Dendritic cells in protoporphyric liver

Number of
NLDC-145-positive DC/mm2

In the control liver, NLDC-1458
positive hepatic DC were located mainly in the portal area and were rarely
7
seen in the parenchyma (Fig. 3a). The
density of these hepatic DC gradually
6
decreased with the duration of griseo5
fulvin feeding (Fig. 4). After 6 weeks,
the DC disappeared in most of the4
portal area (Fig. 3b), and the density of3the hepatic DC was less than 10% ofthat in the controls (Fig. 4). However,2
the index of the number of DC in thewhole liver showed a different course1of changes, increasing slightly until 40weeks, but falling sharply at 6 weeks
0
1
2
3
4
6
8
C
(Fig. 5).
Week
On the other hand, F4/80-positive
cells in the control liver were scattered Fig. 4. Changes in the density of NLDC-145-positive DC inin the hepatic parenchyma and the por- the liver after griseofulvin feeding. The density is signifital area (Fig. 6a). Accumulation of cantly decreased after 1 week. Data are expressed as mean ±macrophages in the portal area was ob- SD. Bars represent SD. C, 13-week-old control mice.
vious at 3 weeks of griseofulvin feeding, but the increase in the density ofKupffer cells in the sinusoidal area was
In the spleen and lung, no significant difrelatively gradual (Fig. 7). At 8 weeks on the ferences were noted in the distribution ofgriseofulvin diet, Kupffer cells were sometimes NLDC-145-positive DC between experimental
hypertrophic, and clusters of Kupffer cells often and control mice (Figs. 8 and 9). These DC
contained brown pigment deposits (Fig. 6b). In were located mainly in the periarterial
this stage, the density of Kupffer cells was lymphoid sheath in the spleen, and studded thesignificantly increased (Fig. 7).
lung parenchyma.

Index of the numberof DC in the whole liver

2

1

0

0

1

2

3

4

6

8

C

Week
Fig. 5. Changes in the index of the number of DC in the whole liver after griseofulvin feeding. The meanvalue of the products [cell density (/mm2) by liver weight (g)] was calculated in each stage, and the ratio of thevalue to that of the 5-week-old controls was evaluated. After 6 weeks, the index falls sharply. C, 13-week-old
control mice.

11

R. Kashiwagi et al.

weeks. These results suggested that the decrease in density of hepatic DC was due to hepatomegaly until 4 weeks of treatment, and wassubstantial after 6 weeks.
In contrast to the decrease in number ofhepatic DC, there were no definite changes inthe distribution of NLDC-145-positive DC in
the spleen or lung. This implied that the decrease in number of NLDC-145-positive DC
was specific in the liver. To evaluate these observations, we considered the results of previous reports showing the behavior of circulating
DC and turnover time of tissue DC. Intravenously transferred DC migrate mainly to theliver and spleen, and partly to the lungs andother organs (Fossum, 1988; Kupiec-Weglinski
et al., 1988; Suda et al., 1996). A physiologicalstudy further showed that the turnover time ofmouse splenic DC is 8–11 days (Steinman et al.,1974). If the decrease of hepatic DC
was due to a reduction in number ofcirculating DC, splenic DC shouldhave decreased in number in the laterstage. However, splenic DC showedno definite changes after 8 weeks ofgriseofulvin feeding, suggesting thatthe number of circulating DC does notdecrease in protoporphyric mice atleast until about 2 months. Accordingly, it is reasonable to speculate thatsome changes that occurred in the injured liver caused the specific decreasein hepatic DC number in protoporphyric mice.
The liver is an important organ forblood DC to migrate to the lymph.
Blood DC attach to the vessel wall inthe sinusoidal area, pass through thespace of Disse to the connective tissue
stroma of either the portal or hepaticvein area, and then enter the initial
lymphatic ducts accumulating in theregional hepatic lymph nodes via theafferent lymph (Kudo et al., 1997). It

Discussion
This study showed a decrease in density of
NLDC-145-positive hepatic DC and an increase in that of F4/80-positive Kupffer cells ingriseofulvin-induced protoporphyric liver injury.
The decrease in density of hepatic DC indicated a reduction of hepatic DC per unit volumeof the liver. However, this change does not imply a decrease in number in the whole liver.
Hepatomegaly, an increase in the volume of theliver, might be responsible for the decrease indensity of hepatic DC. Therefore, we calculated the index of the number of DC in the whole
liver. Hepatic DC number in the whole liverappeared to increase slightly until 4 weeks ofgriseofulvin treatment and fell sharply after 6abFig. 6. Immunostaining of the liver with monoclonal antibody F4/80 for Kupffer cells (red). a: A 5-week-old
control mouse. F4/80-positive Kupffer cells are distributed in the sinusoid. × 250. b: A mouse treated withgriseofulvin for 8 weeks. F4/80-positive Kupffer cells are hypertrophic and markedly increased in number.
Note clusters of Kupffer cells containing brown pigment deposits (arrows). × 250.

