In situ distribution of enolase isozymes in chronic liver disease

In Situ Distribution of Enolase Isozymes in Chronic Liver Disease Yoshihide Fukuda, M.D., Yuuji Miyazawa, M.D., Masami Imoto, M.D., Yasuo Koyama, M.D., Isao Nakano, M.D., Hiroshi Nagura,

THE AMERICAN JOURNAI OV GASTROKNTEROLOGV

Copyri^t © 1989 by Am CoU of Gastroenterology

Vol.84 No 6 1989 Primed in U.S.A.

In Situ Distribution of Enolase Isozymes in

Chronic Liver Disease

Yoshihide Fukuda, M.D., Yuuji Miyazawa, M.D., Masami Imoto, M.D., Yasuo Koyama, M.D.,

Isao Nakano, M.D., Hiroshi Nagura, M.D., and Kanefusa Kato, M.D

Second Department of Internal Medicine and Insitulefor Disease Mechanism and Control Nagoya University School of Medicine, Nagoya, and Department of Biochemistry Institute for Developmental Research Aichi Prefectural Colony.

kasugai Japan

Liver biopsy specimens with or without chronic liver

diseases were examined immunohistochemically to

de-termine the distrihution of enolase isozymes (a, 0, and

y) In normal liver, a-enolase was positively stained in

almost all hepatocytes and bile duct cells, p- and

y-enolases were localized in hepatocytes and bile duct

cells, respectively Electron microscopic studies

re-vealed that Kupffer cells and sinusoidal endotbelial cells

had both a- and 7-enolases In chronic active hepatitis

and cirrhosis, proliferated biliary ductular cells had

both a- and -y-enolases, but did not express /?-enoIase

This is almost the same localization pattern of enolase

isozymes as in preexisting bile duct cells Y-Enolase

was detected in some hepatocytes in eight of 12 cases

with chronic active hepatitis and six of 12 cases with

cirrhosis These hepatocytes were small, showed a

cob-blestone pattern, and binucleate cells were frequent On

the other hand, rosette-formed hepatocytes adjacent to

a regenerating bulging lobule were not stained for

7-enolase These results suggest that regenerating

hepa-tocytes have 7-enolase and that, with maturation,

hep-atocytes lose it

INTRODUCTION Enolase is a glycolytic enzyme distributed in all

mam-malian cells (1) It is a dimer composed of three distinct

subunits, a, /3, and y «-Enolase is the major form in

the liver Small amounts of ^- and 7-enolases are also

detectable in the liver {2, 3) The /3-subunit (/3-enolase)

is distributed mainly in the skeletal muscle as a/3 and

/3i3 forms, whereas the 7-subunit (7-enolase) is present

predominantly in the brain and neuroendocrine cells

as «7 and 77 forms (2) We previously proposed that

the quantification of these isozymes in serum is useful

as a disease marker for patients with myocardial

infarc-tion, myopathy (4), and neuroblastoma (5)

In addition to practical use as a diagnostic tool,

enolase isozymes have received attention as markers

Received Sept 22 1988; revised Feb 9 1989; accepted Feb 22.

1989.

for cell differentiation (2, 6) Niimi et al (7) reported

altered distribution of enolase isozymes in the bronchial mucosa with proliferated and metaplastie lesions Hep-atocellular regeneration and biliary ductular prolifera-tion are common phenomena in chronic active hepa-titis and cirrhosis For these conditions, however, the isozyme distribution patterns of enolase have not yet been reported

The present study clarifies the immunohistochemical distribution of enolase isozymes in various cells ofthe liver with or without chronic liver diseases, and dis-cusses the biological significance ofthe appearance and disappearance of enolase isozymes in hepatocytes and bile duct cells in diseased liver

MATERIALS AND METHODS

Tissue specimens

Tissue specimens were obtained by either needle biopsy or surgical wedge resection from 24 patients with chronic active hepatitis (eight cases of hepatitis B and four cases of non-A, non-B hepatitis) and nonal-coholic cirrhosis (12 cases) Histologically normal Hver tissues were obtained by biopsy for diagnostic purpose from five patients undergoing surgery for abdominal tumors A portion of each specimen was immediately fixed in periodate-lysine-4% paraformaldehyde (8), then washed in increasing concentrations of sucrose in phosphate-buffered saline (PBS) The fixed tissues were embedded in Tissue-Tek OCT compound (Miles Phar-maceutical, Naperville, IL) and frozen in ethanol with

dry ice They were cut into sections 6 tixn thick on a

cryostat microtome

Antibodies

Antibodies specific to «- )3-, and 7-subunits of hu-man enolase were raised in New Zealand white rabbits and purified as reported previously (4, 9) The Fab' fragments of the purified antibody IgG were labeled with horseradish peroxidase (10) In control experi-ments, the Fab' fragments of non-immune rabbit IgG were also labeled with horseradish peroxidase

