Ultrastructure of the hepatic sinusoids in the tropical splenomegaly syndrome

638 TRANSACTIONSOF THE ROYAL SOCIETYOF TROPICAL 2~SIEDICINEAND HYGIENE. Vol. 67. No. 5. 1973. ULTRASTRUCTURE

OF T H E H E P A T I C S I N U S O I D S IN T H E T R O P I C A L SPLENOMEGALY SYNDROME

D. J. FLUCK, M. S. R. H U T T , * DIANA M. F L U C K AND P. C. S T U I V E R t

IVellcome Electron Microscope Unit, Makerere University, Karnpala Department of Pathology, Makerere University Medical School, Kampala Department of Medicine, Mulago Hospital, Kampala There is accumulating evidence that the tropical splenomegaly s y n d r o m e - - T S S - seen in Africa, New Guinea and other tropical areas, represents an unusual host-parasite reaction to malaria, and that prolonged antimalarial therapy will cause regression of the spleen in most cases. (WATSON-WILLIaMS and ALLAN, 1968; SA6OE, 1970; STUIWR et al., 1971; LOW~NTHALet al., 1971). The usual diagnostic features of TSS are a high level of IgM (WELLS, 1968; SAGOE, 1970; ZIEGLFRet al., 1969), a high titre of malarial antibodies (GEBBm et al., 1964) and lymphocytic infiltration of the hepatic sinusoids--HSL--(MagSDEN et al., 1965; MaRSI)EN et al., 1967; PITNEY et al., 1968). While this last feature is nearly always present in Ugandan cases it is less constant in cases from West Africa (SAGOE, 1970) and the degree of H S L is not always related to spleen size (MaRSDEN et al., 1967). The histopathological features of the liver have been described by HtrTT, 1966; MaRSDEr~ et al., 1967; and PITNEY et al., 1968 and these authors have emphasized the presence of mature lymphocytes in the liver sinusoids associated with Ktipffer cell hyperplasia and hypertrophy. Malarial pigment is not usually present in the Kiipffer cells and haemosiderin is only occasionally seen. The findings in the liver sinusoids are almost unique though recently they have been reported in Felty's syndrome (BLENDIS et al., 1970). It was felt that ultrastructural studies might throw new light on these changes and possibly relate them to the more recent concept that TSS represents an immune complex disease (ZIEGLER, 1972). The present report is based on an electron microscope study of liver biopsies taken from patients within the group studied by STUIVERet al., 1971. Materials and m e t h o d s Liver specimens were taken with a Menghini needle from 29 patients admitted to the Mulago Hospital, Kampala, with TSS. Bleeding tendencies were excluded by appropriate laboratory tests. One piece of the liver tissue was fixed in 10% formol saline and paraffin sections 5 ~t thick were stained with haematoxylin and eosin, by a silver impregnation method for reticulin and by Perl's reaction for iron. Other pieces of the biopsy were fixed in 2.5% glutaraldehyde in cacodylate buffer at pH 7-4 for 4 hours at 4°C. They were washed overnight at 4°C. in a similar buffer containing 6.8% sucrose and were then post-fixed in 1% Palade's osmium for 1 hour at 4°C. The fixed material was dehydrated through a graded ethanol series, the middle stages of which contained 1% uranyl acetate, was briefly washed in xylene and then infiltrated with increasing concentrations of araldite in xylene. The sample was finally embedded in pure araldite and *Now Professor of Geographical Pathology, Department of Morbid Anatomy, St. Thomas's Hospital Medical School, London, S.E.1. Supported by the Wellcome Trust. tNow at Harbour Hospital, Rotterdam, The Netherlands.

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polymerized at 60°C. The following complete resin mixture was used at the infiltration and embedding stages :--10 ml. of a 27 : 23 mixture of araldite resin CY 212: Hardener HY 964; 0.5 ml. methyl nadic anhydride; 0.2 ml. accelerator DY 064. Sections were obtained with either a Porter-Blum MT-1 or MT-2 ultramicrotome using glass knives. Thin sections stained with 2% uranyl acetate in absolute methanol and Reynold's lead citrate were examined in a Zeiss EM 9S-2 electron microscope at an accelerating voltage of 60kV. Thick sections of approximately 3000 A were stained for light microscopy with 0.5% toluidine blue in 1% borax. Results

