. Fixed material was subse-quently embedded in paraffin wax and sections were cutat 5 [± thickness. The method followed with cold-ethanol fixed tissue was that of Sainte-Marie (1962).Unfixed, frozen material was cut in a cryostat cabinetand then rapidly dried before further treatment.

Sections of Pig Aorta were cut from cold-ethanol fixedtissue.

Embryo Pig Tissue was obtained from a litter in utero.

Fluorescent Staining was done by the sandwich tech-nique. Antibody to pig laryngeal cartilage CMP wasobtained in rabbits (Loewi and Muir, 1965). This anti-serum, at a dilution of 1:5 in Coons's buffered saline was

applied to sections for 30 min., followed by washing for30 min. After this, fluorescein-conjugated goat-anti-rabbit y-globulin was layered on the sections for 30 min.,followed by another wash of at least 30 min. Controlstreated with an unrelated rabbit antiserum, were includedin every experiment.

Alcian Blue Method for acid polysaccharides was usedon sections of fixed or unfixed tissue. Varying molaritiesof MgCl2 were used, the critical point being different forthe several acid mucopolysaccharides (Scott and Dorling,1965).

ResultsCostal Cartilage.-Sections treated with antibody

showed specific staining of the rims of lacunaesurrounding chrondrocytes (Fig. 1, opposite). Thisregion was unstained in control sections, whereassome staining of chondrocytes was frequently seen incontrol (Fig. 2, opposite) as well as in antibody-treated sections. The zone surrounding chondrocyteclusters, beyond the stained rim, was often, but notalways, seen to be unstained as in Fig. 1. Thiscontrasts strongly with the alcian blue stained section(Fig. 3, opposite) of costal cartilage taken from thesame small block of tissue and cut and prepared inthe same way as the fluorescent section. Here thecircum-lacunar space is strongly stained, in contrastto the next zone, known as the interterritorialregion. This showed stippled staining withantibody, whilst being virtually unstained by alcianblue. This contrasted staining was, however, notso well marked in other types of cartilage or even inall parts of sections of costal cartilage examined.The effect of pre-treatment of sections with hyalur-onidase (Seravac Laboratories, Ltd.) also showeddifferent results when examined by antibody andalcian blue-staining. Treatment for 2 hrs causedmarked generalized increase of fluorescent staining,while a regional staining distribution could still berecognized. Some alcian blue staining was retainedin the lacunar regions. After 24 hrs, however,alcian blue was not taken up, while fluorescencewas very bright and generalized. These results areillustrated in Figs 4 and 5 (opposite) and Figs 6 and7 (overleaf).

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CHONDROMUCOPROTEIN IN CARTILAGE

Fig. 1.-Fresh-frozen section of pigcostal cartilage, treated with rabbitantibody to pig CMP, followed byfluorescein-conjugated goat anti-

rabbit globulin. x 160.

Fig. 2.-As Fig. 1, but treated with rabbit Fig. 3.-As Fig. 1, stained with alcian blue,antibody to dextran. x 160. 0 5 M MgCl2. x 84.

P. -.i.eI-If

t..*.

....

...I.. M

'I

Fig. 4.-Fresh-frozen section of pig costal cartilage,treated withhyaluronidase for 2 hrs followed byCMP

antibody and conjugate. x 200 approx.

For control purposes, some sections were incu-bated in saline for 24 hrs; this did not affect alcianblue staining and only slightly increased the intensityof fluorescent staining. Hyaluronidase treatmentdid not cause any non-specific uptake of fluorescent

Fig. 5.-As Fig. 4, stained with alcian blue, 05 MMgCI2. x 100 approx.

conjugate in control sections treated with rabbit anti-dextran or other rabbit antisera.

Articular Cartilage.-Articular cartilage showedspecific lacunar rim staining by antibody, as did

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ANNALS OF THE RHEUMATIC DISEASES

Fig. 6.-Fresh-frozen section of pig costal cartilage,treated with hyaluronidase for 24 hrs followed byCMP antibody and conjugate. x 200 approx.

costal cartilage (Fig. 8). This was particularlystrongly demonstrated in the deeper layers adjoiningbone (Fig. 9). The distinction between the circum-lacunar spaces and the interterritorial regions wasnot marked, although it could be made out here and

Fig. 7.-As Fig. 6, stained with alcian blue, 0 5 MMgCk2. x 100 approx.

there in sections. A line of staining on the surfaceof the cartilage was usually seen (Fig. 8). Such aline could occasionally be seen in control sections(Fig. 10), though there it was less strong.

