JOURNALOF PATHOLOGY, VOL.
160: 223 230 ( 1990)
DISTRIBUTION O F NON-LYMPHOID, INFLAMMATORY CELLS IN CHRONIC HBV INFECTION JOOST J . VAN DEN OORD, RITA D E VOS, FABIO FACCHETTI, JAN DELABIE, CHRIS DE WOLF-PEETERS AND VALEER J. DESMET
Department of Pathology. Luhorutory of Histoc~lieniistryand Cytochwiistry, University Hospitol St Rutuel, Catholic University of Liweti, Leuveri, Belgium Rrc.eived 21 August 1989 Accepted 29 November 1989
SUMMARY Non-lymphoid cells play a key role in the initiation and maintenance of cellular immune responses. Using in-situ immunohistochemical techniques and a panel of monoclonal antibodies (mcabs) reactive with BS-fixed, paraffincrnbedded liver biopsies. we analysed the non-lymphoid cell component in inflammatory infiltrates in 20 cases of chronic hepatitis B virus (HBV) infection. In addition. lymphocyte subsets and HLA-DR antigens were studied. Mcab KPI labelled scattered Kupffer cells. which variably expressed HLA-DR antigens. Their random distribution and lack of significant topographical association with lymphocytes suggest that classical Kupffer cells d o not play a major role in cell-mediated immune reactions. On the other hand. mcab Mac387 was unreactive with normal liver tissue but labelled HLA-DR dendritic cells in areas of intralobular inflammation. On (immuno)electron microscopy, these Mac387+ dendritic cells were situated in the Disse space. where they formed close contacts with lymphocytes. Similar dendritic cells were situated at the edge of portal tracts in cases of chronic active. but not chronic persistent hepatitis. lmmunostaining on serial frozen sections revealed their close topographical association with cytotoxic/suppressor T-cells. suggesting that Mac387 + HLA-DR + dendriticcells play an immunomodulatory role in theeffector arm of the cellular immune response that takes place in the periphery of portal tracts and the lobular parenchyma, and that involves activation and proliferation of cytotoxic T cells. Finally. large Mac387 - HLA-DR + dendritic cells expressing the LN2 marker were situated amidst helperiinducer T-cells in the centre of severely inflamed portal tracts. In analogy with their role in the lymph node. these cells are likely to act as accessory cells in the afferent arm of the immune response that involves antigen presentation to helper T-cells which subsequently assist in the generation of plasma cells. According to the types of dendritic cells and their associated T-cell subsets. different functional domains involved in the afferent and efferent limbs of the immune response can be distinguished in liver tissue involved by chronic HBV inl'ection.
+
KI:Y WORl>S--LiVCr.
hepatitis B virus. monocytes. accessory cells. immunology. immunohistochemistry.
INTRODUCTION Various clinical a n d experimental studies have yielded circumstantial evidence that the immune response. rather than the virus itself, plays a key role in the pathogenesis and outcome of chronic liver disease induced by the hepatitis B virus (HBV).' Addressee
for correspondence: J. J. van den Oord,
Department of Pathology. Laboratory of Histochemistry and Cytochemistry. University Hospital St Rafael. Catholic University OF Leuven. Leuven. Belgium. 0022-341 7 90'030223-08 $05.00 0 1990 by John Wiley & Sons. Lid.
Recent immunohistochemical studies o n the phenotype of the inflammatory cells have confirmed this view and have demonstrated a predominance of T-lymphocytes in the periportal and lobular parenchyma, stressing the role of T-cell mediated cellular immune reactions in chronic hepatitis B.'-' The initiation and maintenance of cellular immune responses depend on the presence and activity of non-lymphoid. 'accessory' cells.' In the afferent limb, these cells present foreign antigens or immunogenic parts thereof to antigen-specific
