Lactoferrin and lysozyme in the intrahepatic bile duct of normal livers and hepatolithiasis

Journal of Hepatology, 1992; 15: 147-153 @ 1992 Elsevier Science Publishers B.V. All rights

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147

HEPAT 01055

actoferri

Katsuhiko

Saito and Yasuni Nakanuma

Second Department of Pathology, Kaltazawa Universiy School of Medicine, Kana:abra, Japan (Received 18 May 1991)

Lactoferrin and lysozyme have bactericidal activities and are responsible for mucosal defense against local bacterial infections. To assess the local defense mechanisms in the intrahepatic biliary tree. we studied the distribution of lactoferrin and lysozyme immunohistochemically in 14 normal autopsy livers and in 29 surgically resected and two autopsy livers of hepatolithiasis. In the latter, bacterial infection was constantly found. Lactoferrin and lysozyme were detected in low doses and in specific areas in the intramural and extramural glands of certain normal livers. In contrast, in hepatolithiasis, the incidence of lactoferrin- and lysozyme-positive cases significantly increased both in the intramural glands (94% and 77% of 31 cases, respectively) and in the extramural glands (72% and 48% of 29 cases, respectively) @ < 0.01) in the stone-containing bile ducts. These glands proliferated considerably in the stone-containing bile ducts and were stained more widely and inteilsely than in normal livers. These data suggest that these proliferated peribiliary glands in the stone-containing bile ducts produce and secrete significant amounts of lactoferrin and lysozyme. Increased production and secreiion of lactoferrin and lysozyme suggests activated local defense mechanisms against bacterial infection in the stone-containing bike ducts, and may be beneficial for inhibition of the growth of calculi and prevention of the suppurative inflammation.

Lactoferrin (Lf). a non-enzymatic protein, was discovered in cow’s milk (l), and has bactericidal or bacteriostatic activities (2,3) due to its high association with iron and sequestration of iron from bacteria, which is essential for bacterial growth (3,4). Lysozyme (Ly) is a bacteriolytic enzyme (muramidase) in human serum and secretions (5), and acts through the dissolution of N-acetylglucosaminyl-N-acetylmuramic acid linkage in the cell walls of susceptible bacteria. Lf and Ly, secreted from the mucosal surface, contribute to non-specific local defense mechanisms against bacterial infection (5-8). It has been immunohistochemically shown that these proteins are detected in various human tissues (g-12), including the lactating breast (9,10), the bronchial (13,14), salivary (l&16), and gastric glands (17), and the pancreatic acinus (18). As for the biliary system, Masson et al. (6) Correspondence: Kstslhiko

Saito. M.D.. Second Department

showed that normal hepatic bile contains small amounts of Lf at concentrations of 0.01-0.04 mg/ml, although its origin remains unclear. In hepatolithiasis, bile stasis and bacterial infection of the intrahepatic bile ducts are important in the pathogenesis of intrahepatic calculi. especially calcium bilirubinate stones (19-21). /3-Glucuronidase from bacteria, especially Escherichia coli, is regarded as the key enzyme in the development of calcium biiirubinate stones (2224). There must be defense mechanism(s) operating in hepatolithiasis and under normal conditions against such bacterial infections in the intrahepatic biliary tree. Although there are a few reports (25,26) about the participation of immunoglobulins and secretory components as local defense mechanisms, no study has been ~-~-i~tsmed on Lf and Ly in the intrahepatic biliary tree.

of Pathology, Kanazawa University School of Medicine, Kanazawa 920. Japan.

IL SAITO

148 In this study we immunohistochemically examined the distribution of Lf and Ly in the intrahepatic biliary tree, and evaluated the participation of these proteins in local defense mechanisms against bacterial infections in hepatolithiasis. The significance of the intrahepatic peribiliary glands, which were recently disclosed in our laboratory (27), in this local defense system were also examined.

