AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 8, Number 12, 1992 Mary Ann Liebert, Inc., Publishers
Simian
Immunodeficiency Virus (SIVsm) Infection of Cynomolgus Monkeys: Effects on Follicular Dendritic Cells Lymphoid Tissue PIET
JOLING,1 DISA
in
WICHEN,1 HENK K. PARMENTIER,1 PETER BIBERFELD, BÖTTIGER,3 JÜRG TSCHOPP,4 LOUK H.P.M. RADEMAKERS,1
DICK F. VAN
and HENK-JAN
SCHUURMAN1
ABSTRACT We studied follicles in sections of lymph nodes and spleen from cynomolgus monkeys (Macaca fascicularis) after infection with simian immunodeficiency virus (SIVsm), by (immuno)histology and (immunogold) electron microscopy. Also isolated follicular dendritic cells (FDC) were investigated. Histology showed ranged from follicular hyperplasia to follicle fragmentation. FDC showed desmin and vimentin, characteristic of mesenchymal cells. Except for two animals who got experimental chemotherapy in the first postinfection period, the cells expressed SIV gag p28 protein. Electron microscopy showed SIVsm-like particles in the germinal centers. A number of cell types in the germinal center, including FDC, showed tubuloreticular structures, indicative of a-interferon synthesis during an antiviral response. In immunogold electron microscopy, SIV p28 label was observed on the surface of FDC, on SIVsm-like particles, and in the cytoplasm of macrophages. A relatively high density of CD8-positive cells (T cytotoxic-suppressor phenotype) was observed around and in germinal centers, especially areas depleted of FDC. Cells immunoreactive for serine esterase granzyme-B, a protein occurring in granules of cytotoxic cells, occurred around germinal centers, but not in germinal centers at areas where FDC and SIV p28 label localized. This argues against a role of cytotoxic T cells in mediating follicle destruction.
INTRODUCTION
in solitary cells in the germinal center,6~9 and in diffuse signal of low density is observed in areas with FDC.y The analysis of FDC isolated in suspension6 has supported the concept that FDC bind HIV-1, and subsequently can become infected and actively produce the virus. This suggestion has been confirmed by in vitro infection of FDC isolated from uninfected tissue.10 In contrast, a role of the host response to the virus in germinal ' '~12 center destruction has been claimed. The HIV-1-fragmencan tated follicle manifest the accumulation by T cells of the cytotoxic suppressor (CD8) phenotype. 'A The cytotoxic potential of these cells has been suggested by the finding that some germinal center cells show mRNA encoding serine esterase mRNA
occurs
some cases a
I (HIV-1)
Human immunodeficiency virus type 1 is often associated with persistent generalized lymphadenopathy. The major lymph node abnormality is follicular hyperplasia, with histology showing germinal centers of bizarre shape, and mantle zones of irregular size. The framework of follicles, comprising follicular dendritic cells (FDC), is disrupted, fragmentated, and indented. At the subcellular level, FDC show abnormalities indicative of an altered physiologic stage.5 A role of HIV-1 in mediating FDC abnormalities can be suggested. HIV-1 virions and HIV-1 antigens are observed in the labyrinth of dendritic protrusions of FDC.1'68 Also, HIV-1 NFECTION with
'Departments of Pathology and Internal Medicine, University Hospital, Utrecht, The Netherlands. 2lmmunopathology Laboratory, Karolinska Institute, Stockholm, Sweden. 'Department of Virology, National Bacteriology Laboratory. Stockholm, Sweden,
institute of Biochemistry, University of Lausanne, Epalinges, Switzerland. 2021
JOLING ET AL.
2022
granzyme-B," an enzyme present in cytoplasmic granules of cytotoxic T lymphocytes and natural killer cells.I3"15 However, in similar cases examined by our laboratory, we have been unable to document the granzyme-B protein itself in the germinal center.16 There are few animal models for HIV-1 infection.'7 Out of these, simian immunodeficiency virus (SIV) infection in cynomolgus monkeys (Macaca fascicularis) has shown its value in studies on pathogenesis and experimental antiviral chemotherapy.'822 The histopathological changes in lymph nodes of SIV-infected monkeys resemble those in HIV-1 infection in humans.22~27 In the present study, we investigated these abnormalities in detail. Follicles were analyzed for SIV antigen and lymphocytes with cytotoxic granules. FDC were characterized for dendritic cell antigen, vimentin, and desmin, the latter two being contractile elements and markers for mesenchymal cells. In transmission electron microscopy tubuloreticular structures were present in various cells after infection with SIVsm.
MATERIALS AND METHODS Animals and tissues The study included five cynomolgus monkeys (Macaca fascicularis). Clinical and laboratory data are presented in Table 1. Infection was performed with SIVsm, isolate SMM3, which was
propagated by Dr.
P.
in human
peripheral
blood
lymphocytes (provided
Fultz, Yerkes Primate Research Center, Atlanta). The
animals were inoculated intravenously with 10—105 animal doses of SIVsm (SIV-SMM3).'9'2' Thereafter, some animals received experimental chemotherapy (indicated in Table 1). Animals were killed according to the experimental protocol, and were without overt pathology. This was except for animal L63-91, which was autopsied because of severe disease. This animal showed hyperplasia of gut-associated lymphoid tissue, chronic interstitial pneumonitis, and Kupffercell hyperplasia in liver. Other organs investigated showed no pathologic changes.
Table 1.. Clinical
and
Laboratory Data
Spleen
and
lymph
nodes
were
obtained
during
small part was processed for transmission electron microscopy. Finally, part of spleen and lymph nodes were subjected to isolation of FDC.
Isolation
of FDC
procedure described by us previously was applied.28 and lymph node was minced in small fragments (about 3 Spleen mm3); after removal of most leukocytes, a collagenase digestion (15 U/ml) was performed for 30 min at room temperature, followed by 1 g sedimentation on a discontinuous gradient of 1.5%, 2.5%, 5.0%, and 7.5% (wt/vol) bovine serum albumin (BSA), respectively. The interphase between 2.5% and 5% BSA was harvested, and layered onto a discontinuous Percoll (Pharmacia, Uppsala, Sweden) gradient of 1.070, 1.060, and 1.030 mg/ml. After centrifugation at 1200 g for 20 min, the FDCenriched population were harvested from the 1.030-1.060 interphase. Cytological inspection of cytocentrifuge preparations stained by May Grünwald-Giemsa showed mainly cells with the morphology of FDC, either solitary or clustered with lymphoid cells. Cells with a macrophage-like cytology were almost absent, and a few solitary lymphocytes were observed. The
Immunoh is toehem is try Mouse monoclonal antibodies applied were anti-DRC-1 (Dakopatts, Glostrup, Denmark) or anti-Ki-M4 (Behringwerke, Marburg-Lahn, Germany) directed to follicular dendritic cells,
anti-Leu-2a (CD8) (Becton Dickinson, Mountain View, CA) directed to T cells of cytotoxic-suppressor phenotype, and a rabbit antiserum to serine esterase granzyme-B present in granules of T cytotoxic cells and natural killer cells. To detect SIV antigen, the monoclonal antibody SF4 towards gag p28 protein was applied (Biotech Research Laboratories, Rockville, MD).
of the
Cynomolgus Monkeys Investigated SIV
Days
Code
L341-90
L342-90
Sex
postinfection
F
366
M
239
L343-90
M
239
L344-90
M
239
L63-91
F
82
Chemotherapy
Clinical
Blood
symptoms
parameters
Histology
CD4 0.65, CD4/8 1.35
Hyperplasia
CD4 1.0 CD4/8 0.29 CD4 1.3 CD4/8 1.5 CD4 0.3 CD4/8 0.2
Hyperplasia, fragmentation Hyperplasia, fragmentation Hyperplasia, fragmentation Hyperplasia, atrophy
LAS,
None
splenomegaly, weight loss
DDI 0-10 days AZT 9 wk DDI 0-10 days FLT 9 wk d-4T 0-10 days
LAS,
Linomide -4 to +4
Weight loss, wasting syndrome
days
Abbreviations: F, female; M, male; DDI,
thymidine; LAS, lymphadenopathy syndrome.
life and at
autopsy. Part of lymphoid tissue was fixed in formalin and processed for conventional histopathology. Another part was snap-frozen and stored at —70°C for immunohistochemistry. A
splenomegaly
None
None
Ag
in follicles
Isolated FDC
Tissue section
+
+
+
+
dideoxyinosine; AZT, azidothymidine; FLT, '3-fluorothymidine; d-4T, deoxy-4CD4 counts are presented in 109/liter.
