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Internalization of CD4 molecules in human T-cells demonstrated by immuno-electron microscopy. J.J. Wang 1'4, C. Hu 1, F. Lee 2, M.F. Shaio 3, and L.K. Chen 2.
Histochemistry (1992) 97 : 51 59

Histochemistry © Springer-Verlag 1992

Internalization of CD4 molecules in human T-cells demonstrated by immuno-electron microscopy J.J. Wang 1'4, C. Hu 1, F. Lee 2, M.F. Shaio 3, and L.K. Chen 2 1 Department of Biology and Anatomy, Department of Biology and Anatomy, National Defense Medical Center, POB 90048-502, Taipei, Taiwan, Republic of China (10700) 2 Department of Microbiology and Immunology, 3 Department of Parasitology and Tropical Medicine, National Defense Medical Center, and Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, Republic of China Accepted October 8, 1991

Summary. Internalization of CD4 molecules on human CD4-enriched T-cells was demonstrated by immunocytochemical electron microscopy. CD4 + T-cell subclones were obtained from normal human peripheral blood, followed by one-way M L C screening and co-culturing with IL-2. Fixed and non-fixed T-cell samples were indirectly immunolabeled with mouse anti-human CD4 monoclonal antibody and goat anti-mouse IgG conjugated with peroxidase. Unfixed T-cells were immunolabeled at 4 ° C and then re-incubated for 5-45 min at 37 ° C. The selected CD4 + T-cell subclones showed strong CD4 binding on the cell surface after IL-2 incubation. However, fresh T-cells, monocytes, bone marrow cells and CD8 + T-cells all stained negative for CD4. The distribution of CD4 molecules on the fixed cell surface showed a homogeneous pattern. Capping and internalization of CD4-antibody-peroxidase complexes from the cell surfaces were observed follow a pathway of receptor-mediated endocytosis in unfixed T cells. Endocytotic vesicles, vacuoles of diverse sizes and shapes near the cell membrane or deep in the cell center were found to contain CD4 molecules. Negatively stained Golgi saccules were observed up to 45 rain after re-incubation. These results suggest that increased CD4 molecules can be induced on the surface of normal human T-cells in vitro. Internalization and accumulation of CD4 molecules occurred in CD4-enriched T-cells with IL-2 pretreatment.

Introduction CD4 (Cluster differentiation type 4) antigen is a glycoprotein of molecular weight 55 kDa expressed on the surface of T-helper cells. CD4 plays an important role in the activation and functions of normal T-cells in association with the major histocompatibility complex type II antigen (Blue et al. 1987; Gay et al. 1987; Bottomly Offprint requests to: J.J. Wang

1988). H u m a n immunodeficiency virus (HIV) infection through its bindings receptor, CD4 molecule, is the major entrance of HIV to the target cells (Dalgleish et al. 1984; Maddon et al. 1986; Asjo et al. 1987). T helper cells and monocytes/macrophages expressing CD4 on their surface, are major targets for HIV (Klatzman et al. 1984; Ho et al. 1986; Nicholson et al. 1986; Maddon et al. 1986; Gendelman et al. 1989; Kazazi et al. ]989; Traunecker et al. 1989; Buonocore and Rose 1990; Valentin et al. 1990; Westervelt et al. 1991). The binding of HIV to CD4 molecules is specific. Buonocore and Rose (1990) recently demonstrated that intracellular CD4 molecules can block the transportation of HIV glycoproteins during the protein synthesis. A region on the exterior envelope glycoprotein, gp 120 of HIV particles is able to bind to CD4 (Habeshaw et al. 1990), and forms a CD4-gp120 complex (McClure et al. 1988). Furthermore, the terminal V1 domain of CD4 molecules extrudes out of the T-cell surface, has been identified as the binding site for HIV (Arthos et al. 1989). After HIV binding, CD4 molecules are no longer detectable on the T-cell surface (McDougal et al. 1986; Schnittman et al. 1989). Down regulation and possible internalization of CD4 molecules in T-cells exposed to phorbol esters has been demonstrated by Hoxie et al. (1986). This observation is consistant with the increase of intracellular CD4gpl20 complexes in HIV-infected cells reported by Stevenson et al. (1988). Entrance of HIV into T-cells through receptor-mediated endocytosis has been suggested (Stein et al. 1987; Long and Jacobson 1989). Unlike monocytes, which expressed less CD4 molecules than T-cells and serve as reservoir for HIV replication, CD4 + T-helper cells were depleted by HIV infection (Nicholson et al. 1986). The significance of internalization of CD4 receptor accompany with HIV in the human host target cells is unclear. Capping of CD4 molecules on the surface of human peripheral lymphocytes induced by monoclonal antibodies has been observed by Pavan et al. (1990). However, the internalization pathway and location of internalized CD4 molecules in CD4 + T-cells is still obscure.

52 A series o f p a t h o l o g i c c y t o k i n e , i n c l u d i n g i n t e r l e u k i n - 2 (IL-2) i n d u c t i o n b y H I V h a s b e e n r e v i e w e d b y R o s e r b e r g a n d F a u c i (1990) recently. T h e n u m b e r o f i n t e r l e u k i n - 2 r e c e p t o r s ( I L - 2 R ) f o u n d o n T-cells i n d i c a t e s I L - 2 p r o m o t e d T-cell p r o l i f e r a t i o n (Toribio et al. 1989). H I V g p l 2 0 a n d lectin m o l e c u l e s w h i c h m i m i c the a c t i o n s o f l y m p h o k i n e s i n c r e a s e d the e x p r e s s i o n o f I L - 2 R o n the cell surface (Barnes 1988; B o t t o m l y 1988). F u r t h e r exp e r i m e n t s s h o w e d t h a t e l e v a t e d levels o f s o l u b l e I L - 2 R in the s e r u m o f H I V - i n f e c t e d p a t i e n t s m a r k an i n c r e a s e o f I L - 2 d e p e n d e n c y o f infected C D 4 + T-cells (Barnes 1988; R e d d y a n d G r i e c o 1988). O u r p r e v i o u s w o r k also s h o w I L - 2 c a n l e a d to a high e x p r e s s i o n o f C D 4 m o l e cules o n the c u l t u r e d h u m a n T - h e l p e r cells ( C h e n et al. 1986). T h e a i m o f the p r e s e n t e x p e r i m e n t is to use I L - 2 t r e a t e d C D 4 - e n r i c h e d h u m a n T-cells w i t h o u t H I V infect i o n to d e t e c t the in situ C D 4 l o c a t i o n s at the s u b c e l l u l a r level.