Materials and Methods The intrahepatic biliary tree was defined as the bile ducts proximal to the hepatic duct confluence. These were classified into the right and left hepatic ducts, segment ducts (the first major branches of each hepatic duct), area ducts (the first major branches of each segment duct), and their finer branches according to Healey and Schroy (28). In this study, hepatic, segment, and area ducts were collectively termed ‘intrahepatic large bile ducts’, The wall of these bile ducts consisted of a hypocellular fibrous band lined by a layer of columnar epithelia. The periductal tissue formed a loose connective tissue around the bile duct wall. Intrahepatic peribiliary glands were defined as intramural and extramural according to Terada et al. (27). The former were confined within the duct wall and the latter were present in the periductal tissue. Extramural glands were composed of acini arranged in a lobule, and intramural were tubular glands with few branchings in both normal livers and hepatolithiasis. These intramural and extramural glands were ccmposed of serous and mutinous acinar cells. Serous acinar cells have little cytoplasm and the nuclei are centrally located. Mutinous acinar cells have clear and ample cytoplasm (mucus) with basically situated nuclei. We obtained 31 livers with hepatolithiasis (29 surgically resected livers and two autopsy livers), with calcium bilirubinate stones in the intrahepatic large bile ducts, from our University Hospital and affiliated hospitals. The mean age was 60.6 years (37-85) and 18 cases were female. As a control, 14 normal autopsy livers were selected from our recent autopsy files (mean age 60.1 years, seven females). In cases of hepatolithiasis, one or more sections were obtained from stone-containing large bile ducts. In control livers, sections were taken from the intrahepatic large bile ducts of both lobes. Tissue was fixed in 10% neutral buffered formalin and embedded in paraffin. Several serial sections (4 pm in thickness) were cut from each paraffin block for light-microscopic study (H & E, periodic acid-Schiff reaction after diastase digestion (dPAS), alcian-blue at pH 1.0 and 2.5 (AB pH 1.0 and 2.5), and high iron diamine (HID)), and also for immu-

and Y. NAKANUMA

nohistochem.J staining. Immunohistochemical staining for Lf and Ly were performed on deparaffinized sections by the avidin-biotin peroxidase complex method (29). After endogenous peroxidase activity was blocked with 0.3% H,Oz-methanol, sections were treated with 1% bovine serum albumin to reduce background staining, rabbit anti-Lf and -Ly antisera (Dakopatts, Glostrup, Denmark) diluted in 1:lOO in phosphate buffer solution (PBS) for 30 min, biotinylated swine anti-rabbit Ig antiserum (Dakopatts) at 1:400 dilution for 30 mitt, and then avidin-biotin peroxidase complex (Vecter Lab, Burlingame, CA, U.S.A.). Peroxidase activity was demonstrated using 0.03% H,O,3,3’-diaminobenzidine tetrahydrochloride (DAB) (Sigma Chemical Co., St Louis, MO, U.S.A.) for 5 min. Several staining and specificity controls were performed: Positive stain for Lf or Ly was abolished when PBS or nonimmune sera were used as a first layer and following preincubation of diluted primary antisera with 1 mg of pure Lf and Ly (Sigma Chemical Co.) per 5 ml of rabbit antisera at 1:lOO dilution. No positive stain was obtained when H,O, without DAB nor DAB without H,O, was applied to demonstrate peroxidase activity. Positive reactions were graded as follows: no positive reaction in any cells, negative; positive cells less than half the cells examined, weakly positive; positive cells more than half the cells examined, highly positive. The statistic analysis between normal livers and livers with hepatolithiasis was conducted with the Wilcoxon’s rank sum test, and p values of less than 0.05 were considered significant.

Results Normal liver The intrahepatic large bile duct of normal livers had a few glandular elements within the bile duct wall (intramural gland) in 12 cases and in the periductal connective tissue (extramural gland) in all 14 cases (Fig. 1). Lf was positive in the intramural gland in two out of 12 cases (16.7%), and in the extramural gland, in only one of 14 cases (7.1%) (Table 1). The positive reaction was weak and localized, when present (Fig. 2). Ly-positive cells were also weak and were found locally in the intramural gland in two cases (16.7%) and in the extramural gland in three cases (21.4%) (Table 2, Fig. 3). The lining epithelia of the intrahepatic large bile ducts were negative for Lf in all cases and only weakly positive for Ly in three cases (21.4%). Hepatocytes were negative for Lf and also Ly.

LACTOFERRIN

AND LYSOZYME

IN THE LIVER

149

Fig. 1. The intrahepatic large bile duct of the normal iiver has a few glandular elements within the bile duct wall (intramural glands; curved arrows) and in the periductal connective tissue (extramural glands: straight arrows) forming lobular patterns. L: bile duct lumen. H.E., x40.

