Summary. Aprotinin (Trasylol| a polypeptide protease inhibitor from bovine orga~ns, has been labelled with fluorescein isothiocyanate to produce a fluorecent ...
Histochemie 34, 77--84 (1973) 9 by Springer-Verlag 1973
Fluorescent-Labelled Aprotinin: A New Reagent for the Histochemical Detection of Acid Mucosubstances J. A. Kiernan *
Department of Anatomy, University of Cambridge, Cambridge, England R. W. S t o d d a r t Strangeways Research Laboratory, Cambridge, England Received October 10, 1972
Summary. Aprotinin (Trasylol| a polypeptide protease inhibitor from bovine orga~ns, has been labelled with fluorescein isothiocyanate to produce a fluorecent conjugate (FLA). The conjugate h0~s been applied to paraffin sections of rat tissues and has been shown to act as a specific fluorochrome for acidic mucosubstances. There is reason to believe that FLA stains carbohydrates which owe their acidity to the presence of carboxyl groups. The histochemical findings are described and discussed and it is also suggested that attachment of aprotinin to extracellular polysaccharides in vivo may limit the therapeutic usefulness of the drug. Introduction
Aprotinin is a polypeptide inhibitor of proteoly~ic enzymes extracted from bovine organs. The inhibitor forms complexes with trypsin, chymotrypsin, some kallikreins and some plant proteases (see Vogel, Trautschold and Werle, 1968, for biochemical data). The aprotinin molecule contains four lysine residues which are not i m p o r t a n t to the inhibitory properties and can, therefore, be labelled without loss of activity by conjugation with fluorescein isothiocyanate. I t was at first hoped t h a t the labelled inhibitor might be used to localize proteolytic enzymes in frozen sections of animal tissues, but no evidence of such specificity was obtained. I n s t e a d it was observed t h a t the labelled inhibitor principally stained components of tissues k n o w n to be rich in acid mucosubstanees. A biochemical s t u d y b y Stoddart and K i e r n a n (1973b)has indicated t h a t labelled approtinin forms complexes with those sugars, polysaccharides and glycoproteins which owe their acidity to carboxyl groups. The histochemieal use of the new reagent is described below and followed b y a discussion of the possible significance of the findings in relation both to histochemical practice and to the pharmacological properties of aprotinin. Materials and Methods
Preparation o/ Reagent. A solution of aprotinin, as marketed for clinical use as Trasylol| was supplied by Bayer Pharmaceuticals Ltd., Haywards Heath, Sussex, England, in ampoules * Present address: Department of Anatomy, Health Sciences Centre, University of Western Ontario, London 72, Ontario, Canada.
78
~I. A. Kiernan and II. W. Stoddart :
containing about 15 mg of the polypeptidc dissolved in 10 ml of 0.9% NaCl. For fluorescent labelling, 0.2 ml of N NaOH were added to 20 mf of the aprotinin solution to raise the pH to 9.5 10.0. 0.6 mg of fluorescein isothioeyanate, isomer I (dissolved in 0.2-0.3 ml of acetone) were slowly added, with constant stirring at 0 ~ C. The mixture was left for 16-18 h at 4" C and then transferred to dialysis tubing and dialysed against three changes of 21 of water containing 1% polyvinylpyrrolidone (M. W. 10000) at. 4 ~ C for a total of 48 h. After dialysis, the conjugated aprotinin solution was sterilized by Millipore filtration into glass ampoules, which were sealed and stored at 4 ~ C. The conjugate will be referred to as fluoreseein-labelled aprotinin (FLA). I t has a p H of 4,5 5.5. }'or comparative purposes, one batch of 20 ml of aprotinin solution was similarly conjugated with rhodanfine B isothiocyanate. Staining atut Microscopy. Paraffin sections (mounted on slides) of a variety of r a t tissues (fixed in Carnoy or neutral 4 % formMdehyde) were de-waxed in xylene and h y d r a t e d through graded alcohols. Autofluoreseence was suppressed by immersing the slides for 5 nl in 0.5% osmium tetroxide and washing in running water for at least 4 h. The sections were stained under lying drops of