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Accessory cells and macrophages in the histopathology of SIVsm-infected cynomolgus monkeys
Res. Virol. 1993, 144, 81-92
(~) INSTITUTPASTEUR/ELSEVIER Paris 1993
Accessory cells and macrophages in the histopathology of SIVsm-infected cynomoigus monkeys E. Kaaya (i, 4), S.-L. Li (i), H. Feichtinger (I, 3), I. Stahmer (1), p. P u t k o n e n (2), E. M a n d a c h e 0), E. Mgaya (4), G. Biberfeld (2) and P. Biberfeld (I) (*)
(1) Immunopathology Laboratory, Department o f Pathology, Karolinska Institute, 10401 Stockholm, (2j Department o f Immunology, National Bacteriological Laboratory, Stockholm, (3) Department o f Pathology, University o f Innsbruck (Austria) and (4j Department o f Pathology, Muhimbili University College o f Health Sciences, Dar-es-Salaam (Tanzania) SUMMARY
Thirty-three out of 39 cynomolgus monkeys (Macaca fascicularis) infected with SlVsm (strain SMM-3) developed various pathologies similar to those seen in human AIDS. Lymphadenopathy was frequently seen (72 %) and was characterized by hyperplasia followed by involution of follicle/germinal centres due to follicular dendritic cell (FDC) destruction corresponding to the degree of immunodeficiency. Various organs such as the lungs, liver, central nervous system, kidneys, gastrointestinal tract, cardiovascular system and adrenals showed histopathological changes with prominent monocyte/macrophage and multinucleated giant cell formation. Eighteen (54 %) monkeys presented with extranodal malignant lymphoma (ML) associated with marked CD4 decrease and destruction of follicular architecture. The high frequency of ML, giant cell disease and lymph node changes seen in the present SlV model provides an attractive system to elucidate the role of FDC and monocytes/macrophages in the pathogenesis of these cohditions in common with HIV infection and human AIDS.
Key-words: AIDS, SIV, Follicular dendritic cell, Macrophage; Histopathology, Animal model, Electron microscopy.
INTRODUCTION Acquired immunodeficiency syndrome (AIDS) in man is aetiologically linked to the lymphotropic lentiviruses designated HIV1 and HIV2. Apart from their cytopathic effects on CD4 + T cells, in vitro and in vivo studies have suggested that other cells, particularly accessory ceils, may also become targets for productive or latent HIV infection (Biberfeld et ai., 1986; Langhoff et al., 1991 ; Macatonia et al., 1990; Racz et al., 1985 ; Stahmer et al., 1991) resul-
Received October 1 I, 1992. ~'~Corresponding author.
ting in a complex pathology involving various tissues during progression to AIDS. During the last few years, several attempts have been made to establish animal models for studies of the pathogenic mechanisms involved in AIDS and for development and testing of vaccines and new therapies (Letvin, 1990; 1992). The chimpanzee, gibbon ape rabbit and SCID-hu mouse have also been shown to be susceptible to infection with HIV1, but neither immunodeficiency nor disease has been reported in
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E. K A A Y A E T A L .
these species (Letvin, 1990; 1992). A s i a n m a c a q u e s can be persistently infected with some strains o f H I V 2 b u t w i t h o u t d e v e l o p m e n t o f A I D S - l i k e disease ( P u t k o n e n et al., 1990). H o w e v e r , such m o n k e y s are susceptible to i n f e c t i o n with s o m e strains o f s i m i a n i m m u n o d e f i c i e n c y virus (SIV), a Ientivirus p h y l o g e netically related to H I V (Desrosiers, 1990). T h e y o f t e n d e v e l o p an A I D S - l i k e disease a n d m a y therefore represent a relevant m o d e l for H I V - r e l a t e d cond i t i o n s (Letvin, 1990; Letvin a n d King, 1990). W e ( P u t k o n e n et al., 1989) and others (Fultz et al., 1986) have recently d e s c r i b e d such a m o d e l o f A I D S in c y n o m o l g u s m o n k e y s infected with S I V s m . In the present s t u d y , we r e p o r t on the s p e c t r u m o f p a t h o logical changes in a c o h o r t o f 33 c y n o m o l g u s m o n keys ( M a c a c a f a s c i c u l a r i s ) e x p e r i m e n t a l l y infected with S I V s m (strain S M M - 3 ) , a n d d e m o n s t r a t e t h a t m a j o r pathological a n d i m m u n o p a t h o l o g i c a l features involving several o r g a n systems are closely related to the infection a n d / o r d e s t r u c t i o n b y S I V o f accessory a n d m o n o n u c l e a r p h a g o c y t i c cells.
MATERIALS AND METHODS
Monkeys, SIV infection and follow-up
Autopsy and histopathology Autopsies were performed immediately on monkeys sacrificed at extremis, or within 12 h for the few which died spontaneously. Specimens were partly frozen and partly fixed in B5 and Carnoy's fixatives and processed for conventional histopathological evaluation of haematoxylene and eosine (H&E) sections. Selected organ sections were stained with periodic acid-Schiff, Grocott's methenamine silver technique, Congo red, ZiehI-Nielsen (ZN), Gram stain and Giemsa.
Immunohistopathology and electron microscopy (EM) Immunostaining was performed on frozen sections or on Carnoy's fixed material. Frozen sections were cut, acetone-fixed and preserved at - 70°C until used. The following monoclonal antibodies (mAb) were used for the immunostaining of SIV gag antigen (mAb : SFS-6FS ; Biotech. Res. Lab. ; RIC7 ; LTCB/NIH), follicular dendritic cells (mAb KiM4; Behring), B cells (mAb L26, CD20; Dako), (mAb FS-I 1-13, CD45RA; Serotec), CD4 ÷ T cells (mAb OKT4; Ortho), macrophages (mAb KiM6, CD68, Boeh. Mann) and CD8 + T cells (mAb Leu2a and mAb Leu5b, CD2; Becton and Dickinson). The avidin biotin peroxidase complex (ABC) and the alkaline phosphatase anti-alkaline phosphatase (APAAP) methods were used as previously described (Biberfeld et al., 1986).
Thirty-nine wild caught cynomolgus monkeys (table I) were intravenously inoculated with SIVsm (strain SMM-3 from Drs. P. Fultz and H. McClure, Yerkes Primate Research Center, Atlanta, GA, USA) and propagated in human PBL as previously described (Putkonen et al., 1989). The monkeys were inoculated with different doses ranging from undiluted supernatants to 101-105 monkey infectious doses (MIDso) of uncloned virus. The monkeys were kept at the Primate Centre of the National Bacteriological Laboratory (SBL) according to the guidelines of the Swedish ethical committee. They were anaesthetized by ketamine at intervals for physical examination and collection of blood and biopsies for virological, immunological and histopathological studies, as previously described (Putkonen et al., 1989; 1990). Animals in extremis were euthanized by intravenous injection of pentobarbital sodium.
For electron microscopy (EM) tissue biopsies were fixed in 2 % paraformaldehyde/2.5 070 glutaraldehyde, post-fixed in I 070 osmium tetroxide dehydrated in graded ethanol and embedded in agar-100. Sections were examined in a "Jeol EM-100S" transmission electron microscope.
ABC = APAAP = CIP = CNS = CVS = EM = FA = FD = FDC = FF = FH = GC = GCD = GIT = H&E =
LN = mAb = MAI = MALT = MGC = MID = ML = MNC = Mo/Mac = PBL = RF = SBL = SIV = VLP = ZN =
avidin-biotin peroxidase complex. alkaline phosphatase anti-alkaline phosphatase. chronic interstitial pneumonia. central nervous system. cardiovascular system. electron microscopy. follicular atrophy. follicular depletion. follicular dendritic cell. follicular fragmentation. follicular hyperplasia. giant cell. GC disease. gastrointestinal tract. haematoxyline and cosine.
RESULTS
Clinical and immunological manifestations Clinical features o f infected m o n k e y s ~ e s u m m a rized in t a b l e I. T h i r t y - t h r e e (85 % ; males = 16, females = 17) m o n k e y s were e u t h a n i z e d d u e to
lymph node. monoclonal antibody. Mycobacterium avium-intracellulare. mucosal-associated lymphoid tissue. multinucleated GC. monkey infectious dose. malignant lymphoma. mononuclear cell. monocyte/macrophage. peripheral blood lymphocyte. retroperitoneal fibromatosis. Swedish (national) Bacteriological Laboratory. simian immunodeficiency virus. virus-like particle. Ziehl-Nielsen.
HISTOPA THOLOGY OF SIVsm-INFECTED CYNOMOLGUS MONKEYS
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Table I. Clinical and immunological manifestations of SIV infection in cynomolgus monkeys.
