The Spleen in Acquired Immunodeficiency Syndrome (AIDS)

Path. Res. Pract. 183, 425-433 (1988)

The Spleen in Acquired Immunodeficiency Syndrome (AIDS) * Stephan Falk, Hartmut Muller and Hans-Jochen Stutte Senckenbergisches Zentrum der Pathologie, Klinikum der J. W. Goethe-Universitat Frankfurt, Frankfurt am Main, West Germany

SUMMARY

72 spleens from AIDS patients were studied by histologic and immunohistochemical methods. Apart from the opportunistic infections and malignancies typically occuring in AIDS patients (malignant lymphomas, Kaposi's sarcoma, mycobacterial infections, cryptococcosis, cytomegalovirus infections) morphological changes probably directly attributable to HIV infection were noted that progressed from follicular hyperplasia with nearly normal periarteriolar lymphoid sheaths to atrophy of follicles and severe cellular depletion of T cell areas with concomitant plasmacytosis and occurrence of small giant cells. At the same time the number of Protein S-100 positive cells markedly increased; in some cases. almost all pulp cord macrophages expressed the antigen. In addition, the sinus endothelia strongly expressed protease inhibitors. Introduction

An infection by the human immunodeficiency virus (HIV) leads to profound morphological alterations in lymphatic organs, bone marrow and brain. While the spectrum of changes associated with HIV-infection in lymph nodes 2, 6, 10, It,' 16, 18 thymus l7 , and bone marrow4, 12, 14, 15 has been extensively studied, a histopathological appraisal of the spleen in AIDS is still lacking. Therefore, we have systematically evaluated spleens obtained from HIVinfected persons by histological and immunohistochemical methods.

tochemical reactions were performed using a modified immunoalkaline phosphatase technique. Among the primary antisera were antibodies against T lymphocytes (clone UCHL-1, Dako, Hamburg, FRG), B lymphocytes (KiB 3, a kind gift from Prof. M. R. Parwaresch, Kiel), S-100 protein, lysozyme, alpha-1antichymotrypsin, alpha-I-antitrypsin, immunoglobulins as well as kappa and lambda light chains. Furthermore, the absolute amount of splenic white and red pulp as well as of residual structures (large vessels, trabeculae) was calculated from planimetric data obtained by a 100 point grid eyepiece at a magnification of x 100 and multiplied by splenic weight. All results were then evaluated for possible relationships with the clinical course (e. g. opportunistic infections, anaemia, thrombocytopenia) and/or autopsy data and compared to the results of identical studies in 10 age- and sex-matched, HIV-negative controls.

Material and Methods 72 spleens from patients who were seropositive for antibodies against HIV by either ELISA or Western blot technique and who suffered from full-blown AIDS according to CDC criteria 19 were obtained at autopsy. The spleens were weighed and sectioned at 0.5 cm intervals to demonstrate focal lesions. Tissue blocks from different sites were fixed in either 4% buffered formaline or in 4% paraformaldehyde and embedded in paraffin wax. In addition to conventional histological stains (hematoxilin-eosin, Giemsa, PAS, Gomori's silver and Prussian blue) immunohis• Dedicated to Prof. K. Hubner on the occasion of his 60th birthday. © 1988 by Gustav Fischer Verlag, Stuttgart

Results The vast majority of the spleens studied was enlarged; splenic weights ranged from 70 to 1650 g with a median of 370 g. Largest weights were reached in spleens affected by malignant tumors arising as a consequence of AIDS, i. e. by either malignant lymphomas (immunocytoma, polymorphic subtype - three cases-; immunoblastic lymphoma -one case-; Hodgkin's disease, lymphocyte depletion subtype -one case-) or by Kaposi's sarcoma (KS; -four cases-). Malignant lymphomas showed yellowish-white nodular infiltrates frequently coalescing into larger tumor masses 0344-0338/88/0183-0425$3.50/0

426 . S. Falk, H. Muller and H.J. Stutte Table 1. Opportunistic infections and malignancies in the spleen in AIDS (n = 72) Entity Non Hodgkin's lymphoma Hodgkin's disease Kaposi's sarcoma Mycobacterial infections Cryptococcosis Cytomegalovirus infection

Spleen involvement 4 1 4 5 2

19

Number of cases 8

1

32

5

4 41

N.B. The spleens of AIDS patients in numerous instances exhibited more than one opportunistic infections or malignancies. The most prevalent combinations were malignant lymphoma and Kaposi's sarcoma (two cases) and cytomegalovirus infection with any other malignancy or infection (12 cases). The occurrence of multiple secondary complications of HIV induced immunodeficiency is highly typical of AIDS patients (5).

