an immunocytoghemical study on secretory mechanism of iga in human pancreas

Acta Pathol. Jpn. 35/11 : 87-101, 1985

AN IMMUNOCYTOCHEMICAL STUDY ON SECRETORY MECHANISM OF IgA

IN HUMAN PANCREAS

Hideki SAITO, Takeshi KASAJIMA*, and Hiroshi NAQURA* *

The Second Department of Internal Medicine, School of Medicine, Yamagata University, Yamagata

‘The Smnd Department of Pathology, School of Medicine, Yamagata University, Yamagata

a * Laboratory of Germfree Life Research, Institute for Diseuse Mechanism and Control, School of Medicine,

Nagoya University, Nagoya

To analyze the secretory mechanism of immunoglobulins into pancreatic secretion, pancreatic juice IgA was measured by single radial immunodiffusion and characterized immunochemically, and immunoglobulins, J chain and secretory component (SC) were localized immunocytochemically in pancreatic tissues by the peroxidase-labeled antibody method. The concentration of IgA was 0.0225 mg/ml with a range of 0.010 to 0.049. IgA associated with SC was demonstrated immunochemically in three cases. IgA, SC and J chain were demonstrated immunocytochemically in the pancreatic duct epithelium with the features characteristic of endocytic, SC-mediated transfer of IgA. The results of the present study suggested that transfer of IgA linked by J chain and SC across ductal epithelial cells, but not acinar cells occurred in human pancreas. ACTA PATHOL. JPN. 35 : 87- 101, 1985.

Introduction It is well known that secretory immunoglobulin A (sIgA) is the most predominant

class of immunoglobulins present in certain external ~ e c r e t i o n s . ~ ~ * ~ ~ Moreover, sIgA plays the most important role of immunological reactions on the mucous membranes. That is, sIgA has a defense mechanism against a “hostile” environment which includes inflammatory agents other pathologic factors existing in external secretions and on mucosal surfaces.20 sIgA consists of two molecules of IgA joined by the peptide J chain and a glycopeptide secretory component ( SC).2*20,26.40*44 Recent immunocytochemical studies on several mucosa, especially on the i n t e ~ t i n e , ~ , ~ ~ biliary and salivary glands,37 have defined many steps in assembly and transfer of sIgA into

Accepted for publication April 3, 1984.

Mailing address: Hideki SAITO, The Second Department of Pathology, School of Medicine, Yamagata University, Yamagata 990-23, JAPAN.

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HX IgA IN HUMAN PANCREAS Acta Pathol. Jpn.

external fluid. In gut mucosa, the IgA molecule and J chain are synthesized in plasma cells in the lamina propria. On the other hand, SC is synthesized in the glandular epithelial cell^,^.^^ and is located on the baso-lateral surfaces of the epithelial cells. SC has a function as a receptor for the J chain-linked IgA dimer.6*9s33 SC-IgA complexes are endocytosed and transported through the cytoplasm to the luminal s ~ r f a c e . ~ , ~ ~

Recently, there have been several descriptions on the biochemical analysis of immunoglobulins in human pancreatic juice with or without pancreatic d i ~ ~ r d e r ~ . ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ These studies have demonstrated that human pancreatic juice contained detectable levels of IgA, IgM, and IgG. SUWAKI detected SC in pancreatic juice and reported that the greater amount of SC in pancreatic juice was bound to IgA dimer ( s I ~ A ) . ~ ' This suggests that secretory immunity plays an important role in pancreas, as well as in the intestine and biliary tracts.

However, there are very few reports on immunological studies regarding localization of immunoglobulins in human pancreas. TOURVILLE et al . failed to demonstrate IgA, IgM, and IgG using the immunofluorescent technique in the glandular and ductal epithelial cells in which SC was ob~erved.'~ On the other hand, NAGURA et al. demonstrated the localization of IgA and SC in the ductal epithelial cells of pancreas obtained from patients with ~holelithiasis.~~

The purpose of this paper is to confirm whether sIgA is present in human pancreatic juice or not, and to search the localization of SC, IgA, IgM, IgG, and J chain in surgical specimens of human pancreas by the peroxidase-labeled antibody method at the light and electron microscopic levels. Moreover, the mode of cellular pathway of immunoglobulins into pancreatic juice was investigated and discussed.