Alcian blue (Fig. 11, opposite) showed a lacunar

Fig. 8.-Fresh-frozen section of pig arti- Fig. 9.-Same section as Fig. 8, another Fig. 10.-As Fig. 8, but treated withcular cartilage from ankle joint, treated field, bordering underlying bone. rabbit anti-dextran serum. x 160.with CMP antibody and conjugate. x 160. x 160.

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CHONDROMUCOPROTEIN IN CARTILAGE 531

rim accentuation, but therewas, except in the deeper layers,considerable interterritorialstaining also. In the deeperlayers, interterritorial regionswere only slightly stained.Hyaluronidase treatment led togreat generalized increase influorescent staining as withcostal cartilage, while abolish-ing alcian blue uptake (Figs 12and 13).

Fresh-frozen section of pig articular cartilage, stained with alcian blue, 05 M MgCI2.x 125.

W.~ ~ ~ ~ ~

W~~~~~

4.~~~~~~~~~~~~~~~~~~4

~~~~~~~~~~~~~~~~~~~~~~~~~~~.i ....... =c8..C-I

.SES.~~~~~~~~~~~~~~~~~~~~......

Fig. 12.-Fresh-frozen section of pig articular cartilage, treated with Fig 13 As Fig 12 stained with alcian blue, 0 5 M MgCI2.hyaluronidase for 24 hrs, followed by CMP antiserum and conjugate. X 125.

x 240 1

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ANNALS OF THE RHEUMATIC DISEASES

Tracheal Cartilage.-In addition to lacunar rimstaining, there was widespread stippled fluorescenceof cartilage matrix with only indistinct territorialdistribution (Fig. 14). A control section is shownin Fig. 15. Alcian blue showed fairly uniformstaining of matrix (Fig. 16).

Embryonic Cartilage.-Sections taken from afemur showed bright fluorescence of lacunar rimsas well as fairly uniform stippled staining of sur-rounding matrix (Fig. 17). Alcian blue showedfairly heavy uptake by the cartilage matrix withoutmuch regional accentuation (Fig. 18).

Fig. 14.-Pig tracheal cartilage, treatedwith CMP antiserum and conjugate.

x 160.

Fig. 15.-As Fig. 14, control section,treated with antiserum to dextran.

x 160.

Fig. 16.-As Fig. 14, stained with alcianblue, 0 5 M MgCI,. x 84.

Fig. 17.-Cold-ethanol fixed section of pig embryo cartilage,treated with CMP antiserum and conjugate. X 240.

Fig. 18.-As Fig. 17, stained with alcian blue, 0 5 M MgCl2.x 125.

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CHONDROMUCOPROTEIN IN CARTILAGE

Other Tissues.-Amongst other tissues in whichCMP could be demonstrated by the fluorescent anti-body method was aorta. Interfibrillar material ofthe aortic media, as well as the intima, showedfluorescent staining (Fig. 19). A control section isshown in Fig. 20. Chemical analysis of aorta hasshown the presence of considerable amounts of acidmucopolysaccharides, some or all of which areprobably in protein combination (Meyer, 1964).

Controls.-Apart from sections treated with un-related rabbit antisera, which served as controls onevery occasion on which fluorescent staining wascarried out, experiments were performed in whichthe antibody had been absorbed. Such absorptionwith CMP led to complete absence of fluorescentstaining. Absorption of anti-serum with pig serumor with collagen, however, did not affect the fluores-cent staining of cartilage or other tissues. Fluores-cent conjugates other than those directed against theantibody in the sandwich also failed to stain. Thespecificity of antibodies to CMP is further describedin an earlier paper (Loewi and Muir, 1965).

Discussion

The results show that tissues rich in CMP arecapable of being stained by antibody to CMP, usingthe fluorescent method. The antibody used in thepresent work was made against CMP from pigtrachea, the acid polysaccharide constituent ofwhich was predominantly chondroitin sulphate(Muir, 1958). We have previously presented evi-dence (Loewi and Muir, 1965) showing that thisantibody is directed against the protein constituentof CMP. This is presumably also the site ofreaction in tissue sections. We have, however, atpresent no evidence as to the possible reaction ofantibody with proteins bound to other acid poly-saccharides, such as keratosulphate. The alcianblue technique, as described by Scott and Dorling(1965), allows the differentiation of acid polysac-charides from other negatively charged materials andthe distinction between various acid polysaccharidesby the use of different salt concentrations. In theexperiments reported here, 0 5 M MgCl2 has beenused. At this molarity, only chondroitin sulphate,