FLA for 1 m at room temperature. The solution of FLA could be diluted with an equM volume of water with little loss of potency, b u t fourfold dilution resulted in distinctly weaker fluorescence. After staining, the slides were washed in three changes of distilled water for a total of 5 m, dehydrated through graded alcohols, cleared in xylene and mounted in DePeX. Slides were examined in a Reictlert "' B i n o l u x " nficroscope with excitation by blue-violet (350-450 nm) light (Filter: BS12/6 ram) and with an SP3 barrier filter between objective and eyepiece. Control Procedures. The FLA reagent was used alone a n d after mixture with equal volumes of 0.5 M sodium aeetate-hydrnchloric acid and tris-inaleate buffers over a range from pH 2.0-9.0. The effects of varying ~he ionic strength of the reagent were assessed by staining with nfixtures of FLA with equal volumes of solutions of NaCI and Na2SO a to give filial eoncentrations of these salts froln 0.125 1.0 M. Sections were stained after aeetylation in 40 % acetic anhydride in anhydrous pyridine for 18 h at 22 ~ () and after " m e t h y l a t i o n " (Sorvari and Stoward, 1970) in 0.1 N HC1 in methanol for 4 h at 60 ~ 12. Other seetions were stained after incubation according to Thompson (1966) for 24 h at. 37 ~ C in ovine testicular hyaluronidase (0.5 nlg/ml), bacterial/3-glucuronidase (4.0 rag/ nil), pectinase (4.0 mg/ml), Clostridb~m per]ringens neuraminidasc (0.3 mg/ml), cullagcnase (4.0 mg/ml) a n d bovine pancreatic deoxyribonuclease (0.5 mg/ml). For purposes of comparison, slides were also stained with rhodamine B-labelled aprotinin, fluorescein-labelled eoneanavalin A (FITC-Con A) (Stoddart and Kiernan, 1973 a), Alcian bhle 8GX at pH 2.5 and 1.0, t~luidine bhle O, maxilon blue t/,L and the periodic acid-Sehiff (PAS) routine (methods as given by Pearse, 1968).
Results The pre-trcatment with osmium tetroxide completely abolished autofluorescence, i n d i c a t i n g t h a t all t h e f l u o r e s c e n c e o b s e r v e d i n t h e s t a i n e d s e c t i o n s c o u l d b e a t t r i b u t e d t o t h e a t t a c h m e n t of F L A . T h e a p p e a r a n c e s i n d i f f e r e n t o r g a n s a r e s u m m a r i z e d i n T a b l e 1 a n d i l l u s t r a t e d i~l Figs. 1-4. Control Proced'ares. F l u o r e s c e n t . s t a i n i n g w i t h F L A c o u l d b e e f f e c t e d o v e r a r a n g e of p H f r o m 4 . 0 - 8 . 0 . W i t h i n t h i s r a n g e t h e p H i n f l u e n c e d t h e i n t e n s i t y of
Fig. 1. Tongue, showing fluorescence of mast cells and of reticulin surrounding muscle fibres. Stained with FLA ; x 350 Fig. 2. Kidney, showing fluorescence of glomerular, capsular and tubular basement inembranes and weaker fluorescence in cytoplasm and brush borders of tubular epithelium. Stained with FLA ; X 200
Fluorescent-Labelled Apl~tinin
79
Fig. 3. P i n n a of ear, showing intense fluorescence of cartilage matrix and weaker fluorescence of dermal collagen and mast cells. Stained witli F L A ; X 100 Fig. 4. Submaxillary salivary gland, showing fluorescence in a niucous acinus and in serous secretors cells. Stained with rhodamine B-labelled aprotinin; • 150
80
. l . A . Kiernan and R. W. S t o d d a r t : Fluorescent-I:abelled Aprotinin
Table 1. Fluorescence after staining with FLA, compared with other staining properties (0 = o r t h o c h r o m a s i a ; M = metaehromasia) Organ
Component stained
FLA
Salivary
Mucousaeini
+++
glands
Serous cells
L+
Tongue
Stomach
~ ++
PAS
++
Aleian blue
Aleian blue
p H 2,5
p H 1.0
+
+ +
+
+
+
Mast cells
+++
I ++
--
+ +
Collagen fibres
+
t @
+4
+
Retieulin around muscle fibres
+
+ + +
+
+
-++
~+
M
Mast cells
+
--
-t +
Epithelial surface
+ +
+
+
-
Some epithelial nuclei
+
--
--
O
Epithelial basement membrane
+ +
I:
+
--
Surface muein
i +
t --
++
~=
+q-+
++
q
Partly 0 Partly M
-- +
+
--
--
+ t
+ +
M
+ +
t +
M
( ' o l l a g e n fibres
+ +
+ + +
S u b m u c o s a l and serosal rm~st ceils
+
+ + --
Goblet, cells
+:l +
++
Epithelial "brusb borders"
+_
4- +
Collagen a n d
g
+ ~,
--
~+
--
I
+++ q ~
++-I ',
+
?
