pancreatic secretory immunoreactivity · 792 the table shows the results of the immuno-...

J Clin Pathol 1986;39:786-793

Pancreatic endoproteases and pancreatic secretorytrypsin inhibitor immunoreactivity in human PanethcellsM BOHE,*$ A BORGSTROM,*T C LINDSTROM,t K OHLSSONIFrom the *Departments ofSurgery, tPathology, and tSurgical Pathophysiology, University ofLund, MalmoGeneral Hospital, Malmo, Sweden

SUMMARY Normal and metaplastic gastrointestinal mucosa obtained at surgical resection werestudied by light microscopy, using the unlabelled antibody enzyme method for immuno-histochemical staining of lysozyme, pancreatic endoproteases, and pancreatic secretory trypsininhibitor (PSTI). Paneth cells in the mucosa ofnormal small intestine, gastric mucosa with intestinalmetaplasia, and colonic metaplastic mucosa were found to contain anionic trypsin, cationic trypsin,lysozyme, and PSTI immunoreactivity, but not chymotrypsin and elastase immunoreactivity.Normal gastric and colonic mucosa and some goblet cells in the small intestine showed positivePSTI immunoreactivity but no endoprotease immunoreactivity. The presence of immunoreactivetrypsin and immunoreactive PSTI in the Paneth cells, which are of secretory type, probably indi-cates an important extrapancreatic source of these proteins rather than a storage of endocytosedmaterial.

The Paneth cells originally described by Schwalbe in18721 and later by Paneth in 18882 are normallyfound at the base of the crypts of Lieberkuhn in theduodenum, jejunum, and ileum. Paneth cells are alsofound in metaplastic areas in gastric or colonicmucosa.3 They are ultrastructurally similar to secre-tory cells and like the pancreatic acinar cells, withapically located large cytoplasmatic granules. Amongproposed properties and functions are the abilities tosecrete immunoglobulins and lysozyme into the gutlumen and to eliminate metals from the mucosa intothe luminal contents.4

Using an immunohistochemical technique, wefound a trypsin like immunoreactivity in the Panethcells in the duodenum, the small intestine, and inmetaplastic areas in the gastric mucosa in man.5 Thepurpose of this investigation was to further character-ise the trypsin like immunoreactivity and to determineif the Paneth cells also contain other pancreatic endo-proteases as well as the pancreatic secretory trypsininhibitor (PSTI).

Accepted for publication 7 January 1985

Material and methods

TISSUE SAMPLESThe material comprised different resection specimenstaken during operation from various types of surgicalcases. The specimens were fixed in 10% buffered for-malin and were imbedded in paraffin. The followingtypes of mucosa were represented: normal mucosafrom stomach, duodenum, jejunum, ileum, and colon;colonic mucosa from cases with ulcerative colitis,containing glands with Paneth cell metaplasia; gastricmucosa of intestinal metaplastic type; and, in addi-tion, colonic adenoma with Paneth cell metaplasia.

SPECIFIC MATERIALDEAE-Sephadex, Sepharose 4B, and prepacked PD-10 columns were obtained from Pharmacia FineChemicals, Uppsala, Sweden. Trasylol was providedby Bayer AG, West Germany. Swine antirabbit IgG,peroxidase and antiperoxidase (PAP), and normalswine serum were obtained from Daco Immu-noglobulins, Copenhagen, Denmark. Di-iso-propyl-fluorophosphate (DFP) was obtained from SigmaChemicals (St Louis, United States of America).

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Trypsin and PSTI in Paneth cells

Pancreatic juice was obtained from patients whohad had the main pancreatic duct drained after pan-creatic surgery. The pancreatic juice was collected intubes on ice and frozen within two hours at -20°C.Human cationic trypsin and human PSTI werepurified at the laboratory.67

ANTISERARabbit antisera against cationic trypsin, chymo-trypsin, elastase 2, and PSTI were produced at thelaboratory.5 78 Specific rabbit antihuman lysozymeserum was obtained from Professor Carl-Bertil Lau-rell, Malm6, Sweden.

