Yonago Acta medica 2001;44:7–16

Immunohistochemical Study of Dendritic Cells and Kupffer Cells in
Griseofulvin-Induced Protoporphyric Mice
Ryota Kashiwagi, Toshiyuki Kaidoh and Takao Inoué
Second Department of Anatomy, Faculty of Medicine, Tottori University, Yonago 683-0826
JapanTo determine whether protoporphyric liver injury affects hepatic dendritic cells (DC)and Kupffer cells, we examined liver tissues of griseofulvin-induced protoporphyric
mice using histological and immunohistochemical methods. After 1 week of griseofulvinfeeding, the protoporphyric liver showed prominent hepatomegaly and a gradual increase in histopathological changes such as hepatocellular hypertrophy, focal necrosis
and brown pigment deposits. After 4 weeks of treatment, marked ductular reaction
was observed in the liver tissue. Immunohistochemical analyses indicated that the density of NLDC-145-positive hepatic DC gradually decreased during griseofulvin feeding.
However, the index of the number of DC in the whole liver appeared to fall sharply
after 6 weeks. In contrast, the density of F4/80-positive Kupffer cells gradually increased
during griseofulvin feeding. In the spleen and lung, no significant differences werenoted in the distribution of NLDC-145-positive DC between experimental and control
mice. These results suggested that griseofulvin-induced protoporphyria leads to a
specific decrease in the density of hepatic DC due to hepatomegaly until 4 weeks oftreatment and is substantial after 6 weeks. This substantial decrease of hepatic DC
might have been induced by some alterations in protoporphyric liver injury including
ductular reaction at a later stage in this experiment. Since hepatic DC were reduced innumber, they seemed to have no significant relation to the progression of griseofulvininduced protoporphyric liver injury. However, the decrease of hepatic DC might affectthe cellular immune response in protoporphyria.
Key words: dendritic cell; griseofulvin; immunohistochemistry; Kupffer cell; protoporphyria

et al., 1989; Woltsche et al., 1991; Cadrin et al.,1995). However, these studies have focused onchanges in hepatocytes or Kupffer cells, so little
is known about changes in dendritic cells (DC)in the liver.
DC are professional antigen-presentingcells, which contribute to the initiation of T
cell-mediated immune responses. DC areintimately related to the liver in both normal
and pathological conditions. Recently, DC
have been shown to migrate from the blood tothe lymph via the hepatic sinusoids (Matsuno etal., 1996; Kudo et al., 1997). It appears that thisis the major migration pathway for DC from theblood, and Kupffer cells play an important role

Although human porphyrias are often associated with the development of liver injury, they
are relatively uncommon, preventing adequate
investigation in patients. Griseofulvin, an antifungal drug, is known to inhibit hepatic ferrochelatase activity (Lochhead et al., 1967) anddisturb porphyrin metabolism in the mouse (DeMatteis and Rimington, 1963). This experimental mouse model shows liver injury withaccumulation of brown protoporphyrin crystals,
resembling human erythropoietic protoporphyria. This mouse model has therefore beenused to study the pathogenesis of protoporphyric liver injury (Matilla and Molland, 1974;
Gschnait et al., 1975; Denk et al., 1979; Kawahara

Abbreviations: BSA, bovine serum albumin; DC, dendritic cells; PBS, phosphate-buffered saline7R. Kashiwagi et al.

in this migration (Matsuno et al., 1996; Suda etal., 1996; Kudo et al., 1997). In addition, DC
have been suggested to infiltrate into the livertissues and then play a pivotal role in the development of chronic inflammatory liver diseases such as primary biliary cirrhosis, chronic
hepatitis type B and type C, and autoimmune
hepatitis (van den Oord et al., 1990; Kaji et al.,
1997; Shinomiya et al., 1998; Tanimoto et al.,1999). However, there have been no attempts
to quantify changes in hepatic DC or Kupffer
cells in protoporphyric liver injury. Therefore,
we examined changes in the numbers of thesecells in griseofulvin-induced protoporphyric
mice using immunohistochemistry to determine
whether protoporphyric liver injury affects
these cells and to speculate on the pathologicalrole of these cells, particularly hepatic DC, in
protoporphyric liver injury.