FOLLICULAR DENDRITIC CELLS
IN
SIV,m-INFECTED
To demonstrate contractile elements,
a rabbit anti-desmin and monoclonal anti-vimentin antibody were used (Eurodiagnostics, Apeldoorn, The Netherlands). Immunohisto- and
mouse
cytochemistry
was
performed by
a
three-step immunoperoxi-
dase method. Frozen sections (6 p,m) or cytocentrifuge preparations of isolated cells were fixed for 10 min in acetone at room temperature. After the first incubation with monoclonal antibody at predetermined optimal dilution, the second step included rabbit anti-mouse immunoglobulins and the third step swine anti-rabbit immunoglobulins (both antibodies were conjugated to horseradish peroxidase, Dakopatts). In case of a primary rabbit antibody, only the second step with swine anti-rabbit immunoglobulins was performed. Peroxidase activity was visualized using 3-amino-9-ethylcarbazole and H202 as substrate, in 0.1 M acetate buffer, pH 4.8. All incubation media were supplemented with 10% (wt/vol) human serum albumin and 10% (vol/vol) heat-inactivated human AB serum. The preparations were slightly counterstained with hematoxylin. Nonspecific labeling was controlled by omission of the first antibody or by replacement of the first antibody by an irrelevant one. Under these conditions, no immunolabeling product was observed. Polymorphonuclear granulocytes (when present) gave a reaction product due to endogenous peroxidase activity. For the simultaneous detection of desmin on one hand, and vimentin or FDC antigen on the other hand, two-color immunofluorescence was applied. Acetone-fixed sections were incubated with mouse antibodies DRC-1, KÍ-M4, or anti-vimentin. The second incubation was done with fluorescein isothiocyanate-conjugated goat anti-mouse antibody (Nordic Immunological Laboratories, Tilburg, The Netherlands). Subsequently, the sections were incubated with rabbit anti-desmin antibody, and thereafter with goat anti-rabbit antibody conjugated with tetramethyl rhodamine isothiocyanate. Sections were embedded in PBS-glycerol (1/9 vol/vol, pH 8.0) and analyzed using a Laborlux 12 fluorescence microscope equipped with HBO 100 epi-illumination, 25/0.60 or 50/1.00 water immersion objectives and 12.5x oculars.
Electron
microscopy
Conventional transmission electron microscopy. Small pieces of spleen or lymph node were fixed by immersion in 2.5% (vol/vol) glutaraldehyde (GA) and 2% (wt/vol) paraformaldehyde (PFA) in 0.1 M Na-cacodylate buffer (pH 7.4) supplemented with 250 p,M CaCl2 and 500 p.M MgCl2 for at least 2 h. Isolated cells were fixed by resuspension in 2% GA in 0.1M cacodylate buffer (pH 7.4) for at least 2 h, then resuspended in 100% AB serum and pelletted by centrifugation. After removal of the supernatant the pellet was fixed in 2% GA in cacodylate buffer for at least 16h. After washing pellets or tissue specimens were postfixed in 1% Os04 in the same buffer supplemented with 500 p,M CaCl, and 1 mM MgCI2, and embedded in Epon according to routine procedures. One-p.m thick epon sections were stained with pararosanilin and méthylène blue.29 Ultrathin sections of areas of interest were collected on pioloform-coated grids, contrasted with 3% magnesiumacetate (45 min, 63°C), followed by Reynolds lead citrate (10 min, room temperature), and examined in a Philips 201C electron microscope. Immunogold electron microscopy.1" Small specimens of tissue or isolated cells were fixed in 4% PFA in 0.1 M phosphate
MONKEYS
2023
buffer, pH 7.4, for at least 2 h
at 4°C, and washed twice in this buffer supplemented with 0.15% glycine. Tissue blocks and cell pellets were embedded in 10% gelatin, impregnated overnight at 4°Cwith 1.8 M sucrose containing 15% poly(vinylpyrrolidone), and mounted on copper holders and frozen in liquid nitrogen. Ultrathin cryosections (80 nm) were cut at —I I0°C and then thawed and transferred to carbon Formvar-coated copper grids. In the immunogold labeling procedure,,0 the grids were incubated with the anti-SIV p28 antibody at optimal dilution, followed by a rabbit anti-mouse immunoglobulin antibody (Dakopatts), and then with protein A-gold complex. The cryosections were embedded and contrasted in medium consisting of 1.8% methylcellulose and 3% uranyl acetate, pH 7.0. The sections were examined in a Jeol 1200 EX electron microscope. Controls included the omisión of the anti-SIV p28 antibody or replacement by an irrelevant monoclonal antibody. Under these conditions there was no immunogoldsignal observed.
RESULTS
Histopathology and immunohistochemistry Conventional histopathology revealed changes in lymphoid follicles ranging from follicular hyperplasia (Fig. la) to follicular fragmentation, and general lymphoid atrophy of the white pulp in spleen (Fig. lb). This histological appearance was confirmed by immunohistology with anti-DRC-1 or anti-KiM4. Staining varied between a continuous fine reticular pattern in hyperplasia, a varying extent of indentations and fragmentations of the network in fragmentation, and labeling of only part of the original network with only remnants of FDC in atrophy. The immunolabeling for SIV p28 gag protein co-localized with the FDC network (Fig. 2a and b). On cytocentrifuge preparations, SIV p28 gave surface immunolabeling of FDC, located either solitary or in clusters with lymphoid cells (Fig. 3). This concordance in immunolabeling between anti-DRC I/antiKi-M4 and anti-SIV p28 was observed for animals L341-90, L344-90, and L63-91. For animals L342-90 and L343-90, cytological preparations of FDC were not immunolabeled by the anti-SIV p28 antibody, and tissue sections showed only a faint labeling of germinal centers. Both cytologie preparations and germinal centers in tissue sections showed intense labeling by anti-DRC-1 or anti-Ki-M4 antibodies. FDC were additionally characterized in two-color immunofluorescence using combinations of anti-desmin on the one hand, and anti-vimentin or anti-DRC-1/anti-Ki-M4 on the other. Anti-desmin immunolabeled stroma throughout the tissue section, in reticular staining patterns of interfollicular and follicular areas. In follicles, the desmin staining pattern was similar to that of anti-DRC-l/anti-Ki-M4 but less extensive (Fig. 4a and b). Anti-vimentin labeled scattered cells outside follicles that were desmin-negative. In follicles, the anti-vimentin staining pattern was almost identical to that of anti-desmin (Fig. 4c and d). These data indicate that FDC in germinal centers express both vimentin and desmin, and thus are mesenchymal cells. Serial sections were immunolabeled for anti-DRC-1, CD8, and antibody to serine esterase granzyme-B (Fig. 5). CD8 revealed immunoreactive cells at a high density in interfollicular areas and at a lower density in germinal centers. In germinal
2024
JOLING ET AL. :.-'
s
a
.
it-
>?
TjPBie1«
n«H
•'!"?* •
if FIG. 1.
Conventional histology of lymphoid tissue from cynomolgus monkeys after SIVsm infection, (a) Lymph node from animal L341-90 showing hyperplasia of the lymphoid follicle (F). (b) Spleen of animal L63-91 showing atrophy of the white pulp. Frozen tissue section, hematoxylin and eosin stain-
ing,
FIG. 2.
Serial sections of frozen splenic tissue from animal L341-90, labeled by anti-DRC-1 (a) and anti-SIV gag p28 (b). SIV p28 localizes in a similar pattern in follicle germinal centers
anti-DRC-1 immunolabeling product, Immunoperoxidase labeling, x70. as
but is less extensive.
X70.