Fig. 1. Kaposi's sarcoma of the spleen presenting as isolated purplish nodules of up to 0.8 cm in diameter.

with extensive necrosis; microscopically and with respect to their infiltration patterns they did not differ from lymphomas in HIV negative patients. KS occurred either as isolated dark nodules (Fig. 1; two cases) or replaced most of the splenic parenchyma (two cases). Microscopically, formations of highly cellular spindle cell components interspersed with vascular structures of variable diameter (Fig. 2) were evident that possessed ill-defined margins and freely invaded the red pulp. However, splenomegaly was not restricted to spleens affected by neoplastic infiltrates; it was also common in mycobacterial infections of the organ. Large spleens with weights of up to 700 g and a nodular surface (Fig. 3) were pathognomonic of mycobacterial histiocytosis. This form of mycobacterial infection with absence of epitheloid or giant cells of the Langhans type and only occasional areas of caseous necrosis was observed in four cases and microscopically showed bloated macrophages with masses of PAS- and Ziehl-Neelsen positive so-called atypical mycobacteria that upon culture yielded M. avium-intracellulare (Fig. 4). Small white lesions corresponding to miliary tuberculosis with typical central caseous necrosis and surrounding epitheloid and Langhans cells were found in one case only. An additional patient exhibited several scarred nodules that bore witness to an old disseminated infection by M. tuberculosis .that had responded to appropriate therapy. In two cases small white lesions of up to 0.5 em in diameter corresponded to foci of cryptococcal infections that showed budding yeasts either freely within the pulp cords or in the cytoplasm of macrophages (Fig. 5). The most frequent opportunistic infection, i. e. cytomegalovirus infection (19 cases) did not cause macroscopically evident lesions in the spleen and by itself apparently did not lead to splenic enlargement. Table 1 summarizes the occurrence of opportunistic infections and malignancies in the spleen of AIDS patients. Apart from these alterations attributable to opportunistic infections or malignancies, spleens from HIV-infected

Fig. 2. Kaposi's sarcoma: predominance of spindle cells and irregular vascular formations. H & E. X256.

The Spleen in AIDS . 427

persons without secondary pathologic lesions also showed slight to moderate splenomegaly (weight: 70 to 460 g, median: 280 g) and exhibited several conspicuous changes that were related to the duration of AIDS. Specifically, spleens from patients who had died in the early stage of AIDS, i. e. individuals succumbing to one of the initial manifestations of the (usually Pneumocystis carinii pneumonia or CNS toxoplasmosis) exhibited large follicles with prominent germinal centers and broadened marginal zones that partly were not well demarcated from the surrounding perifollicular region (Fig. 6). These contained large amounts of B lymphocytes and their precursors while the germinal centers also harbored increased

Fig. 3. Nodular cut surface of the spleen in mycobacterial histiocytosis; the size of each nodule may reach 2 cm in diameter.

Fig. 4. PAS-positive, acid-fast mycobacteria in swollen macrophages. Ziehl-Neelsen. X 1280.

numbers of UCHL-l positive T lymphocytes and lysozyme positive macrophages. With progression of the immunodeficiency the number of B lymphocytes diminished, the size of the follicles and the number of germinal centers was reduced; fibrin deposits appeared. The structure of the follicles was eventually destroyed; in many cases only frayed remnants of the erstwhile population of B lymphocytes were visible (Fig. 7), and irregular hyalinizations corresponding to the so-called burnt-out germinal center-like structures in lymph nodes appeared that corresponded to end-stage AIDS. The surrounding tissue contained only a few Band T lymphocytes as well as macrophages and plasma cells. Germinal centers could not

428 . S. Falk, H. Miiller and H.J. Stutte

be found. The T cell-dependent areas, i. e. the periarteriolar lymphoid sheaths (PALS) also exhibited a cellular depletion that corresponded to the syndrome's duration. While patients who died within one to eight weeks after