AFFINITY PURIFICATION OF ANIONIC TRYPSINON SEPHAROSE CONJUGATED TRASYLOLTrasylol (150 mg) was coupled to 15 g cyanogen bro-mide activated Sepharose 4B, according to the manu-facturer's instructions. Pancreatic juice (50 ml) from apatient with a high concentration of anionic tryp-sinogen and only a small concentration of cationictrypsinogen was applied to the sepharose linked tra-sylol column (2 5 x 10cm), equilibrated with Trishydrochloric acid buffer -0 I M, containing 01Msodium chloride at pH 8. After thorough washingwith the starting buffer, until absorbance at 280 nmreached zero, the anionic trypsinogen was eluted withO-O1M formic acid containing 02M sodium chlorideat pH 3 0. The fractions containing trypsinogen weredesalted immediately on PD- 10 columns, equilibratedwith OOOIM hydrochloric acid, and lyophilised. Thepurified anionic trypsinogen showed only one proteinband on agarose gel electrophoresis at pH 8-6: afterautoactivation this band has a slightly less anionicmobility. The anionic trypsin was inactivated withDFP by adding 1O 4ul DFP (I M/1) in iso-propranol toI ml of anionic trypsin (I mg/ml) in hydrochloric acidOOOIM/l. After the addition of DFP the pH wasadjusted to 7-0 by adding 100pI Tris buffer, IM/l.

Antiserum against human anionic trypsin(ogen)was produced by immunising rabbits with humananionic trypsin inactivated with DFP and emulsifiedwith Freund's adjuvant. Immunoelectrophoresis

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against human pancreatic juice before and after acti-vation showed one strong anionic precipitate, corre-sponding to anionic trypsinogen and trypsin. Some-times a faint precipitate was seen, corresponding tocationic trypsin(ogen), probably indicating a crossreaction between the two trypsins, as has beenreported previously.9

IMMUNOADSORPTION OF RABBIT ANTISERAAGAINST HUMAN ANIONIC AND CATIONICTRY PSINSHuman cationic trypsin (8-8 mg) inactivated by DFPwas conjugated to I g of Sepharose 4B, and 15 mgsimilarly inactivated human anionic trypsin was cou-pled to 1-5 g of Sepharose 4B, according to the manu-facturer's instructions. Two separate columns, 0-9 x7 cm and 0 9 x II cm, were equilibrated in a glycinebuffer, 01 M/l, containing sodium chloride 01 5M/I,ethylene diamino tetra-acetate 0-005M/1, and sodiumnitrate 0 02% at pH 6 8. Rabbit antiserum againsthuman anionic trypsin (25 ml) was applied to thesepharose cationic DFP-trypsin column. The columnwas then washed with the same glycine buffer. Thefractions containing protein were pooled and appliedto the sepharose anionic DFP-trypsin column. Thecolumn was washed with the same glycine buffer untilabsorbance at 280 nm was below 01. Then thespecific antibodies against human anionic trypsinwere eluted with a glycine buffer, 01 M/l, containingsodium chloride 0-1 SM/1, sodium thiocyanate 3 5M/1,ethylene diamino tetra-acetate 0 005M/l, and sodiumnitrate 0 02% at pH 7 3. The absorption at 280 nm ofthe effluent was measured and the fractions contain-ing antibody were pooled and dialysed against water.The antiserum against human cationic trypsin was

processed in the same way; first being filtered throughthe sepharose-anionic DFP-trypsin column, with theeffluent from this column then being applied tothe Sepharose-cationic-DFP-trypsin column. Afterwashing with the starting buffer to an absorption at280 nm of less than 0-1 the specific antibodies againsthuman cationic trypsin were eluted with the bufferdescribed above. The fractions containing antibodies

Table Pancreatic endoproteases and PSTI immunoreactivity in Paneth cells in investigated specimens is'ith positiveimmunohistochemical staining

Anionic trypsin Cationit trypsin PSTI ChYmotrYpsin Elastase

Normal mucosa:Duodenum 414 4/4 4/4 0/4 0/4Jejunum 4/4 4/4 4/4 0/4 0/4Ileum 4/4 4/4 4/4 0/4 0/4