601

Immunoh istochem istry

The direct peroxidase-labeled antibody method was

used for the immunohistochemical staining, with minor

modifications (11),

Cryostat sections for light microscopy were treated

with 100% methanol containing 0.1% hydrogen

per-oxide for 20 min to inactivate endogenous peroxidase

Sections were reacted with the peroxidase-labeled

anti-body for 16 h at 4°C, then dipped in 0,025%

diamino-benzidine (DAB) solution containing 10 mM sodium

azide for 5 min and counterstained with methyl green

Cryostat sections adjacent to those taken for light

microscopy were treated similarly through the antibody

incubation steps, and the sections were postfixed in 1 %

glutaraldehyde in PBS The postfixed sections were

washed and incubated with 0.025% DAB solution

with-out hydrogen peroxide for 15 min, and then with

0.025% DAB solution containing 10 mM hydrogen

peroxide for 10 min The sections were washed, reacted

with 2% osmium tetroxide in PBS, dehydrated in

graded ethanols, and embedded in Epon Ultrathin

sections were viewed with a Hitachi H-300 electron

microscope

RESULTS

Light microscopy

Histologically normal liver a-Enolase was found in

almost all hepatocytes and epithelial cells of hile

duc-tules and interlobular and septal bile ducts in all five

cases The intensity of the staining of this antigen in

bile duct cells was greater than that in hepatocytes (Fig

1) Zonal distribution in the hepatic lobules was not

found |3-Enolase was positively stained in hepatocytes,

but was not detected in epithelial cells of bile ducts in

various sizes It was more strongly stained in

hepato-cytes located in zone 1 than in zone 3 (Fig 2) The

y-subunit was not detected in hepatocytes, except one

case which showed a weakly positive stain of a few

hepatocytes This form of enolase was identified for the

most part in bile duct cells and intrahepatic nerve fibers

(Fig 3), as previously reported (12, 13) Enolase

iso-zymes of sinusoidal lining cells were difficult to detect

at the light microscopic level

The results of the distribution of the isozymes in

various cells are summarized in Table 1

Chronic active hepatitis and cirrhosis Staining

pat-terns of a- and ^-enolases were almost the same in

chronic active hepatitis and cirrhosis as in normal liver

tissues Biliary ductular proliferation was found in liver

tissues from the patients with chronic active hepatitis

and cirrhosis, a- and 7-enolases were positive in these

proliferated biliary ductular cells, whereas /3-enolase was

completely negative (Figs 4 and 5)

Distribution of 7-enolase was altered in chronic liver

Figs, i-3, Immunoperoxidase staining for a-enolase {Fig I), 0-enolase {Fi}; 2) and 7-0-enolase l,Fi,^ 3) in normal liver «-EnoIase is positive in hepatocytes and bile duct cells (arrowheads) /j-Enolase is

positive in hepatocytes but negative in bile duct cells 7-Enolase is seen in bile duct cells (wrrtnv/icaJ) and nerve fibers (tJ/row.v) (xl45).

TABLE I

Distribution of Three Enotase Isozymes in Various Cells of Normal

Liver

Hepatocyte + + -*

Bile duct cell + - + Kupffer cell + - + Endothelial cell + — + Nerve fiber - - +

* One case showed weakly positive staining of a few hepatocytes.

disease, ln six of eight cases with hepatitis B, two of four cases with non-A, non-B hepatitis, and six of 12 cases with cirrhosis, 7-enoiase was stained in hepato-cytes (Table 2) Hematoxylin and eosin staining

re-June 1989 ENOLASE ISOZYMES IN LIVER DISEASE 603

Figs 4 and 5 Immunoperoxidase staining for o-enolase {Fi}> 4)

and /5-cnolase (Fig 5) in chronic active hepatitis Both enolases are

seen in hepatocytes t»-Eno!ase is also positive in proliferated biliary

ductular cells {arrowheads), but 0 is negative in these cells Adjoining

sections (X145).