Electron
FDC-containing areas were identified by anti-DRC-1 immunolabeling and SIV p28 labeling (intense staining was
centers,
noted for animals L341-90, L344-90, and L63-91). In these the density of CD8+ cells was lower than in areas that wereDRC-1 negative. Granzyme-B + cells were present solitary in interfollicular areas. Occasionally a positive cell was observed in indentations or fragmentations of germinal centers, where there was no DRC-1 labeling. Areas immunolabeled by anti-DRC-1 or anti-SIV p28 did not show cells immunoreactive for granzyme-B. These data indicate the presence of CD8+ cells in high proportions in spleen and lymph nodes, in particular in interfollicular areas. Here, part of the cells are granzyme-B + The lowest density of CD8+ cells is observed in FDC-containing areas in germinal centers, and here cells are not labeled by areas,
.
antibody to granzyme-B.
microscopy
and lymph nodes, follicles were present for each The observations are presented separate for spleen (Figs. 6, 7) and lymph node. The follicles in spleen generally did not show the architecture of well-developed germinal centers surrounded by a lymphocyte corona. The main lymphoid population in follicles comprised small lymphocytes. A few atypical blast cells were observed that showed irregular shaped nuclei or even binucleated nuclei. Their presence indicated germinal center cell differentiation. The stroma consisted of FDC, which were classified for differentiation aspects.5'31 In the center of the follicle, there were a few differentiated FDC with electron-dense In
spleen
case.
deposits, presumably immune-complex depositions (Fig. 6a). Within these depositions virions resembling SIVsm particles were present (Fig. 6b), in particular, for animals L341-90, L344-90, and L63-91. In case L63-91, a large group of SIV™
FOLLICULAR DENDRITIC CELLS
in
SIV
-INFECTED MONKEYS
2025
Immunogold electron microscopy SIV p28 immunogold electron microscopy was performed on sections of spleen and purified FDC preparation of spleen. A labeling product was observed on electron-dense deposits on the surface of FDC. In the periphery of the follicle, p28 immunogold label was present in macrophages having a well-developed rough endoplasmic reticulum and showing lysosomes and tubuloreticular structures (Fig. 8). In FDC-enriched cell suspensions, immunogold labeling was present on virus-like structures between the cells (Fig. 9).
DISCUSSION
FIG. 3. Cytocentrifuge preparation of follicular dendritic cells isolated from spleen of animal L341-90, labeled by antiSIV gag p28. An FDC in a cluster with lymphocytes is shown, with labeling product on the FDC. Immunoperoxidase labeling, X492.
virions was observed around atypical blast cells (Fig. 7a and b). In this case, the majority of the FDC had a less differentiated ultrastructure with only few villous extensions, absence of electron-dense surface depositions, and concentrations of intermediate filaments in the cytoplasm. Paracrystalline arrays resembling so-called tubuloreticular structures32 were regularly observed in the cytoplasm of FDC (Fig. 6a and c), both for FDCs with the ultrastructural features of differentiated cells and undifferentiated cells. It was also observed in some atypical cells with a lymphoblastoid morphology, and in some small lymphocytes. Electron microscopy of FDC-enriched cell populations prepared from spleen showed a similar ultrastructural morphology as FDC in tissue sections. Along the plasma membrane, SIVsm virions were regularly observed (animals L341 -90, L344-
90, andL63-91).
In lymph nodes (data not illustrated), irregularly indented follicles and follicles with large mantle zones were observed. Germinal center characteristics were evident, with blast cells, centrocytes, FDC, and starry-sky macrophages.31 There were solitary viral particles, exclusively in the protrusions of FDC. In the mantle zone, FDC showed characteristics of undifferentiated cells and contained large aggregates of intermediate filaments. Tubuloreticular structures were seen in a variety of cell types. including FDC in the mantle zone and germinal center, blast cells and centrocytes in the germinal center, and small lymphocytes in mantle zones. In interfollicular areas, these structures were present in plasma cells, small lymphocytes, blasts, fibroblastic reticulum cells, and endothelial cells of capillaries and high endothelial venules. Tubuloreticular structures were not observed in germinal center macrophages.
SIVsm infection in cynomolgus monkeys resembles HIV-1 infection in humans in a number of aspects.I7-18-20-22-27 The histological alterations, including follicular hyperplasia, fragmentation and indentation, and finally general lymphoid atrophy '~3 (Fig. 1), are similar to those described after HIV-1 infection, and confirm earlier data on SIV-infection in monkeys by e.g. the group from Harvard Medical School.22-25 In the follicles, SIVsm virions and SIV gag p28 protein localize in the labyrinth of FDC (Figs. 2 and 6b),25 which was confirmed by the analysis of isolated FDC (Figs. 3 and 9). The isolation procedure28 yielded suspensions that were highly enriched in FDC, manifesting SIVsm particles (electron microscopy) and SIV gag p28 (immunocytochemistry, all but two animals, Table 1). In lymph nodes from HIV-1-infected patients, the architecture and cellular subset composition of interfollicular areas are largely*" similar to those in the normal reactive lymph node. CD4 T cells occur in somewhat lowered density, and CD8+ T cells are found scattered in the interfollicular area.1'3'5 In contrast, the high density of CD8+ cells in interfollicular areas in lymphoid tissue from SIVsm-infected monkeys (Fig. 5b) exceeded that in HIV-1-infected lymph nodes. It rather resembled that observed in some viral infections in humans (H.-J. Schuurman, unpublished observations). CD8+ cells also occurred in the indentations and fragmentations of follicles, and in the germinal center composed of FDC (DRC-1 or Ki-M4 staining, Fig. 5b).+ This might have a pathogenetic significance, inasmuch as CD8 cells include cytotoxic cells which have been implicated in follicular center destruction."12 However, in germinal centers there were no cells immunoreactive for serine esterase granzyme-B, a component of granules in cytotoxic cells. Such cells actually were observed in the interfollicular area. In this respect, the labeling of serine esterase granzyme-B in the SIVMI, situation resembles that in HIV-1-infected lymph nodes. These observations contrast with a report" that cells expressing mRNA of serine esterase occur in lymphoid follicles of HIV-1-infected patients. We elsewhere have ascribed this discrepancy to the sensitivity and specificity of the methods applied, and a difference in study material.33 There are no studies in the literature resolving this discrepancy by the simultaneous detection of mRNA and protein in the same tissue or tissue section. Awaiting such studies, it may be suggested that mRNA'+ cells in germinal centers produce serine esterase at levels that are too low to be detectable by immunohistochemistry, or that the protein after secretion diffuses in the tissue
JOLING ET AL.
2026
Frozen section of spleen from animal L63-91, subjected to two-color immunofluorescence. (a) Anti-DRC-1 (fluorescein isothiocyanate label) and (b) anti-desmin (tetramethyl rhodamine isothiocyanate label), (c) Anti-vimentin (fluorescein isothiocyanate label) and (d) anti-desmin (tetramethyl rhodamine isothiocyanate label). Most follicular dendritic cells identified by anti-DRC-1 (a) are labeled by anti-desmin (b). Vimentin immunoreactivity (c) coincides with desmin immunoreactivity (d). Immunofluorescence labeling, X244.
FIG. 4.
resulting in a too low loc'al concentration to be detectable by immunohistochemistry. It remains to be established whether the protein at such low local concentration in tissue has sufficient biological activity to exert its role in cytotoxicity. Our findings, both in HIV-1 infection and in SIVsm infection, do not support the hypothesis that cytotoxic cells play a main role in follicle center
destruction."'2
FDC in SIVsm-infected tissue resembled FDC in HIV-1 infection in electron microscopy5 and phenotypic markers.5 The cells showed positivity for vimentin and desmin (two-color immunofluorescence, Fig. 4), indicative of a mesenchymal origin. In humans FDC in the follicle mantle zone expressed both markers.34 An interesting observation was the presence of tubuloreticular structures in a number of cell types, including FDCs (Fig. 6a and c). These structures have been observed after retroviral infection,35"37 mainly in lymphoid cells and endothelial cells, but not in FDC. Their presence has been associated with the synthesis of a-interferon by the cell itself, or with the reaction of the cell to the presence of a-interferon in the local
reaction to viral infection associated with a-interferon In the present study the structures were not only observed in cells of hematopoietic lineage, but also in FDC that are of mesenchymal origin, even those with a less differentiated morphology present in the mantle zone. We conclude that FDC already early in their differentiation from mesenchymal cells contribute to the response to viral infection. The cells can be infected by the virus,6'8"10 and it remains to be established whether the cells are able to produce interferon. The SIVsm infection model in monkeys enables to evaluate treatment. Two of the animals studied (L342-90 and L343-90) received dideoxyinosine, in combination with azidothymidine or '3-fluorothymidine during the first 9 wk after infection (Table 1). At autopsy, 25 wk later, these animals showed the highest values of CD4+ cells and CD4/8 ratio in blood, suggestive of the beneficial effect of prior treatment. Interestingly, these animals had much less SIVsnl identified in follicular areas as determined by SIV immunocytochemistry on tissue sections and isolated cates a
synthesis.
FOLLICULAR DENDRITIC CELLS
J>5-
**
IN
SIVsm-INFECTED MONKEYS
2027
a
t
a
PC. s#
»
r*
•F r «
'*# i
'
? f
'• '
e -
#**
4
PC*'"
FIG. 6.
