Detection of SIV in rhesus monkey thymus stroma cell cultures

(~) INSTITUTPASTEUR/ELsEVIER Paris 1994

Res. Virol. 1994, 145, 239-244

Detection of SIV in rhesus monkey thymus stroma cell cultures J.G. Mfiller O) (*), S. Czub 0), A. Marx (l), R. Brinkmann (2), R. Plesker (3) and H.K. Mfiller-Hermelink 0) Institute o f ¢o Pathology and ~) Virology, University o f Wiirzburg (Germany), and t3) Paul-Ehrlich-Institut, Langen (Germany)

SUMMARY To clarify the pathogenesis of SlY-induced thymus atrophy, the presence of SlY within thymus stromal cell cultures (epithelial cells, IDC, macrophages or fibroblasts) was investigated. The material studied consisted of 15 thymus specimens of rhesus macaques infected with SlVmac251 (2-4 months postinoculation). No viral antigen was detected, either in the cultures, by immunohistochemistry, or in cell culture supernatants, by ELISA (p17 antigen), and no viral RNA was detected by in situ hybridization. Only after coculture with the C 8 1 6 6 cell line, was virus detected in 2 out of 15 stroma cultures. The fact that the virus could only be detected after several passages of coculture with the C 8 1 6 6 cell line indicates that the virus exists in the thymus stroma cells in the form of proviral DNA. The infection of thymus stromal cells may contribute to the destruction of the thymus microenvironment and to the SlV-induced thymus atrophy.

Key-words: SIV, Thymus, AIDS; Atrophy, Detection, Stroma cell cultures, FJhesus monkey, Epithelial cells, Proviral DNA.

INTRODUCTION The production of naive T cells by the thymus requires a supply of prethymic stem cells from the bone marrow and an intact thymic microenvironment. The latter is provided by different types of epithelial ceils, macrophages, interdigitating dendritic cells (IDC), B cells as well as mesenchymal cells (Anderson et al., 1993 ; Jenkinson et al., 1992). The role of these cells in the homing, proliferation, selection and maturation processes is little understood but seems to require physical cell contact as well as the production of various cytokines (Le et al., 1991 ; Wolf and Cohen, 1992; Moore et al., 1993). In the context of the HIV

Received February 23, 1994. (*) Corresponding author.

infection, destruction of the epithelial cells of the thymus microenvironment was suggested in humans (Savino et al., 1986; Papiernik et al., 1992) and was demonstrated in the experimental model of SIV infection of rhesus macaques (Mfiller et ai., 1993). However, the mechanism of this atrophy and destruction of the epithelial cells remains unclear. One possibility is the infection of the thymus epithelial cells by HIV or SIV. Numazaki et al. (1989) reported the successful HIV1 infection of purified human thymus epithelial cells in vitro. These results could not be reproduced by Schnittman et al. (1991), but in later experiments the same group reported a productive HIVI infection of human thymus epi-

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thelial cells after intrathymic injection of HIV1 into human thymus implants in Scid-hu mice (Stanley et al., 1993a,b). To test this in the primarily immunocompetent host in vivo, we investigated thymus specimens from SIV-infected rhesus macaques, for the presence of SIV in thymus stroma cell cultures.

MATERIALS

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METHODS

Thirteen rhesus monkeys from the Paul-Ehrlich-Institut and two rhesus monkeys from the German Primate Center, G6ttingen (8 juvenile (2-4 years) and 7 adult animals), were infected intravenously with SIVmac251/32H. The animals belonged to different experiments, and only the thymus was used for this experiment. All animals were killed 2-4 months postinfection, prior to any clinical disease. At autopsy, no signs of immunodeficiency (atrophy of the lymph nodes, opportunistic infections) or other diseases were found. Thymus specimens from 2 non-SIV-infected juvenile control monkeys (M. nemestrina) were obtained from Dr. A.D. Osterhaus, Bilthoven, The Netherlands. Thymus stroma cell cultures were prepared by a fourstep mechanical and enzymatic tissue dissociation. Step 1 : after mechanical disintegration into pieces smaller than 0.1 cm, the cells in the supernatant were washed with PBS and grown in plastic culture dishes in DMEM with 10 % FCS and 1 % "PenStrep". Step 2: the remaining pieces were digested for 30 min at 37°C with 1 % collagenase/ hyaluronidase (Sigma, Munich) and further disintegrated by forced pipetting; the cells in the supernatant were washed with PBS and put into culture. Step 2 was repeated twice (steps 3 and 4). SIV antigen in the supernatant was detected by p27 ELISA (Coulter Corp, Luton, UK). Infectious virus particles were detected by coculture with the C8166 cell line. The thymus stroma cell cultures were passaged 1:2 into 50-ml culture flasks, and to one flask, 4 x 104 exponentially growing C8166 cells were added. As control, 4 x 10 4 C8166 cells were put into a third flask and processed as described above. The cultures were scored microscopically every second day for the presence of giant cells, for 3-6 weeks. After each one-week period, the cultures were passaged 1:5. For immunohistochemistry and in situ hybridization, thymus stroma cells were grown on "LAB TAK" chamber slides (Miles Scientific, Naperville, IL). Antibodies against cytokeratin 8+18 (3513Hll, Enzo), vimentin (4313H12, Enzo), CD68 (KiMlp, Kiel) and CD3 (FNIS, Margret Jonker, Amsterdam) were stained by an indirect immunofluorescence method using fluorescein isothyocyanate (FITC). Antibodies KK8 (recognizing SIVenv gp120) and KK20 (recognizing SIV gp160/41;