Animals and SIV infection:
- infection - animal weight (age) -
-
sex
- time of infection (days)
SIV SMM-3 (undiluted to 10-105 MIDs0) 1.8-6.125 kg male 16; female 17 mean 431 (+ 214); range 58-905
Clinical manifestations:
- weight loss (°70) - lymphadenopathy - ML - diarrhoea - cachexia - splenomegaly - respiratory symptoms
mean 24/33 12/33 12/33 10/33 5/33 2/33
19.2 (_+ 11); range 2-44 (73 %) (36 %) (36 %) (30 %) (15 %) (6 %)
Immunological manifestations:
- PBL-CD4 conts (x 109/1) - CD4/CD8 :
mean 0.13 (+ 0.1); range 0.02-0.5 mean 0.23 (_+ 0.19); range 0.01-0.7
MID~o = monkey infectious dose/50.
disease after 58 to 905 days, were included in the study. Six monkeys without clinical manifestations are still alive two to four years post-inoculation. A variable degree of weight loss was seen in all monkeys and severe cachexia in 10 (30 %). Twenty-four (73 %) monkeys had terminal lymphadenopathy. Five monkeys (15 %) presented with clinically palpable spleens. Diarrhoea as the dominant clinical manifestation was noted in 12 (36 %) monkeys. Two (6 %) monkeys had neurological symptoms including ataxia and paraplegia, one with central nervous system (CNS) lymphoma (ML) and another with giant cell (GC) encephalopathy. Extranodal malignant lymphoma (ML) developed in 12 (36 %) monkeys, in 3 (9 %) of which it was the only presenting disease manifestation. Thirty-two animals, all with clinical disease, showed variable immunosuppression, as determined by CD4 counts and CD4/CD8 ratios of peripheral blood lymphocytes (PBL). All monkeys were seropositive for SIV antibodies (Putkonen et al., 1992) and SIV antigens were demonstrable by immunohistochemistry in all tested lymph nodes (LN) and spleens.
Histopathology
Table II summarizes the main pathological findings in the monkeys. Saiient changes seen in the lymphoid organs, lungs, liver, kidneys, CNS, gas-
trointestinal tract (GIT) and cardiovascular system (CVS) are reported below. Lymphoid tissues including M A L T
Follicles/germinal centres of LN, spleen and the mucosa-associated lymphoid tissue (MALT) showed prominent changes, usually in the form of follicular hyperplasia (FH) in early stages of infection. During disease progression with decreasing CD4 + PBL counts, a gradual involution of follicles/germinal centers was usually seen and defined as follicular fragmentation (FF), follicular atrophy (FA) and follicular depletion (FD) in accordance with previously described changes in HIV PGL (Porwit et al., 1989) (table III; fig. 1 and 2a). Although the histopathology of lymphoid tissues varied between different monkeys (table III) the degree of involution appeared to correlate with a decrease in CD4 (fig. 1). The spleen often showed a variable spectrum of FH to FA changes. At early stages of infection, paxacortical expansion, angiogenesis and sinus histiocytosis were observed to a variable degree in the parafollicular areas and sinuses of LN. Angiogenesis became more pronounced with progression of follicular involution from early (FF) to late (FA and FD) stages. Likewise, in the red pulp of the spleen, hyperplastic changes were observed in 18 (54 e/0) monkeys. Most thymuses were involuted, but in a few instances, non-involuted, remaining thymus tissue showed regressive changes.
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Table H. Histopathology in cynomolgus monkeys infected with SIVsm (SMM3). D
Organ/. system and histopathology Frequency (%) Systemic lymphadenopathy GCD ML RF Lung CIP ML GIT enteritis GCD ML Kidney interstitial MNC infiltrate glomerulitis focal glomerulosclerosis ML CNS GC ML Liver MNC infiltrates Kupffer cell hyperplasia GCD fatty change ML CVS MNC infiltrates GCD fibrinous pericarditis ME Oral cavity oral hairy leukoplakia
33 18/33 15/33 1/33
(100) (54) (45) (6)
24/33 5/33
(73) (15)
13/32 13/32 9/32 13/32
(41) (41) (27) (41)
13/33 6/33 5/33 6/33
(39) (18) (15) (18)
8/32 2/32
(25) (6)
21/33 9/33 5/33 1/33 3/33
(64) (27) (18) (3) (9)
12/32 3/32 1/32 2/32
(38) (9) (3) (6)
1733
(3)
t
o
m 0
•
FH
T
"
FH FF
!
FF
"
I
FF FA
"
I
•
FA
I
•
FA FD
Hist~ogy
FD
grade
Fig. 1. LN histopathology in relation to CD4 values in infected cynomolgus monkeys• FH = follicular hyperplasia, FF = follicular fragmentation, FA = follicular atrophy, FD = follicular depletion.
Fig. 2. Examples of histopathological changes in organs of cynomolgus monkeys infected with SIVsm (see table II for frequencies); (H&E stained paraffin sections). a) MGC (arrow heads) and atrophic MALT (arrow) in the gut of an infected monkey; x 250. b) Lung showing CIP with septal thickening and MNC infiltration ; x 400. c) Lung with MGC as part of GCD manifestation; x 160. d) Lung involvement of generalized RF; x 100. e) Brain with MNC and MGC (arrow) aggregate; x 400. f) Liver showing centrilobular vacuolar degeneration and MGC; x 160. g) Kidney with MNC infiltration and glomerulosclerosis; x 250. h) Lung with pulmonary vascular occlusion and recanalization; x 360.
Table IlL Occurrence of LN follicular changes, ML and GCD in SIV-infected cynomolgus monkeys at autopsy. Morphology (') FH/FF FF
FA FD
Number (o70) 8 4 15 6
(24) (12) (45) (18)
ML no. (o70) 3 4 7 1
(20.0) (26.7) (46.7) (6.6)
GCD no. (%) 3 1 12 2
(17) (6) (66) (11)
(*) Classified as previously described (Porwit et al., 1989) into follicular hyperplasia (FH), follicular fragmentation (FF), follicular atrophy (FA) and follicular depletion (FD).
L,-
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E. KAA YA E T AL.
Lungs
The lungs showed frequent and extensive histopathology. Twenty-four (73 070) monkeys had changes consistent with chronic interstitial pneumonia (CIP) with patchy to diffuse distribution. In 15 (45 070)animals, changes were seen predominantIy as a thickening of the alveolar septa and mononuclear cell (MNC) infiltration with a variable degree of desquamative changes in association with intraalveolar proliferation of macrophages and obliteration of alveolar spaces (fig. 2b). Another type of histopathology was found in nine (27 %) monkeys consisting of CIP with multinucleated GC (MGC) diffusely spread in the lungs (fig. 2c). Focal emphysema was also frequently observed, apparently secondary to inflammatory obstructive changes. One monkey had extensive systemic involvement of various tissues including the lung by fibromatous lesions (LN, gut, skin) reminiscent of generalized retroperitoneal fibromatosis (RF) (fig. 2d). Five monkeys had lymphomatous infiltrates in the pleura and peribronchially as part of a generalized ML spread.
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sisted mainly of perivascular MNC and MGC infiltrates (fig. 2e). Brain parenchyma changes were subtle and often consisted of diffuse or more focal increases in MNC. Clearly defined focal gliosis was seen in only one monkey. However, no characteristic features of nodular microglial SIV encephalitis were found. In four spinal cords examined, no vacuolar myelopathy was observed. No evidence of cytomegalovirus or toxoplasmosis were observed in the brains examined. Two animals had primary brain lymphomas, one with spinial cord involvement.
Liver
Twenty-one (64 070)monkeys had MNC infiltrates of the liver, mainly in portal but also in intralobular areas, associated with Kupffer cell hyperplasia and mild siderosis (table II). Involvement of the liver in systemic GCD was seen in five (18 070) cases (fig. 2f). Three monkeys had focal ML infiltrates of the liver. Apart from two cases with zonal fatty change and vacuolar degeneration, no cytopathic or necrotic hepatocellular changes were observed.
Kidney
In thirteen (39 070) monkeys the GIT showed a variable degree of chronic enteritis and/or colitis with or without sloughing of the mucosa. Acid-fast bacilli were identified in the gut of one animal which also had GC disease (GCD). AFB were, however, not seen in other tissues with GCD involvement. Besides the giant cells and a scanty lymphocytic infiltrate, there were no characteristic tuberculoid features and therefore this lesion was diagnosed as Mycobacterium avium-intracellulare (MAD infection (fig. 3).
Renal changes consisted mostly of focal interstitial MNC infiltrates (13/33; 39 07o), focal chronic glomerulitis (6/33; 18 070) and glomerulosclerosis, sometimes with periglomerular fibrosis (5/33 ; 15 070) (fig. 2g). Involvement of the kidney by GCD was rare and mainly seen as perivascular infiltrates. ML involved the kidneys of eight monkeys (24 070).