Electron microscopy of FDC in the center of a follicle spleen from animal L344-90. (a) Electron micrograph showing extensions (arrow) covered with electron-dense depositions, reflecting immune complexes. In the cytoplasm aggregates of tubuloreticular structures are seen (arrowheads), x 13,500. (b) At higher magnification, a SIVsm virion (arrow) in the labyrinth in
FIG. 5.
Serial sections of frozen splenic tissue from animal L341-90, labeled by anti-DRC-1 (a), CD8 (cytotoxic-suppressor T-cell phenotype) (b), and antibody to serine esterase Granzyme-B (c). CD8+ cells are present in relatively high density in the paracortex (PC) and in follicles (F) at indented areas where follicular dendritic cells (DRC-1 labeling) are absent (asterisk). In the areas labeled by anti-DRC-1 there are only a few CD81+ cells. There are a few granzyme-B+ cells around the follicle (indicated by an arrow), but not in areas containing FDC in DRC-1 labeling. Most dark spots around follicles in c represent endogenous peroxidase activity, and not specific immunolabeling by the antibody to granzyme-B. Immunoperoxidase labeling, X130.
of villous extensions of FDC, within electron-dense (immune complex) deposits, is shown (arrow). X77.000. (c) Part of a primitive FDC located at the periphery of the follicle, showing a large tubuloreticular structure with a paracrystalline substructure,
x15,964.
JOLING ET AL.
2028
*-
n
m.
FIG. 8. SIV gag p28 immunogold electron microscopy of a follicule in spleen from animal L63-91. The cytoplasm of a macrophage is shown, with p28 colloidal gold label presumably associated with membranes of rough endoplasmic reticulum and
Golgi complex,
x
14,580.
FIG. 7. Electron microscopy of a follicle in spleen from animal L63-91. (a) A binucleated atypical blast cell is surrounded by groups of virions (arrow). In adjacent sections this cell contained tubuloreticular structures. There are ringlike structures and other vesicle malformations in the cytoplasm resembling swollen mitochondria (arrowhead). Asterisk: FDC labyrinth. X8,600. (b) At higher magnification, the SIVsm virions (arrows) accumulated near the plasma membrane of the atypical blast cell are shown, x31,720.
FDC. Because of the number of animals studied, the signifiof these observations is unknown but warrants further
cance
study.
In conclusion, this study on lymphoid tissue of SIVsminfected cynomolgus monkeys shows the similarities between the animal infection and HIV-1 infection in humans. It underlines our previous suggestion that the immune response to the virus, i.e., cytotoxic cells, may not be involved in the destruction of follicles. Rather the virus and viral proteins themselves may play a role in this process. The reduced presence of viral protein in follicles in conditions of prior chemotherapy indicates a potential value of the experimental monkey model, because it is not readily possible to perform a similar study in patients after HIV-1 infection.
FIG. 9. SIV gag p28 immunogold electronmicroscopy of a FDC isolated from spleen of animal L341-90. Part of the villous extensions of the cell is shown, with immunogold labeling of a
SIVsnl-like particle (arrow). x61,000. (Inset) Higher magnifiSIVsm-like particle. x58,560.
cation of the
Biberfeld was supported by the Swedish Medical Research Council. The authors thank Jolanda Krijnen and Timo Meerloo for expert technical assistance.
REFERENCES ACKNOWLEDGMENTS The work in the Dutch group was supported by the Dutch Ministry of Health, as part of the National Program on AIDS Research, RGO/WVC grant 88-79/89005. The group of Dr.
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FOLLICULAR DENDRITIC CELLS
in
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2. Ost A, Baroni CD, Biberfeld P, Diebold J, Moragas A, Noël H, Pallesen G, Rácz P, Schipper M, Tenner-Rácz K, and Van den Tweel JG: Lymphadenopathy in HIV infection: histological classification and staging. Acta Pathol. Microbiol. Scand. 1989;Suppl 8:7-15. 3. Schuurman H-J, Kluin PM, Gmelig-Meyling FHJ, van Unnik JAM, and Kater L: Lymphocyte status of lymph node and blood in acquired immunodeficiency syndrome (AIDS) and AIDS-related complex disease. J Pathol 1985;147:269-280. 4. Biberfeld P, Porwit-Ksiazek A, Böttiger B, Morfeldt-Mánson L, and Biberfeld G: lmmunohistopathology of lymph nodes in HTLVIII infected homosexuals with persistent adenopathy or AIDS. Cancer Res 1985;45 Suppl:4665s-4670s. 5. Rademakers LHPM, Schuurman H-J, De Frankrijker JF. and Van Ooyen A: Cellular composition of germinal centers in lymph nodes after HIV-1 infection: evidence for an inadequate support of germinal center B lymphocytes by follicular dendritic cells. Clin Immunol Immunopathol 1992;62:148-159. 6. Parmentier HK, van Wichen DF, Sie-Go DMDS, Goudsmit J, Borleffs JJC, and Schuurman H-J: HIV-1 infection and virus production in follicular dendritic cells in lymph nodes. A case report, with analysis of isolated follicular dendritic cells. Am J Pathol 1990;137:247-251. 7. Schuurman H-J, Krone WJA, Broekhuizen R, and Goudsmit J: Expression of RNA and antigens of human immunodeficiency virus type-1 (HIV-1) in lymph nodes from HIV-1 infected individuals. Am J Pathol 1988;133:516-524. 8. Biberfeld P, Chayt KJ, Marselle LM, Biberfeld G, Gallo RC, and Harper ME: HTLV-III expression in infected lymph nodes and relevance to pathogenesis of lymphadenopathy. Am J Pathol
1986;125:436-442. Herbst H, Niedobitek G, Foss H-D, and Stein H: Follicular dendritic cells are a major reservoir for human immunodeficiency virus type 1 in lymphoid tissues facilitating infection of CD4+ T-helper cells. Am J Pathol 1992;140:15-22. 10. Stahmer I, Zimmer JP, Ernst M, FennerT, Finnern R, Schmitz H, Fiad H-D, and Gerdes J: Isolation of normal human follicular dendritic cells and CD4-independent in vitro infection by human immunodeficiency virus (HIV-1). Eur J Immunol 1991 ;21:1873— 1878. 11. Devergne O, Peuchmaur M, Crevon M-C, Trapani JA, Maillot M-C, Galanaud P, and Emilie D: Activation of cytotoxic cells in hyperplastic lymph nodes from HIV-infected patients. AIDS 9.
Spiegel H,
1991;5:1071-1079. 12. Laman JD, Claassen E, Van Rooijen N, and Boersma WJA: Immune complexes on foHicular dendritic cells as a target for cytolytic cells in AIDS. AIDS 1989;3:543-548. 13. Masson D, and Tschopp J: A family of serine esterases in lytic granules of cytolytic T lymphocytes. Cell 1987;49:679-685. 14. Krähenbühl O, Rey C, Jenne D, Lanzavecchia A, Groscurth P, Carrel S, and Tschopp J: Characterization of granzymes A and B isolated from granules of cloned human cytotoxic T lymphocytes. J Immunol 1988;141:3471-3477. 15. Peters PJ, Borst J, Oorschot V, Fukuda M, Krähenbühl O, Tschopp J, Slot JW, and Geuze HJ: Cytotoxic T lymphocyte granules are secretory lysosomes, containing both perform and granzymes. J Exp Med 1991 ; 173:1099-1109. 16. Parmentier HK, Van Wichen DF, Peters PJ, Tschopp J, De Weger RA, and Schuurman H-J: No histological evidence for cytotoxic T cells in destruction of lymph-node follicle centers after HIV infection. AIDS 1991;5:778-780. 17. Letvin NL. Animal models for AIDS. Immunol Today 1990; 11:322-326. 18. Feichtinger H, Putkonen P, Paravicini C, Li SL, Kaaya EE, Böttiger D, Biberfeld G, and Biberfeld P: Malignant lymphomas in
2029
infected with simian immunodeficiency viAm J Path 1990;137:1311-1315. 19. Putkonen P, Warstedt K, Thorstensson R, Benthin R, Albert J, Lundgren B, Öberg B, Norrby E, and Biberfeld G: Experimental infection of cynomolgus monkeys (Macaca fascicularis) with simian immunodeficiency virus (SIV5m). AIDS 1989;2:359-365. 20. Desrosiers RC, and Ringler DJ: Use of simian immunodeficiency viruses for AIDS research. Intervirology 1989;30:301-312. 21. Putkonen P. Thorstensson R, Albert J, Hild K, Norrby E, Biberfeld P, and Biberfeld G: Infection of cynomolgus monkeys with HIV-2 protects against pathogenic consequences of a subsequent simian immunodeficiency virus infection. AIDS 1990; 4:783-789. 22. Simon MA, Chalifoux LV, and Ringler DJ: Pathologic features of SIV-induced disease and the association of macrophage infection with disease evolution. AIDS Res Human Retroviruses 1992; 8:327-337. 23. Chalifoux LV, Ringler DJ, King NW, Sehgal PK, Desrosiers RC, Daniel MD, and Letvin NL: Lymphadenopathy in macaques experimentally infected with the simian immunodeficiency virus (SIV). Am J Pathol 1987;128:104-110. 24. Chalifoux LV, King NW, Daniel MD, Kannagi M, Desrosiers RC, Sehgal PK, Waldron LM, Hunt RD, and Letvin NL: Lymphoproliferative syndrome in an immunodeficient rhesus monkey naturally infected with an HLTV-III-like virus (STLV-III). Lab Invest
cynomolgus monkeys rus.