224
1. J . V A N DEN OORD E T A L .
T-lymphocytes in the context of self major histocompatibility complex ( M H C ) products or HLA antigens: in the efferent limb of the cellular immune response. these cells modulate the activation and proliferation of antigen-specific cytotoxic T-cells. Experimental studies have revealed that the immune response to both the he atitis B surface depen(HBs) antigen (Ag)‘ and HBeAg’ IS T-cell . dent and requires accessory cells.’ ‘ . I 2 However. the exact nature of these accessory cells and their distribution in HBV-infected liver tissue are still unknown. In the present study. we have searched for non-lymphoid cells in the inflammatory infiltrates in chronic hepatitis B using monoclonal antibodies recognizing different subsets of monocyte-derived cells in paraffin-embedded material. In addition. the microenvironment in which these cells are located was studied with a panel of antibodies directed to €3- and T-lymphocytes. and HLA-DR antigens.
R..
MATERIALS AND METHODS Twenty liver biopsies from patients with serologically and immunohistochemically proven HBV infection were used for this study. They comprised ten cases of chronic active hepatitis B with variable degrees of periportal inflammation, four cases of chronic persistent hepatitis B, and six cases of posthepatitic B cirrhosis. For comparison. a series of ten liver biopsies without obvious changes were also studied. All samples were received fresh and divided into three parts. A representative part was fixed in B5 fixative. embedded in paraffin. and used for both routine histology and immunohistochemistry. Another part was snap-frozen in liquid nitrogencooled isopentane and stored at - 75 C until used for immunohistochemistry. Finally. a small part of each sample was processed for electron microscopy and. in one case. for immuno-electron microscopy according to a previously described method.l7 In all cases. a three-step indirect immunoperoxidase procedure was performed on serially cut, €35-fixed tissue sections. Following inhibition of endogenous peroxidase in a solution of methanol and H,O,. the tissue sections were incubated with the foliowing monoclonal antibodies (mcabs): MTI (Biotest Seralc. Brussels. Belgium: diluted 1 :10). reacting with the 100, 110. and 190 k D molecule(s) present on T-cells, mononuclear phagocytes. myeloid cells. and erythrocyte precursors;“ M B2 (Biotest: diluted l:!O), defining the 31-35 k D molecule present on all B-cells except plasma cells;’5
LN2 (Biotest; diluted 1 : lo), identifying a 3 1 35 kD molecule on B-cells. macrophages. and interdigitating reticulum cells;” TALlB5 (a kind gift of Sir Walter Bodmer, Imperial Cancer Research Fund, London, U.K.), defining the invariant part of HLADR alpha chains;” KPl (a generous gift of Dr D. Y . Mason (Nuffield Department of Pathology, Oxford, U.K.), identifying mononuclear phagocytes;Ix and Mac387 (Dakopatts sia, Copenhagen. Denmark; diluted 1:200). reacting with monocytes and some monocyte-derived cells, but not with classical macrophages.” The latter antibody required prior trypsinization of the tissue section according to standard procedures. The secondary and tertiary antibodies consisted of peroxidaseconjugated rabbit anti-mouse and peroxidaseconjugated swine anti-rabbit immunoglobulins (Ig), respectively. S-100 protein was detected with the three-step unlabelled peroxidase-antiperoxidase (PAP) method using rabbit anti-S-100 antibody. swine anti-rabbit Ig and rabbit PAP complex (all obtained from Dakopatts sia). Analysis ofT-cell subsets was carried out on serial frozen sections of five cases with a similar three-step indirect immunoperoxidase method. CD4 + helper/ inducer T-cells were detected with a mixture of monoclonal antibodies OKT4 (Ortho Pharmaceuticals. Raritan. NJ; diluted 1:20) and Leu3a (Becton-Dickinson, Sunnyvale, CA; diluted I :20);” CD8 + suppressor/cytotoxic T-lymphocytes were identified with monoclonal antibody OKT8 (Ortho Pharmaceuticals). All incubations were carried out for 30 min at room temperature, and followed by a wash in three changes of phosphate-buffered saline (PBS). pH 7.4. Peroxidase activity was detected with 3.3’-diaminobenzidine and H,O,. Controls. which were invariably negative, consisted of omission of primary or secondary antibody. and of substitution of the primary antibody by non-immune mouse ascites. RESULTS