Fig. 19.-Cold ethanol-fixed section of pig aorta, treated withCMP antiserum and conjugate. x 240.

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Fig. 20.-Control to Fig. 19, treated with anti-dextran andconjugate. x 240.



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ANNALS OF THE RHEUMATIC DISEASESkeratosulphate, and heparin are stained. Thus,combining the fluorescent and alcian blue techniques,it seems possible that both the protein and poly-saccharide of CMP would be demonstrable. Asshown in the accompanying illustrations, in manysections of cartilage, there was some degree ofinverse relationship between the results of stainingby the two methods. The interterritorial regionshowed fluorescent staining, but little alcian blueuptake while there was often a peri-lacunar zonewhich showed the reverse relationship. The effectof hyaluronidase was to cause widespread increaseof fluorescent staining while alcian blue uptake wasabolished. The reactivity of antibody with CMP isgreatly increased by prior hyaluronidase treatmentof CMP; this is presumably due to degradation andremoval of polysaccharide and freeing of reactivesites on the protein moiety ofCMP (Loewi and Muir,1965). This explanation can also account for in-creased staining of sections by fluorescence, whilepolysaccharide loss has led to diminution or lack ofalcian blue uptake. The different results with thetwo methods in the peri-lacunar and interterritorialregions could be explained on a similar basis: arelatively polysaccharide-rich and protein-poorcomplex, or at least one with polysaccharide groupscovering protein combining sites might be present inthe peri-lacunar region, while a reverse relationshipmight apply in the interterritorial region. On theother hand, it must be remembered that polysac-charides have excluded volume (Laurent and Ogston,1963) which would lead to failure to penetrate,especially by a molecule of the size of y-globulin.Thus, a region such as the peri-lacunar one which isstained heavily by alcian blue might be relativelyunstained by the fluorescent technique because theantibody might fail to penetrate (Scott, 1965). Itwas thought that treatment of cartilage, especiallyfixation, would have an effect on staining, andnearly all the work reported here was thereforecarried out both on alcohol-fixed and fresh-frozenmaterial. In general, there was found to be nodifference in the results obtained on the two typesof sections by either staining method, with theexception that the effect of hyaluronidase was moremarked on fresh-frozen than on fixed tissue. It wasalso noted that cold formaldehyde-ethanol-fixedmaterial showed a rather more even distribution anddeeper staining by alcian blue; such fixation, how-ever, was unsuitable for the fluorescent antibodytechnique.

SummaryAntibody to porcine cartilage chondromucopro-

tein has been used to stain sections of costal,

articular, and tracheal cartilage by the fluorescentantibody technique. The results have been com-pared with alcian blue uptake under controlledconditions.The presence of chondromucoprotein was shown

in the lacunar rim. In the surrounding matrix,fluorescent staining was less marked, but wasparticularly associated with the interterritorialregions. Alcian blue staining gave, in some in-stances, the reverse of the fluorescent stainingpattern, in that there was strong staining of a fairlywide peri-lacunar zone, but very little uptake ininterterritorial regions.

Treatment of sections with hyaluronidase greatlyincreased fluorescent antibody staining both inintensity and extent. At the same time alcian blueuptake was abolished. The action of hyaluronidasewas most marked on unfixed sections.The hyaluronidase effect may be attributed to