1986;55:43-50. 25.
26.
Ringler DJ, Wyand MS, Walsh DG, MacKey JJ, Chalifoux LV, Popovic M, Minassian AA, Sehgal PK, Daniel MD, Desrosiers RC, and King NW: Cellular localization of simian immunodeficiency virus in lymphoid tissues. I. Immunohistochemistry and electron microscopy. Am J Pathol 1989;134:373-383. Li SL, Kaaya EE, Feichtinger H, Putkonen P, Parravicini C, Böttiger D, Biberfeld G, and Biberfeld P.: Monocyte/macrophage giant cell disease in SIV-infected cynomolgus monkeys. Res Virol 1991;142:173-182.
27. Letvin NL, and King, NW: Immunologie and pathologic manifestations of the infection of Rhesus monkeys with simian immunodeficiency virus of macaques. J AIDS 1990;3:1023-1040. 28. Parmentier HK, Van der Linden JA, Krijnen J, Van Wichen DF, Rademakers LHPM, Bloem AC, and Schuurman H-J: Human follicular dendritic cells: isolation and characteristics in situ and in suspension. Scand J Immunol 1991;33:441-452. 29. SatoT, and Shamoto M: A simple rapid polychrome stain forepoxy embedded tissue. Stain Technol 1973;48:223-227. 30. Meerloo T, Parmentier HK, Osterhaus ADME, Goudsmit J, and Schuurman H-J: Modulation of cell surface molecules during HIV-1 infection of H9 cells. An immunoelectron microscopic study. AIDS 1992; 6(10):1105-1116. 31. Rademakers LHPM: Dark and light zones of germinal centres of the human tonsil: an ultrastructural study with emphasis on heterogeneity of follicular dendritic cells. Cell Tissue Res 1992; 269:359368. 32. Luu J, Bockus D, Remington F, Bean MA, and Kammar SP: Tubuloreticular structures and cylindrical confronting cisternae: a review. Hum Pathol 1989;20:617-627. 33. Parmentier HK, and Schuurman H-J: Cytotoxic CD8+ T cells in lymph node follicle centres after HIV-1 infection? A reply. AIDS
1992;6:334-335. 34. Rademakers LHPM, Van Wichen DF, and De Weger RA: Immunohistochemical localization of intermediate filament and contractile proteins in the tonsil with reference to follicular dendritic cells. In: Lymphatic Tissues and In Vivo Immune Responses. Ezine S, Berrih-Aknin S, and Imhof B (eds). Marcel Dekker, New York, 1991, pp. 721-725. 35. Onerheim RM, Wang N-S, Gilmore N, and Jothy S: Ultrastructural markers of lymph nodes in patients with acquired immunodefi-
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36.
ciency syndrome and in homosexual males with unexplained persistent lymphadenopathy. Am J Clin Pathol 1984;82:280-288. Kostianovsky M, Kang YH, and Grimley PM: Disseminated tubuloreticular inclusions in acquired immunodeficiency syndrome
(AIDS). Ultrastruct Pathol 1983;4:331-336. 37. Schiaffino E, Bestetti-Bosisio M, Toia G, Onida L, Riboli P, and Schmid C: Ultrastructural alterations and virus-like particles in lymph nodes of drug addicts with lymphadenopathy syndrome (LAS). Pathol Res Pract 1986;181:755-760. 38. Grimley PM, Davis GL, Kang Y-H, Dooley JS, Strohmaier J, and Hoofnagle JH. Tubuloreticular inclusions in peripheral blood
JOLING ET AL. mononuclear cells related to Lab Invest 1985;52:638-649.
systemic therapy
with a-interferon.
Address
reprint requests to: P. Joling, Ph.D. Department of Pathology University Hospital,
P.O. Box 85.500 3508 GA Utrecht, The Netherlands.
Simian
Immunodeficiency Virus (SIVsm) Infection of Cynomolgus Monkeys: Effects on Follicular Dendritic Cells Lymphoid Tissue PIET
JOLING,1 DISA
in
WICHEN,1 HENK K. PARMENTIER,1 PETER BIBERFELD, BÖTTIGER,3 JÜRG TSCHOPP,4 LOUK H.P.M. RADEMAKERS,1
DICK F. VAN
and HENK-JAN
SCHUURMAN1
ABSTRACT We studied follicles in sections of lymph nodes and spleen from cynomolgus monkeys (Macaca fascicularis) after infection with simian immunodeficiency virus (SIVsm), by (immuno)histology and (immunogold) electron microscopy. Also isolated follicular dendritic cells (FDC) were investigated. Histology showed ranged from follicular hyperplasia to follicle fragmentation. FDC showed desmin and vimentin, characteristic of mesenchymal cells. Except for two animals who got experimental chemotherapy in the first postinfection period, the cells expressed SIV gag p28 protein. Electron microscopy showed SIVsm-like particles in the germinal centers. A number of cell types in the germinal center, including FDC, showed tubuloreticular structures, indicative of a-interferon synthesis during an antiviral response. In immunogold electron microscopy, SIV p28 label was observed on the surface of FDC, on SIVsm-like particles, and in the cytoplasm of macrophages. A relatively high density of CD8-positive cells (T cytotoxic-suppressor phenotype) was observed around and in germinal centers, especially areas depleted of FDC. Cells immunoreactive for serine esterase granzyme-B, a protein occurring in granules of cytotoxic cells, occurred around germinal centers, but not in germinal centers at areas where FDC and SIV p28 label localized. This argues against a role of cytotoxic T cells in mediating follicle destruction.
INTRODUCTION
in solitary cells in the germinal center,6~9 and in diffuse signal of low density is observed in areas with FDC.y The analysis of FDC isolated in suspension6 has supported the concept that FDC bind HIV-1, and subsequently can become infected and actively produce the virus. This suggestion has been confirmed by in vitro infection of FDC isolated from uninfected tissue.10 In contrast, a role of the host response to the virus in germinal ' '~12 center destruction has been claimed. The HIV-1-fragmencan tated follicle manifest the accumulation by T cells of the cytotoxic suppressor (CD8) phenotype. 'A The cytotoxic potential of these cells has been suggested by the finding that some germinal center cells show mRNA encoding serine esterase mRNA
occurs
some cases a
I (HIV-1)
Human immunodeficiency virus type 1 is often associated with persistent generalized lymphadenopathy. The major lymph node abnormality is follicular hyperplasia, with histology showing germinal centers of bizarre shape, and mantle zones of irregular size. The framework of follicles, comprising follicular dendritic cells (FDC), is disrupted, fragmentated, and indented. At the subcellular level, FDC show abnormalities indicative of an altered physiologic stage.5 A role of HIV-1 in mediating FDC abnormalities can be suggested. HIV-1 virions and HIV-1 antigens are observed in the labyrinth of dendritic protrusions of FDC.1'68 Also, HIV-1 NFECTION with
'Departments of Pathology and Internal Medicine, University Hospital, Utrecht, The Netherlands. 2lmmunopathology Laboratory, Karolinska Institute, Stockholm, Sweden. 'Department of Virology, National Bacteriology Laboratory. Stockholm, Sweden,
institute of Biochemistry, University of Lausanne, Epalinges, Switzerland. 2021
JOLING ET AL.