In normal liver tissue. mcab KPI identified scattered cells in the sinusoids. These cells were also labelled with mcab LN2, variably expressed HLADR antigens and frequently contained phagocytosed material in their cytoplasm. According to their morphology and distribution, these cells were considered to represent classical Kupffer cells. Mcab Mac387, on the other hand, was virtually unreactive with normal liver tissue and labelled only
NON-LYMPH0111 CELLS IN HBV-INFECTED I,IVER
monocytes and polymorphonuclear granulocytes in the sinusoids. Few scattered MTI + T-lymphocytes were observed in portal tracts and in the lobular parenchyma. In addition to its reactivity with few. scattered B-lymphocytes in portal tracts. mcab MB2 labelled bile duct( ule)s. some periportal hepatocytes. and slender sinusoidal lining cells. different from the KPI cells and presumably corresponding to endothelial cells. S-1 00 protein-positive cells were completely lacking in normal liver tissue. In chronic hepatitis and cirrhosis. variable numbers of M T I + T-lymphocytes were observed in areas of focal intralobular and periportal inflammation where they were admixed with small numbers of KPI + cells. Mcab Mac387 labelled two populations of mononuclear cells. One population consisted of scattered small. round, darkly stained cells in the sinusoids; the other population consisted of medium-sized to large cells with an irregular nuclear outline and abundant cytoplasm which often displayed dendritic features. On serial sections, cells with similar morphology and distribution were found t o carry large amounts of HLADR antigens. These cells will be further referred t o as 'dendritic cells'. In all cases. pale dendritic Mac387 + HLA-DR cells were abundant in areas of focal intralobular inflammation where they expressed LN2. and where they were admixed with variable numbers of MTI + T-lymphocytes (Fig. 1 ): immunostaining for Mac387 and anti-T-cell subset antibodies on serial frozen sections revealed these T-cells to belong largely to the CD8 + suppressor!cytotoxic T-cell subset. In portal tracts, Mac387-t HLAD R + pale dendritic cells showed either a scattered distribution or were preferentially situated in a ringlike distribution at the border between the portal tract and parenchyma (Fig. 2) where they were admixed with MTI + T-lymphocytes; on serial frozen sections. these T-cells were also found to belong mainly to the C D 8 + suppressoricytotoxic T-cell subset (Fig. 3). This preferential distribution of Mac387 + dendritic cells at the margin of portal tracts was observed only in cases of chronic active hepatitis. Both in areas of spotty intralobular inflammation and in inflamed portal tracts. the pale dendritic Mac387+ cells were admixed with variable numbers of small. round, darkly stained Mac387 + cells. Another type of large, dendritic cell with irregular nuclear contour. open chromatin. and abundant cytoplasm was situated in the centre of inflamed portal tracts (Fig. 4). These dendritic cells were
225
+
+
Fig. I-Area of spolty necrosis in a case of chronic active hepatitis B. showing many Mac387 + mononuclear cells amidst lymphocytes. Immunoperoxidase Fig. ?-Mac387+ dendritic cells are distributed at the outer margin of the portal tract in this case o f chronic active hepatitis B. Scattered darkly stained monocytes are seen throughout the parenchyma. whereas Kupffer cells are unreactive. Immunoperoxidase
unreactive with mcab Mac387 and occasionally expressed the S- 100 protein; they showed strong positivity for the LN2 marker and carried large amounts of HLA-DR antigens. The associated MTI + T-cells in these areas belonged mainly to the CD4 + helper,'inducer subset. Well-formed lymphoid follicles composed of MB2+ B-lymphocytes were observed in three cases of chronic active hepatitis and two cases of active cirrhosis. All other cases of chronic hepatitis showed variable numbers of scattered MB2+ B-cells in their portal tracts. In areas of inflammation. mcab LN2 diffusely labelled the cytoplasm of variable numbers of hepatocytes (Fig. 5 ) . Staining for HLA-DR antigens on serial sections revealed membranous positivity on hepatocytes in eight cases; in only a minority of these cases was concordance between LN2 and HLA-DR reactivity and distribution found.