Materials and Method

1. Immunochemical study of pancreatic juice IgA Pancreatic juice was collected from four patients with secretin stimulation a t the time of

endoscopic retrograde cholangiopancreatography (ERCP) and was also obtained surgically from one patient who underwent pancreatoduodenotomy due to gastric cancer. For the qualitative and quantitative analysis of IgA and SC, pancreatic juice was concentrated by vacuum dialysis until a 10 to 50-fold concentration was obtained. Next, the concentrated juice was tested by double immunodiffusion and immunoelectrophoresis for IgA and for the possible association of IgA with SC. Antibody wells or troughs were filled with either gammaglobulin fractions of commercial rabbit anti- human SC, IgA, IgM IgG (Calbiochem, Behringer Corp., San Diego, CA, and/or DAKOPATTS a/s Denmark). IgA and SC were measured by single radial immunodiffusion technique following MancinLz8 Purified human myeloma 7 S IgA, human colostral 11 S IgA and SC prepared in our laboratory were used as standard. 2. Immunocytochemical study on human pancreatic tissues

Tissue specimens : Pancreatic tissues were obtained from eight patients by surgical removal during operation of gastric cancer without pancreatic disorders.

Antibody : Rabbit anti-human SC, EgA, IgM, and Igcf were purchased from Behringer-Hoechst and/or DAKOPATTS. Antisera for the J chain were prepared by hyper-immunization of rabbits with purified pr~te in .~ ' Antibody specificity was certified by immunoelectrophoresis and immunodiffusion in agarose gel. Fab or Fab' fragments of gammaglobulins were labeled with horseradish peroxidase (HRP) following Wilson and Nakane's method.46 For use on control experiments, the Fab' fragmenb

35(1) : 1985 H. SAITO, T . KASAJIMA, AND H. NAQURA 89

of non-immune rabbit gammaglobulins were also labeled with HRP. The fresh tissue specimens were immediately h e d in periodate-lysin-4yo

paraformaldehyde (4yoPLP)29 for 6 hours at 4°C and were washed in increasing concentrations of sucrose in PBS. The specimens were frozen in OCT compound (Lab Tek Products, Naperville 11. U. S.A.) and were cut 6 micron thick with a cryostat, placed on glass slides coated with egg albumin and rapidly dried in room air. Sections for light microscopy were treated with 0.005 M periodic acid and 0.003 M sodium borohydride in order to inactivate endogenous peroxidse.zz After the inactivation, the sections were immersed in 10% non-immune rabbit serum, and reacted with the HRP-labeled antibodies for 12 hours a t 4°C. Control sections were treated either with the HRP-Fab fragmenb of non-immune rabbit gammaglobulins of with conjugated antibodies after absorption with appropriate antigens. After washing with PBS, the sections were reacted with Karnovski’s 3-3 diaminobenzidin (DAB) solution containing 0.01 M sodium azide and then counterstained with methyl green.

For electron microscopic observation, the staining process was almost the same as that for light microscopy, except for the treatment with periodic acid and sodium borohydride. After reaction with HRP-antibodies, the sections were refixed with 1% glutaraldehyde in PBS and incubated sequentially in DAB solution without hydrogen peroxide containing 0.01 M sodium azide with 1% dimethyl sulfoxide (DMSO), and then in the complete Karnovski’s DAB solution containing hydrogen peroxide. After specific staining was confirmed, the sections were fixed in 2% osmium tetraoxide in 0.1 M phosphate buffer, dehydrated in a graded alcohol, and embedded in Quetol 812. Ultrathin sections were observed with a Hitachi HS-9 electron microscope.

In addition, for the detection of J-chain whose antigenic determinants were masked in IgA or IgM, cryostat sections were treated with 6 M urea in glycine-HCI buffer (pH 3.2) for 20 hours at 4°C before the immuno~taining.~~

Immunocytochemisty :

Results Immunochemical study of pancreatic juice.