CVS CNS
Changes in the cerebrum, cerebellum and meninges were seen in 8/32 (25 070)monkeys and con-
A variable degree of peri- and myocardial MNC infiltration was often observed. GCD involved the pericardium and perivascular areas in the myocardium of three animals (9 07o). Pronounced fibrinous
Fig. 3. MGC with cytoplasmic acid-fast bacilli (MAD exclusively in the gut of a monkey with
generalized GCD (ZN; × 200). Fig. 4. Adjacent frozen section of LN immunostained with: (a) mAb Kim4showing fragmented FDC
and (b) anti-SIVgag showing expression of viral antigen in the remaining FDC (APAAP ; × 200). Fig. 5. Small gut immunostained with anti-SIVgag (mAb RIC7) showing virus antigen in the remnant of MALT (Carnoy's flied paraffin section, ABC peroxidase; x 500).
H I S T O P A T H O L O G Y OF SIVsm-INFECTED C Y N O M O L G U S M O N K E Y S
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E. K A A Y A E T A L .
pericarditis was observed in the one monkey with RD-type disease. In a few instances, vascular lesions of the lung were observed which consisted of occlusive arteriopathy characterized by marked vascular wall thickening and occlusive intimal proliferation (fig. 2h). Occasionally, peripherally disseminated thrombosis with recanalization was observed. Two monkeys had myocardial and pericardial ML spread. Endocrine system No significant alterations were seen in the adrenals and pancreas except for one case of GCD associated with adrenal medullary MGC formation. Immunohistopathology Follicular size, form and cellular density was evaluated by immunostaining with antibodies to follicular dendritic cells (FDC) (KiM4). The observed changes were characteristic and clearly correlated with the four histopathological patterns found in the LN and spleen (see above). In FH, a strong and uniform reticular staining of the FDC network in hyperplastic follicles was seen. This was replaced by the characteristic focal and diffuse decrease in immunostaining of the FDC network resulting in a fragmented pattern of the FF stage (fig. 4a). Only remnants of KiM4 + cells were seen at the FA and virtually absent at the FD stages. Staining for T-cell markers showed an increased infiltration of CD8 + ceils in the follicles during the follicular fragmentation stage, while CD4 cells were scanty. In the FD stage, most lymphocytes of the LN were CD8 +. LN with severe GCD showed virtually no staining with KiM4. Corresponding to the extension and pattern of the FDC network, staining with anti-SIVgag showed strong and diffuse staining at the FH stage, becoming more irregular and fragmented during FF and FA (fig. 4b). At the FD stage, SIV staining was virtually absent. Intense staining for SIV was also seen in the cytoplasm of MGC, sinusoidal macrophages and in the germinal centres of MALT (fig. 5). EM Conspicuous changes were observed in the FDC from late stages of SIV infection compared to early stages (fig. 6). Thus at the FF-FA stage, most FDC showed cytopathic changes with fragmentation and swollen dendrites, disconnection of desmosomes, cytoplasmic vesicles and forthright karyorrhexis. Large amounts of virus-like particles (VLP) were usually associated with the fragmented reticular network, but no clear evidence of virus budding from FDC membranes was observed. Most of the VLP
profiles associated with FDC showed considerable variation in size and form, density and definable core, and often appeared embedded in proteinaceous material with a poorly defined surface membrane. These changes suggested that many VLP were undergoing disintegration and/or were associated with antibodies. EM of MGC in LN of monkeys with GCD corroborated previous findings indicating a monocyte/macrophage (Mo/Mac) origin of these cells (Li et ai., 1991), displaying well developed Golgi zones with associated vesicular and vacuolar structures. Most MGC showed obvious intravacuolar accumulation of mature SIV particles (fig. 7), but no evidence of virus maturation or budding at the level of the cell membrane, nor did they show evidence of phagocytic activity. ML Fifteen (45 %) monkeys developed ML which clinically were fast-growing and mostly disseminated in extranodal organs (table I and II). They showed considerable morphological heterogeneity, but all were of high malignant grade and of B-cell origin (Feichtinger et al., 1990). Immunohistochemical, molecular biology and EM studies revealed the presence of an Epstein-Barr-like herpes virus in twelve lymphomas studied as reported elsewhere (Feichtinger et al., 1992a, b). The majority of the animals with ML presented with low CD4 values and advanced involution of lymph node B-cell areas (table III). GCD Systemic MGC formation was found in 18 (54 %) monkeys distributed in various organs (table II; Li et aL, 1991). Most monkeys with GCD had'low CD4 values and lymph node involution beyond FF (table III). The GC showed a marked histological variability with regard to size, number and distribution of nuclei. Immunohistologically, they were of Mo/Mac origin and usually positive for anti-SIVgag (Li et al., 1991). DISCUSSION Most observations on the pathology of SIVinfected cynomolgus monkeys in the present study indicate several common features between the SIV and HIV infections in monkey (Letvin and King, 1990; Simon et al., 1992) and man (Biberfeld et al., 1986; 1987; Levine, 1992; Racz et al., 1985 ; Sharer and Cho, 1989) respectively, suggesting that the same or similar pathogenic mechanisms are important for the development of AIDS in both species. Histopathological changes were most frequently
H I S T O P A T H O L O G Y OF SIVsm-INFECTED C Y N O M O L G U S M O N K E Y S
Fig. 6. Electron micrograph from LN showing: (a) swollen and fragmented FDC in late stage disease and numerous virus particles ( x 31,000; bar = 0.5 ~m), (b) characteristic VLP ( x 126,000; bar = 0.1 ~m), (c) virus particles between dendrites ( x 85,000; bar = 0.I ~a'n). Fig. 7. Electron micrograph from LN showing GC with cytoplasmic tubular and vacuolar structures containing SIV particles with no evidence of budding at the cell membrane (× 20,000; bar = 0.5 ~m). Insert: Intravacuolar accumulation of mature SIV particles (× 57,000; bar = 0.1 I~m).
89
90
E. K A A Y A E T A L .
observed in the lymphoid system, lungs, liver, CNS, kidneys and GIT, hut also in CVS and adrenals. Interestingly, the frequently observed CIP was not associated with opportunistic infections, as also previously indicated (Baskerville et al., 1992), but in contrast to findings in human AIDS (Ramaswamy et al., 1985) and some other SIV/rhesus monkey studies (Letvin and King, 1990; Simon et ai., 1992). The observed pathological manifestations in monkey livers were mainly restricted to the portal tracts as infiltrates of MNC and MGC, which is concordant with observations in other experimental SIV infections (Gerber et al., 1991), but has only occasionally been described in human AIDS (Kahn et al., 1991 ; Lebovics et al., 1985). Also, the non-specific interstitial and glomerular changes observed in this study are similar to previously observed renal lesions in humans (Cohen et al., 1988), rhesus and cynomolgus monkeys (Baskerville et al., 1990; Simon et al., 1992). The encephalopathy observed in the SIVsminfected cynomolgus monkeys was usually not associated with opportunistic infections but characterized by the conspicuous formation of MGC and MNC as previously described in HIV infection (Koenig et al., 1986; Sharer and Cho, 1989) and in studies of SIV-infected rhesus monkeys (Letvin and King, 1990; Simon et al., 1992). Furthermore, MGC and MNC infiltrates were also more frequently seen in the GIT, CVS and adrenals of the cynomolgus monkeys in comparison to findings in other simian (Simon et al., 1992) and human (Koenig et al., 1986) AIDS studies. In the lymphoid system the conspicuous and consistent changes observed in the B-cell compartment characterized by the early hyperplasia and by the progressive involution of the germinal centres in parallel with the destruction of the FDC network are virtually similar to changes previously described in HIV lymphadenopathy (Biberfeld et ai., 1986; 1987; Racz et al., 1985) and also in rhesus monkeys (Letvin and King, 1990; Simon et al., 1992). In vivo susceptibility of FDC to HIV infectibn has been suggested from biopsy studies (Amstrong and Home, 1984; Biberfeld et al., 1986; Ringler et al., 1989) and was recently confirmed in vitro (Stahmer et al., 1991). Consequently, the observed FDC destruction during SIV infection most likely indicates similar pathophysiologic mechanisms in virus/FDC interaction of HIV and SIV. Pathogenic similarities are also probably reflected by the terminal T-cell depletion and neo-angiogenesis seen in SIVsm-infected monkeys as well as in human AIDS lymph nodes (Biberfeld et al., 1987). Systemic MGC formation (GCD), as previously reported (Li et al., 1991), appeared to he more frequent in the present cynomolgus SIV infections compared to other monkey (Letvin et al., 1990; Simon et al., 1992) and human studies (Sharer and Cho,
1989). Syncytia or MGC formation in vitro is regarded as an important cytopathological quality of HIV isolates (Valentin, 1992), but the in vivo significance remains unclear. Thus the present SIV/cynomolgus system appears particularly suitable for studies of the mechanisms involved in MGC formation in vivo. The present demonstration of SIV antigens (Li et al., 1991) and viral particles in these MGC in vivo, extends previous findings in humans (Koenig et al., 1986) and in rhesus monkeys (Ringlet et al., 1989; Simon et ai., 1992) and suggests an important role for Mo/Mac in the pathogenesis and spread of HIV/SIV infections in relation to the cell tropism of the virus. AIDS patients are at high risk to develop ML (Levine, 1992) which also have occasionally been reported in monkey studies (Baskerville et al., 1990; King et al., 1983 ; Simon et al., 1992). In our cynomolgus series, a very high frequency of ML was noticed as previously reported (Feichtinger et al., 1990). Of particular interest is the marked resemblance between these ML and those of human AIDS (Levine, 1992) with regard to extranodal distribution, including CNS, high grade morphology and association with an Epstein-Barr-like virus (Feichtinger et al., 1990; 1992a; 1992b). The wide anatomical dissemination of the HIV/SIV-associated ML could reflect the destruction of the FDC network with impaired GC-homing and growth control of B cells (Biberfeld et ai., 1986). In conclusion, SIVsm-infected cynomolgus monkeys develop a wide range of pathological changes, mostly similar to those seen in HIV infection and in other SIV/rhesus macaque settings. In the SIV/cynomolgus system, opportunistic infections were rarely seen despite the development of p r o n o u n c e d immunodeficiency and various histopathologies. Furthermore, a high frequency of ML and systemic MGC were noted. These pathobiological features are likely related to primate species and virus strain characteristics (Fultz and Anderson, 1990; Simon et al., 1992), as well as differences in environmental conditions. The various disease manifestations observed in our cynomolgus monkey series appear to involve direct viral interactions, particularly with cells of the immune system including FDC and Mo/Mac, whose functional impairment and/or destruction most likely influence pathogenesis and disease progression as well as the development of lymphomas. This experimental SIV-cynomolgus infection therefore appears particularly useful as a model for studies of the role of FDC and Mo/Mac in the pathogenesis of malignancies and other p~tthologies associated with HIV/SIV infections.