J . J . V A N DEN OUR11 FT AL.
DISCUSSION
Fig. 3 serial frozen sections of a case ofchronic active hepatitis H. stained uirh ( a ) Mac3X7. (b)OKTX.and (c)amixtureofOKT4
and Leu 3a. l a ) Mac3X7f dendritic cells are present at the inargin o f the portal tract. as well as in scattered areas of spotty iiecrosis in t h e lobular parenchyma. ( b ) CDX+ cytotoxic \uppressor T-cells show a similar distribution. whereas ( c ) C’D1+ helper inducer T-cclls form H nodule in the portal rr,ic‘t (‘Lrrou) that is devoid of Mac3X7 + dendritic cells. Immunoperoxidase
On immuno-electron microscopy, mcab Mac387 labelled oval to irregular stellate cells with cytoplasmic extensions of variable thickness and length (Fig. 6). lmmunoreactivity was observed in the entire hyaloplasm including the cytoplasmic dendrites or extensions. These dendritic immunoreactive cells were situated in the Disse space and occasionally in the sinusoids. differed from endothelial cells. and were especially numerous in areas of spotty and piecemeal necrosis. Their nucleus showed variable indentations. and the heterochromatin was distributed as clumps along the nuclear membrane a n d as small dots in the nucleoplasm. Their cytoplasm contained many small organelles. Typical Kupffer cells in the sinusoids and the endothelial cells in the Disse spaces were unreactive with mcab Mac387.
Using mcabs reactive with paraffin-embedded tissue. we have analysed the cellular composition of inflammatory infiltrates in chronic HBV infection a n d have observed subsets of non-lymphoid cells that differ in their immunophenotype and distribution. Mcab KPI identified classical Kupffer cells, scattered throughout the liver lobule. as well as occasional plump macrophages in the portal tracts in both normal liver and in chronic hepatitis B. These immunoreactive cells were LN2-positive. variably expressed HLA-DR antigens, and frequently contained debris in their cytoplasm. According to their phenotype. distribution, and lack of apparent relationship with lymphocytes. K P I + macrophages are more likely to be involved in phagocytosis of various substances, rather than to play an accessory role for T-lymphocytes in lobular or portal inflammatory processes. In contrast. two types of cells with dendritic morphology were found in close association with subsets of T-lymphocytes. Although we did not perform double staining immunohistochemistry. the phenotype of these dendritic cells could be well defined on serially stained sections, since the use of BS-fixed. paraffin-embedded tissue guaranteed a well-preserved morphology in combination with a good immunohistochemical reactivity. In the centre of heavily inflamed portal tracts. large HLA-DR LN2 + dendritic cells that occasionally expressed S- 100 protein but lacked reactivity for Mac387 were situated amidst C D 4 + helperiinduced T-cells. In the lymph node. dendritic cells with a similar phenotype and micro-environment correspond to interdigitating reticulum cells.” These cells are situated in the paracortex and are considered to participate in the afferent limb of the cellular immune response by presentation of foreign antigens in association with HLA-DR antigens to helper,’ inducer T-cells.” We suggest that the H L A - D R + L N 2 + Mac387- dendritic cells in the centre of inflamed portal tracts perform a similar antigenpresenting function and are involved in the generation of immunoregulatory helper and suppressor T-cells. These T-cells subsequently assist in the maturation of B-cells into immunoglobulinsecreting plasma cells and modulate the effector function of C D 8 + cytotoxic T-cells. Unlike Flavell or u / , I y we did not observe reactivity of mcab Mac387 with classical Kupffer cells. These discrepancies can be explained by the
+
YON-I.YMPHOII> CEI.1.S IN HBV-INFF