Single radial immunodiffusion demonstrated that pancreatic juice from each patient contained detectable levels of IgA. The concentration of IgA in the five pancreatic juice samples was 0.010-0.049 mg/ml (mean f standard deviation : 0.0225 f

Table 1. IgA and SC Content of Human Pancreatic Juice

Patient Age Sex No. Diagnosis’

1 60 M Chr. pancreatitis



(suspected)

(suspected)

0.0135 ND

0.010 0.96

0.016 1.50 4 54 F Pancreatic carcinoma 0.024 ND 5 70 F Nomal pancreas 0.049 1.50

(Catheterization after pancreatoduodenotomy for gastric cancer)

ND : not done * : The diagnostic criteria were : ( 1 ) a distinct picture of chronic panmeatitis on

histological examination (2) a definite calcific density in the pancreas in the X-ray examination, and (3) a marked decrease in pancreatic exocrine function. Clinically established chronic pancreatitis satisfied these criteria : cases falling short of these criteria were designated as suspected chronic pancreatitis.

90 IgA IN HUMAN PANCREAS Acta Pathol. Jpn.

0.031). SC was detected in pancreatic juice from patients 2, 3, and 5 (9.6-15.0 pg/ml) (Table 1).

Double gel diffusion studies in pancreatic juice from patients 2, 3 and 5 also showed detectable IgA and SC (Fig. la). The precipitin pattern suggested the exis- tence of IgA associated with SC. IgG also was detectable, but IgM was virtually undetectable with this technique (Fig. lb).

Immunocytochemist y . Light microscopy : There were almost the same findings for eight cases of

pancreatic tissues at the light microscopic level. SC was exclusively detected in epithelial cells lining the large and small interlobular ducts, especially located along the lateral cell margin (Fig. 2a). Faint immunoreactivity for SC was observed in some epithelial cells of intralobular ducts and in some centroacinar cells. On the other hand, no SC was found in acinar and islet cells (Fig. 2a). IgA was also seen in the epithelial cells lining the interlobular ducts, principally along the lateral cell margins, similar to SC (Figs. 2 a and 2 b). Only a few plasma cells containing IgA and none containing IgG and IgM were detected in the interstitium. In additon, IgA was stained extracellularly in the interstitium and in blood capillaries (Fig. 2b). J chain could be detected in ductal epithelial cells after acid-urea treatment similar to

Fig. la. Double immunodiffusion analysis of pancreatic juice from patient 2, 3 (wells 2, 3). a : anti-IgA, b : anti-SC, c : control (myeloma 75 IgA), S : normal human serum. Precipitin patterns indicate the presence of IgA associated SC in pancreatic juice. lb. Double immunodiffusion analysis of pancreatic juice from patient 5 to each class of immunoglobulin and SC. 5 : Pancreatic juice from patient 5, SC : anti-human SC, sA : anti-human sIgA (anti-human colostrum 11s IgA), A : anti-human IgA, M: anti-human IgM, G : anti-human IgG. Precipitin patterns indicate the presence of IgA (sIgA), IgM and SC. Fuse line between IgA (sIgA) and SC suggests that IgA in pancreatic juice is associated with SC (sIgA).

35(1) : 1985 H. SAITO, T. KASAJIMA, AND H. NAGIJRA 91

IgA. Intensity of positive staining of J chain in the interstitium and blood capillaries was increased after acid-urea treatment (Figs. 2 c, 2 d).

IgG and IgM were hardly seen in ductal and acinar epithelial cells, but sometimes were faintly identified in the interstitium and along the luminal surfaces of the capillary endothelium in addition to IgA and J chain (Fig.2). No reaction w&s observed in the sections treated with HRP-labeled non-immune rabbit gamma-

Fig. 2. Light micrograph of human pancreas reacted with peroxidase (HRP)-labeled anti-SC

Reaction products of diaminobenzidine indicating the site of SC are present in ductal

IgA is present along the margins of ductal epithelial cells, in connective tissue and

J chain staining is faint in connective tissues and blood capillaries of the interstitiurn. In the section treated with acid-urea, J chain appears on the margins of ductal

[a], anti-IgA [b] and anti-J chain [c, d] antibodies. 2a. epithelial cells of small interlobular duct (arrow) and intralobular duct (arrow head) 2b. blood capillaries of the interstitium. 2c. 2d. epithelial cells (arrow). x 300.