DMEM FCS FITC IDC

= = = =

Dulbecco's minimal essential medium. foetal calf serum. fluorescein isothiocyanate. interdigitating dendritic cell.

K. Kent, AIDS Reagent Project) were used to detect SIV proteins. To detect the intracellular antigens, the medium was removed, the slides were rinsed with PBS, fixed with 3.5 % formaldehyde for l0 rain and incubated with 0.25 % Triton-Xl00 for 15 min. SIV RNA was detected by in situ hybridization, using a 527-bp gag RNA probe prepared as described earlier (Mfiller et al., 1993). The probe was labelled with 35S-,~UTP (New England Nuclear) with the T7 and SP6 polymerase (Promega). The cells were fixed in acetone and postfLxed in 5 % paraformaldehyde. Anti-sense and sense probes hybridized overnight at 45°C. The slides were washed twice with 0.1 × SSC at 60°C, followed by washing with 2xSSC containing 10~tg/ml RNase A and l Unit/ml RNase T (Promega).

RESULTS All animals were SIV-infected as demonstrated by seroconversion and virus reisolation from the peripheral blood. No intercurrent diseases were found at autopsy; 5 of 8 juvenile monkeys developed cortical atrophy of the thymus (fig. lb) and 3 showed a normal thymus structure (fig. la). The adult animals exhibited an age-related thymus involution. The stroma cell cultures were composed of about 30-50 % epithelial cells growing in solid formation (fig. 2a), and they stained positive for cytokeratin in immunohistochemistry. The remaining spindle cells (fig. 2b) were only positive for vimentin and occasionally for CD68. The culture remained stable for around 2 months. In later passages, the epithelial cells and macrophages disappeared, leaving behind fibroblast-like cells. In all SIV-infected animals,, the thymocytes died within one week o f culture and were washed out. At the same time, no remaining CD3-positive or panLeu-positive cells were detected (fig. 2a,b). In cultures of 2 non-SIV-infected control monkeys, thymocytes (CD3-positive) which adhered to the stromal cells were seen up to 4-5 weeks of culture (fig. 2c). Virus was only detected by cocultivation with the C8166 cell line for at least 4-6 weeks in 2 of the juvenile rhesus monkeys with cortical atrophy. In these cultures, a cytopathogenlc effect occurred, and the existence of SIV was proven by p27 ELISA, P C R and electron microscopy (fig. 2d).

ISH PBS SIV SSC

=

= = =

hybridization. phosphate-buffered saline. simian immunodeficiency virus. standard saline citrate.

in s i t u

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Fig. 1. SIV-induced thymus atrophy. A) Size of the thymus in the controls and also in 3 juvenile SIV-infected macaques. B) 5 of the SIV-infected rhesus macaques exhibited atrophy of the thymus cortex. Haematoxylin + eosin, x 45.

DISCUSSION SIV infection of thymus stroma cells occurred in vivo. However, the amount of SIV was very low, and no viral RNA was detectable by in situ hybridization of these cultures. Moreover, no viral protein was detected by immunohistochemistry. In view of the negative results of immunohistochemistry, ELISA and in situ hybridization, the coculture experiments were carried out. In 2 cases; virus was detectable after 3-4 weeks including 2 passages. This parallels the results of Brinkmann et ai. (1993) and indicates the presence of proviral DNA and a low rate of virus replication within the thymus stromal cells. An earlier start of the coculture experiments or PCR analysis for proviral DNA might result in a higher rate of SIVpositive thymus stromal cell cultures. Up to now, no identification of the cell type carrying the virus was possible. The low amount of virus in the thymus stroma cells of SIV-infected macaques seems to contrast with the recent data of Stanley et aL (1993a,b) of a productive infection of human thymus epithelial ceils by HIVI. They used human thymus specimens, which were transplanted into Scid mice and infected in-