2022
granzyme-B," an enzyme present in cytoplasmic granules of cytotoxic T lymphocytes and natural killer cells.I3"15 However, in similar cases examined by our laboratory, we have been unable to document the granzyme-B protein itself in the germinal center.16 There are few animal models for HIV-1 infection.'7 Out of these, simian immunodeficiency virus (SIV) infection in cynomolgus monkeys (Macaca fascicularis) has shown its value in studies on pathogenesis and experimental antiviral chemotherapy.'822 The histopathological changes in lymph nodes of SIV-infected monkeys resemble those in HIV-1 infection in humans.22~27 In the present study, we investigated these abnormalities in detail. Follicles were analyzed for SIV antigen and lymphocytes with cytotoxic granules. FDC were characterized for dendritic cell antigen, vimentin, and desmin, the latter two being contractile elements and markers for mesenchymal cells. In transmission electron microscopy tubuloreticular structures were present in various cells after infection with SIVsm.
MATERIALS AND METHODS Animals and tissues The study included five cynomolgus monkeys (Macaca fascicularis). Clinical and laboratory data are presented in Table 1. Infection was performed with SIVsm, isolate SMM3, which was
propagated by Dr.
P.
in human
peripheral
blood
lymphocytes (provided
Fultz, Yerkes Primate Research Center, Atlanta). The
animals were inoculated intravenously with 10—105 animal doses of SIVsm (SIV-SMM3).'9'2' Thereafter, some animals received experimental chemotherapy (indicated in Table 1). Animals were killed according to the experimental protocol, and were without overt pathology. This was except for animal L63-91, which was autopsied because of severe disease. This animal showed hyperplasia of gut-associated lymphoid tissue, chronic interstitial pneumonitis, and Kupffercell hyperplasia in liver. Other organs investigated showed no pathologic changes.
Table 1.. Clinical
and
Laboratory Data
Spleen
and
lymph
nodes
were
obtained
during
small part was processed for transmission electron microscopy. Finally, part of spleen and lymph nodes were subjected to isolation of FDC.
Isolation
of FDC
procedure described by us previously was applied.28 and lymph node was minced in small fragments (about 3 Spleen mm3); after removal of most leukocytes, a collagenase digestion (15 U/ml) was performed for 30 min at room temperature, followed by 1 g sedimentation on a discontinuous gradient of 1.5%, 2.5%, 5.0%, and 7.5% (wt/vol) bovine serum albumin (BSA), respectively. The interphase between 2.5% and 5% BSA was harvested, and layered onto a discontinuous Percoll (Pharmacia, Uppsala, Sweden) gradient of 1.070, 1.060, and 1.030 mg/ml. After centrifugation at 1200 g for 20 min, the FDCenriched population were harvested from the 1.030-1.060 interphase. Cytological inspection of cytocentrifuge preparations stained by May Grünwald-Giemsa showed mainly cells with the morphology of FDC, either solitary or clustered with lymphoid cells. Cells with a macrophage-like cytology were almost absent, and a few solitary lymphocytes were observed. The
Immunoh is toehem is try Mouse monoclonal antibodies applied were anti-DRC-1 (Dakopatts, Glostrup, Denmark) or anti-Ki-M4 (Behringwerke, Marburg-Lahn, Germany) directed to follicular dendritic cells,
anti-Leu-2a (CD8) (Becton Dickinson, Mountain View, CA) directed to T cells of cytotoxic-suppressor phenotype, and a rabbit antiserum to serine esterase granzyme-B present in granules of T cytotoxic cells and natural killer cells. To detect SIV antigen, the monoclonal antibody SF4 towards gag p28 protein was applied (Biotech Research Laboratories, Rockville, MD).
of the
Cynomolgus Monkeys Investigated SIV
Days
Code
L341-90
L342-90
Sex
postinfection
F
366
M
239
L343-90
M
239
L344-90
M
239
L63-91
F
82
Chemotherapy
Clinical
Blood
symptoms
parameters
Histology
CD4 0.65, CD4/8 1.35
Hyperplasia
CD4 1.0 CD4/8 0.29 CD4 1.3 CD4/8 1.5 CD4 0.3 CD4/8 0.2
Hyperplasia, fragmentation Hyperplasia, fragmentation Hyperplasia, fragmentation Hyperplasia, atrophy
LAS,
None
splenomegaly, weight loss
DDI 0-10 days AZT 9 wk DDI 0-10 days FLT 9 wk d-4T 0-10 days
LAS,
Linomide -4 to +4
Weight loss, wasting syndrome
days
Abbreviations: F, female; M, male; DDI,
thymidine; LAS, lymphadenopathy syndrome.
life and at
autopsy. Part of lymphoid tissue was fixed in formalin and processed for conventional histopathology. Another part was snap-frozen and stored at —70°C for immunohistochemistry. A
splenomegaly
None
None
Ag
in follicles
Isolated FDC
Tissue section
+
+
+
+
dideoxyinosine; AZT, azidothymidine; FLT, '3-fluorothymidine; d-4T, deoxy-4CD4 counts are presented in 109/liter.
FOLLICULAR DENDRITIC CELLS
IN
SIV,m-INFECTED
To demonstrate contractile elements,
a rabbit anti-desmin and monoclonal anti-vimentin antibody were used (Eurodiagnostics, Apeldoorn, The Netherlands). Immunohisto- and
mouse
cytochemistry
was
performed by
a
three-step immunoperoxi-
dase method. Frozen sections (6 p,m) or cytocentrifuge preparations of isolated cells were fixed for 10 min in acetone at room temperature. After the first incubation with monoclonal antibody at predetermined optimal dilution, the second step included rabbit anti-mouse immunoglobulins and the third step swine anti-rabbit immunoglobulins (both antibodies were conjugated to horseradish peroxidase, Dakopatts). In case of a primary rabbit antibody, only the second step with swine anti-rabbit immunoglobulins was performed. Peroxidase activity was visualized using 3-amino-9-ethylcarbazole and H202 as substrate, in 0.1 M acetate buffer, pH 4.8. All incubation media were supplemented with 10% (wt/vol) human serum albumin and 10% (vol/vol) heat-inactivated human AB serum. The preparations were slightly counterstained with hematoxylin. Nonspecific labeling was controlled by omission of the first antibody or by replacement of the first antibody by an irrelevant one. Under these conditions, no immunolabeling product was observed. Polymorphonuclear granulocytes (when present) gave a reaction product due to endogenous peroxidase activity. For the simultaneous detection of desmin on one hand, and vimentin or FDC antigen on the other hand, two-color immunofluorescence was applied. Acetone-fixed sections were incubated with mouse antibodies DRC-1, KÍ-M4, or anti-vimentin. The second incubation was done with fluorescein isothiocyanate-conjugated goat anti-mouse antibody (Nordic Immunological Laboratories, Tilburg, The Netherlands). Subsequently, the sections were incubated with rabbit anti-desmin antibody, and thereafter with goat anti-rabbit antibody conjugated with tetramethyl rhodamine isothiocyanate. Sections were embedded in PBS-glycerol (1/9 vol/vol, pH 8.0) and analyzed using a Laborlux 12 fluorescence microscope equipped with HBO 100 epi-illumination, 25/0.60 or 50/1.00 water immersion objectives and 12.5x oculars.
Electron
microscopy
Conventional transmission electron microscopy. Small pieces of spleen or lymph node were fixed by immersion in 2.5% (vol/vol) glutaraldehyde (GA) and 2% (wt/vol) paraformaldehyde (PFA) in 0.1 M Na-cacodylate buffer (pH 7.4) supplemented with 250 p,M CaCl2 and 500 p.M MgCl2 for at least 2 h. Isolated cells were fixed by resuspension in 2% GA in 0.1M cacodylate buffer (pH 7.4) for at least 2 h, then resuspended in 100% AB serum and pelletted by centrifugation. After removal of the supernatant the pellet was fixed in 2% GA in cacodylate buffer for at least 16h. After washing pellets or tissue specimens were postfixed in 1% Os04 in the same buffer supplemented with 500 p,M CaCl, and 1 mM MgCI2, and embedded in Epon according to routine procedures. One-p.m thick epon sections were stained with pararosanilin and méthylène blue.29 Ultrathin sections of areas of interest were collected on pioloform-coated grids, contrasted with 3% magnesiumacetate (45 min, 63°C), followed by Reynolds lead citrate (10 min, room temperature), and examined in a Philips 201C electron microscope. Immunogold electron microscopy.1" Small specimens of tissue or isolated cells were fixed in 4% PFA in 0.1 M phosphate
MONKEYS
2023
buffer, pH 7.4, for at least 2 h
at 4°C, and washed twice in this buffer supplemented with 0.15% glycine. Tissue blocks and cell pellets were embedded in 10% gelatin, impregnated overnight at 4°Cwith 1.8 M sucrose containing 15% poly(vinylpyrrolidone), and mounted on copper holders and frozen in liquid nitrogen. Ultrathin cryosections (80 nm) were cut at —I I0°C and then thawed and transferred to carbon Formvar-coated copper grids. In the immunogold labeling procedure,,0 the grids were incubated with the anti-SIV p28 antibody at optimal dilution, followed by a rabbit anti-mouse immunoglobulin antibody (Dakopatts), and then with protein A-gold complex. The cryosections were embedded and contrasted in medium consisting of 1.8% methylcellulose and 3% uranyl acetate, pH 7.0. The sections were examined in a Jeol 1200 EX electron microscope. Controls included the omisión of the anti-SIV p28 antibody or replacement by an irrelevant monoclonal antibody. Under these conditions there was no immunogoldsignal observed.