160: 223 230 ( 1990)
DISTRIBUTION O F NON-LYMPHOID, INFLAMMATORY CELLS IN CHRONIC HBV INFECTION JOOST J . VAN DEN OORD, RITA D E VOS, FABIO FACCHETTI, JAN DELABIE, CHRIS DE WOLF-PEETERS AND VALEER J. DESMET
Department of Pathology. Luhorutory of Histoc~lieniistryand Cytochwiistry, University Hospitol St Rutuel, Catholic University of Liweti, Leuveri, Belgium Rrc.eived 21 August 1989 Accepted 29 November 1989
SUMMARY Non-lymphoid cells play a key role in the initiation and maintenance of cellular immune responses. Using in-situ immunohistochemical techniques and a panel of monoclonal antibodies (mcabs) reactive with BS-fixed, paraffincrnbedded liver biopsies. we analysed the non-lymphoid cell component in inflammatory infiltrates in 20 cases of chronic hepatitis B virus (HBV) infection. In addition. lymphocyte subsets and HLA-DR antigens were studied. Mcab KPI labelled scattered Kupffer cells. which variably expressed HLA-DR antigens. Their random distribution and lack of significant topographical association with lymphocytes suggest that classical Kupffer cells d o not play a major role in cell-mediated immune reactions. On the other hand. mcab Mac387 was unreactive with normal liver tissue but labelled HLA-DR dendritic cells in areas of intralobular inflammation. On (immuno)electron microscopy, these Mac387+ dendritic cells were situated in the Disse space. where they formed close contacts with lymphocytes. Similar dendritic cells were situated at the edge of portal tracts in cases of chronic active. but not chronic persistent hepatitis. lmmunostaining on serial frozen sections revealed their close topographical association with cytotoxic/suppressor T-cells. suggesting that Mac387 + HLA-DR + dendriticcells play an immunomodulatory role in theeffector arm of the cellular immune response that takes place in the periphery of portal tracts and the lobular parenchyma, and that involves activation and proliferation of cytotoxic T cells. Finally. large Mac387 - HLA-DR + dendritic cells expressing the LN2 marker were situated amidst helperiinducer T-cells in the centre of severely inflamed portal tracts. In analogy with their role in the lymph node. these cells are likely to act as accessory cells in the afferent arm of the immune response that involves antigen presentation to helper T-cells which subsequently assist in the generation of plasma cells. According to the types of dendritic cells and their associated T-cell subsets. different functional domains involved in the afferent and efferent limbs of the immune response can be distinguished in liver tissue involved by chronic HBV inl'ection.
+
KI:Y WORl>S--LiVCr.
hepatitis B virus. monocytes. accessory cells. immunology. immunohistochemistry.
INTRODUCTION Various clinical a n d experimental studies have yielded circumstantial evidence that the immune response. rather than the virus itself, plays a key role in the pathogenesis and outcome of chronic liver disease induced by the hepatitis B virus (HBV).' Addressee
for correspondence: J. J. van den Oord,
Department of Pathology. Laboratory of Histochemistry and Cytochemistry. University Hospital St Rafael. Catholic University OF Leuven. Leuven. Belgium. 0022-341 7 90'030223-08 $05.00 0 1990 by John Wiley & Sons. Lid.
Recent immunohistochemical studies o n the phenotype of the inflammatory cells have confirmed this view and have demonstrated a predominance of T-lymphocytes in the periportal and lobular parenchyma, stressing the role of T-cell mediated cellular immune reactions in chronic hepatitis B.'-' The initiation and maintenance of cellular immune responses depend on the presence and activity of non-lymphoid. 'accessory' cells.' In the afferent limb, these cells present foreign antigens or immunogenic parts thereof to antigen-specific