degradation and removal of polysaccharideproviding for increased access of antibody to theprotein moiety of chondromucoprotein. Regionalstaining differences could be due to different com-position of the chondromucoprotein polymer in thevarious territories, but could also be accounted forby the property of excluded volume.

I thank Mr. J. Dorling for performing alcian bluestains and Dr. J. E. Scott for helpful discussions. Dr.H. Muir provided the chondromucoprotein for immuniza-tion.

REFERENCESLaurent, T. C., and Ogston, A. G. (1963). Biochem. J.,

89, 249.Loewi, G., and Muir, H. (1965). Immunology, 9, 119.Meyer, K. (1964). "Small Blood Vessel Involvement in

Diabetes Meilitus", American Institute of Bio-logical Sciences, p. 193.

Muir, H. (1958). Biochem. J., 69, 195.Sainte-Marie, G. (1962). J. Histochem. Cytochem., 10,

250.Scott, J. E. (1965). Ann. rheum. Dis., 24, 184.

and Dorling, J. (1965). Histochemie, 5, 221.

Localisation de La chondromucoprot6ine dans le cartilageRisuMit

On s'est servi de l'anticorps contre la chondromuco-proteine du cartilage du porc pour colorer des coupes ducartilage costal, articulaire et tracheal par le procede defluorescence. On a compar6 les resultats avec ceux obtenusavec l'absorption du bleu d'alcian dans des conditionsdeterminees.On a demontre la presence de la chondromucoprot6ine

dans le rebord lacunaire. Dans la matrice environnantela coloration fluorescente etait moins marquee, maiselle se trouvait associee tout particulierement avec desregions interterritoriales. Le bleu d'alcian donnait

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CHONDROMUCOPROTEIN IN CARTILAGEquelquefois une image opposee A celle produite par lamethode de fluorescence: une coloration forte et assezetendue de la zone perilacunaire, mais tres faible dans lesregions interterritoriales.

Lorsqu'on traitait les coupes par l'hyaluronidase,la coloration fluorescente de l'anticorps devenait plusintense et plus etendue, tandis que l'absorption du bleud'alcian cessait. L'action de l'hyaluronidase etaitplus accentuee sur des coupes non fixees.

L'effet de l'hyaluronidase peut etre attribue A ladegradation et l'enlevement du polysaccharide, ce quifacilite l'acces de l'anticorps A la portion prot6ique dela chondromucoproteine. Les differences regionales decoloration pourraient etre dues A une compositiondifferente du polymere chondromucoproteique enterritoires differents ou bien a la propriete du volumeexclu.

Localizaci6n de la condromucoproteina en el cartilago

SUMARIOEl anticuerpo contra la condromucoproteina del

cartilago porcino fue empleado para colorar cortes del

cartilago costal, articular y traqueal por el pro cedimientode fluorescencia. Se compararon los resultados conlos obtenidos con la absorpci6n del azul alciano encondiciones determinadas.

Se demostro la presencia de la condromucoproteinaen el borde lacunar. En la cercana matriz la coloraci6nfluorescente fue menos marcada, pero se vio asociadamuy particularmente con regiones interterritoriales. Elazul alciano daba a veces un cuadro opuesto al producidopor el metodo de fluorescencia: una coloraci6n fuerte ybastante extensa, de la zone perilacunar pero muy debilen las regiones inter-territoriales.

Al tratar los cortes con hialuronidasa, la coloraci6nfluorescente del anticuerpo se volvia mas intensa ymas amplia, mientras que la absorpci6n del azul alcianocesaba. La acci6n de la hialuronidasa fue mas marcadaen cortes sin fijar.

El efecto de la hialuronidasa puede atribuirse a ladegradacion y a la eliminaci6n del polisaccarido, facili-tando asi el acceso del anticuerpo a la porci6n proteinicade la condromucoproteina. Las diferencias regionalesde coloraci6n pudieran deberse a una composici6ndiferente del polimer condromucoproteinico en variosterritorios o, quizAs, a la propiedad del volumen excluido.

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LOCALIZATION OF CHONDROMUCOPROTEIN IN CARTILAGE · greatly increased by prior hyaluronidase treatment ofCMP;this is presumablydueto degradation and removal of polysaccharide and freeing