Light microscopy Examination of paraffin embedded sections confirmed the observations of previous workers in Uganda. There is dilatation of the liver sinusoids which are lined by hyperplastic Kiipffer cells with a variable infiltration of the sinusoids by mature lymphocytes. Although some parenchymal cell changes are occasionally seen, this appears to be no more frequent than in liver biopsies from other patients in Uganda without overt liver disease. Changes in the portal areas in TSS are similar to those seen in biopsies from other groups of Ugandan Africans. It has been customary to grade liver biopsies according to the average concentration of lymphocytes in the liver sinusoids over the whole section (MARSDEN et al., 1965; 1967) and in this series all grades were seen. Although grading is subjective, repeat observations on biopsies have given reasonably reproducible results. Examples of biopsies prepared for electron microscopy but examined as 0.3 sections by light microscopy are shown in Figs. 1-3. The first is representative of the few cases in which clinical splenomegaly is present, but the liver biopsy shows only occasional lymphocytes in the sinusoids with minor degrees of Kiipffer cell hypertrophy or hyperplasia. Fig. 2 shows the biopsy appearances in the sinusoids of the liver graded as 1 on light microscopy. In this thin section the sinusoids contain numerous nucleated cells some of which are K~ipffer cells and some lymphocytes. In both Figs. 1 and 2 there are examples of apparently empty vesicular structures within the sinusoids. Fig. 3 is typical of a biopsy graded as 3 on light microscopy. The characteristic TSS lymphocytosis is present and the cells are packed closely together so that little cell-free plasma remains. The Kiipffer cells and/or endothelial cells are both hypertrophied and hyperplastic and this combined with the presence of lymphocytes in the sinusoids has led to almost complete obliteration of the lumen so that little cell free plasma and no red cells are seen. No malarial pigment is present in the Kiipffer cells. Examination of the thin resin embedded sections shows that the Kiipffer cells are more hypertrophied and hyperplastic than is apparent on examination of standard paraffin sections and these cells and the infiltrating lymphocytes appear to fill the sinusoid lumen in grade 2 and 3 biopsies. In addition, empty vesicular bodies of uncertain origin may contribute significantly to the sinusoidal changes. These structures appear to be disrupted after formol-saline fixation and paraffm embedding. There is an approximately inverse relationship between the numbers of lymphocytes and of red cells within the sinusoids, and the number of red cells seen gives a good indication of the degree of sinusoidal involvement.

Electron microscopy Normal hepatic sinusoids are lined by heterogeneous groups of ceils which have been identified on the basis of their nltrastructural characteristics as phagocytic, endothelial or protein-secretory in nature (ScHAFFNERet al., 1963; MCGEE and PATRICK, 1972). Perisinusoidal cells lying in the space of Disse are probably the origin of the connective tissue fibrils of the sinusoids (McGEE and PATRICK, 1972) though because of their lipid