Fig. 5. Focus of cryptococcosis with encapsulated yeasts of varying size in the pulp cords. Alcian-blueIPAS. X512.

their initial AIDS manifestation usually exhibited PALS that could not be distinguished from those of age-matched controls, in later stages cell-depleted PALS with only a few T lymphocytes besides B lymphocytes, plasma cells and macrophages could be found. A conspicuous feature, however, was the occurence of small giant cells with compact multilobated nuclei negative for any of the antibodies used and that were morphologically distinct from megakaryocytes (Fig. 8). Independent of the duration of the disease and of opportunistic infections or malignancies the splenic red pulp in 40% of the cases contained clusters of polyclonal plasma cells that occured not only in the vicinity of trabeculae but were also observed deep within the red pulp. 10% of the spleens studied contained extramedullary haematopoiesis; erythroblasts and granulopoietic precursors were visible within the sinus and the pulp cords, respectively. Although the extent of splenic haematopoiesis was variable, some cases showed large clusters especially of erythroblasts; we could not ascertain a common cause for the occurence of extramedullary haematopoiesis in spleens from AIDS patients. The sinus endothelia exhibited prominent nuclei and showed enhanced expression of protease inhibitors such as alpha-I-antitrypsin and -1-antichymotrypsin (Fig. 9). The production of these substances was apparently also increased in other vascular structures, since we observed hyaline bodies in the endothelia of splenic veins that immunohistochemically proved to be aggregates of alphaI-antichymotrypsin. These phenomena again were not related to age or sex of the patients, nor was there a relationship with the duration of the disease or with secondary complications. The splenic red pulp also showed several other conspicuous features: with increasing size of the spleen the relative and the absolute amount of red pulp rose concominantly at the expense of the white pulp that was reduced from a mean of 21 ± 9% in early stage AIDS to 4 ± 2%

Fig. 6. Follicular hyperplasia with prominent germinal centers in early AIDS. Giemsa. X 112.

The Spleen in AIDS . 429 (mean of 15 cases, in the rest determination was impossible because of poor cellularity) in end-stage AIDS. The mean diameter of the sinus and the widths of the pulp cords increased; in many cases we observed a congestion of the red pulp by erythrocytes. At the same time the number of lysozyme positive macrophages in the expanded pulp cords rose; however, we could not demonstrate a direct relationship with duration of the disease or opportunistic infections and/or malignancies. Moreover, there was a highly prominent erythrophagocytosis by. intrasinusoidal and pulp cord macrophages that appeared in 12% of the cases and was accompanied by a more or less pronounced siderosis of macrophages and, to a lesser extent of sinus endothelia. This erythrophagocytosis was neither related to anaemia or to blood transfusions some patients had received because of bone marrow failure after chemotherapy for malignant lymphoma or KS. In another 8% we found signs of an increased thrombocyte sequestration in the spleen, i. e. granular PAS-positive cytoplasmic inclusions in foamy macrophages. Again, a relationship with clinically manifest severe thrombocytopenia could not be established, although some AIDS patients had experienced episodes of thrombocytopenia earlier in the course of their disease.

Fig. 8. Small giant cell with compact nucleus and scant cytoplasm in the immediate vicinity of a PALS showing severe cellular depletion. UCHL-l. X256.

Fig. 7. Frayed remnants of a splenic follicle with loss of zonal arrangement and cellular depletion. KiB3, APAAP. X256.

Most conspicuous, however, was an increase in the number of S-100 positive cell. While in control spleens only scattered cells within the PALS and the pulp cords expressed this antigen (Fig. 10) spleens from patients who had died soon after the first AIDS manifestation contained larger numbers of cells staining for this antigen; in these cases the cells in many instances morphologically did not correspond to interdigitating reticulum cells with typical indented nuclei and cytoplasmic processes that were stained in controls. Rather, a number of cells in the marginal zones and even within the cords expressed the antigen; in most AIDS patients who had survived for more than 25 weeks numerous cells within the pulp cords were reactive for S-100. In eight cases almost all pulp cord macrophages expressed S-100 protein; a cause could not be ascertained (Fig. 11). Sheathed capillaries appeared normal in early stages of AIDS; neither their number nor the composition of the surrounding cells that chiefly consisted of T lymphocytes and macrophages was altered. In later stages, however, we observed a progressive cellular depletion with the occurence of S-l00 protein positive macrophages and of plasma