+ ]+
--
villous stroma and outer layers Glycogen in hepatoeytes (removed by amylase) Capsular collagen t'tnd b a s e m e n t membranes of duets and blood vessels
~
M
q I+
r c t i e u l i n of
Liver
Tohfidine blue O and maxilon blue R L
+++
--
Collagen and reticulin fibres and b a s e m e n t i n e l n branes
Mueosa] m a s t cells Intestine
FITCCon A
+
+
Mostly-
-
--
--
Table 1 (continued) Organ
Component stained
FLA
FITCCon A
PAS
Alcian blue p H 2.5
Alcian blue pH 1.0
Toluidine blue 0 and maxilon blue R L
Kidney
Basement membrane of glomerulus
5- 5- 5-
+ 5-
5- 5-
5- 5-
--
Slight 0
Basement membrane of Bowman's capsule
+ 5-
5- +
5-
--
--
--
Basement membrane of tubules
+ +
+ +
+
+
--
--
" B r u s h borders" of tubular epithelium
5- 5-
+ +
5- +
--
--
--
Cytoplasm of tubular epithelial cells
5-
--
+
--
--
0
External
Cartilage
5-5-5-
+
++5-
5-5-5-
5-5-+
M
ear
Collagen fibres of dermis
5-
5- +
5- +
+
--
--
Basement membranes 5of hair follicles, sebaceous glands and blood vessels
5-
5-
-~
Mast cells
5- 5-
+ 5-
--
A- 5- 5-
+ + +
M
Mast cells
-+-5-5-
+-kA-
--
+5-+
5-5-5-
M
Collagen and reticulin fibres
+ +
5- 5-
5- 5-
5-
Reticular fibres surrounding cardiac muscle fibres
5-
~- 5-
+ +
5-
--
Mast cells
5- 5- 5-
+ 5- 5-
--
5- 5-
5- q-
Generally distributed fluorescence in neuropil ; probably extracellular
§ +
5- 5-
5- 5-
§
--
Junctions between ependymal cells
+ +
5-
5-
-
--
Basement membrane of choroid plexus
5- +
5- 5- 5-
5- 5- 5-
5-
5-
" B r u s h border" at ventricular surface of choroidal ependyma
+ 5-
5- 5- 5-
5- 5-
5-
--
Occasional cell nuclei, especially in white matter
+
--
--
--
~Iesentery
Myocardium
Brain
6
H i s t o c h c m i e , Bd. 34
Slight 0
M
O
82
J.A. Kiernan and R. W. Stoddart:
fluorescence only very slightly; the optimal appearances were obtained with the reagent at p H 6.0. The presence of inorganic salts inhibited staining to an extent proportional to the concentration, and NaC1 and Na~SO~ caused almost complete suppression at 0.5 M. The most strongly fluorescent structures, such as intestinal goblet, cells and cartilage matrix, were most resistant to increases in the ionic strength of the reagent. After acetylation, most cell nuclei became FLA-positive, but the attachment of the fluorochrome was otherwise unaffected. The reactivity of nuclei was reduced, but not, abolished, by treatment of acetylated sections with deoxyribonuclease before staining. " M e t h y l a t i o n " prevented all staining by FLA, except in intestinal goblet cells, which remained weakly positive. Ityaluronidase and /~-glucuronidase had no effect on FLA-induced fluorescence. Incubation with neuraminidase resulted in greatly reduced fluorescence in serous and mucous salivary acini, intestinal goblet cells and gastric surface inucin and abolished the fluorescent staining of epithelial and vascular basement membranes. Collagenase prevented the staining of collagen in (~arnoy-fixed (but caused only slight suppression in formaldehyde-fixed) material. Pectinasc treatment resulted in somewhat reduced fluorescence of mucus and collagen and, like acetylation, caused some nuclei to fluoresce with FLA. This induced nuclear fluorescence was most conspicuous in the liver, which normally showed only a weak positive reaction in the capsular connective tissue and basement membrane. The conjugate of aprot.inin with rhodamine B displayed staining properties (Fig. 4) identical with those of the conjugate with fluorescein, except t h a t the emitted light, was orange instead of yellow. The staining of tissues by other methods is summarised in 'Fable 1. Discussion