640

ULTRASTRUCTURE OF THE HEPATIC SINUSOIDS IN TROPICAL SPLENOMEGALY

content some authors have caUed these fat storage cells (ITO and NEMOTO, 1952). It appears likely that Kiipffer ceUs are derived, hke other macrophages, from the monocytes produced by the marrow (VAN FURTH, 1970) though some authors suggest the endothelial cells are capable of phagocytosis. Fig. 4, which is taken from a typical biopsy of grade 2 TSS shows almost complete filling of the sinusoidal lumen by cells. In the centre is a normal lymphocyte L2 and lying above and below this is the cytoplasm of 2 ceils L1 and L3 which are possibly of the lymphoid series though their nuclei are not seen. L3 shows mitochondria in the cytoplasm and swollen endoplasmic reticulum suggesting this may be an 'activated' ceil. 3 other types of cell are seen in this section. A thin rim of endothelial ceil cytoplasm lining the sinusoid hes adjacent to the space of Disse and the hepatic parenchymal ceU at the bottom of the picture. 2 ceils, KC1 and KC2, fill a large part of the sinusoidal lumen. The plasma membranes of these ceUs (designated here for descriptive purposes as K.C. or Kiipffer cells) are characterized by a labyrinthine network of processes which must considerably extend the surface area of the cells. The channels between adjacent processes can be seen in cross section, sometimes lying deep within the cells. In the extracellular space around and between the processes there is electron dense material D. The third group of cells in the sinusoids which we refer to as M cells (macrophages) are different in many ways from those just described. The extensive network of plasma membrane processes is not a feature of these cells. The background to the M cell cytoplasm is considerably less dense than that of the KC cells and examples of the microfibres normally present in Kiipffer cell cytoplasm are infrequent. In addition there are fewer mitochondria and rough endoplasmic reticulum elements in the M cell. The golgi apparatus is, however, considerably expanded and is associated with an increase in the number of small coated vesicles. The numbers of large coated vesicles are increased and there is evidence of phagocytic activity. The nuclei of both Kiipffer ceils and M cells appear similar. There are frequent instances, in some parts of the sinusoidal system, of partially empty M cells. In these cells, or parts of cells, there tends to be an absence of all organelles with the exception of large coated vesicles. Such cells show a structure very similar to that described by GOLDBERGand GREEN (1959) and regarded by them as characteristic of antigen-antibody-complement interation at the cell surface. Figs. 4 and 5 both show the compactness of the sinusoidal blocks and the infrequency of areas of cell-free plasma. The occasional non-cellular areas apparently contain an electron-dense deposit, as at D in these figures. Fig. 5 shows 2 Kiipffer cells, KC3 and KC4, characterized by the complex proliferation and processes of their surface membranes, large mitochondria, and the fine fibrillar background to the cytoplasm. As in Fig. 4 these ceils show little evidence of phagocytic activity. The cell M2 has most of the characteristics previously described, although there is rather more rough endoplasmic reticuhim and rather less golgi apparatus. The surface membrane does not show proliferation and the fine fibrillar background seen in the KC cell cytoplasm is absent. Such a cell suggests that Kiipffer cells and M cells seen in this situation are functional diversifications of the same cell-type. A quite typical Kiipffer cell is shown in Fig. 6. The nucleus is irregnlar in shape. As in the previous examples there is little evidence of phagocytic activity. The manner in which the membrane protiferations enclose large areas of the dense plasma is clearly shown and it is apparent that the membrane extensions occur primarily on those surfaces of the cell that face the sinusoidal lumen rather than those closely applied to the hepatic cells, or in this instance, the fat containing perisinusoidal c¢11 within the Disse space. Some endothelial ceil cytoplasm can be seen lining the opposite side of the sinusoid.

FIG. 1. Hepatic

sinusoid

Grade 0. The lumen conta

FIG. 2. Hepatic sinusoid Grade 1. The lumen is partly filled with lymphoid and Kiipffer cells. Toluidine blue x 1536.

veral red cells and the nucleus of one Kiipffer ., **on

cell.

FIG. 3. Hepatic sinusoid Grade 3. The lumen is obliterated by lymphoid and Kiipffer cells. Toluidine blue x 1536.

FIG. 4. Hepatic sinusoid Grade 2. The lumen of the sinusoid is filled with lymphoid cells Ll, 2 and 3, Kiipffer cells KC1 and KC2 with complex surface membranes and a macrophage Ml with coated vesicles and an expanded golgi apparatus. x 5,250.

FIG. 5. Hepatic sinusoid Grade 2. The lumen cells KC3 and KC4 and a phagocytic

of the sinusoid contains two Kiipffer macrophage M2. x 9,200.

FIG. 8. Hepatic

FIG. 9. Hepatic

sinusoid

Grade 2. Activated lymphocyte lying surrounded of a Kiipffer cell. x 18,600.

sinusoid Grade 2. Plasma cell in sinusoid v __ --.

by complex

surface membranes

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641

Fig. 7 shows a lymphocyte adjacent to a Kiipffer cell on one side and a macrophage on the other. The high ribosomal content of the lymphocyte and the expansion of its perinuclear space suggest that this cell is within the 'activated lymphocyte' group. It can be seen that the contact area of the lymphocyte with the Kiipffer cell and the M cell is different. Thus the cytoplasmic membranes of the M cell and the lymphocyte are closely applied and in several places seem to merge. The cytoplasm of the adjacent cells is distinguished by the large coated vesicles which are absent in the lymphocyte. By contrast, the extensions of the Kiipffer cell are seen to stop short of the lymphocyte and the cytoplasmic membranes of each cell are clearly distinguished. This figure also shows, at higher magnification, the expanded golgi apparatus and the large numbers of both large and small coated vesicles characteristic of the M cell. Fig. 8 again shows the lack of contact between the complex cytoplasmic process of a Kiipffer cell and an adjacent activated lymphocyte. The latter, in this instance, showing further metabolic expansion of the perinuclear space. Occasionally mature plasma cells are seen in the sinusoids. In Fig. 9 a typical cell lies close to the sinusoidal edge and it is apparent that the extensive rough endoplasmic reticulum and the golgi systems are in an advanced state of activity. In all the sections examined the space of Disse appeared to be narrowed. There did not appear to be any increase in connective tissue fibrils in the sinusoids. Discussion