224
1. J . V A N DEN OORD E T A L .
T-lymphocytes in the context of self major histocompatibility complex ( M H C ) products or HLA antigens: in the efferent limb of the cellular immune response. these cells modulate the activation and proliferation of antigen-specific cytotoxic T-cells. Experimental studies have revealed that the immune response to both the he atitis B surface depen(HBs) antigen (Ag)‘ and HBeAg’ IS T-cell . dent and requires accessory cells.’ ‘ . I 2 However. the exact nature of these accessory cells and their distribution in HBV-infected liver tissue are still unknown. In the present study. we have searched for non-lymphoid cells in the inflammatory infiltrates in chronic hepatitis B using monoclonal antibodies recognizing different subsets of monocyte-derived cells in paraffin-embedded material. In addition. the microenvironment in which these cells are located was studied with a panel of antibodies directed to €3- and T-lymphocytes. and HLA-DR antigens.
R..
MATERIALS AND METHODS Twenty liver biopsies from patients with serologically and immunohistochemically proven HBV infection were used for this study. They comprised ten cases of chronic active hepatitis B with variable degrees of periportal inflammation, four cases of chronic persistent hepatitis B, and six cases of posthepatitic B cirrhosis. For comparison. a series of ten liver biopsies without obvious changes were also studied. All samples were received fresh and divided into three parts. A representative part was fixed in B5 fixative. embedded in paraffin. and used for both routine histology and immunohistochemistry. Another part was snap-frozen in liquid nitrogencooled isopentane and stored at - 75 C until used for immunohistochemistry. Finally. a small part of each sample was processed for electron microscopy and. in one case. for immuno-electron microscopy according to a previously described method.l7 In all cases. a three-step indirect immunoperoxidase procedure was performed on serially cut, €35-fixed tissue sections. Following inhibition of endogenous peroxidase in a solution of methanol and H,O,. the tissue sections were incubated with the foliowing monoclonal antibodies (mcabs): MTI (Biotest Seralc. Brussels. Belgium: diluted 1 :10). reacting with the 100, 110. and 190 k D molecule(s) present on T-cells, mononuclear phagocytes. myeloid cells. and erythrocyte precursors;“ M B2 (Biotest: diluted l:!O), defining the 31-35 k D molecule present on all B-cells except plasma cells;’5
LN2 (Biotest; diluted 1 : lo), identifying a 3 1 35 kD molecule on B-cells. macrophages. and interdigitating reticulum cells;” TALlB5 (a kind gift of Sir Walter Bodmer, Imperial Cancer Research Fund, London, U.K.), defining the invariant part of HLADR alpha chains;” KPl (a generous gift of Dr D. Y . Mason (Nuffield Department of Pathology, Oxford, U.K.), identifying mononuclear phagocytes;Ix and Mac387 (Dakopatts sia, Copenhagen. Denmark; diluted 1:200). reacting with monocytes and some monocyte-derived cells, but not with classical macrophages.” The latter antibody required prior trypsinization of the tissue section according to standard procedures. The secondary and tertiary antibodies consisted of peroxidaseconjugated rabbit anti-mouse and peroxidaseconjugated swine anti-rabbit immunoglobulins (Ig), respectively. S-100 protein was detected with the three-step unlabelled peroxidase-antiperoxidase (PAP) method using rabbit anti-S-100 antibody. swine anti-rabbit Ig and rabbit PAP complex (all obtained from Dakopatts sia). Analysis ofT-cell subsets was carried out on serial frozen sections of five cases with a similar three-step indirect immunoperoxidase method. CD4 + helper/ inducer T-cells were detected with a mixture of monoclonal antibodies OKT4 (Ortho Pharmaceuticals. Raritan. NJ; diluted 1:20) and Leu3a (Becton-Dickinson, Sunnyvale, CA; diluted I :20);” CD8 + suppressor/cytotoxic T-lymphocytes were identified with monoclonal antibody OKT8 (Ortho Pharmaceuticals). All incubations were carried out for 30 min at room temperature, and followed by a wash in three changes of phosphate-buffered saline (PBS). pH 7.4. Peroxidase activity was detected with 3.3’-diaminobenzidine and H,O,. Controls. which were invariably negative, consisted of omission of primary or secondary antibody. and of substitution of the primary antibody by non-immune mouse ascites. RESULTS