IgA IN HUMAN PANCREAS Actu Pathol. Jpn.

35(1) : 1985 H. SAITO, T. KASAJIMA, AND H. NAGURA 93

Fig. 4. Terminal portion of pancreatic duct system reacted with HRP-anti-SC. SC is present on lateral plasma membranes of intralobular duct epithelial cells (arrow) and centroacinsr cells (CA). Note no SC is identified in acinar cells (AC). ~ 3 , 3 0 0 .

globulins or in those treated with HRP-labeled antibodies absorbed by the appropriate antigens.

Electron microscopy : Immunoelectron microscopy of eight specimens supported the findings of the light microscopic findings. In the epithelial cells lining the interlobular ducfs, SC was identified in association with baso-lateral plasma membranes, endocytotic invaginations, and cytoplasmic vesicles. In addition, SC was localized in the protein synthetic organelles, such as perinuclear spaces, rough endoplasmic reticulum and Golgi apparatuses (Fig. 3). Furthermore, in a few epithelial cells of the intralobular ducts and centroacinar cells, in which faint staining of SC was observed by the light microscopic immunocytochemist ry, SC was identified along the external surfaces of the basolateral plasma membranes and in part of perinuclew spaces (Fig. 4). No SC was detected in the cytoplasm of the acinar cells or in islet cells.

Fig. 3. Electron micrograph of epithelial cells of the interlobular duct [a], supranuclear region [b, c] of duct epithelial cells reacted with HRP-anti-SC. SC is present in perinuclesr spaces (PN), cisternae of the endoplwmic reticulum (rER), the Golgi complexes (a), the cytoplasmic vesicles (arrow) and on lateral plasma membranes (LPM). In small interlobular duct epithelium, SC is also present on apical plasma membranes (APM) and ductal lumen. 3a. ~ 5 , 8 0 0 , 3b. ~12,500, 3c. x14,OOO.


Fig. 6. Electron micrograph of interlobular duct epithelial cells reacted with HRP-anti-J chain [a], HRP-anti-J chain after acid-urea treatment [b] and HRP-anti-IgM [c]. 6a, b. Epithelial cells exhibit no evidence of J chains when the acid-urea treatment is omitted, although J chains are present on lateral plasma membranes (arrow) and cytoplasmic vesicles (V) after the acid-urea treatment. 6c. Note small amount of IgM detected on lateral plasma membranes (arrow) of interlobular duct epithelial cells.

~9,000.

x 10,700.

Like SC, IgA was detected along the external surfaces of the basolateral plasma membranes and in the endocytotic invaginations of ductal epithelial cells (Fig. 5). In the apical portion of the cytoplasm, there were several IgA-containing vesicles which occasionally appeared to be lying near the apical plasma membrane and at the base of microvilli, and open into the lumen (Fig. 5b). However, there was no evidence of the

Fig. 5. Electron micrograph of interlobular duct epithelial cells [a, b] and capillary vessels in the interstitium [c] reacted with HRP-anti-IgA. 5a. IgA is present on lateral plasma membranes (arrow) and cytoplasmic vesicles (V). x 7,500. 5b. In apical portion, IgA is present in cytoplasmic vesicles which appeared to be lying right beneath the apical membrane between bases of microvilli (V), and opening into the lumen (arrow head). Note no IgA extending apically beyond the tight junction (arrow). x 20,000. 5c. IgA is also present within the lumen of blood capillaries in interstitium, in endocytic vesicles of the endothelial cells (V), and in the intercellular spaces between them (arrow). x 9,300.

96 IgA IN HUMAN P A N C R E A S Acta Pathol. Jpn.

presence of IgA on the lateral plasma membrane beyond the tight junctions (Fig. 5b). IgA was not present in the protein synthetic organelles of ductal epithelial cells. IgA was detected on the basement membrane of the epithelial cells and on collagen fibers of the interstitium. Moreover, i t was also present within the lumen of the periacinar and periductal capillaries, in endocytotic vesicles of endothelial cells and the intercellular spaces betweem them (Fig. 5c). IgA was not detected in the cytoplasm of either acinar cells or islet cells.