Hepatomegaly is found in the majority of cases of TSS and usually parallels the degree of splenomegaly (/VlaRSDEN et al., 1965; MUSTAFA, 1965; PRYOR, 1967). The characteristic findings on light microscopy of the liver, obtained at either biopsy or post-mortem, are lymphocytic infiltration of the sinusoids and Kiipffer celi hypertrophy and hyperplasia (HUTT, 1966). Parenchymal cell change is not a feature at this level and other findings such as portal infiltration and fibrosis are no more common than in individuals without splenomegaly living in the same area (FAWDRY, 1955; MARSDEN et al., 1967; PRYOR, 1967; PITNEY et al., 1968). Light microscopy of thin resin sections, whilst confirming the lymphocytosis and the lack of erythrocytes within the sinusoids, particularly emphasizes that the extent of Kiipffer cell hypertrophy is greater than is normally apparent after routine paraffin preparation. The majority of hepatic sinusoids of TSS grade 1 and above appear filled by the numerous hypertrophied Kiipffer cells and the infiltrating lymphocytes, so that cell-free plasma and red cells are rarely seen. Electron microscopy shows that the sinusoids are often filled with cells of different morphological types. Lymphocytes, some of which have features of "activation" are present in the central parts of the sinusoids and very occasional plasma cells are seen. The cells described as hypertrophic and hyperplastlc Kiipffer cells by light microscopy show 2 different morphological types that we have called KC (Kiipffer cell) and M (macrophage). The former do not show phagocytosis and are characterized by a network of processes arising like thin villi from the surface membrane. The M cells are characterized by phagocytosis with numerous large coated vesicles and frequently an empty appearance to much of the cytoplasm. Occasional cells with intermediate features are seen and it seems probable that the KC and M cells are functional diversifications of the same cell. The work of VAN FURTH (1970) suggests that the Kiipffer cells of liver sinusoids are derived from the monocytes of the blood produced by bone marrow. The functional and ultrastructural features of the KC cells are similar to those of dendritic reticular cells, which suggests that they may play a r61e in the trapping of antigen.

642

ULTRASTRUCTURE OF THE HEPATIC SINUSOIDS IN TROPICAL SPLENOMEGALY

SZAr~Land HANNA(1968) studied the distribution of 12Sl~labelled antigen in mouse spleen by autoradiographic techniques and electron microscopy. They showed that within 4 days the labelled antigen was localized between the membrane extensions of the dendritic cells which were in close association with immunoblastic cells. At no time was a significant amount of label within the cytoplasm of these cells. In their experiments the extensive membrane proliferation was short lived and restricted to the time period when antigen can be detected. The presence of lymphoid ceils associated with hypertrophy and hyperplasia of the Kiipffer cells is a characteristic feature of TSS in most parts of the world. A similar association of lymphoid cells and macrophages is present in the spleen and the hyperplasia of these 2 groups of ceils constitutes the basic pathology. In chronic lymphatic leukaemia lymphocytes accumulate in the portal areas rather than the sinusoids and their presence in liver sinusoids, in the absence of gross liver damage, is only reported in the tropical splenomegaly syndrome and Felty's syndrome. Splenectomy in TSS does not alter the appearance in the liver and these changes must therefore be due to the same mechanisms that produce the splenic change. The association of macrophages and lymphoid ceils in these organs suggests that they are the sites of an immunological reaction and that the responsible agents are circulating in the blood stream. The high malarial antibody titre, high IgM levels and constant response to antimalarial therapy suggest that this condition is due to repeated malarial infection though with extremely low and intermittent parasitaemia. It has been suggested that the fundamental abnormality in these cases is a genetically determined proneness to overproduce IgM to a variety of antigens (ZIEGLERand STUIWR, 1972). The other manifestations of an abnormal immunological response include the presence of rheumatoid factor (SHAPERet al., 1968) and of cryoprotein immune complexes (ZIF.GL~.R, 1972). Immunofluorescent studies have shown the presence of IgM and, to a lesser extent, IgG and Ca on the Kiipffer cells of the liver in cases of TSS but not in controls. The electron microscopy findings in the liver sinusoids could be explained by postulating that macromolecular immune complexes are taken up by the blood vascular macrophages (macrophages and Kiipffer cells within the vascular system of the liver and spleen). These complexes may initially be due to a malarial antigen but changes in the globulin molecule may lead to auto-antibody formation. This is associated with the development of a cellular immune reaction as shown by the numerous lymphocytes in the sinusoids of the liver a n d spleen. The significance of this response is uncertain, as is the whole r61e of cellular immunity in malarial infection.