In normal liver tissue. mcab KPI identified scattered cells in the sinusoids. These cells were also labelled with mcab LN2, variably expressed HLADR antigens and frequently contained phagocytosed material in their cytoplasm. According to their morphology and distribution, these cells were considered to represent classical Kupffer cells. Mcab Mac387, on the other hand, was virtually unreactive with normal liver tissue and labelled only
NON-LYMPH0111 CELLS IN HBV-INFECTED I,IVER
monocytes and polymorphonuclear granulocytes in the sinusoids. Few scattered MTI + T-lymphocytes were observed in portal tracts and in the lobular parenchyma. In addition to its reactivity with few. scattered B-lymphocytes in portal tracts. mcab MB2 labelled bile duct( ule)s. some periportal hepatocytes. and slender sinusoidal lining cells. different from the KPI cells and presumably corresponding to endothelial cells. S-1 00 protein-positive cells were completely lacking in normal liver tissue. In chronic hepatitis and cirrhosis. variable numbers of M T I + T-lymphocytes were observed in areas of focal intralobular and periportal inflammation where they were admixed with small numbers of KPI + cells. Mcab Mac387 labelled two populations of mononuclear cells. One population consisted of scattered small. round, darkly stained cells in the sinusoids; the other population consisted of medium-sized to large cells with an irregular nuclear outline and abundant cytoplasm which often displayed dendritic features. On serial sections, cells with similar morphology and distribution were found t o carry large amounts of HLADR antigens. These cells will be further referred t o as 'dendritic cells'. In all cases. pale dendritic Mac387 + HLA-DR cells were abundant in areas of focal intralobular inflammation where they expressed LN2. and where they were admixed with variable numbers of MTI + T-lymphocytes (Fig. 1 ): immunostaining for Mac387 and anti-T-cell subset antibodies on serial frozen sections revealed these T-cells to belong largely to the CD8 + suppressor!cytotoxic T-cell subset. In portal tracts, Mac387-t HLAD R + pale dendritic cells showed either a scattered distribution or were preferentially situated in a ringlike distribution at the border between the portal tract and parenchyma (Fig. 2) where they were admixed with MTI + T-lymphocytes; on serial frozen sections. these T-cells were also found to belong mainly to the C D 8 + suppressoricytotoxic T-cell subset (Fig. 3). This preferential distribution of Mac387 + dendritic cells at the margin of portal tracts was observed only in cases of chronic active hepatitis. Both in areas of spotty intralobular inflammation and in inflamed portal tracts. the pale dendritic Mac387+ cells were admixed with variable numbers of small. round, darkly stained Mac387 + cells. Another type of large, dendritic cell with irregular nuclear contour. open chromatin. and abundant cytoplasm was situated in the centre of inflamed portal tracts (Fig. 4). These dendritic cells were
225
+
+
Fig. I-Area of spolty necrosis in a case of chronic active hepatitis B. showing many Mac387 + mononuclear cells amidst lymphocytes. Immunoperoxidase Fig. ?-Mac387+ dendritic cells are distributed at the outer margin of the portal tract in this case o f chronic active hepatitis B. Scattered darkly stained monocytes are seen throughout the parenchyma. whereas Kupffer cells are unreactive. Immunoperoxidase
unreactive with mcab Mac387 and occasionally expressed the S- 100 protein; they showed strong positivity for the LN2 marker and carried large amounts of HLA-DR antigens. The associated MTI + T-cells in these areas belonged mainly to the CD4 + helper,'inducer subset. Well-formed lymphoid follicles composed of MB2+ B-lymphocytes were observed in three cases of chronic active hepatitis and two cases of active cirrhosis. All other cases of chronic hepatitis showed variable numbers of scattered MB2+ B-cells in their portal tracts. In areas of inflammation. mcab LN2 diffusely labelled the cytoplasm of variable numbers of hepatocytes (Fig. 5 ) . Staining for HLA-DR antigens on serial sections revealed membranous positivity on hepatocytes in eight cases; in only a minority of these cases was concordance between LN2 and HLA-DR reactivity and distribution found.