RESULTS
Histopathology and immunohistochemistry Conventional histopathology revealed changes in lymphoid follicles ranging from follicular hyperplasia (Fig. la) to follicular fragmentation, and general lymphoid atrophy of the white pulp in spleen (Fig. lb). This histological appearance was confirmed by immunohistology with anti-DRC-1 or anti-KiM4. Staining varied between a continuous fine reticular pattern in hyperplasia, a varying extent of indentations and fragmentations of the network in fragmentation, and labeling of only part of the original network with only remnants of FDC in atrophy. The immunolabeling for SIV p28 gag protein co-localized with the FDC network (Fig. 2a and b). On cytocentrifuge preparations, SIV p28 gave surface immunolabeling of FDC, located either solitary or in clusters with lymphoid cells (Fig. 3). This concordance in immunolabeling between anti-DRC I/antiKi-M4 and anti-SIV p28 was observed for animals L341-90, L344-90, and L63-91. For animals L342-90 and L343-90, cytological preparations of FDC were not immunolabeled by the anti-SIV p28 antibody, and tissue sections showed only a faint labeling of germinal centers. Both cytologie preparations and germinal centers in tissue sections showed intense labeling by anti-DRC-1 or anti-Ki-M4 antibodies. FDC were additionally characterized in two-color immunofluorescence using combinations of anti-desmin on the one hand, and anti-vimentin or anti-DRC-1/anti-Ki-M4 on the other. Anti-desmin immunolabeled stroma throughout the tissue section, in reticular staining patterns of interfollicular and follicular areas. In follicles, the desmin staining pattern was similar to that of anti-DRC-l/anti-Ki-M4 but less extensive (Fig. 4a and b). Anti-vimentin labeled scattered cells outside follicles that were desmin-negative. In follicles, the anti-vimentin staining pattern was almost identical to that of anti-desmin (Fig. 4c and d). These data indicate that FDC in germinal centers express both vimentin and desmin, and thus are mesenchymal cells. Serial sections were immunolabeled for anti-DRC-1, CD8, and antibody to serine esterase granzyme-B (Fig. 5). CD8 revealed immunoreactive cells at a high density in interfollicular areas and at a lower density in germinal centers. In germinal
2024
JOLING ET AL. :.-'
s
a
.
it-
>?
TjPBie1«
n«H
•'!"?* •
if FIG. 1.
Conventional histology of lymphoid tissue from cynomolgus monkeys after SIVsm infection, (a) Lymph node from animal L341-90 showing hyperplasia of the lymphoid follicle (F). (b) Spleen of animal L63-91 showing atrophy of the white pulp. Frozen tissue section, hematoxylin and eosin stain-
ing,
FIG. 2.
Serial sections of frozen splenic tissue from animal L341-90, labeled by anti-DRC-1 (a) and anti-SIV gag p28 (b). SIV p28 localizes in a similar pattern in follicle germinal centers
anti-DRC-1 immunolabeling product, Immunoperoxidase labeling, x70. as
but is less extensive.
X70.
Electron
FDC-containing areas were identified by anti-DRC-1 immunolabeling and SIV p28 labeling (intense staining was
centers,
noted for animals L341-90, L344-90, and L63-91). In these the density of CD8+ cells was lower than in areas that wereDRC-1 negative. Granzyme-B + cells were present solitary in interfollicular areas. Occasionally a positive cell was observed in indentations or fragmentations of germinal centers, where there was no DRC-1 labeling. Areas immunolabeled by anti-DRC-1 or anti-SIV p28 did not show cells immunoreactive for granzyme-B. These data indicate the presence of CD8+ cells in high proportions in spleen and lymph nodes, in particular in interfollicular areas. Here, part of the cells are granzyme-B + The lowest density of CD8+ cells is observed in FDC-containing areas in germinal centers, and here cells are not labeled by areas,
.
antibody to granzyme-B.
microscopy
and lymph nodes, follicles were present for each The observations are presented separate for spleen (Figs. 6, 7) and lymph node. The follicles in spleen generally did not show the architecture of well-developed germinal centers surrounded by a lymphocyte corona. The main lymphoid population in follicles comprised small lymphocytes. A few atypical blast cells were observed that showed irregular shaped nuclei or even binucleated nuclei. Their presence indicated germinal center cell differentiation. The stroma consisted of FDC, which were classified for differentiation aspects.5'31 In the center of the follicle, there were a few differentiated FDC with electron-dense In
spleen
case.
deposits, presumably immune-complex depositions (Fig. 6a). Within these depositions virions resembling SIVsm particles were present (Fig. 6b), in particular, for animals L341-90, L344-90, and L63-91. In case L63-91, a large group of SIV™
FOLLICULAR DENDRITIC CELLS
in
SIV
-INFECTED MONKEYS
2025
Immunogold electron microscopy SIV p28 immunogold electron microscopy was performed on sections of spleen and purified FDC preparation of spleen. A labeling product was observed on electron-dense deposits on the surface of FDC. In the periphery of the follicle, p28 immunogold label was present in macrophages having a well-developed rough endoplasmic reticulum and showing lysosomes and tubuloreticular structures (Fig. 8). In FDC-enriched cell suspensions, immunogold labeling was present on virus-like structures between the cells (Fig. 9).
DISCUSSION
FIG. 3. Cytocentrifuge preparation of follicular dendritic cells isolated from spleen of animal L341-90, labeled by antiSIV gag p28. An FDC in a cluster with lymphocytes is shown, with labeling product on the FDC. Immunoperoxidase labeling, X492.
virions was observed around atypical blast cells (Fig. 7a and b). In this case, the majority of the FDC had a less differentiated ultrastructure with only few villous extensions, absence of electron-dense surface depositions, and concentrations of intermediate filaments in the cytoplasm. Paracrystalline arrays resembling so-called tubuloreticular structures32 were regularly observed in the cytoplasm of FDC (Fig. 6a and c), both for FDCs with the ultrastructural features of differentiated cells and undifferentiated cells. It was also observed in some atypical cells with a lymphoblastoid morphology, and in some small lymphocytes. Electron microscopy of FDC-enriched cell populations prepared from spleen showed a similar ultrastructural morphology as FDC in tissue sections. Along the plasma membrane, SIVsm virions were regularly observed (animals L341 -90, L344-
90, andL63-91).
In lymph nodes (data not illustrated), irregularly indented follicles and follicles with large mantle zones were observed. Germinal center characteristics were evident, with blast cells, centrocytes, FDC, and starry-sky macrophages.31 There were solitary viral particles, exclusively in the protrusions of FDC. In the mantle zone, FDC showed characteristics of undifferentiated cells and contained large aggregates of intermediate filaments. Tubuloreticular structures were seen in a variety of cell types. including FDC in the mantle zone and germinal center, blast cells and centrocytes in the germinal center, and small lymphocytes in mantle zones. In interfollicular areas, these structures were present in plasma cells, small lymphocytes, blasts, fibroblastic reticulum cells, and endothelial cells of capillaries and high endothelial venules. Tubuloreticular structures were not observed in germinal center macrophages.