Summary In Ugandan cases of TSS electron microscopy shows that the hepatic sinusoids are filled by Kiipffer ceils and lymphocytes. The Kiipffer ceils are both hyperplastic and hypertrophied, much of the latter being due to extensive proliferation of their surface membranes similar to that of antigen trapping dendritic macrophages from germinal centres. The membrane extensions are apparently functioning as a trapping zone which accumulates an electron-dense deposit on the cell surface. There is no evidence that this deposit is taken up into the cell. A group of cells described here as M cells, though basically similar to Kiipffer cells, lack many of the Kiipffer cell characteristics and in particular the extensive plasma membrane development. Such cells show considerable evidence of uptake of material via large coated vesicles and have fewer mitochondria and less endoplasmic reticulum than the Kiipffer cells. They probably represent a different functional development from the Ktipffer cells or possibly

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643

from a common stem cell. Many of the lymphocytes show, by their ribosome content, expanded perinuclear space, and increased numbers of endoplasmic reticulum elements and mitochondria, that they are not small lymphocytes but are of the activated lymphocyte series. The general appearance is consistent with a complex immunological reaction occurring in the liver sinusoids. REFERENCES BLENDIS, L. M., ANSELL, I. D., JONES, K. L., HAMILTON, E. & WILLIAMS, R. (1970). Br. reed. jr., 1, 131. FAWDRY, A. L. (1955). Trans. R. Soc. trop. 214ed. Hyg., 49, 387. GEBBIE, n . A. i . , HAMILTON,P. J. S., HUTT, M. S. R., MARSDEN, P. D., VOLLER, A. & WILKS, N. E. (1964). Lancet, 2, 392. GOLDBERG,G. & GREEN, H. (1959). ft. exp. Med., 109, 505. HurT, M. S, R. (1966). In: Pathology Annual, Vol. 1, p. 250, Ed. Sommers, S. C. New York: Appleton-Cemury-Croffs. ITO, T. & NEMOTO, M. (1952). Okafimas Folia Anat. flap., 24, 243. LOWENTHAL,M. N., O'RIORDAN,E. C. & HtrrT, M. S. R. (1971). Br. reed. jr., 1, 429. MARSDEN, P. D., HtYrT, M. S. R., WILKS, N. E., VOLLER,A., BLACKMAN,V., SHAH, K. K., CONNER, D. H., HAMILTON, P. J. S., BANWELL, J. G. & LUNN, H. F. (1965). Ibid., 1, 89. ~ , CONNER, D. H., VOLLER, A., KmLLY, A., SCHOFIELD,F. D. & HtrrT, M. S. R. (1967). Bull. Wld Hlth Org., 36, 901. MCGEE, J. O'D. & PATRICK,R. S. (1972). Lab. Invest, 96, 429. MODY, N. J. (1970). M.D. Thesis, Makerere University, Kampala. MUSTAFA, D. (1965). ft. trop. Med. Hyg., 68, 183. PITNEY, W. R., PRYOR, D. S. & TAIT SMITH, A. (1968). jr. Path. Bact., 95, 417. PRYOR, D. S. (1967). Quart. jr. Med., 36, 321. SAGOE,A-S. (1970). Br. med. jr., 3, 378. SCI-IAFFNER,F., BARK& T. & POPPER, H. (1963). Exp. Molec. Pathol., 2, 419. SHAPER, A. G., KAPLAN, M. H., MODY, lq. J. & MClNTYRE, P. A. (1968). Lancet, 1, 1342. STUIVER, P. C., ZIEGLER, J. L., WOOD, J. B., MORROW, R. H. & HUTT, M. S. R. (1971). Br. med. jr., 1, 426. SZAKAL, A. K. & HANNAN, M. G. (1968). Exp. Molec. Path., 8, 75. VAN FURTH, R. (1970). In: Mononuclear Phagocytes, Ed. Van Furth, R. Oxford: Blackwell. WATSON-WILLIAMS,E. J. & ALLAN, N. C. (1968). Br. med. jr., 4, 793. WELLS, J. V. (1968). Clin. exp. Immunol., 3, 943. ZIEGLER, J. L., COHEN, M. H. & HlYrT, M. S. R. (1969). Br. med. jr., 4, 15. (1972). Ibid., 3, 79. - & STUIVER,P. C. jr. din. Invest., in press.