TABLE 2

Distribution ofy-Subunil in Hepatocytes*

Degree of Reactiv-ity

Normal liver

CAHB

CAH NANB

Cirrhosis

5 8 4 12

+ 0 6

2

6

+

1 1

2

-4 1 0 I

* -f, hepatocytes positive; ±, some weakly positive cells; - , aimost

all hepatocytes negative; CAH B, chronic active hepatitis B; CAH

NANB, chronic active ncn-A, non-B hepatitis.

vealed irregular and non-uniform distribution of

necro-sis, inflammation, and regeneration from one lobule to

the next (Fig 6) Regenerating hepatocytes., showing a

cobblestone appearance with thickening ofthe liver-cell

plates, were small in size and lacked lipofuscin

Some-times these grouped cells compressed the adjacent liver

tissue In the serial sections, 7-enoiase-positive

hepato-cytes were generally small and showed a cobblestone arrangement Binucleate cells were sometimes detected

in the lobule In contrast, rosette-formed hepatocytes neighboring these bulging lobules lacked 7-enoIase (Fig 7) The intensity ofthe 7-enolase staining varied among

pseudolobules in cirrhotic livers \

Control stainings

No reaction was observed in the sections treated with the horseradish peroxidase-labeled non-immune rabbit Fab' fragments

Electron microscopy

Intraceliular localizations of enolase isozymes in hep-atocytes and bile duct cells were almost the same The isozyme was detected electron microscopically in free and membrane-bound ribosomes and the cytoplasm (Fig 8)

Both a- and 7-enolases were detected in Kupffer cells

and sinusoidal endothelial cells, whereas ^-enolase was not demonstrated in these cells (Fig 9) Nerve fibers

FIG 6 Histology of a patient with chronic active hepatitis Regen-erating hepatocytes show a cobblestone appearance in the right half

of the photograph In the left half, damages hepatocytes are seen grouped in rosettes Hematoxylin and eosin, xl 15,

FIG 7 Imtnunoperoxidase staining for 7-enolase in chronic ac-tive hepatitis y-Enolase is stained in regenerating hepatocytes In the right side ofthe photograph, whereas it is negative in neighboring hepatocytes (xl 15).

Fici 8 rt-Enolase is distributed m free (*) and bound (arrowhead)

ribosomes, and in the cjloplasm of proliferated biliary ductular cells

(x9,7OO).

FiCi 9 /^-Enolase is stained in ribosomes of the hepatocyte (//),

but not demonstrated in the endothelial cell (£) or the Kupffer cell

(A')(x6.650).

showed diffuse staining of 7-enoiase in their axons

These results are summarized in Table I

DISCUSSION

We describe, immunohistochemically., the cellular

distribution of the various isozymes of enolase in

nor-mal human Hver and in chronic active hepatitis and

nonalcoholic cirrhosis In normal liver, we found

a-enolase widely distributed in hepatocytes, biliary

duc-tular cells, Kupffer cells, and endothelial cells; /3-enolase

was present only in hepatocytes, and 7-enolase in

bili-ary ductular cells and sinusoidal lining cells In

agree-ment with these observations, previous work from our

laboratory found ^-enolase in normal biliary epithelial

cells (12) Thus, the immunohistologic distribution of

the enolase isozymes corresponds roughly with

bio-chemical analyses that have found that a-enolase and

/?-eno!ase predominate over 7-enolase in normal liver (2)

In diseased liver, our most important fmding was that 7-enoIase was often present in hepatocytes Viral hepatitis is characterized pathologically by three major morphological components: cytologic damage, inflam-matory reaction, and hepatocellular regeneration In chronic iiver disease, these reactions vary considerably from one lobule to the next, whereas they are generally uniform both among lobuies and inside the lobule in acute hepatitis (14) 7-Enolase-positive hepatocytes had morphologic features of regeneration—that is, cobble-stone configuration and obscure cell plates (15) These features contrasted with those of damaged hepatocytes, which were grouped in rosettes and were negative for 7-enoIase Thus, 7-enoIase may be a marker of regen-erating hepatocytes

The clinical applicability of immunohistochemical localization of enolase isozymes remains to be estab-lished However, we speculate tbat it could be used as

a marker of the regenerating capability of hepatocytes after toxic or viral injury to the Hver, or ofthe degree

of activity of viral hepatitis

ACKNOWLEDGMENT This study was supported in part by Grant-in-Aid for Encouragement of Young Scientists ofthe Ministry of Education, Science, and Culture of Japan

Reprint requests: Yoshihide Fukuda, M.D Second Department

of Internal Medicine Nagoya University School of Medicine, 65 Tsunimai-cho, Showa-ku Nagoya 466, Japan.

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