Metaplastic mucosa:Stomach 4/4 4/4 4/4 0/4 0/4Colon (ulcerative colitis) 5/6 5/6 5/6 0/6 0/6Adenoma (Paneth cell metaplasia) 1/1 1/1 1/1 0/1 0/1


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Bohe, Borgstrom, Lindstriim, Ohlsson

A --

B -

+ tFig. 1 Precipitation patterns obtained by immunoelectrophoresis ofpancreatic juice with (A) rabbit anticationic trypsinserum, (B) rabbit antianionic trypsin serum, and (C) rabbit anti-PSTI serum. Arrow denotes application points.

.. ._.,1%

Fig. 2 Normaljejunal mucosa. Lysozyme shown withinPaneth cells by immunohistochemical staining. x 160.

Fig. 3 Normaljejunal mucosa. Complete blocking ofstaining reaction using absorbed antisera against anionictrypsin. x 160.

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Trrpsin anid PSTI in Patinth (cel/s 789

17~~ ~ ~ ~ ~ ~ ~~A

V&V

; .,ri '¶+

cte

*F.ig., t$4.

- F *J:''- i,tL~" v * ,F. 6

Fig.4

Fig. 6

4,v g.wN,cFig. 4 gastric mucosaswithfocalintestinalmetaplasia. Anionic trypsin shown within

Panethcellsby immunohistochemical staining.(Haematoxylin and eosin.) x 160.Fig. 5 Gastricmucosaoffundustype with*;3> f;;- E _- ~~~~~~~~~~~~~~chronic gastritis and severe intestinal

metaplasia. PSTIshownwithinPaneth cells

*mu(arrowhe ) aswemll as in normal glands^s - _ - ~~~~~~~~~~~(Haematoxylin and eosin.) x 160.

w z~~~~~trpi shown within Paneth cellsb

F Nz.S

Fig. 5


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Bohe, Borgstrom, Lindstrom, Ohlsson

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a .%

Fig. 8 Normaljejunal mucosa. Anionic trypsin shown withinPaneth cells by immunohistochemical staining. x 640.

and peroxidase complexes were used in the dilutionsof 1/50. All reagents were applied for 30 minutes atroom temperature in a humidified chamber.

Fig. 7 Normaljejunal mucosa. Cationic trypsin shownwithin Paneth cells by immunohistochemical staining. x 160.

were pooled and dialysed against water.The immunoadsorbed specific rabbit antibodies

against human anionic and cationic trypsins werechecked by immunoelectrophoresis against humanpancreatic juice. The antibodies were used in dilutionsmade from a stock solution of 2 mg/ml.

IMMUNOHISTOCHEMICAL METHODS

The tissue samples were stained with haematoxylinand eosin and analysed with light microscopy. Theperoxidase-antiperoxidase (PAP) method describedby Sternberger et al'0 with the slight modificationalready mentioned," was used for localisation of thedifferent pancreatic endoproteases, PSTI, andlysozyme. The antisera were used in serial dilutions1/100 to 1/2000. Controls were performed with eachnew staining series using the antisera after immuno-sorbent absorption with their appropriate antigensand non-immune rabbit serum. Swine antirabbit IgG

Results

After immunoabsorption of the anionic and cationictrypsin antisera no cross reactivity between them wasseen, and only one distinct precipitation line was seenon immunoelectrophoresis for each antiserum(Fig. 1).

Fig. 9 Normal duodenal mucosa. PSTI shown in Panethcells as well as goblet cells by immunohistochemical staining.(Haematoxylin and eosin.) x 1600.

Fig. 10 Colonic mucosa in a case ofulcerative colitis withintestinal metaplasia containing Paneth cells. Anionic trypsinshown within Paneth cells by immunohistochemical staining.x 160.

Fig. 11 Colonic adenoma containing Paneth cells. Anionictrypsin shown within Paneth cells by immunohistochemicalstaining. (Haematoxylin and eosin.) x 160.

Fig. 12 Colonic adenoma containing Paneth cells. PSTIshown within Paneth cells by immunohistochemical staining.(Haematoxylin and eosin.) x 160.