J . J . V A N DEN OUR11 FT AL.
DISCUSSION
Fig. 3 serial frozen sections of a case ofchronic active hepatitis H. stained uirh ( a ) Mac3X7. (b)OKTX.and (c)amixtureofOKT4
and Leu 3a. l a ) Mac3X7f dendritic cells are present at the inargin o f the portal tract. as well as in scattered areas of spotty iiecrosis in t h e lobular parenchyma. ( b ) CDX+ cytotoxic \uppressor T-cells show a similar distribution. whereas ( c ) C’D1+ helper inducer T-cclls form H nodule in the portal rr,ic‘t (‘Lrrou) that is devoid of Mac3X7 + dendritic cells. Immunoperoxidase
On immuno-electron microscopy, mcab Mac387 labelled oval to irregular stellate cells with cytoplasmic extensions of variable thickness and length (Fig. 6). lmmunoreactivity was observed in the entire hyaloplasm including the cytoplasmic dendrites or extensions. These dendritic immunoreactive cells were situated in the Disse space and occasionally in the sinusoids. differed from endothelial cells. and were especially numerous in areas of spotty and piecemeal necrosis. Their nucleus showed variable indentations. and the heterochromatin was distributed as clumps along the nuclear membrane a n d as small dots in the nucleoplasm. Their cytoplasm contained many small organelles. Typical Kupffer cells in the sinusoids and the endothelial cells in the Disse spaces were unreactive with mcab Mac387.
Using mcabs reactive with paraffin-embedded tissue. we have analysed the cellular composition of inflammatory infiltrates in chronic HBV infection a n d have observed subsets of non-lymphoid cells that differ in their immunophenotype and distribution. Mcab KPI identified classical Kupffer cells, scattered throughout the liver lobule. as well as occasional plump macrophages in the portal tracts in both normal liver and in chronic hepatitis B. These immunoreactive cells were LN2-positive. variably expressed HLA-DR antigens, and frequently contained debris in their cytoplasm. According to their phenotype. distribution, and lack of apparent relationship with lymphocytes. K P I + macrophages are more likely to be involved in phagocytosis of various substances, rather than to play an accessory role for T-lymphocytes in lobular or portal inflammatory processes. In contrast. two types of cells with dendritic morphology were found in close association with subsets of T-lymphocytes. Although we did not perform double staining immunohistochemistry. the phenotype of these dendritic cells could be well defined on serially stained sections, since the use of BS-fixed. paraffin-embedded tissue guaranteed a well-preserved morphology in combination with a good immunohistochemical reactivity. In the centre of heavily inflamed portal tracts. large HLA-DR LN2 + dendritic cells that occasionally expressed S- 100 protein but lacked reactivity for Mac387 were situated amidst C D 4 + helperiinduced T-cells. In the lymph node. dendritic cells with a similar phenotype and micro-environment correspond to interdigitating reticulum cells.” These cells are situated in the paracortex and are considered to participate in the afferent limb of the cellular immune response by presentation of foreign antigens in association with HLA-DR antigens to helper,’ inducer T-cells.” We suggest that the H L A - D R + L N 2 + Mac387- dendritic cells in the centre of inflamed portal tracts perform a similar antigenpresenting function and are involved in the generation of immunoregulatory helper and suppressor T-cells. These T-cells subsequently assist in the maturation of B-cells into immunoglobulinsecreting plasma cells and modulate the effector function of C D 8 + cytotoxic T-cells. Unlike Flavell or u / , I y we did not observe reactivity of mcab Mac387 with classical Kupffer cells. These discrepancies can be explained by the
+
YON-I.YMPHOII> CEI.1.S IN HBV-INFF