Hi,stochemistry. I t can be seen from 'Fable 1 that., in general, the structures which fluoresced after treatment with FLA wcre those which also stained with Alciar~ blue 8GX at. p H 2.5. However, the intensity of fluorescence did not always parallel the depth of staining with Alcian blue. All metachromatic components of tissues gave particularly intense fluorescence with FLA. Fluorescence occurred in m a n y tissue elements which were PAS-positive and in some which were PAS-negative. Only glycogen could be detected by the P A t method but not with FLA. Fluorescein-labclled concanavalin A combines with polysaccharidcs containing hexose residues in the ~-D-arabino configuration and parallels, in general, staining by the P A t method (Stoddart and Kiernan, 1973a). The control procedures indicated t h a t FLA had certain properties in common with cationic dyes, in that staining could be abolished by prior " m e t h y l a t i o n " of the sections and was less intense in solutions of high ionic strength. FLA differs from Alcian blue and other basic dyes in t h a t fluorochroming is optimal at p H 6.0 and is possible on the alkaline side of neutrality. Alcian blue at p I I 2.5 stains polysaccharides which owe their acidity to both carboxyl and snlphate groups, while at. p i t 1.0, only the sulphated mucosubstanccs are alcianophilic (Quintarelli, Scott and Dellovo, 1964). The sulphated muco-
Fluorescent-LabelledAprotinin
83
substances, however, also contain carboxyl groups so it is not possible on the basis of the present observations to state that FLA does not react with sulphated as well as with carboxylated polysaccharides. However, tests in vitro by Stoddart and Kiernan (1973 b) have indicated that FLA cannot combine with carbohydrates such as dextran sulphate which owe their acidity exclusively to sulphate radicals. Neither will FLA stain acidic proteins or nucleic acids. The staining of nuclei by FLA after acetylation (which blocks amino and hydroxyl groups) remains to be explained. It is conceivable that acylation of the basic proteins of the nucleus, such as histones, might unmask the presence of acidic glycoproteins stainable with FLA. The partial suppression of FLA stainability of nuclei by treatment of acetylated sections with deoxyribonuelease could indicate an association between DNA and such acidic glycoproteins. The fluorescent staining of collagen matched in intensity the fluorescence after staining with fluorescein-labelled concanavalin A, a reagent which stains mucosubstances containing glucosyl and man~osyl residues. Acidic sugars associated with collagen fibres include D-glucuronic acid of chondroitin sulphates A and C, i-iduronic acid of chondroitin sulphate B and sialic acids of glycoproteins (Brimacombe and Webber, 1964) which are presumably responsible for the affinity of FLA for collagen-rich regions. The granules of mast cells are basophilic and metachromatic owing to their content of heparin, a polysaccharide containing both carboxyl and sulphate radicals (Brimacombo and Wobber, 1964). However, these granules also contain proteolytie enzymes resembling trypsin and chymotrypsin (Lagunoff, 1968), and it is possible that in this instance the fluorescent staining may partially represent attachment of aprotinin to a pretense. It seems unlikely that the enzymes would remain active after alcoholic dehydration and paraffin embedding, though they might still have intact binding properties. The antiproteolytic potency of aprotinin has been shown to be unaltered by conjugation with fluorescein isothiocyanate (Stoddart and Kiernan, 1973b). The process of histochemieal staining by FLA is reminiscent of that by labelled concanavalin A, a lectin (carbohydrate-binding protein of plant origin) which attaches specifically to certain hexose residues (see Stoddart and Kiernan, 1973a). It