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Fig. 9 Fig. 11

Fig. 12

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The Table shows the results of the immuno-histochemical staining. The Paneth cells were

identified on the basis of histological criteria and bytheir immunohistochemical staining for lysozyme

(Fig. 2). A positive brown staining for the two tryp-

sins and for PSTI was evident in Paneth cells of vari-ous locations. Control sections with normal rabbitsera or antisera previously absorbed against theirappropriate antigens yielded negative results. (Fig. 3).No pancreatic chymotrypsin or elastase immuno-reactivity could be shown in any control specimen.

STOMACHIn normal gastric mucosa no trypsin immuno-reactivity was identified. In areas of intestinal meta-plasia with Paneth cells a distinct trypsin immuno-reactivity of both anionic and cationic types was

identified in the Paneth cells (Fig. 4). The Paneth cellsalso contained PSTI immunoreactive material (Fig.5). PSTI immunoreactive material was also seen insome areas of normal mucosa.

DUODENUM, JEJUNUM, AND ILEUM

In all specimens analysed a distinct positive anionicand cationic trypsin immunoreactivity was seen in thePaneth cells, both in the basal and apical parts of thecells (Figs. 6, 7, 8). PSTI immunoreactivity was seen

in some Paneth cells and also in goblet cells in thebasal parts of the glandular crypts of Lieberkuhn(Fig. 9).

COLONIn the normal colonic mucosa a positive PSTIimmunoreactivity was seen mainly in the basal part ofthe crypts. In the normal colonic mucosa no Panethcells were identified, and no pancreatic endoproteaseimmunoreactivity was seen. In metaplastic areas ofthe mucosa in cases with ulcerative colitis, containingPaneth cells, positive anionic and cationic trypsin as

well as PSTI immunoreactivity were identified withinthe cells (Fig. 10). In one case of adenoma withPaneth cell metaplasia distinct anionic and cationictrypsin immunoreactivity were identified (Fig. 11).PSTI immunoreactive material was also stored in var-

ious parts of the adenoma (Fig. 12).

Discussion

In a recent study using immunohistochemicalmethods,5 we reported on trypsin like immuno-reactivity in the human Paneth cells in normalmucosa and in metaplastic areas of gastric mucosa. Inthe present study it was necessary to eliminate thecross reacting antibodies from each antiserum as thetwo trypsins show some cross reactivity. This was

accomplished using an immunoabsorption technique

Bohe, Borgstrom, Lindstrom, Ohisson

with the two isolated antigens bound in a solid phase.Applying the respective specific antibodies, weshowed that the trypsin like immunoreactivity in thePaneth cells was of anionic and cationic types. Thepositive brown staining was completely abolished inthe controls using the specific antisera previouslyabsorbed with their appropriate antigens, furtherconfirming the specificity of the reaction for anionictrypsin and cationic trypsin.The Paneth cells of all tissues studied were easily

identified according to their histological criteria andby their immunochemical staining for lysozyme. Inaddition to Paneth cells of metaplastic and normaltypes, immunoreactive PSTI was seen in areas of nor-mal gastric and colonic mucosa and in some gobletcells in the basal parts of the crypts of Lieberkuhn inthe small intestine. No immunoreactive trypsin wasidentified in the goblet cells. Thus immunoreactivetrypsin was always accompanied by immunoreactivePSTI, fitting the general concepts of Laskowsky:'2tissues that contain a protease also always contain theappropriate inhibitor. Further direct isolation andcharacterisation of these immunoreactivities willprobably clarify the presence of PSTI in some cellswithout apparent trypsin content.A common observation in published reports is the