430 . S. Falk, H. Miiller and H.]. Stutte cells in the vicinity of sheathed capillaries whose numbers, however, did not appreciably change. Table 2 summarizes the morphological findings in spleens from AIDS patients presumably directly related to HIV-infeetion. Discussion The present study of morphological alterations in spleens from HIV-infeeted persons draws upon a large and representative sample of specimens that encompass more than 10% of all autopsies of AIDS patients performed in West Germany until October 1, 1987 (Prof. E. Bl. Helm, personal communication). Since spleens· from patients with AIDS of variable duration (less than one to 122 weeks) were available for detailed histological analysis, a major drawback of the study i. e. the lack of spleens removed surgically from symptom-free, HIV-infectedpersons and from patients with either persistent generalized lymphadenopathy (PGL) or AIDS-related complex (ARC), could be partially overcome. Thus, truly early manifestations of HIV infection in the spleen could not, unlike in the more accessible lymph nodes, be investigated. Since the definition of AIDS uses arbitrary criteria (e. g. Pneumocystis carinii pneumonia or KS fulfil the criteria, although

Fig. 10. In early AIDS the number of S-100 positive cells is slightly increased; many correspond to interdigitating cells. S100. X256.

Fig. 9. Sinus endothelia strongly express protease inhibitors. Alpha-1-anti-chymotrypsin. X 512.

they may develop in the setting of low-normal T helper/ inducer cell counts) an analysis of specimens from a large group of persons with AIDS of variable duration would be expected to reveal different tissue changes if HIV-infection caused progressive morphological alterations in the spleen. This hypothesis was proven to be correct; histological as well as immunohistochemical methods show that the different lymphatic compartments of the spleen undergo progressive changes whose extent is primarily dependent on the duration of the full-blown syndrome. Splenic follicles that constitute the B lymphocyte areas in early stages show a florid follicular hyperplasia with prominent germinal centers and an abundance of B lymphocytes and their precursors. This finding reminds of the changes observed in lymph nodes from PGL patients 13 ; further alterations in spleen morphology in the course of the disease also bear resemblance to the lymph node pathology i~ HIV infection. Notably, progressive cellular depletion in B as well as in T lymphocyte areas occurs; the former is associated with a disruption of the follicular dendritic cells' network within the germinal centers. The deposition of fibrin within erstwhile germinal centers that may signify the demise of follicular dendritic cells 8 was observed in all our cases. Due to lack of electron microscopic or immunohistochemical studies of follicular dendritic cells in spleens

The Spleen in AIDS· 431

obtained at autopsy we cannot conclusively prove that these accessory cells of B lymphocytes are among the primary targets of HIV in the spleen as it has been assumed for lymph nodes ll . However, demonstration of viral parti-

Fig. 11. In AIDS of long duration numerous pulp cord and sinus macrophages express S-100 protein. APAAP, X256.

Fig. 12. End-stage AIDS with severe cellular depletion and patchy hyalinizations of erstwhile germinal centers (l') PAS. X256.

cles and antigens on the surface of this cell type 16 render this possibility highly likely; damage to dendritic cells may then be caused either by a direct cytopathic effect of the HIV or by autoaggression mediated by cytotoxic T lymphocytes and macrophages whose numbers we found to be increased in the germinal centers and that may recognize HIV antigens on the dendritic cells' surface. In any case, the terminal phase of HIV infection leads to a disintegration and profound cellular depletion of the follicles that are accompanied by the development of hyalinizations and a concomitant decrease in the number of T lymphocytes within the PALS. The latter phenomenon has to be ascribed to a direct cytopathic effect of HIV, since the number of T lymphocyte accessory cells apparently is not decreased (see below). T as well as B cell areas in the terminal stage of AIDS are depleted, and in a few cases, almost devoid of lymphocytes; instead macrophages and plasma cells are present. Thus in end-stage AIDS the spleen in respect to its lymphatic component exhibits the non-specific features of a severe combined immunodeficiency (Fig. 12) that in many cases may not be distinguished from e. g. effects of cytotoxic chemotherapy on morphological grounds. However, there are aspects that distinguish HIV induced changes in the spleen from alterations caused by other disease processes. These differences are primarily evident in the monocyte/macrophage system of the spleen. First of all, the number of macrophages as assessed by staining with an antibody against lysozyme is slightly to moderately increased in AIDS patients; this phenomenon is not related to the duration of the illness, to opportunistic infections or to malignancies. Secondly, the phagocytic activity of the pulp cord and the intrasinusoidal macrophages is increased in at least 12% of the cases that showed marked erythrophagocytosis and siderosis without apparent cause. At the same time, however, the activation of macrophages to effector cells is impaired either for lack of suitable lymphokines produced by T helper/inducer cells or because of direct HIV infection of