Occasionally, a small amount of IgM was associated with the ductal cells as in the case of IgA (Fig. 6c). The ultrastructural localization of J chain in ductal epithelial cells after acid-urea treab ment mostly corresponded to that of IgA (Figs. 6a, b). IgM and IgG as well as IgA and J chain were located on the luminal surface and in micropinocytotic vesicles of capillary endothelial cells. IgM and IgG were also found as diffusely distributed patterns through the connective tissue between the capillary basement membrane and the basis of adjacent ductal epithelial cells.

However, no IgG was observed in these epithelial cells.

Discussion The present study confirmed immunochemically that human pancreatic juice

contained detectable levels of sIgA, and also showed immunocytochemically that human pancreas had the SC-mediated transport mechanism of IgA through the ductal epithelial cells.

Our immunochemical analysis ascertained that most of the IgA in human pancreatic juice linked SC and the antigenic configuration was characteristically not different from that of sIgA. SUWAKI has demonstrated with gel filtration column chromatogra- phy that the major part of SC in pancreatic juice was bound to IgA dimer." This result coincided with that of our study. BRASHER et aL3 reported that the molecular weight of IgA in human pancreatic juice was determined to be approximately 155,000 daltons and SC was absent from human pancreatic juice. It might be possible that IgA monomer detected in that report was derived from fragments of denaturated sIgA in pancreatic juice or from serum IgA intermingled with pancreatic juice, during collection, storing, and analysis.

Quantitative values of pancreatic juice IgA were widely variable, ranging from 0. 010 to 0.049 mg/ml. CLEMENTE and SARLES found an increased level of immunoglobulins in pancreatic juice of patients with chronic pancreatiti~.~ Moreover, SOTO et aL40 and GOODALE et aLL5 pointed out the high concentration of immunoglobulins recognized in pancreatic juice of pancreatic cancer patients. These results are in agreement with variations in the IgA level in the present study, because the pancreatic juice samples in the present study were collected from patients with and without pancreatic disorders. This means that the IgA level in the pancreatic juice may vary depending on the condition of the pancreas, and the variation in the IgA level is a possible evidence that IgA in the pancreas plays an important role in the occurrence and development of various pancreatic diseases.

35(1) : 1985 H. SAITO, T. KASAJIMA, AND H. NAOURA 97

The immunocytochemical findings suggested that J chain-linked IgA dimer is transported into pancreatic juice by SC-mediated endocytotic transfer through ductal epithelial cells of the human pancreas, similar to the process in the intestine, biliary tracts, and salivary glands. In many reports the cellular pathway of IgA translocation has been described electron-microscopically in detai1.5.33.35-37 SC was idetified only in ductal epithelial cells, in agreement with an immunofluorescence study by TOURVILLE et aZ.45 The electron microscopical findings regarding SC in protein synthetic organelles, namely perinuclear spaces, rough endoplasmic reticulum, and Golgi complexes of the ductal epithelial cells, provide the evidence that these cells are responsible for synthesis of SC. SC is also localized on the external surfaces of the basolateral plasma membranes and cytoplasmic vesicles where IgA and J chain are found. An extensive parallelism between the location of SC and IgA linked by J chain would lead to the conclusion that the SC-mediated transport of IgA occurred through ductal epithelial cells. However, the acinar cells and islet cells of Langerhans do not participate in IgA transport, in contrast to the serous acinar cells of salivary

TOURVILLE et aZ., using immunofluorescence technique, reported no localization of IgA or of IgM and IgG in ductal and acinar cells of the human pancrew. Although, at the light microscopic level, we also found that IgA was not detected in some ductal epithelial cells where SC was present, small amount of IgA was detected in the cytoplasm or basolateral plasma membranes on our careful electron microscopical observation.