Hepatolithiasis The bile duct wall showed marked fibrous thickening with variable degrees of chronic inflammatory changes, and the intramural (especially mucous type) and extramural glands (mucous and serous types) proliferated markedly in the stone-containing bile ducts, as previously reported (20,21,30). The lining epithelia oi’ the stonecontaining bile ducts were eroded in places and the remaining epithelia showed regeneration or hyperplasia (Fig. 4). The mucous acinar cells outnumbered the serous cells in the stone-containing bile ducts, especially in the intramural glands. Lf was positive in the intramural glands in 29 of 31 livers with hepatolithiasis (93.5%) and in the extramural gland in 21 of the 29 cases (72.4%) (Table 1). While the majority of Lf-positive cases were rather weakly stained in the extramural gland, the presence of immunoreactive cells in the intramural gland was high (Fig. 5). While the cytoplasm of serous gland epithelium was diffusely posTABLE 1 The incidence and the gravity of lactoferrin-positive

cases in normal liver and hepatolithiasis

Normal liver (no. of cases) _~_____ ~______~ lntrahepatic peribiliary gland __-extramural intramural gland gland Highly positive case5 Weakly positive cases Negative cases

(3 2 10

itive in both the intramural and extramural glands (Fig. 6A), reaction products of Lf were restricted to the peripheral rim avoiding mucus-positive area in mutinous acinar cells (Fig. 6B). The covering epithelia of stonecontaining bile duct were positive in 17 cases (54.8%). most of which was focal and weak (12/17 cases) and reaction products were intracytoplasmic (Fig. 6C). A few inflammatory cells infiitrating the bile duct wali were also positive. Most of these were neutrophilic granulocytes. Ly was positive in the intramural glands of 24 of the 3 1 cases (77.4%) and in the extramural glands of 14 of the 29 cases (48.3%) (Table 2). Although positive reactions to Ly in the intrahepatic biliary gland were lower than for Lf, the distribution pattern and intensity of Ly was similar to Lf. The staining patterns in serous and mucinous acinar cells were also similar to Lf (see above) (Fig. 7). Weak positive reactions were found in the covering epithelia of the stone-containing bile ducts in 11 cases (35.5%) as seen in Lf.

~__Covering epithelium of the bile duct

0 1 13

7.1 16.7 Incidence of positive case (clc) ~________ -*p < 0.01 for statistic analysis benveen normal liver and hepatolithiasis.

-____ 0 0 14 0

-.-

Hepatolithiasis (no. of cases) _ ___~ __-~ Intrahepatic peribiliary gland intramural gland*

extramural gland*

21 8 2

2 19 8

93.5

-

72.4 ____

___ Covering epithelium of the bile duct

__~ 5 12 14 ~-~

54.8 ___

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LACTOFERRIN

AND LYSOZYME

IN THE LIVER

Fig. 6. Higher magnification of immunohistochemical staining for lactoferrin in the epithelia of hepatolithiasis. (A) in:l~amural serous glands are frequently positive for lactoferrin. The cytoplasm of positive cells is diffusely and strongly positive. (B) The epithelia of intramural mucous glands are also intensely positive, but the positive site is restricted to peripheral rim except mucus containing area. (C) The covering epithelia of the bile duct are positive focally and weakly with diffuse cytoplasmic staining. A, B and C, immunostain for lactoferrin (ABC method) and hematoxylin. x 172. Fig.7. Higher magnification of immunohistochemical staining for lysozyme in hepatolithiasis in the epithelia of intramural serous glands (A) and intramural mucous glands (B). Lysozyme is intensely stained in the same pattern as lactoferrin. Immunostain for lysozyme (ABC method) and hematoxylin. x172.

T.4BLE 2 The incidence and the gravity of lysozyme-positive

cases in normal liver and hepatolithiasis _~___ Normal liver (no. of cases) Hepatolithiasis

___

..~

~_~

Intrahepatic peribiliary gland intramural gland

extramural gland

Highly positive cases Weakly positive cases Negative cases

0 2 10

0 3 11

incidence of positive case (‘%)

16.7

21.4

*p c 0.01 for statistic analysis between normal liver and hepatolithiasis.