432 . S. Falk, H. Miiller and H.J. Stutte

CD 4 positive macrophages. This lack of activation is best illustrated by the occurrence of disseminated mycobacterial infections (so-called mycobacterial histiocytosis 9 ) that show masses of mycobacteria propagating within the cytoplasm of bloated macrophages that are unable to transform into mycobactericidal epitheloid or giant cells of the Langhans type. Another, less well understood phenomenon consists of the expression of 5-100 protein by a majority of phagocytosing macrophages of the pulp cords that was predominantly found in the advanced stages of AIDS and that was not associated with a demonstrable cause. Although an increase of S-100 positive cells was already noted in patients with short duration of the syndrome, many of these cells exhibited typical morphological features of interdigitating reticulum cells - a phenomenon that has been described for lymph nodes of PGL patients 13 • In later stages, however, S-100 expression was no longer restricted to this cell type, and the reasons why pulp cord and intrasinusoidal macrophages that normally are negative for the antigen 3 express S-100 remain speculative. Since HIV infection presumably is present from the onset of the disease and therefore is not easily imaginable to cause de novo changes of cellular phenotypes in later stages, it appears as if 5-100 expression by macrophages may reflect a "functional" status. This assumption is supported by the fact that sinus endothelia in AIDS also express increased amounts of functionally important substances, i. e. protease inhibitors such as alpha-l-antichymotrypsin and -antitrypsin, although this finding is not related to the stage of the disease and its significance as well as its cause remain to be determined. Apart from these morphological and immunohistochemical pecularities of the spleen in AIDS there are other details worth mentioning. In contrast to the lymph nodes obtained from the patients at autopsy the angioar-

Table 2. Morphologic findings in spleens from AIDS patients probably attributable to HIV infection Early stages

Late stages

Follicular hyperplasia with enlarged germinal centers and broadened marginal zones

Cellular depletion and disruption of follicles, hyalinizations of erstwhile germinal centers

Slight reduction of T lymphocytes in PALS; occasional plasma cells

Severe cellular depletion of PALS with occurrence of small giant cells and' numerous plasma cells

Normal number and cellularity of sheathed capillaries

Cellular depletion and occurrence of plasma cells and S-100 positive cells near sheathed capillaries

Slight increase in the number Occurrence of large numbers of of S-100 positive interdigitat- S-100 positive macrophages in ing cells; some macrophages the pulp cords and sinus are also positive Strong expression of protease inhibitors (alpha-1-antitrypsin/alpha-1-antichymotrypsin) by sinus endothelia

chitecture of the spleens was preserved, i. e. the sometimes highly irregular capillary proliferations could not be demonstrated. Whether this finding be.ars any significance with respect to the histogenesis of KS from capillary endothelia7 and the relatively infrequent KS infiltration of the spleen (four cases out of 32 lymphadenopathic KS) also remains to be determined. In summary, the spleen in AIDS exhibits HIV-induced morphologic alterations affecting both the lymphatic compartment and the mononuclear phagocyte system that show, for the most part, progressive changes whose degree is predominantly influenced by the duration of the illness. Special features, such as the expression of S-l 00 protein or of HIV antigens l that may be elucidated by immunohistochemical methods may serve to distinguish HIV-induced alterations from the non-specific morphological appearance of a severe combined immunodeficiency even in the end-stage of the retroviral infection. Acknowledgement The authors wish to express their gratitude to Drs. H. R. Brodt and C. Friebe, Department of Internal Medicine, University of Frankfurt for the provision of clinical data.