Acknowledgements Three monkeys were obtained for necropsy from B. Orberg and Disa B6uiger (SBL). Reinhold Bentin was responsible for the
HISTOPATHOLOGY OF SIVsm-INFECTED CYNOMOLGUS MONKEYS
necropsies. The skillful technical assistance of Angelina de Santiago, R. Gantschnig and Marianne Ekman is highly appreciated. Supported by Swedish Medical ResearchCouncil and Swedish Cancer Foundation. E.E. Kaaya was partly supported by the Swedish Agencyfor ResearchCooperationwith DevelopingCountries (SAREC) and ICSC World Laboratory project MCD2. H. Flichtingerwas partly supported by the EEC concerted action "Pathophysiology and Immunology of HIV-related diseases. Ingrid Stahmer, VisitingScientist, was supported by a grant from the "DeutscheAIDS-Stipendiumprogramm'.E. Mandache, visiting scientist from Victor Babes Institute, Bucharest,was supported by the Swedish Medical Research Council.
Cellules accessoires et macrophages dans la pathologie de macaques infect~ par le SIV SMM-3 Trente-neuf macaques (Macacafascicularis) infectds par le virus d'immunoddficience simien (SIV SMM-3) ont dtd surveillds pendant deux anndes et demi. Trente-trois animaux ont dtd examinds apr~s autopsie selon les manifestations d'immunoddficience et de pathologie. Six animaux ont survdcu sans ddvelopper de maladie. La majorit6 des macaques ddploient, pendant l'infection, une lymphaddnopathie caractdristique e t / o u des manifestations histopathologiques dans la plupart des organes, surtout les poumons, le syst~me gastro-intestinal, les reins, le cerveau, le foie et le syst~me cardio-vasculaire. L'histopathologie des tissus involuds montre un r61e important joud par les cellules accessoires dendritiques folliculaires et les monocytes macrophages dans les diverses manifestations, en particulier dans la formation systdmique de cellules gdantes multinucldes. Quinze (45 %) des animaux ont aussi ddveloppd des tumeurs malignes lymphocytaires apparemment associ6es avec un dtat plus important d'immunoddficience et d'involution ganglionnaire. Ces tumeurs ressemblent dtroitement aux lymphomes du SIDA humain du point de vue clinique et biologique. La ressemblance entre des manifestations pathologiques ainsi que des lymphomes chez les macaques infectds par le SIV SMM3 et ceux du SIDA humain rdv~le l'importance de ce module animal pour l'dtude des divers m6canismes de la pathogen~se de l'infection par le VIH. Mots-cids: SIDA, SIV, Cellule dendritique folliculaire, Macrophage; Histopathologie, Mod61e animal, Microscopie dlectronique.
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Baskerville, A., Ramsay, A.D., Addis, B.J., Dennis, M.J., Cook, R.W., Cranage, M.P. & Greenaway, P.J. (1992), Interstitial pneumonia in simian immunodeficiency infection. J. Path., 167, 241-247. Baskerville, A., Ramsay, A., Cranage, M.P., Cook, N., Cook, R.W., Dennis, M.J., Greenaway, P.J., Kitchin, P.A. & Stott, E.J. (1990), Histopathological changes in simian immunodeficiency virus infection. J. Path., 162, 67-75. Biberfeld, P., Chayt, K.J., Marselle, L.M., Biberfeld, G. & Harper, M.E. (1986), HTLV-III expression in infected lymph nodes and relevance to pathogenesis of lymphadenopathy. Amer. d. Path., 125, 436-442. Biberfeld, P., Ost, A., Porwit, A. b Sandstedt, B., Pallesen, G., B6ttiger, B., Morfeldt-Manson, L. & Biberfeld, G. (1987), Histopathology and immunopathology of HTLV/LAV-related lymphadenopathy and AIDS. Acta Path. Microbiol. Immunol. Scand., 95, 47-65. Cohen, A.H. & Nast, C.C. (1988), HIV-associated nephropathy. A unique combined glomerular tubular and interstitial lesion. Modern Path., 1, 87-97. Desrosiers, R.C. (1990), The simian immunodeficiency viruses. Ann. Rev. Immunok, 8, 557-578. Feichtinger, H., Kaaya, E., Putkonen, P., Li, S.L., Ekman, M., Gendelman, R., Biberfeld, G. & Biberfeld, P. (1992a), Malignant lymphoma associated with human AIDS and with SIV-induced immunodeficiency in macaques. AIDS Res Hum. Retroviruses, 8,339-348. Feichtinger, H., Li, S.L., Kaaya, E., Putkonen, P., Grunewald, K., Weyrer, D., B6ttiger, D., Ernberg, I., Linde, A., Biberfeld, G. & Biberfeld, P. (1992b), A monkey model for Epstein-Barr-virus-associated lymphomagenesis in human-acquired immunodeficiency syndrome. J. exp. Med., 176, 281-286. Feichtinger, H., Putkonen, P., Parravicini, C., Li, S.L., Kaaya, E.E., B6ttiger, D., Biberfeld, G. & Biberfeld, P. (1990), Malignant lymphomas in cynomolgns monkeys infected with simian immun~deficiency virus. Amer. J. Path., 137, 1311-1315. Fultz, P.N. & Anderson, D.C. 0990), The biology and immunopathology of simian immunodeficiency syndrome virus infection. Curr. Opinion Immunok, 2,403-408. Fultz, P.N., McClure, H.M., Anderson, D.C., Swenson, R.B., Anand, R. & Srinivasan, A. (1986), Isolation of a T lymphotropic retrovirus from naturally infected sooty mangabey monkeys (Cercocebus atys). Proc. nat. Acad. Sci. (Wash.), 83, 5286-5290. Gerber, M.A., Chen, M-L., Hu, F.S., Baskin, G.B. & Petrovich, L. (1991), Liver disease in Rhesus monkeys infected with simian immunodeficiency virus. Amer. J. Path., 139, 1081-1088. Kahn, E., Greco, M.A., Daum, F., Magid, M., Morecki, R., Mahnovski, V. & Anderson, V. (1991), Hepatic pathology in paediatrics acquired immunodeficiency syndrome. Hum. Path., 22, 1111-1119. King, N.L., Hunter, R.D. & Letvin, N.L. (1983), Histopathologic changes in macaques with an acquired immunodeficiency syndrome (AIDS). Amer. J. Path., 113, 382-388. Koenig, S., Gendelman, H.E., Orenstein, J., Dal Canto, M., Pezeshkpour, G., Yungbluth, M., Janotta, F., Aksamit, A., Martin, M. & Fauci, A.S. (1986), Detection of AIDS virus in macrophages in brain tissue from AIDS patients with encephalopathy. Science, 233, 1089-1093.