trathymically. This experimental model resulted in large-scale virus replication in the thymocytes and in acute thymus atrophy (Aldrovandi et al., 1993; Bonyhadi et al., 1993) in the absence of an immune response. In the immunocompetent host there is a burst of productively infected cells in the first 1 or 2 weeks of infection, prior to the development of a humoral and a cellular immune reaction. This is seen in HIV-infected humans (Graziosi et al., 1993) and also in SIV-infected rhesus macaques (Miiller and Czub, unpublished results). In contrast to the Scid model, this first line of virus replication ceases with the emergence of the immune reaction in the primarily immunocompetent host. Thereafter, only low amounts of SIV RNA are detectable. From these data it seems that the virus infects many tissues, including cells of the thymus stroma, in the first weeks after inoculation. After the emergence of an immune reaction, the virus then seems to persist in the thymus stroma in the form of proviral DNA. Up to now, few investigations regarding HIV or SIV infection of the thymus in vivo have been performed. Ward et al. (1987) detected, by immunohistochemistry, epitopes of viral proteins in thymic epithelial ceils. In our hands, no convincing immuno-

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Fig. 2. Thymus stromal cell cultures. A and B are from SIV-infected animals, exhibiting solid sheets of epithelial cells (A) and spindle cells (B) without adhering lymphocytes. C shows the culture from one of the controls exhibiting adherent lymphocytes on the epithelial cell layer after 4 weeks of culture. A, B and C unstained, x 880. D: electron microscopy from one successful coculture experiment demonstrated large amounts of virus particles around the C8166 cells. Epon, PbCO3, x 28,800.

SIV IN RHESUS MONKEY

THYMUS STROMA CELL CULTURES

histochemical or in situ hybridization signals, showing an infection of thymic epithelial cells, were obtained. The critical point in in situ hybridization is to identify the infected cells owing to the broad signal produced by 35S-autoradiography. The problem in the immunohistochemistry is a possible cross-reactivity with non-SIV- or HIV-infected human (Parmentier et al., 1992) or rhesus monkey thymus (Miiller et al., 1993). Both the time course and the characteristics of the SIV-induced thymus atrophy in 5/8 juvenile macaques exhibiting an SIVinduced thymus atrophy 2-4 months postinfection resembled those of our earlier experiment (Miiller et al., 1993). In the control thymus cultures, T cells remained adherent to the stroma cells for several weeks. In the SIV-infected cultures, however, with rapid thymocyte death, only the adherent stroma cells remained. There are two possible explanations for this difference in behaviour: (1) SIV-induced rapid thymocyte death by apoptosis in vitro (Banda et al., 1992; Groux et aL, 1992), and (2) an SIV-induced alteration of the thymus stroma cells. Fauci (1993) postulated that the development of the CD4 cell loss in HIV infection is due to a failure in the regenerative capacity of the T-cell system. This might be due to impaired maturation of early T-cell progenitors, either in the bone marrow or in the thymus (Schnittman et al., 1991). One explanation for this could be the impairment of the thymic microenvironment (thymic epithelial cells, fibroblasts, macrophages, interdigitating dendritic cells and others) in its capacity to carry out critical nursing functions for the developing thymocytes (cytokine secretion, class I and II expression, expression of adhesion molecules) (Anderson et al., 1993 ; Jenkinson et al., 1992). This study shows that not only thymocytes are infected in vivo (Kneitz et al., 1993) but also the cells of the thymic microenvironment. This may contribute to the thymus epithelial cell destruction and to the breakdown of intrathymic T-cell proliferation and maturation (Miiller et al., 1993). Further work needs to be done to clarify the amount and type of stromal cells infected by SIV or HIV in viyo in the primarily - - immunocompctent host.

D6tection du virus de rimmunod~ficience simienne dans des cultures de cellules du stroma thymique

Pour 6tudier la pathogen6se de l'atrophie thymique due au SIV, nous avons recherch6 la pr6sence du virus dans des cultures de cellules du stroma thymique (cellules 6pith61iales, interdigit6es, macrophagiques et fibroblastiques). Quinze sp6cimens de thymus de macaques rh6sus ont 6t6 pr61ev6s et 6tu-

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di6s 2 A 4 mois apr~s infection par le SIVmac251. Dans les cultures primaires la pr6sence de l'antig6ne p17 du virus n'a pas 6t6 d6tect~e par immunohistochimie ou par ELISA. Au niveau de la transcription virale, aucun ARNm sp6cifique n'a 6t6 d6tect6 par hybridation in situ. Apr~s co-culture des cellules thymiques avec la lign6e C8166, le virus a ~t~ retrouv6 darts 2 sp6cimens sur 15. Cela indique que l'infection latente du thymus peut ~tre un facteur important de la destruction progressive du microenvironnement thymique. Mots-cl~s: SIV, Thymus, SIDA; Atrophic, I ~ e c tion, Cultures de cellules du stroma, Macaque rh6sus, Cellules 6pith61iales, ADN proviral.

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