SIVsm infection in cynomolgus monkeys resembles HIV-1 infection in humans in a number of aspects.I7-18-20-22-27 The histological alterations, including follicular hyperplasia, fragmentation and indentation, and finally general lymphoid atrophy '~3 (Fig. 1), are similar to those described after HIV-1 infection, and confirm earlier data on SIV-infection in monkeys by e.g. the group from Harvard Medical School.22-25 In the follicles, SIVsm virions and SIV gag p28 protein localize in the labyrinth of FDC (Figs. 2 and 6b),25 which was confirmed by the analysis of isolated FDC (Figs. 3 and 9). The isolation procedure28 yielded suspensions that were highly enriched in FDC, manifesting SIVsm particles (electron microscopy) and SIV gag p28 (immunocytochemistry, all but two animals, Table 1). In lymph nodes from HIV-1-infected patients, the architecture and cellular subset composition of interfollicular areas are largely*" similar to those in the normal reactive lymph node. CD4 T cells occur in somewhat lowered density, and CD8+ T cells are found scattered in the interfollicular area.1'3'5 In contrast, the high density of CD8+ cells in interfollicular areas in lymphoid tissue from SIVsm-infected monkeys (Fig. 5b) exceeded that in HIV-1-infected lymph nodes. It rather resembled that observed in some viral infections in humans (H.-J. Schuurman, unpublished observations). CD8+ cells also occurred in the indentations and fragmentations of follicles, and in the germinal center composed of FDC (DRC-1 or Ki-M4 staining, Fig. 5b).+ This might have a pathogenetic significance, inasmuch as CD8 cells include cytotoxic cells which have been implicated in follicular center destruction."12 However, in germinal centers there were no cells immunoreactive for serine esterase granzyme-B, a component of granules in cytotoxic cells. Such cells actually were observed in the interfollicular area. In this respect, the labeling of serine esterase granzyme-B in the SIVMI, situation resembles that in HIV-1-infected lymph nodes. These observations contrast with a report" that cells expressing mRNA of serine esterase occur in lymphoid follicles of HIV-1-infected patients. We elsewhere have ascribed this discrepancy to the sensitivity and specificity of the methods applied, and a difference in study material.33 There are no studies in the literature resolving this discrepancy by the simultaneous detection of mRNA and protein in the same tissue or tissue section. Awaiting such studies, it may be suggested that mRNA'+ cells in germinal centers produce serine esterase at levels that are too low to be detectable by immunohistochemistry, or that the protein after secretion diffuses in the tissue
JOLING ET AL.
2026
Frozen section of spleen from animal L63-91, subjected to two-color immunofluorescence. (a) Anti-DRC-1 (fluorescein isothiocyanate label) and (b) anti-desmin (tetramethyl rhodamine isothiocyanate label), (c) Anti-vimentin (fluorescein isothiocyanate label) and (d) anti-desmin (tetramethyl rhodamine isothiocyanate label). Most follicular dendritic cells identified by anti-DRC-1 (a) are labeled by anti-desmin (b). Vimentin immunoreactivity (c) coincides with desmin immunoreactivity (d). Immunofluorescence labeling, X244.
FIG. 4.
resulting in a too low loc'al concentration to be detectable by immunohistochemistry. It remains to be established whether the protein at such low local concentration in tissue has sufficient biological activity to exert its role in cytotoxicity. Our findings, both in HIV-1 infection and in SIVsm infection, do not support the hypothesis that cytotoxic cells play a main role in follicle center
destruction."'2
FDC in SIVsm-infected tissue resembled FDC in HIV-1 infection in electron microscopy5 and phenotypic markers.5 The cells showed positivity for vimentin and desmin (two-color immunofluorescence, Fig. 4), indicative of a mesenchymal origin. In humans FDC in the follicle mantle zone expressed both markers.34 An interesting observation was the presence of tubuloreticular structures in a number of cell types, including FDCs (Fig. 6a and c). These structures have been observed after retroviral infection,35"37 mainly in lymphoid cells and endothelial cells, but not in FDC. Their presence has been associated with the synthesis of a-interferon by the cell itself, or with the reaction of the cell to the presence of a-interferon in the local
reaction to viral infection associated with a-interferon In the present study the structures were not only observed in cells of hematopoietic lineage, but also in FDC that are of mesenchymal origin, even those with a less differentiated morphology present in the mantle zone. We conclude that FDC already early in their differentiation from mesenchymal cells contribute to the response to viral infection. The cells can be infected by the virus,6'8"10 and it remains to be established whether the cells are able to produce interferon. The SIVsm infection model in monkeys enables to evaluate treatment. Two of the animals studied (L342-90 and L343-90) received dideoxyinosine, in combination with azidothymidine or '3-fluorothymidine during the first 9 wk after infection (Table 1). At autopsy, 25 wk later, these animals showed the highest values of CD4+ cells and CD4/8 ratio in blood, suggestive of the beneficial effect of prior treatment. Interestingly, these animals had much less SIVsnl identified in follicular areas as determined by SIV immunocytochemistry on tissue sections and isolated cates a
synthesis.
FOLLICULAR DENDRITIC CELLS
J>5-
**
IN
SIVsm-INFECTED MONKEYS
2027
a
t
a
PC. s#
»
r*
•F r «
'*# i
'
? f
'• '
e -
#**
4
PC*'"
FIG. 6.
Electron microscopy of FDC in the center of a follicle spleen from animal L344-90. (a) Electron micrograph showing extensions (arrow) covered with electron-dense depositions, reflecting immune complexes. In the cytoplasm aggregates of tubuloreticular structures are seen (arrowheads), x 13,500. (b) At higher magnification, a SIVsm virion (arrow) in the labyrinth in
FIG. 5.
Serial sections of frozen splenic tissue from animal L341-90, labeled by anti-DRC-1 (a), CD8 (cytotoxic-suppressor T-cell phenotype) (b), and antibody to serine esterase Granzyme-B (c). CD8+ cells are present in relatively high density in the paracortex (PC) and in follicles (F) at indented areas where follicular dendritic cells (DRC-1 labeling) are absent (asterisk). In the areas labeled by anti-DRC-1 there are only a few CD81+ cells. There are a few granzyme-B+ cells around the follicle (indicated by an arrow), but not in areas containing FDC in DRC-1 labeling. Most dark spots around follicles in c represent endogenous peroxidase activity, and not specific immunolabeling by the antibody to granzyme-B. Immunoperoxidase labeling, X130.
of villous extensions of FDC, within electron-dense (immune complex) deposits, is shown (arrow). X77.000. (c) Part of a primitive FDC located at the periphery of the follicle, showing a large tubuloreticular structure with a paracrystalline substructure,
x15,964.
JOLING ET AL.
2028
*-
n
m.
FIG. 8. SIV gag p28 immunogold electron microscopy of a follicule in spleen from animal L63-91. The cytoplasm of a macrophage is shown, with p28 colloidal gold label presumably associated with membranes of rough endoplasmic reticulum and
Golgi complex,
x
14,580.
FIG. 7. Electron microscopy of a follicle in spleen from animal L63-91. (a) A binucleated atypical blast cell is surrounded by groups of virions (arrow). In adjacent sections this cell contained tubuloreticular structures. There are ringlike structures and other vesicle malformations in the cytoplasm resembling swollen mitochondria (arrowhead). Asterisk: FDC labyrinth. X8,600. (b) At higher magnification, the SIVsm virions (arrows) accumulated near the plasma membrane of the atypical blast cell are shown, x31,720.
FDC. Because of the number of animals studied, the signifiof these observations is unknown but warrants further
cance
study.
In conclusion, this study on lymphoid tissue of SIVsminfected cynomolgus monkeys shows the similarities between the animal infection and HIV-1 infection in humans. It underlines our previous suggestion that the immune response to the virus, i.e., cytotoxic cells, may not be involved in the destruction of follicles. Rather the virus and viral proteins themselves may play a role in this process. The reduced presence of viral protein in follicles in conditions of prior chemotherapy indicates a potential value of the experimental monkey model, because it is not readily possible to perform a similar study in patients after HIV-1 infection.
FIG. 9. SIV gag p28 immunogold electronmicroscopy of a FDC isolated from spleen of animal L341-90. Part of the villous extensions of the cell is shown, with immunogold labeling of a
SIVsnl-like particle (arrow). x61,000. (Inset) Higher magnifiSIVsm-like particle. x58,560.
cation of the
Biberfeld was supported by the Swedish Medical Research Council. The authors thank Jolanda Krijnen and Timo Meerloo for expert technical assistance.
REFERENCES ACKNOWLEDGMENTS The work in the Dutch group was supported by the Dutch Ministry of Health, as part of the National Program on AIDS Research, RGO/WVC grant 88-79/89005. The group of Dr.
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systemic therapy
with a-interferon.
Address
reprint requests to: P. Joling, Ph.D. Department of Pathology University Hospital,
P.O. Box 85.500 3508 GA Utrecht, The Netherlands.