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Langhoff, E., Terwilliger, E.F., Bos, H.J., Kalland, K.H., Poznasky, M.C., Bacon, O.M. & Haseltine, W.A. (1991), Replication of human immunodeficiency virus type 1 in primary dendritic cell cultures. Proc. nat. Acad. Sci. 0/Cash.), 88, 7998-8002. Lebovics, E., Thung, S.N., Schaffner, F. & Radensky, P.W. (1985), The liver in the acquired immunodeficiency syndrome: a clinical and histological study. Hepatology, 5, 293-298. Letvin, N.L. (1990), Animal models for AIDS. ImmunoL Today, 11, 322-326. Letvin, N.L. (1992), Animal models for the study of human immunodeficiency virus infections. Curr. opinion Immunol., 4, 481-485. Letvin, N.L. & King, N.W. (1990), Immunologic and pathologic manifestations of the infection of rhesus monkeys with simian immunodeficiency virus of macaques. J. AIDS, 3, 1023-1040. Levine, A.M. (1992), Acquired immunodeficiency syndrome-related lymphoma. Blood, 80, 8-20. Li, S.L., Kaaya, E.E., Feichtinger, H., Putkonen, P., Parravicini, C., B6ttiger, D., Biberfeld, G. & Biberfeld, P. (1991), Monocyte/macrophage giant cell disease in SIV-infected cynomolgus monkeys. Res. Virol., 142, 173-182. Macatonia, S.E., Lau, R., Patterson, S., Pinching, A.J. & Knight, S.C. (1990), Dendritic cell infection, depletion and dysfunction in HIV-infected individuals. Immunology, 72, 38-45. Meltzer, M.S., Skillman, D.R., Gomatos, P.J., Kalter, D.C. & Gendelman, H.E. (1990), Role of mononuclear phagocytes in the pathogenesis of human immunodeficiency virus infection. Ann. Rev. ImmunoL, 8, 169. Porwit, A., B6ttiger, B., Pallsen, G., Bodner, A. & Biberfeld, P. (1989), Follicular involution in HIV lymphadenopathy. Acta. path. Microbiol. Immunol. scand., 97, 153-165. Putkonen, P., Kaaya, E.E., B6ttiger, D., Li, S.L., Ntisson, C., Biberfeld, P. & Biberfeld, G. (1992), Clinical features and predictive markers of disease progression in cynomolgus monkeys experimentally infected with simian immunodeficiency virus.AIDS, 6, 257-263.
Putkonen, P., Thorstensson, R., Albert, J., Hiid, K., Norrby, E., Biberfeid, P. & Biberfeld, G. (1990), Infection of cynomolgus monkeys with HIV-2 protects against pathogenic consequences of a subsequent simian immunodeficiency virus infection. AIDS, 4, 783-789. Putkonen, P., Warstedt, K., Thorstensson, R., Benthin, R., Albert, J., Lundgren, B., Oberg, B., Norrby, E. & Biberfeld, G. (1989), Experimental infection of cynomolgus monkeys (Macaca fascicularis) with simian immunodeficiency virus (SIVsm). J. AIDS, 2, 359-365. Racz, P., Tenner-Racz, K., Kahl, C., Feller,A.C., Kern, P. & Dietrich, M. 0985), The spectrum of morphologicchanges in lymph nodes from patientswith AIDS or AIDS-related complex. Progr. Allergy, 37, 81-181. Ramaswamy, G., Jagadha, V. & Tchertkoff, V. (1985), Diffuse alveolar damage and interstitialfibrosis in AIDS patients without concurrent pulmonary infection. Arch. Path. Lab. Med., 109, 408-412. Ringler, D.J., Wyand, M.S., Walsh, D.G., MacKey, J.J., Chalfoux, L.V., Popovic, M., Minassiah, A.A., Sehgal, P.K., Daniel, M.D., Desrosiers, R.C. & King, N.W. (1989), Cellular localization of simian immunodeficiency virus in lymphoid tissues. Amer. J. Path., 134, 373-383. Sharer, L.R. & Cho, E.S. (1989), Neuropathology of HIV infection: adults versus children.Progr. AIDS. Path., l, 131-141. Simon, M.A., Chalifouix, L.V. & Ringler, D.J. (1992), Pathologic features of SIV-induced disease and the association of macrophage infection with disease evolution. A I D S Res. Hum. Retroviruses, 8, 327-337. Stahmer, I.,Zimmer, J.P., Ernst, M., Fenner, T., Finnern, R., Schmitz, H., Flad, H-D. & Gerdes, J. (1991), Isolation of normal human folliculardendritic cellsand CD4-independent in vitroinfection by human immunodeficiency virus (HIV-I). Europ. J. ImmunoL, 21, 1873-1878. Valentin, A. (1992), Mononuclear phagocytes and HIV-I infection. Thesis, Karolinska Institute, Stockholm, Sweden.
(~) INSTITUTPASTEUR/ELSEVIER Paris 1993
Accessory cells and macrophages in the histopathology of SIVsm-infected cynomoigus monkeys E. Kaaya (i, 4), S.-L. Li (i), H. Feichtinger (I, 3), I. Stahmer (1), p. P u t k o n e n (2), E. M a n d a c h e 0), E. Mgaya (4), G. Biberfeld (2) and P. Biberfeld (I) (*)
(1) Immunopathology Laboratory, Department o f Pathology, Karolinska Institute, 10401 Stockholm, (2j Department o f Immunology, National Bacteriological Laboratory, Stockholm, (3) Department o f Pathology, University o f Innsbruck (Austria) and (4j Department o f Pathology, Muhimbili University College o f Health Sciences, Dar-es-Salaam (Tanzania) SUMMARY
Thirty-three out of 39 cynomolgus monkeys (Macaca fascicularis) infected with SlVsm (strain SMM-3) developed various pathologies similar to those seen in human AIDS. Lymphadenopathy was frequently seen (72 %) and was characterized by hyperplasia followed by involution of follicle/germinal centres due to follicular dendritic cell (FDC) destruction corresponding to the degree of immunodeficiency. Various organs such as the lungs, liver, central nervous system, kidneys, gastrointestinal tract, cardiovascular system and adrenals showed histopathological changes with prominent monocyte/macrophage and multinucleated giant cell formation. Eighteen (54 %) monkeys presented with extranodal malignant lymphoma (ML) associated with marked CD4 decrease and destruction of follicular architecture. The high frequency of ML, giant cell disease and lymph node changes seen in the present SlV model provides an attractive system to elucidate the role of FDC and monocytes/macrophages in the pathogenesis of these cohditions in common with HIV infection and human AIDS.
Key-words: AIDS, SIV, Follicular dendritic cell, Macrophage; Histopathology, Animal model, Electron microscopy.
INTRODUCTION Acquired immunodeficiency syndrome (AIDS) in man is aetiologically linked to the lymphotropic lentiviruses designated HIV1 and HIV2. Apart from their cytopathic effects on CD4 + T cells, in vitro and in vivo studies have suggested that other cells, particularly accessory ceils, may also become targets for productive or latent HIV infection (Biberfeld et ai., 1986; Langhoff et al., 1991 ; Macatonia et al., 1990; Racz et al., 1985 ; Stahmer et al., 1991) resul-
Received October 1 I, 1992. ~'~Corresponding author.
ting in a complex pathology involving various tissues during progression to AIDS. During the last few years, several attempts have been made to establish animal models for studies of the pathogenic mechanisms involved in AIDS and for development and testing of vaccines and new therapies (Letvin, 1990; 1992). The chimpanzee, gibbon ape rabbit and SCID-hu mouse have also been shown to be susceptible to infection with HIV1, but neither immunodeficiency nor disease has been reported in
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these species (Letvin, 1990; 1992). A s i a n m a c a q u e s can be persistently infected with some strains o f H I V 2 b u t w i t h o u t d e v e l o p m e n t o f A I D S - l i k e disease ( P u t k o n e n et al., 1990). H o w e v e r , such m o n k e y s are susceptible to i n f e c t i o n with s o m e strains o f s i m i a n i m m u n o d e f i c i e n c y virus (SIV), a Ientivirus p h y l o g e netically related to H I V (Desrosiers, 1990). T h e y o f t e n d e v e l o p an A I D S - l i k e disease a n d m a y therefore represent a relevant m o d e l for H I V - r e l a t e d cond i t i o n s (Letvin, 1990; Letvin a n d King, 1990). W e ( P u t k o n e n et al., 1989) and others (Fultz et al., 1986) have recently d e s c r i b e d such a m o d e l o f A I D S in c y n o m o l g u s m o n k e y s infected with S I V s m . In the present s t u d y , we r e p o r t on the s p e c t r u m o f p a t h o logical changes in a c o h o r t o f 33 c y n o m o l g u s m o n keys ( M a c a c a f a s c i c u l a r i s ) e x p e r i m e n t a l l y infected with S I V s m (strain S M M - 3 ) , a n d d e m o n s t r a t e t h a t m a j o r pathological a n d i m m u n o p a t h o l o g i c a l features involving several o r g a n systems are closely related to the infection a n d / o r d e s t r u c t i o n b y S I V o f accessory a n d m o n o n u c l e a r p h a g o c y t i c cells.