ultrastructural resemblance between Paneth cells andthe acinar pancreatic cells.4 Several findings alsopoint to a functional resemblance. Paneth cells are ofsecretory type. Pilocarpine has been shown toincrease the secretion from these cells.'3 Trasylol, apotent trypsin inhibitor, increases the granule size inthe Paneth cells.i4 Studies in hamsters have shownthat pancreatic duct ligation was followed by degen-eration of exocrine pancreas and caused a hyper-trophy of Paneth and goblet cells."5 The duodenalmucosa in patients suffering from chronic pancreatitisshowed an appreciable increase in the number ofPaneth cells.'6 These data, as well as our proof thatboth trypsin immunoreactive material and PSTIimmunoreactive material are present in the same typeof cells, argue the case for a production of trypsin andPSTI in the Paneth cells. The findings of both immu-noreactive anionic and cationic trypsin, as well astheir appropriate inhibitor PSTI in Paneth cells, froma physiological and pathophysiological point of view,show that the Paneth cells are probably an importantextrapancreatic source of these enzymes and give afurther indication of the resemblance between Panethcells and acinar pancreatic cells.

This investigation was supported by grants from theSwedish Medical Research Council (project noB86-17X-0390-14A), the Medical Faculty, Universityof Lund, and Malmo General Hospital Foundationagainst Cancer.


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References

1Schwalbe G. Beitrage zur Kenntnis der Drusen in der Darmwan-dungen, in's Besondere der Brunner'schen Drusen. Archiv furMikroskepische Anatomie 1872;8:92-140.

2Paneth J. Ober die secernierenden Zellen des Dunndarm-Epithels.Archiv fur Mikroskepische Anatomie 1888;31:113-91.

'Lewin K. The Paneth cell in disease. Gut 1969;10:804- 11.4Sandow M, Whitehead R. The Paneth cell. Gut 1979;20:420-31.'Bohe M, Borgstrom A, Lindstrom C, Ohlsson K. Trypsin-like

immunoreactivity in human Paneth cells. Digestion 1984;30:271-5.

6Ohlsson K, Skude G. Demonstration and semiquantitative deter-mination of complexes between various proteases and humanM2-macroglobulin. Clin Chim Acta 1976:66:1-7.

'Eddeland A, Ohisson K. Purification and immunochemical quan-titation of human pancreatic secretory trypsin inhibitor. Scand JClin Lab Invest 1978;38:261-7.

8Bohe M, Borgstr6m A, Genell S, Ohlsson K. Determination ofimmunoreactive trypsin, pancreatic elastase and chymotrypsinin extracts of human faeces and ileostomy drainage. Digestion1983;27:8-15.

9Feinstein G, Hofstein R, Koifmann J, Sokolovsky M. Human pan-creatic proteolytic enzymes and protein inhibitors. Isolation andmolecular properties. Eur J Biochem 1974;43:569-8 1.

'0 Sternberger LA, Hardy PH, Cuculis JJ, Meyer HG. The unlabeledantibody enzyme method of immunohistochemistry. Prepara-

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antibody enzyme method of immunohistochemistry. Prepara-tions and properties of soluble antigen-antibody complex(horseradish peroxidase-antihorseradish peroxidase) and its usein identification of spirochetes. J Histochem Cytochem 1970;18:315-33.

"Fryksmark U, Ohisson K, Polling A, Tegner H. Distribution ofantileukoprotease in upper respiratory mucosa. Ann Otol RhinolLaryngol 1982;91:268-78.

12 Laskowsky JRM. Resume in proteinase inhibitors. Fritz, TschescheH, Greene L, Truscheit E, eds. Springer-Verlag, 1974: 679-84.

13Trier JS, Lorenzsonn V, Groehler K. Pattern of secretion ofPaneth cells of the small intestine of mice. Gastroenterologi'1967;53:240-9.

'4Ahonen A, Penttila A. Effect of Trasylol on Paneth cells of themouse. Erperientia 1975;31:577-8.

s Balas D, Senegas-Balas F, Bertrand C, Frexinos J, Ribet A. Effectsof pancreatic duct ligation of the hamster intestinal mucosa.Digestion 1980;20:157-67.

16Senegas-Balas F, Bastie MJ, Balas D, et al. Histological variationsof the duodenal mucosa in chronic human pancreatitis. DigDis Sci 1982;27:917-22.

Requests for reprints to: Dr Mans Bohe, Department ofSurgery, Malm6 General Hospital, S-21401 Malmo,Sweden.


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