munostaining method was as follows. After
blocking nonspecific binding with 1% bovine
serum albumin (BSA) in phosphate-bufferedsaline (PBS, pH 7.4) for 10 min and then withavidin-biotin blocking kit (Vector Laboratories,Burlingame, CA), liver sections were incubatedwith rat anti-mouse macrophage monoclonal
antibody F4/80 (diluted 1:50; Serotec, Oxford,United Kingdom) and those of the spleen andlung were incubated with rat anti-mouse dendritic cell monoclonal antibody NLDC-145 (diluted 1:25; Serotec) for 60 min (Austyn andGordon, 1981; Witmer-Pack et al., 1995). Boundmonoclonal antibody was detected with biotinylated rabbit anti-rat IgG (diluted 1:200 for
F4/80, 1:1000 for NLDC-145; Vector) for 30 min.
The sections were then treated with avidinbiotin-alkaline phosphatase complex (Vectastain
ABC-AP Kit; Vector) for 30 min and labeledcells were colored red with alkaline phosphatase substrate kit I (Vector Red; Vector). Sections were counterstained with hematoxylin andmounted in Aquatex (Merck, Darmstadt, Germany).
Since NLDC-145 recognizes not only DC but
also proliferating bile ductules, we performed
double immunostaining of the liver using antikeratin antibody as follows to distinguish thesestructures. Endogenous peroxidase activity waseliminated by treatment with 1% H2O2 and 0.1%
NaN3 for 10 min. After blocking nonspecificbinding with 1% BSA in PBS for 10 min, thesections were incubated with rabbit polyclonalanti-keratin, Wide Spectrum Screening (diluted1:600; Dako, Carpinteria, CA) for 30 min tolabel the bile ductules (Shiojiri, 1994). The
reaction of the antibody was detected using goatanti-rabbit immunoglobulin conjugated toperoxidase labeled-dextran polymer (DakoEnVision+; Dako) for 30 min and labeled cells
were colored brown with 3,3'-diaminobenzidine
tetrachloride (Dojin Chemical, Kumamoto,Japan). The sections were then treated with 5%
normal rabbit serum in PBS for 20 min and withavidin-biotin blocking kit. Then, they werereacted with NLDC-145 and processed by theindirect immunoalkaline phosphatase method
as described above. On double immunostaining, hepatic DC (red) could be distinguishedfrom bile ductules (brown).

Materials and Methods
Twenty-nine male ICR mice, 5 weeks old andweighing about 30 g, were fed a powdered dietcontaining 3.5% griseofulvin (Sigma ChemicalCo., St. Louis, MO) and water ad libitum. They
were analyzed at 1 (n = 5), 2 (n = 5), 3 (n = 5), 4(n = 5), 6 (n = 5) and 8 (n = 4) weeks after thecommencement of griseofulvin feeding. Eightmale ICR mice, 5 (n = 5) and 13 (n = 3) weeks
old on a powdered diet without griseofulvin,served as controls. The 13-week-old control
mice were the same age as the mice fed griseofulvin for 8 weeks. Animals were weighed andkilled under diethyl ether anesthesia. The liver,spleen and lung were removed and the liver wasweighed. Small pieces of these specimens wereprocessed for histological and immunohistochemical studies.
Liver tissues were fixed in 10% formalin
and processed using standard histological methods to yield paraffin sections 4 µm thick stainedwith hematoxylin and eosin.
Specimens of the liver, spleen and lung weresnap-frozen in OCT medium with liquid nitrogen. Cryostat sections 4 µm thick were fixed in
acetone for 10 min and single or double immunostaining was carried out. The single im8

Dendritic cells in protoporphyric liver

The density of hepatic DC and Kupffer cells
was analyzed using the Mann-Whitney U test.
A P value < 0.05 was taken to indicate statistical significance.