MATERIALS AND METHODS
Monkeys, SIV infection and follow-up
Autopsy and histopathology Autopsies were performed immediately on monkeys sacrificed at extremis, or within 12 h for the few which died spontaneously. Specimens were partly frozen and partly fixed in B5 and Carnoy's fixatives and processed for conventional histopathological evaluation of haematoxylene and eosine (H&E) sections. Selected organ sections were stained with periodic acid-Schiff, Grocott's methenamine silver technique, Congo red, ZiehI-Nielsen (ZN), Gram stain and Giemsa.
Immunohistopathology and electron microscopy (EM) Immunostaining was performed on frozen sections or on Carnoy's fixed material. Frozen sections were cut, acetone-fixed and preserved at - 70°C until used. The following monoclonal antibodies (mAb) were used for the immunostaining of SIV gag antigen (mAb : SFS-6FS ; Biotech. Res. Lab. ; RIC7 ; LTCB/NIH), follicular dendritic cells (mAb KiM4; Behring), B cells (mAb L26, CD20; Dako), (mAb FS-I 1-13, CD45RA; Serotec), CD4 ÷ T cells (mAb OKT4; Ortho), macrophages (mAb KiM6, CD68, Boeh. Mann) and CD8 + T cells (mAb Leu2a and mAb Leu5b, CD2; Becton and Dickinson). The avidin biotin peroxidase complex (ABC) and the alkaline phosphatase anti-alkaline phosphatase (APAAP) methods were used as previously described (Biberfeld et al., 1986).
Thirty-nine wild caught cynomolgus monkeys (table I) were intravenously inoculated with SIVsm (strain SMM-3 from Drs. P. Fultz and H. McClure, Yerkes Primate Research Center, Atlanta, GA, USA) and propagated in human PBL as previously described (Putkonen et al., 1989). The monkeys were inoculated with different doses ranging from undiluted supernatants to 101-105 monkey infectious doses (MIDso) of uncloned virus. The monkeys were kept at the Primate Centre of the National Bacteriological Laboratory (SBL) according to the guidelines of the Swedish ethical committee. They were anaesthetized by ketamine at intervals for physical examination and collection of blood and biopsies for virological, immunological and histopathological studies, as previously described (Putkonen et al., 1989; 1990). Animals in extremis were euthanized by intravenous injection of pentobarbital sodium.
For electron microscopy (EM) tissue biopsies were fixed in 2 % paraformaldehyde/2.5 070 glutaraldehyde, post-fixed in I 070 osmium tetroxide dehydrated in graded ethanol and embedded in agar-100. Sections were examined in a "Jeol EM-100S" transmission electron microscope.
ABC = APAAP = CIP = CNS = CVS = EM = FA = FD = FDC = FF = FH = GC = GCD = GIT = H&E =
LN = mAb = MAI = MALT = MGC = MID = ML = MNC = Mo/Mac = PBL = RF = SBL = SIV = VLP = ZN =
avidin-biotin peroxidase complex. alkaline phosphatase anti-alkaline phosphatase. chronic interstitial pneumonia. central nervous system. cardiovascular system. electron microscopy. follicular atrophy. follicular depletion. follicular dendritic cell. follicular fragmentation. follicular hyperplasia. giant cell. GC disease. gastrointestinal tract. haematoxyline and cosine.
RESULTS
Clinical and immunological manifestations Clinical features o f infected m o n k e y s ~ e s u m m a rized in t a b l e I. T h i r t y - t h r e e (85 % ; males = 16, females = 17) m o n k e y s were e u t h a n i z e d d u e to
lymph node. monoclonal antibody. Mycobacterium avium-intracellulare. mucosal-associated lymphoid tissue. multinucleated GC. monkey infectious dose. malignant lymphoma. mononuclear cell. monocyte/macrophage. peripheral blood lymphocyte. retroperitoneal fibromatosis. Swedish (national) Bacteriological Laboratory. simian immunodeficiency virus. virus-like particle. Ziehl-Nielsen.
HISTOPA THOLOGY OF SIVsm-INFECTED CYNOMOLGUS MONKEYS
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Table I. Clinical and immunological manifestations of SIV infection in cynomolgus monkeys.
Animals and SIV infection:
- infection - animal weight (age) -
-
sex
- time of infection (days)
SIV SMM-3 (undiluted to 10-105 MIDs0) 1.8-6.125 kg male 16; female 17 mean 431 (+ 214); range 58-905
Clinical manifestations:
- weight loss (°70) - lymphadenopathy - ML - diarrhoea - cachexia - splenomegaly - respiratory symptoms
mean 24/33 12/33 12/33 10/33 5/33 2/33
19.2 (_+ 11); range 2-44 (73 %) (36 %) (36 %) (30 %) (15 %) (6 %)
Immunological manifestations:
- PBL-CD4 conts (x 109/1) - CD4/CD8 :
mean 0.13 (+ 0.1); range 0.02-0.5 mean 0.23 (_+ 0.19); range 0.01-0.7
MID~o = monkey infectious dose/50.
disease after 58 to 905 days, were included in the study. Six monkeys without clinical manifestations are still alive two to four years post-inoculation. A variable degree of weight loss was seen in all monkeys and severe cachexia in 10 (30 %). Twenty-four (73 %) monkeys had terminal lymphadenopathy. Five monkeys (15 %) presented with clinically palpable spleens. Diarrhoea as the dominant clinical manifestation was noted in 12 (36 %) monkeys. Two (6 %) monkeys had neurological symptoms including ataxia and paraplegia, one with central nervous system (CNS) lymphoma (ML) and another with giant cell (GC) encephalopathy. Extranodal malignant lymphoma (ML) developed in 12 (36 %) monkeys, in 3 (9 %) of which it was the only presenting disease manifestation. Thirty-two animals, all with clinical disease, showed variable immunosuppression, as determined by CD4 counts and CD4/CD8 ratios of peripheral blood lymphocytes (PBL). All monkeys were seropositive for SIV antibodies (Putkonen et al., 1992) and SIV antigens were demonstrable by immunohistochemistry in all tested lymph nodes (LN) and spleens.
Histopathology
Table II summarizes the main pathological findings in the monkeys. Saiient changes seen in the lymphoid organs, lungs, liver, kidneys, CNS, gas-
trointestinal tract (GIT) and cardiovascular system (CVS) are reported below. Lymphoid tissues including M A L T
Follicles/germinal centres of LN, spleen and the mucosa-associated lymphoid tissue (MALT) showed prominent changes, usually in the form of follicular hyperplasia (FH) in early stages of infection. During disease progression with decreasing CD4 + PBL counts, a gradual involution of follicles/germinal centers was usually seen and defined as follicular fragmentation (FF), follicular atrophy (FA) and follicular depletion (FD) in accordance with previously described changes in HIV PGL (Porwit et al., 1989) (table III; fig. 1 and 2a). Although the histopathology of lymphoid tissues varied between different monkeys (table III) the degree of involution appeared to correlate with a decrease in CD4 (fig. 1). The spleen often showed a variable spectrum of FH to FA changes. At early stages of infection, paxacortical expansion, angiogenesis and sinus histiocytosis were observed to a variable degree in the parafollicular areas and sinuses of LN. Angiogenesis became more pronounced with progression of follicular involution from early (FF) to late (FA and FD) stages. Likewise, in the red pulp of the spleen, hyperplastic changes were observed in 18 (54 e/0) monkeys. Most thymuses were involuted, but in a few instances, non-involuted, remaining thymus tissue showed regressive changes.
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Table H. Histopathology in cynomolgus monkeys infected with SIVsm (SMM3). D
Organ/. system and histopathology Frequency (%) Systemic lymphadenopathy GCD ML RF Lung CIP ML GIT enteritis GCD ML Kidney interstitial MNC infiltrate glomerulitis focal glomerulosclerosis ML CNS GC ML Liver MNC infiltrates Kupffer cell hyperplasia GCD fatty change ML CVS MNC infiltrates GCD fibrinous pericarditis ME Oral cavity oral hairy leukoplakia
33 18/33 15/33 1/33
(100) (54) (45) (6)
24/33 5/33
(73) (15)
13/32 13/32 9/32 13/32
(41) (41) (27) (41)
13/33 6/33 5/33 6/33
(39) (18) (15) (18)
8/32 2/32
(25) (6)
21/33 9/33 5/33 1/33 3/33
(64) (27) (18) (3) (9)
12/32 3/32 1/32 2/32
(38) (9) (3) (6)
1733
(3)
t
o
m 0
•
FH
T
"
FH FF
!
FF
"
I
FF FA
"
I
•
FA
I
•
FA FD
Hist~ogy
FD
grade
Fig. 1. LN histopathology in relation to CD4 values in infected cynomolgus monkeys• FH = follicular hyperplasia, FF = follicular fragmentation, FA = follicular atrophy, FD = follicular depletion.