The present authors could clarify the localization of SC in some part of the perinuclear spaces and basolateral plasma membranes of a few centroacinar cells and intralobular duct epithelial cells as well as in interlobulax duct epithelial cells. It is reasonable, therefore, that centroacinar cells functionally belong to the duct system of the pancreas, because it produces SC. On the other hand, a decrease in the number of SC-positive cells and faint stainablity of SC in centroacinar cells and intralobular duct epithelial cells could be caused by the influence of the shift process during functional differentiation of the duct system of the pancreas. Micropuncture analysis of the pancreas showed that the terminal portion of the duct system could secrete electrolyte^.^^ KODAMA demonstrated that centroacinar cells of the human pancreas had numerous mitochondria and also suggested that the mitochondrion-rich centroacinar cells were the best candidate for bicarbonate s e c r e t ~ r . ~ ~ These findings of a considerably high developmental potentiality of the terminal portion of the human pancreatic duct system support our immunocytochemical observation.

A definite source of IgA in human pancreatic juice has not been established. From the present study, as from previous rep0rts,3~ it is in doubt to consider that the pancreas is the main source of pancreatic juice IgA, because only a few IgA containing plasma cells are present in non-pathological pancreatic tissues. Therefore, IgA from the circulation must account for the source of pancreatic juice IgA. We observed that IgA and J chain were found in the spaces between endothelial cells of blood capillaries, in the endocytotic vesicles of these cells and in the connective tissue between capil-

98 IgA I N HUMAN PANCREAS Acta Pathol. Jpn.

laries and ductal epithelial cells. Several reports supported the selective transfer of serum IgA to bile, milk, and saliva.10,13.16,18,19,23,30,46 We suggest that the exocrine pancreas can also function to clear IgA polymer linked by J chain from circulation, like the liver, mammary, and salivary glands do. However, there have been no reports about these phenomena concerning the pancreas.

The finding of a small amount of IgM associated with ductal epithelial cells is consistent with the evidence that IgM pentamer linked J chain transported in associatiation with SC through the intestinal and biliary e p i t h e l i ~ m , ~ . ~ ~ and that pancreatic juice has a detectable level of IgM.3,8,11*14.38139*40 The absence of detectable amounts of IgG associated with SC-positive ductal epithelial cells suggests that transport of IgG into pancreatic juice could occur by other mechanisms. This is consistent with data showing that IgG enters into external fluid mainly by t ran~udat ion.~~ NAGURA et ~ 1 . ~ ~ presumed that intestinal epithelial transmission of IgG depended mainly on passive leakage through the epithelial cells. It is possible that this phenomena could occur in the pancreas.

Irrespective of the mechanism by which IgA reaches pancreatic juice, its presence in pancreatic juice is, probably, biologically important. The pancreatic juice IgA might contribute to the defense mechanism of the gut and pancreato-biliary tract in collaboration with intestinal and bile IgA,7,27 and the pancreas might function to compensate for the liver, which functions to clear circulating IgA dimer, namely “the liver as an IgA sc~venger”.~~ Furthermore, a recent biochemical analysis of pancreatic juice immunoglobulins has pointed out the participation of immunoglobulins including IgA in the occurrence and development of pancreatic diseases. Therefore, further informations on the relation between pancreatic diseases and pancreatic juice IgA, and also on the localization of IgA, IgM, and IgG in diseased pancreatic tissues is urgently needed.

Ackowledgements : The authors wish to express thanks to Professor M. ISHIKAWA, The Second Department of Internal Medicine, Professor Y. IMAI, The Second Department of Pathology, School of Medicine, Yamagata University, and Professor K. WATANABE, Department of Pathology, School of Medicine, Tokai University and Dr. T. WATANABE, Dr. H. HASEGAWA, Dr. N. KOMATSU, and the other staff members, Cell Biology Laboratory, School of Medicine, Tokai University, for their excellent and valuable advices. We are also indebted to Professor 0. TSUKAMOTO and his staff, The First Department of Surgery, School of Medicine, Yamagata University, and Dr. M. CHIBA, The Surgical Department, Yamagata Prefectual Kahoku Hospital, for their willing consistent to use the pancreas specimens.

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an immunocytoghemical study on secretory mechanism of iga in human pancreas

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