may be that aprotinin behaves as a leetin-like substance of animal origin, with specificity for carboxylie acid-containing polysaccharides. Pharmacological Implications. Aprotinin combines with and inhibits enzymes in vivo as well as in vitro, and has been used therapeutically in acute pancroatitis (Trapnell, 1967) and certain other conditions due to over-activity of susceptible pretenses (Kaller, 1967). However, despite many years of clinical experience, the efficacy of the drug is still not proven. One reason for the failure of aprotinin to gain widespread clinical approval is probably the necessity to use very large doses of an expensive product. Large doses are required both to maintain adequate circulating levels and to compensate for the rapid metabolism and excretion (half-life 30-60 m after intravenous injection) of the drug (Vogel et al., 1968). If aprotinin itself, like FLA, is bound by acidic sugar residues, it is possible that the low circulating levels may be partially a consequence of binding of the inhibitor to such widely distributed extracellular mucosubstancos as hyaluronic acid and the glycoproteins of connective tissue. If much of the aprotimn were sequestered in 6*
84
J.A. Kicrnan and t~. W. Sto(tdart: Fluorescent-Labelled Aprotinin
connective tissue, t h e n less would be available to i n h i b i t enzymes at sites of disease. The above suggestion could be tested b y t r a c i n g a n d comparing the fates of labelled a n d unlabelled a p r o t i n i n in vivo.
Ac~mowled.qements. We are grateful to Mr. d. S. Dixon of the MedieM Department, Bayer Pharmaceuticals Ltd, Haywards Heath, Sussex, for generous gifts of Trasylol, from which we prepared the fluorescent conjugates. Our thanks are also due to Mr. J. Bashford and Miss Christine Golding of the Anatomy Department, Cambridge University, for assistance with photography and histological preparation. R.W.S. thanks the Cancer Research Campaign for a grant.
References Brimacombc, J.S., Webber, J.M.: Mucopolysaccharides. Amsterdam-Lnndon-NewYork: Elsevier 1964. Nailer, H.: Pharmacology of Trasylol. In: New aspects of Trasylol therapy, Book 2, edit,. I:L.Marx, H. lmdahl aml G. L. Haberland, p. 1-8. Stuttgart: Sehattauer 1967. Lagunoff, D.: The properties of mast call prot.eascs. Bioehem. Pharmaeol. 17 Suppl., 221-227 (1968). Pearse, A. G. E.: Histochemistry; theoretical and applied, third edit., vol. 1. Edinburgh: Churchill 1968. Quintarelli, G., Scott, J. E., Dellovo, M.C.: The chemical and histochemieal properties of Alcian blue. lI. Dye binding of tissue polyanions, tfis~ochemie 4, 86 98 (1964). Sorvari, T. E., Stoward, 13. J. : Some investigations of the so-called "methylation" reactions used in mucosubstance histochemistry. I. "Methylation" with methyl iodide, diazomethane, and various organic solvents containing either hydrogen chloride or thionyl chloride. Histochemie 24, 106 113 (1970). Stoddart, l~. W., Kiernan, J.A.: Histoehemical detection of the ~-D-arabinopyranoside configuration using fluorescent-labelled concanavalin A. Histoehemie 88, 87 94 (1973). Stoddart, R. W., Kicrnan, J. A. : Aprotinin : a carbohydrate-binding protein. Histochemie (in press) (1973b). Thompson, S.W.: Selected histochemieM and histopathologieM methods. Springfield, Ill.: Thomas 1966. Trapnell, J. E.: Trasylol in pancrcatitis a clinical review. In: Proceedings of a Symposium on Proteinase Inhibition in Medicine and Surgery. FBA Pharmaceuticals Ltd., Haywards Heath, p. 36~tl (1967). Vogel, I~., Trautschold, I., Werle, E.: Natural proteinase inhibitors. Second (English) edit. NewYork and London: Aer Press 1968. Dr. R, W. Stoddart Strangeways Researcix Laboratory Wort's Causeway Cambridge CBI4RN England