Fig. 2. Examples of histopathological changes in organs of cynomolgus monkeys infected with SIVsm (see table II for frequencies); (H&E stained paraffin sections). a) MGC (arrow heads) and atrophic MALT (arrow) in the gut of an infected monkey; x 250. b) Lung showing CIP with septal thickening and MNC infiltration ; x 400. c) Lung with MGC as part of GCD manifestation; x 160. d) Lung involvement of generalized RF; x 100. e) Brain with MNC and MGC (arrow) aggregate; x 400. f) Liver showing centrilobular vacuolar degeneration and MGC; x 160. g) Kidney with MNC infiltration and glomerulosclerosis; x 250. h) Lung with pulmonary vascular occlusion and recanalization; x 360.
Table IlL Occurrence of LN follicular changes, ML and GCD in SIV-infected cynomolgus monkeys at autopsy. Morphology (') FH/FF FF
FA FD
Number (o70) 8 4 15 6
(24) (12) (45) (18)
ML no. (o70) 3 4 7 1
(20.0) (26.7) (46.7) (6.6)
GCD no. (%) 3 1 12 2
(17) (6) (66) (11)
(*) Classified as previously described (Porwit et al., 1989) into follicular hyperplasia (FH), follicular fragmentation (FF), follicular atrophy (FA) and follicular depletion (FD).
L,-
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E. KAA YA E T AL.
Lungs
The lungs showed frequent and extensive histopathology. Twenty-four (73 070) monkeys had changes consistent with chronic interstitial pneumonia (CIP) with patchy to diffuse distribution. In 15 (45 070)animals, changes were seen predominantIy as a thickening of the alveolar septa and mononuclear cell (MNC) infiltration with a variable degree of desquamative changes in association with intraalveolar proliferation of macrophages and obliteration of alveolar spaces (fig. 2b). Another type of histopathology was found in nine (27 %) monkeys consisting of CIP with multinucleated GC (MGC) diffusely spread in the lungs (fig. 2c). Focal emphysema was also frequently observed, apparently secondary to inflammatory obstructive changes. One monkey had extensive systemic involvement of various tissues including the lung by fibromatous lesions (LN, gut, skin) reminiscent of generalized retroperitoneal fibromatosis (RF) (fig. 2d). Five monkeys had lymphomatous infiltrates in the pleura and peribronchially as part of a generalized ML spread.
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sisted mainly of perivascular MNC and MGC infiltrates (fig. 2e). Brain parenchyma changes were subtle and often consisted of diffuse or more focal increases in MNC. Clearly defined focal gliosis was seen in only one monkey. However, no characteristic features of nodular microglial SIV encephalitis were found. In four spinal cords examined, no vacuolar myelopathy was observed. No evidence of cytomegalovirus or toxoplasmosis were observed in the brains examined. Two animals had primary brain lymphomas, one with spinial cord involvement.
Liver
Twenty-one (64 070)monkeys had MNC infiltrates of the liver, mainly in portal but also in intralobular areas, associated with Kupffer cell hyperplasia and mild siderosis (table II). Involvement of the liver in systemic GCD was seen in five (18 070) cases (fig. 2f). Three monkeys had focal ML infiltrates of the liver. Apart from two cases with zonal fatty change and vacuolar degeneration, no cytopathic or necrotic hepatocellular changes were observed.
Kidney
In thirteen (39 070) monkeys the GIT showed a variable degree of chronic enteritis and/or colitis with or without sloughing of the mucosa. Acid-fast bacilli were identified in the gut of one animal which also had GC disease (GCD). AFB were, however, not seen in other tissues with GCD involvement. Besides the giant cells and a scanty lymphocytic infiltrate, there were no characteristic tuberculoid features and therefore this lesion was diagnosed as Mycobacterium avium-intracellulare (MAD infection (fig. 3).
Renal changes consisted mostly of focal interstitial MNC infiltrates (13/33; 39 07o), focal chronic glomerulitis (6/33; 18 070) and glomerulosclerosis, sometimes with periglomerular fibrosis (5/33 ; 15 070) (fig. 2g). Involvement of the kidney by GCD was rare and mainly seen as perivascular infiltrates. ML involved the kidneys of eight monkeys (24 070).
CVS CNS
Changes in the cerebrum, cerebellum and meninges were seen in 8/32 (25 070)monkeys and con-
A variable degree of peri- and myocardial MNC infiltration was often observed. GCD involved the pericardium and perivascular areas in the myocardium of three animals (9 07o). Pronounced fibrinous
Fig. 3. MGC with cytoplasmic acid-fast bacilli (MAD exclusively in the gut of a monkey with
generalized GCD (ZN; × 200). Fig. 4. Adjacent frozen section of LN immunostained with: (a) mAb Kim4showing fragmented FDC
and (b) anti-SIVgag showing expression of viral antigen in the remaining FDC (APAAP ; × 200). Fig. 5. Small gut immunostained with anti-SIVgag (mAb RIC7) showing virus antigen in the remnant of MALT (Carnoy's flied paraffin section, ABC peroxidase; x 500).
H I S T O P A T H O L O G Y OF SIVsm-INFECTED C Y N O M O L G U S M O N K E Y S
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pericarditis was observed in the one monkey with RD-type disease. In a few instances, vascular lesions of the lung were observed which consisted of occlusive arteriopathy characterized by marked vascular wall thickening and occlusive intimal proliferation (fig. 2h). Occasionally, peripherally disseminated thrombosis with recanalization was observed. Two monkeys had myocardial and pericardial ML spread. Endocrine system No significant alterations were seen in the adrenals and pancreas except for one case of GCD associated with adrenal medullary MGC formation. Immunohistopathology Follicular size, form and cellular density was evaluated by immunostaining with antibodies to follicular dendritic cells (FDC) (KiM4). The observed changes were characteristic and clearly correlated with the four histopathological patterns found in the LN and spleen (see above). In FH, a strong and uniform reticular staining of the FDC network in hyperplastic follicles was seen. This was replaced by the characteristic focal and diffuse decrease in immunostaining of the FDC network resulting in a fragmented pattern of the FF stage (fig. 4a). Only remnants of KiM4 + cells were seen at the FA and virtually absent at the FD stages. Staining for T-cell markers showed an increased infiltration of CD8 + ceils in the follicles during the follicular fragmentation stage, while CD4 cells were scanty. In the FD stage, most lymphocytes of the LN were CD8 +. LN with severe GCD showed virtually no staining with KiM4. Corresponding to the extension and pattern of the FDC network, staining with anti-SIVgag showed strong and diffuse staining at the FH stage, becoming more irregular and fragmented during FF and FA (fig. 4b). At the FD stage, SIV staining was virtually absent. Intense staining for SIV was also seen in the cytoplasm of MGC, sinusoidal macrophages and in the germinal centres of MALT (fig. 5). EM Conspicuous changes were observed in the FDC from late stages of SIV infection compared to early stages (fig. 6). Thus at the FF-FA stage, most FDC showed cytopathic changes with fragmentation and swollen dendrites, disconnection of desmosomes, cytoplasmic vesicles and forthright karyorrhexis. Large amounts of virus-like particles (VLP) were usually associated with the fragmented reticular network, but no clear evidence of virus budding from FDC membranes was observed. Most of the VLP
profiles associated with FDC showed considerable variation in size and form, density and definable core, and often appeared embedded in proteinaceous material with a poorly defined surface membrane. These changes suggested that many VLP were undergoing disintegration and/or were associated with antibodies. EM of MGC in LN of monkeys with GCD corroborated previous findings indicating a monocyte/macrophage (Mo/Mac) origin of these cells (Li et ai., 1991), displaying well developed Golgi zones with associated vesicular and vacuolar structures. Most MGC showed obvious intravacuolar accumulation of mature SIV particles (fig. 7), but no evidence of virus maturation or budding at the level of the cell membrane, nor did they show evidence of phagocytic activity. ML Fifteen (45 %) monkeys developed ML which clinically were fast-growing and mostly disseminated in extranodal organs (table I and II). They showed considerable morphological heterogeneity, but all were of high malignant grade and of B-cell origin (Feichtinger et al., 1990). Immunohistochemical, molecular biology and EM studies revealed the presence of an Epstein-Barr-like herpes virus in twelve lymphomas studied as reported elsewhere (Feichtinger et al., 1992a, b). The majority of the animals with ML presented with low CD4 values and advanced involution of lymph node B-cell areas (table III). GCD Systemic MGC formation was found in 18 (54 %) monkeys distributed in various organs (table II; Li et aL, 1991). Most monkeys with GCD had'low CD4 values and lymph node involution beyond FF (table III). The GC showed a marked histological variability with regard to size, number and distribution of nuclei. Immunohistologically, they were of Mo/Mac origin and usually positive for anti-SIVgag (Li et al., 1991). DISCUSSION Most observations on the pathology of SIVinfected cynomolgus monkeys in the present study indicate several common features between the SIV and HIV infections in monkey (Letvin and King, 1990; Simon et al., 1992) and man (Biberfeld et al., 1986; 1987; Levine, 1992; Racz et al., 1985 ; Sharer and Cho, 1989) respectively, suggesting that the same or similar pathogenic mechanisms are important for the development of AIDS in both species. Histopathological changes were most frequently
H I S T O P A T H O L O G Y OF SIVsm-INFECTED C Y N O M O L G U S M O N K E Y S
Fig. 6. Electron micrograph from LN showing: (a) swollen and fragmented FDC in late stage disease and numerous virus particles ( x 31,000; bar = 0.5 ~m), (b) characteristic VLP ( x 126,000; bar = 0.1 ~m), (c) virus particles between dendrites ( x 85,000; bar = 0.I ~a'n). Fig. 7. Electron micrograph from LN showing GC with cytoplasmic tubular and vacuolar structures containing SIV particles with no evidence of budding at the cell membrane (× 20,000; bar = 0.5 ~m). Insert: Intravacuolar accumulation of mature SIV particles (× 57,000; bar = 0.1 I~m).