12

Dendritic cells in protoporphyric liver

600

Number of F4/80-positive
Kupffer cells/mm2

is probably important for migrationthat the normal hepatic structure andfunction are maintained. Alteration ofliver structure after 4 weeks ofgriseofulvin feeding might interrupt
the migration pathway and subsequently cause the decrease of DC in
the whole liver. After 4 weeks ofgriseofulvin feeding, marked ductularreaction occurred in the injured liver(Shapiro and Wessely, 1984). This
reaction is a characteristic change
observed in the cholestasis of bile ductobstruction (Slott et al., 1990; Desmet,
1995; Roskams and Desmet, 1998).
Recently, Tanimoto et al. (1999) reported that DC numbers were extremely low in the livers of patientswith large bile duct obstruction. Thisimplies that ductular reaction mightcause a decrease in number of DC in
the whole liver. However, ductular reaction has also been shown to occur inother liver diseases in which hepatic
DC increase in number (Popper, 1986;
Roskams and Desmet, 1998). Therefore, we could not conclude that theductular reaction we found led to a decrease of hepatic DC in protoporphyricmice.
DC have been suggested to infiltrate the liver 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). In contrast, hepatic DC seem to have no significant relation to the progression ofgriseofulvin-induced protoporphyric
liver injury as these cells decreased in
number in this type of liver injury.

500

400

300

200

100

0
0

1

2

3

4

6

8

C

Week
Fig. 7. Changes in the density of F4/80-positive Kupffer
cells after griseofulvin feeding. The density of these cells
shows a significant increase at 8 weeks. Data are expressed
as mean ± SD. Bars represent SD. C, 13-week-old control
mice.

a

b

Fig. 8. NLDC-145-positive DC in the spleen of a 5-week-old control mouse (a) and a mouse treated withgriseofulvin for 8 weeks (b). There are no significant differences in the distribution of splenic DC between
the control and experimental animals. The splenic DC are distributed in the periarterial lymphoid sheath(arrows). × 280.

13

R. Kashiwagi et al.

might be due to the decrease in
number of hepatic DC.
In protoporphyric mice, Kupffer
cells are activated to phagocytose protoporphyrin crystals (Gschnait et al.,1975). However, quantitative
analysis of Kupffer cells has not been
performed. There are no precise
markers for mouse Kupffer cells. In
this study, we used a monoclonal
antibody, F4/80, which recognizes
macrophages including Kupffer cells
in mice. To estimate the density ofKupffer cells in the liver, we counted
the F4/80-positive cells only in thesinusoidal area and excluded
macrophages in the portal area. Our
results showed that the density ofKupffer cells gradually increased in
the injured liver. Considering theeffect of hepatomegaly on the densityof Kupffer cells, the number of Kupffer
cells in the whole liver probably increased more significantly than theirdensity revealed. However, we could
not calculate the index of the number
Fig. 9. NLDC-145-positive DC in the lung of a 5-week-oldof Kupffer cells in the whole liver
control mouse (a) and a mouse treated with griseofulvin for 8
from the product of their density by
weeks (b). In these sections, DC show a similar distribution in
the parenchyma (arrows). × 280.
liver weight because they werelocated only in the sinusoidal area.
Recently, it was reported that
These observations raise the question of Kupffer cells are involved in the migration ofwhether the decrease in hepatic DC number af- hepatic DC; i.e. Kupffer cells were shown to befects the immune system in protoporphyric capable of selectively trapping DC from themice. Previous studies showed that griseofulvin- blood (Suda et al., 1996; Kudo et al., 1997). In
treated mice were susceptible to bacterial infec- the present study, since densities of DC andtion (Weston-Hurst and Paget, 1963; Mitchell Kupffer cells showed opposite changes in the
et al., 1973), and suggested a selective depress- injured liver, the decrease of hepatic DC seeming effect of griseofulvin on the cellular im- ed to have no relation to Kupffer cells. Howmune response in mice (Mitchell et al., 1973). ever, we could not eliminate the possibility that
Recently, Matsuno et al. (1996) suggested that Kupffer cells failed to trap circulating DC in the
DC phagocytosed intravenously injected parti- protoporphyric liver. Further studies are needcles in the blood marginating pool and trans- ed to determine whether Kupffer cells in protolocated from the sinusoidal area to the hepatic porphyric mice can trap DC from the blood.
lymph, and these cells might initiate immune
In conclusion, we demonstrated a decrease
responses against pathogenic organisms such as in number of hepatic DC and an increase inbacteria and fungi. The susceptibility to bacte- number of Kupffer cells in protoporphyric liverrial infection and the depression of the cellular injury using an experimental mouse model andimmune response in griseofulvin-treated mice immunohistochemical methods. It seems that

a

b

14

Dendritic cells in protoporphyric liver

fulvin-treated mice. Lab Invest 1989;61:609–
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10 Kudo S, Matsuno K, Ezaki T, Ogawa M. A novel
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the decrease in density of hepatic DC is due tohepatomegaly until 4 weeks of griseofulvintreatment, and is substantial after 6 weeks.
Alteration of the liver structure might cause thissubstantial decrease in number of hepatic DC.
Although hepatic DC seemed to have no relation to the progression of the griseofulvininduced protoporphyric liver injury, their decrease might affect the cellular immune response in protoporphyria.

Acknowledgments: The authors are grateful toProfessor H. Kawasaki of the Second Department ofInternal Medicine and Professor T. Terada of theSecond Department of Pathology, Faculty of Medicine,
Tottori University, for their helpful discussions.
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Received December 18, 2000; accepted December 21, 2000
Corresponding author: Dr. Ryota Kashiwagi

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