Coverine epithelium of the bile duct

(no. of cases)

!ntrahepatic peribiliary gland intramural gland*

extramural gland*

0 3 11

15 9 7

6 8 15

21.4

77.4

48.3

Covering epithelium of the bile duct 2 9 20

.___

3.5. :

152 The stone-containing bile ducts of surgically resected livers and two autopsy livers all contained increased numbers of Lf- and Ly-positive cells. Thus, the immunohistochemistry was satisfactory on all pOSttnOrtem tissues including normal control livers. Statistic study me incidence of Lf- and Ly-positive reaction in both intramural and extramural gland in hepatolithiasis was higher (p c 0.01) than that in normal liver. While no positive reactions of Lf or Ly could be found in the covering epithelium of the intrahepatic large bile ducts in normal liver, Lf and Ly were detected in 54.8% and 35.5% in the covering epithelia in the stone-containing bile ducts, respectively. There were, however, no statistically significant differences Cp > 0.05).

Discussion The present study showed that lactoferrin (Lf) and lysozyme (Ly) were focally distributed in the intrahepatic peribiliary glands but not in the lining biliary epithelia of normal livers. Thus, the intrahepatic peribiliary glands may produce and secrete Lf and Ly physiologically, and these glands may be a source of these proteins in the normal hepatic bile (6). It is possible, therefore, that the peribiliary glands might physiologically participate in a non-specific local defense mechanism against bacterial infection of the biliary tree. Bacterial infection tends to occur in stagnant bile flow in dilated stone-containing ducts (19-24). Several studies showed that many sorts of bacteria were present in the bile of stone-containing intrahepatic bile ducts in hepatolithiasis (19,22,31). The present study showed that both kf and Ly were more intensely and generally positive in the proliferated peribiliary glands in the stone-containing bile ducts in hepatolithiasis. Our findings indicate that these glands, especially the intramural glands, produce and secrete large amounts of Ly and Lf. Thur;; activities of non-specific defense mechanisms mediated by Lf and Ly are markedly increased in the stone-containing tile ducts. Bacterial infection of the intrahepatic bile ducts has at References

SijrensenM, Siirensen SPL. The proteins in whey. C R Trav Lab Carlsberg 1939; 23: 55-99. Arnold RR, Cole MF, McGhee JR. A bactericidal effect for human lactoferrin. Science 1977; 197: 263-5. Bullen JJ. The significanceof iron in infection. Rev Infect Dis 1981; 3: 1127-38.

K. SAITO and Y. NAKAWUMA

least two important implications in the pathogenesis of hepatolithiasis. Firstly, the hydrolysis of the bilirubinglucuronide into free bilirubin and glucuronic acid is mediated $23 &lucuronidase of bacteria origin, especially E. di. This leads to consolidation of calciumbilirubinate stones (22), which represent the majority of intrahepatic calculi (30). Because this study showed that Lf and Ly were increased in the stone-containing bile ducts, increased production of Lf and Ly in hepatolithiasis might suppress further growth of bacteria and possibly help inhibit the further development of intrahepatic calculi. Although recent bacteriologic studies on hepatic bile in hepatolithiasis in Japan revealed many sorts of bacteria (19,22,31), suppurative cholangitis or abscess formation were histopathologically confirmed in only eight out of 273 livers with hepatolithiasis in the series of Japanese Hepatolithiasis Study Group (30). Why did the suppurative changes occur at a lower frequency? The activation of local Lf and Ly defense mechanisms might have contributed to the prevention of secondary suppurative changes within or around stone-containing bile ducts. There was no difference for the staining of Lf between serous type glands and mucous type glands. Our data of serial sections showed cytoplasmic positive staining for both Lf and Ly except in mucus-containing areas within the same epithelium of mucous type glands, which proliferated markedly and actively secreted mucus especially in the intramural gland during hepatolithiasis. The proliferated biliary gland may, thus, be multi-functional (produce and secrete several kinds of mucous glycoproteins (32), Lf and Ly). On the one hand mucus itself is thought to be one of the accelerating factors of formation and growth of stones (30), on th:: other these secretors may serve to protect the mucos:.l surface from injury by pathologic bile or calculi and prevent bacterial infection. In conclusion, increased production and secretion of Lf and Ly in the proliferate,1 inbrahepatic peribiliary glands during hepatolithiasis ~::&yact as a ke) factor in suppressing or inhibiting further bactzrlal infection in the biliary tree, thereby leading tc the inhibition of further stone growth and the occurrence of suppur;tivc changes in the stone-containing bile ducts.

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LACTGFERRIN

AND LYSQZYME

IN THE LIVER

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