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observed in the lymphoid system, lungs, liver, CNS, kidneys and GIT, hut also in CVS and adrenals. Interestingly, the frequently observed CIP was not associated with opportunistic infections, as also previously indicated (Baskerville et al., 1992), but in contrast to findings in human AIDS (Ramaswamy et al., 1985) and some other SIV/rhesus monkey studies (Letvin and King, 1990; Simon et ai., 1992). The observed pathological manifestations in monkey livers were mainly restricted to the portal tracts as infiltrates of MNC and MGC, which is concordant with observations in other experimental SIV infections (Gerber et al., 1991), but has only occasionally been described in human AIDS (Kahn et al., 1991 ; Lebovics et al., 1985). Also, the non-specific interstitial and glomerular changes observed in this study are similar to previously observed renal lesions in humans (Cohen et al., 1988), rhesus and cynomolgus monkeys (Baskerville et al., 1990; Simon et al., 1992). The encephalopathy observed in the SIVsminfected cynomolgus monkeys was usually not associated with opportunistic infections but characterized by the conspicuous formation of MGC and MNC as previously described in HIV infection (Koenig et al., 1986; Sharer and Cho, 1989) and in studies of SIV-infected rhesus monkeys (Letvin and King, 1990; Simon et al., 1992). Furthermore, MGC and MNC infiltrates were also more frequently seen in the GIT, CVS and adrenals of the cynomolgus monkeys in comparison to findings in other simian (Simon et al., 1992) and human (Koenig et al., 1986) AIDS studies. In the lymphoid system the conspicuous and consistent changes observed in the B-cell compartment characterized by the early hyperplasia and by the progressive involution of the germinal centres in parallel with the destruction of the FDC network are virtually similar to changes previously described in HIV lymphadenopathy (Biberfeld et ai., 1986; 1987; Racz et al., 1985) and also in rhesus monkeys (Letvin and King, 1990; Simon et al., 1992). In vivo susceptibility of FDC to HIV infectibn has been suggested from biopsy studies (Amstrong and Home, 1984; Biberfeld et al., 1986; Ringler et al., 1989) and was recently confirmed in vitro (Stahmer et al., 1991). Consequently, the observed FDC destruction during SIV infection most likely indicates similar pathophysiologic mechanisms in virus/FDC interaction of HIV and SIV. Pathogenic similarities are also probably reflected by the terminal T-cell depletion and neo-angiogenesis seen in SIVsm-infected monkeys as well as in human AIDS lymph nodes (Biberfeld et al., 1987). Systemic MGC formation (GCD), as previously reported (Li et al., 1991), appeared to he more frequent in the present cynomolgus SIV infections compared to other monkey (Letvin et al., 1990; Simon et al., 1992) and human studies (Sharer and Cho,
1989). Syncytia or MGC formation in vitro is regarded as an important cytopathological quality of HIV isolates (Valentin, 1992), but the in vivo significance remains unclear. Thus the present SIV/cynomolgus system appears particularly suitable for studies of the mechanisms involved in MGC formation in vivo. The present demonstration of SIV antigens (Li et al., 1991) and viral particles in these MGC in vivo, extends previous findings in humans (Koenig et al., 1986) and in rhesus monkeys (Ringlet et al., 1989; Simon et ai., 1992) and suggests an important role for Mo/Mac in the pathogenesis and spread of HIV/SIV infections in relation to the cell tropism of the virus. AIDS patients are at high risk to develop ML (Levine, 1992) which also have occasionally been reported in monkey studies (Baskerville et al., 1990; King et al., 1983 ; Simon et al., 1992). In our cynomolgus series, a very high frequency of ML was noticed as previously reported (Feichtinger et al., 1990). Of particular interest is the marked resemblance between these ML and those of human AIDS (Levine, 1992) with regard to extranodal distribution, including CNS, high grade morphology and association with an Epstein-Barr-like virus (Feichtinger et al., 1990; 1992a; 1992b). The wide anatomical dissemination of the HIV/SIV-associated ML could reflect the destruction of the FDC network with impaired GC-homing and growth control of B cells (Biberfeld et ai., 1986). In conclusion, SIVsm-infected cynomolgus monkeys develop a wide range of pathological changes, mostly similar to those seen in HIV infection and in other SIV/rhesus macaque settings. In the SIV/cynomolgus system, opportunistic infections were rarely seen despite the development of p r o n o u n c e d immunodeficiency and various histopathologies. Furthermore, a high frequency of ML and systemic MGC were noted. These pathobiological features are likely related to primate species and virus strain characteristics (Fultz and Anderson, 1990; Simon et al., 1992), as well as differences in environmental conditions. The various disease manifestations observed in our cynomolgus monkey series appear to involve direct viral interactions, particularly with cells of the immune system including FDC and Mo/Mac, whose functional impairment and/or destruction most likely influence pathogenesis and disease progression as well as the development of lymphomas. This experimental SIV-cynomolgus infection therefore appears particularly useful as a model for studies of the role of FDC and Mo/Mac in the pathogenesis of malignancies and other p~tthologies associated with HIV/SIV infections.
Acknowledgements Three monkeys were obtained for necropsy from B. Orberg and Disa B6uiger (SBL). Reinhold Bentin was responsible for the
HISTOPATHOLOGY OF SIVsm-INFECTED CYNOMOLGUS MONKEYS
necropsies. The skillful technical assistance of Angelina de Santiago, R. Gantschnig and Marianne Ekman is highly appreciated. Supported by Swedish Medical ResearchCouncil and Swedish Cancer Foundation. E.E. Kaaya was partly supported by the Swedish Agencyfor ResearchCooperationwith DevelopingCountries (SAREC) and ICSC World Laboratory project MCD2. H. Flichtingerwas partly supported by the EEC concerted action "Pathophysiology and Immunology of HIV-related diseases. Ingrid Stahmer, VisitingScientist, was supported by a grant from the "DeutscheAIDS-Stipendiumprogramm'.E. Mandache, visiting scientist from Victor Babes Institute, Bucharest,was supported by the Swedish Medical Research Council.
Cellules accessoires et macrophages dans la pathologie de macaques infect~ par le SIV SMM-3 Trente-neuf macaques (Macacafascicularis) infectds par le virus d'immunoddficience simien (SIV SMM-3) ont dtd surveillds pendant deux anndes et demi. Trente-trois animaux ont dtd examinds apr~s autopsie selon les manifestations d'immunoddficience et de pathologie. Six animaux ont survdcu sans ddvelopper de maladie. La majorit6 des macaques ddploient, pendant l'infection, une lymphaddnopathie caractdristique e t / o u des manifestations histopathologiques dans la plupart des organes, surtout les poumons, le syst~me gastro-intestinal, les reins, le cerveau, le foie et le syst~me cardio-vasculaire. L'histopathologie des tissus involuds montre un r61e important joud par les cellules accessoires dendritiques folliculaires et les monocytes macrophages dans les diverses manifestations, en particulier dans la formation systdmique de cellules gdantes multinucldes. Quinze (45 %) des animaux ont aussi ddveloppd des tumeurs malignes lymphocytaires apparemment associ6es avec un dtat plus important d'immunoddficience et d'involution ganglionnaire. Ces tumeurs ressemblent dtroitement aux lymphomes du SIDA humain du point de vue clinique et biologique. La ressemblance entre des manifestations pathologiques ainsi que des lymphomes chez les macaques infectds par le SIV SMM3 et ceux du SIDA humain rdv~le l'importance de ce module animal pour l'dtude des divers m6canismes de la pathogen~se de l'infection par le VIH. Mots-cids: SIDA, SIV, Cellule dendritique folliculaire, Macrophage; Histopathologie, Mod61e animal, Microscopie dlectronique.
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