The number of NLDC-145-positive DC wasquantified by counting cells per unit area on thesections in a blinded fashion, and the density ofthese cells was determined. Since the protoporphyric liver showed prominent hepatomegaly,
we considered that hypertrophy of hepatocytes
decreased the density of NLDC-145-positive
DC. Therefore, we calculated the index of thenumber of DC in the whole liver as follows. We
multiplied cell density (/mm2) by liver weight(g) in each mouse and calculated the meanvalue of the products in each stage. Then theratio of the value to that of the 5-week-old
controls was evaluated. On the other hand, toquantify the density of Kupffer cells, we counted the number of F4/80-positive cells only inthe sinusoidal area and excluded macrophages
in the portal area in randomly selected fields.
Since Kupffer cells were counted only in thesinusoidal area, we could not estimate thechanges in Kupffer cell number in the wholeliver. In these analyses, only the labeled cells
where the nucleus could be identified werecounted.

Results

WeightGriseofulvin-induced protoporphyric mice
showed retardation of growth. Mean bodyweight of these mice at 8 weeks was about 80%of that of the 13-week-old controls (Fig. 1).
These mice showed prominent hepatomegaly,
with the liver being dark brown in color. The
livers of the mice fed griseofulvin for 8 weeks
were about 3.6-fold as heavy as those of the 13week-old controls (Fig. 1).
The intensity of histopathological changes
in the liver gradually increased over the experimental period (Fig. 2). Hepatocytes showed
nuclear and cytoplasmic hypertrophy, andvacuolated cytoplasm. Focal necrosis in theparenchyma occurred at all stages. Brown pigment deposits appeared 1 week after the commencement of griseofulvin feeding. They accumulated in hepatocytes, Kupffer cells, portal
macrophages, bile ducts and bile ductules. Plugs of the brown pigment fre(g)50quently obstructed the bile ducts andductules. Proliferation of bile ductules was obvious at 3 weeks and be40
came marked after 4 weeks. This wasaccompanied by a variable number ofconnective tissue elements and infil30
tration of neutrophils and mononuclear cells as reported previously(Weston-Hurst and Paget, 1963;
20BW
Gschnait et al., 1975). These changes,
LW
the proliferation of bile ductules accompanied by inflammatory infiltra10tion and connective tissue elements,
are similar to so-called “ductular reaction”. After 4 weeks of griseofulvin
00
1
2
3
4
68C
feeding, this ductular reaction becamea marked pathological change in theWeek
injured liver. It extended from theFig. 1. Body weight (BW) and liver weight (LW) in control
portal tracts toward neighboring porand griseofulvin-treated mice. Data are expressed as mean ±tal tracts and penetrated the parenSD. Bars represent SD. C, 13-week-old control mice, idenchyma between the liver cell plates.
tical in age to the mice treated with griseofulvin for 8 weeks.
9R. Kashiwagi et al.

Fig. 2. Light micrographs of the liver of amouse fed griseofulvin for 8 weeks. Hematoxylin and eosin. a: Brown pigment deposits are accumulated in the liver tissue.
Central (arrow) and interlobular (arrowhead) veins are visible. × 120. b: Hepatocytes show hypertrophy. The proliferationof bile ductules is accompanied by inflammatory infiltrate and connective tissueelements, similarly to the so-called “ductular reaction”. It penetrates the parenchyma between the liver cell plates (arrowheads). Note brown pigment deposits in thebile ductules (arrows). × 250.

abFig. 3. Double immunostaining of the liverwith monoclonal antibody NLDC-145 (red)and polyclonal anti-keratin, Wide Spectrum
Screening (brown). a: A 5-week-old control mouse. Several NLDC-145-positive
dendritic cells (DC) are visible in the portal
area (arrows). × 500. b: A mouse treatedwith griseofulvin for 8 weeks. The numberof NLDC-145-positive DC (arrow) is obviously decreased. Proliferating bile ductules
are colored brown. × 500.

ab10