References 1 Baroni CD, Pezzella F, Mirolo M et al (1986) Immunohistochemical demonstration of p24 HTLVIII major core protein in different cell types within lymph nodes from patients with lym.phadenopathy syndrome. Histopathology 10: 5-13 2 Biberfeld P, Porwit-Kwiazek A, Battiger B et al (1985) Immunohistopathology of lymph nodes in HTLV-III infected homosexuals with persistent adenopathy or AIDS. Cancer Res 45: 46655-4670s 3 Carbone A, Manconi R, Poletti A et al (1985) S-100 protein immunostaining in cells of dendritic morphology within reactive germinal centers by ABC immunoperoxidase method. Virch Arch (Pathol Anat) 406: 27-32 4 Delacretaz F, Perey L, Schmidt PM et al (1987) Histopathology of bone marrow in human immunodeficiency virus infection. Virch Arch A 411: 543-551 5 Falk S, Schmidts HL, Miiller H et al (1987) Autopsy findings in AIDS - a histopathological analysis of fifty cases. Klin Wochenschr 65: 654-663 6 Guarda LA, Butler 11, Mansell P et al (1983) Lymphadenopathy in homosexual men. Morbid anatomy with clinical and immunological correlations. Am J Clin Pathol 79: 559-563 7 Hashimoto H, Miiller H, Falk S, Stutte HJ (1987) Histogenesis of Kaposi's sarcoma associated with AIDS; a histologic, immunohistochemical and enzyme histochemical study. Path Res Pract 182: 658-668 8 Heusermann U (1982) Follikelfibrin - ein degenerativer Prozeg der dendritischen Retikulumzellen. Verh dtsch Ges Path 66: 600 9 Kaiserling E, Miiller-Hermelink KH, Lennert K (1980) Zur Morphologie und Pathogenese der mykobakteriellen Histiozytose. Verh dtsch Ges Path 64: 392-396 10 Mathur-Wagh U, Enlow RW, Spigiand I et al (1984) Longitudinal study of persistent generalized lymphadenopathy in homosexual men: relation to acquired immunodeficiency syndrome. Lancet I: 1033-1038

The Spleen in AIDS . 433 11 Miiller H, Falk S, Stutte HJ (1986) Accessory cells as primary target of human immunodeficiency virus HIV infection. J Clin Pathol 39: 1161 12 Miiller H, Weybora W, Falk S et al (1987) Bioptische Knochenmarksbefunde bei der HIV-Infektion. Verh Dtsch Ges Path 71, 477 13 Miiller H, Falk S, Stutte HJ (1987) Immunohistochemical investigations oflymph nodes in LAS and AIDS patients. Cancer Detect Prev, Suppl. 1, 577-582 14 Namiki TS, Boone DC, Meyer PR (1987) A comparison of bone marrow findings in patients with acquired immunodeficiency syndrome (AIDS) and AIDS related conditions. Hematol Oncol 5: 99-106 15 Osborne BM, Guarda LA, Butler 11 (1984) Bone marrow biopsies in patients with acquired immunodeficiency syndrome. Hum Path 15: 1048-1053

16 Racz P, Tenner-Racz K, Kahl C et al (1984) Spectrum of morphologic changes of lymph nodes from patients with AIDS or AIDS-related complex. Progr Allergy 37: 81-181 17 Savino W, Dardenne M, Marche C et al (1986) Thymic epithelium in AIDS: an immunohistologic study. Am J Pathol 122: 302-307 18 Turner RR, Meyer PR, Taylor CR et al (1987) Immunohistology of persistent generalized lymphadenopathy. Evidence for progressive lymph node abnormalities in some patients. Am J Clin Pathol 28: 10-19 19 World Health Organisation (1986) WHO/CDC case definition for AIDS. Wkly Epid Rec 61: 69-76

Received November 5, 1987 . Accepted in revised form January 18, 1988

Key words: Human immunodeficiency virus - Lymphocytes - Spleen - S-100 Protein - AIDS Dr. S. Falk, Zentrum der Pathologie, Theodor-Stern-Kai 7, D-6000 Frankfurt 70, West Germany