mark) at the titer of 1:10. Goat anti-mouse IgG conjugated with fluorescent isothiocyanate (FITC, Janssen, Belgium) at the titer of 1:20 was used as a label for fluorescent microscopy. The cells were observed under Zeiss fluorescent microscope without further incubations. Unfixed cells were immunolabeled as above at 4° C with antihuman CD4 MAb followed by goat anti-mouse IgG (H&L) conjugated with peroxidase (PO, 1/1000) (Janssen, Belgium). The cells were reincubated at 37°C for periods of 5 to 45 min and then fixed in 2% glutaraldehyde at room temperature for 30 rain. For the fixed groups, cell suspensions were washed twice with D-PBS and then fixed as above. After washed overnight in the same buffer, the cells were incubated in 2% BSA, and then immunostained with the primary antibody and then the secondary antibody conjugated with PO. After color reactions with diaminobenzidine (DAB) and hydrogen peroxide (H202), the samples were post-fixed in 1% aqueous osmium tetroxide at 4° C for i h. Cell pellets were obtained by centrifugation at 250 g for 10 min and processed for electron microscopy (Wang et al. 1990).

Control groups for immunocytochemistry Materials and methods

Preparation of T-cell clones by micromanipulation Lymphocytes were isolated by Ficoll-Hypaque (Pharmacia, Sweden) density gradient centrifugation from fresh blood donated by healthy young male donors. Cells were sensitized in a one-way mixed lymphocyte culture (MLC) for 6 days against specific allogenic peripheral blood lymphocytes. After 6 days of culture, the primed cells were restimulated for an additional 6 days with the specific stimulating cells in the presence of IL-2-enriched (20%) conditioned RPMI culture medium containing 20% human serum (HS). The primed cells were then suspended in 5% HS/RPMI medium and cell suspensions were transfered into a 100 mm glass petri dish (Gibco, New York). Single cells were selected with a fine capillary pipette under an inverted light microscope under aseptic conditions. Selected cells in the pipette were discharged into a second petri dish containing 5 ml of fresh culture medium to make sure that only one cell was selected. The single cell was then transferred into an individual well of a microtiter 96-well-plate (Coster, Cambridge, MA) containing 1[00 I-tlof RPMI and 20 % IL-2-conditioned medium. Irradiated (2000rads) mononuclear cells (5x 104 cells/ 50 gl) from specific donor were added to each well. A subsequent clonal ceil expansion was maintained according to previous methods.

Preparation of IL-2-enriched culture medium Ficoll-Hypaque-purified splenic mononuclear cells were cultured for 36-48 h in a complete medium containing 3% heat-inactivated pooled human serum, 1% phorbol 12-myristate 13-acetate (PMA, Sigma) and PMA-M (Gibco, New York) and the resulting supernatants were used as a source of IL-2.

ImmunojTuorescent microscopy and immuno-electron microscopy CD4 + and CD8 + T-cell subclones were obtained from cloning of primed cultures. Fresh peripheral lymphocytes, bone marrow cells from normal young male adults and a patient with histiocyte lymphoma were obtained. Cultured and fresh unfixed cells in suspension were washed twice with Dutbecco's phosphate-buffered saline (D-PBS), pH 7.4, and then blocked with 2% bovine serum albumin (BSA) at 4° C. The cells were incubated with mouse antihuman CD4 monoclonal antibody (MAb) (IgG1, Dakopatts, Den-

The endogeneous peroxidase activity was not blocked before immunostaining in this experiment. The unfixed and fixed CD4 + T-cells were used for immunolabeling without primary antibody incubation as one of the controls. Another monoclonal antibody against Dengue-2 virus was used to test the specific bindings of secondary antibody on the T-cells from healthy and non-Dengue virus infected individuals. Besides, CD8 + T cells, mixed CD4 + and CD8 + T cells and other freshly prepared human cells, such as: splenic mononuclear cells, monocyte/macrophages, histiocyte lymphoma and normal bone marrow cells were collected and fixed for CD4-immunostaining.

Results S u b c l o n e s o f C D 4 + a n d C D 8 + T - h e l p e r cells were o b t a i n e d a n d successfully e x p a n d e d . I m m u n o f l u o r e s c e n t s t a i n i n g s h o w e d t h a t u n f i x e d C D 4 ÷ T-cells were positively s t a i n e d b y f l u o r e n c e n c e i s o t h i o c y a n a t e ( F I T C ) . P a t c h e s o f f l u o r e n c e n t labelings were s h o w n o n the cell surfaces a n d p o s s i b l y in the c y t o p l a s m (Fig. 1 A). T h e s a m e cell a r e a u n d e r the light m i c r o s c o p e s h o w e d t h a t o n l y one cell was n e g a t i v e l y s t a i n e d ( a r r o w h e a d in Fig. 1 B). T h e r e were a b o u t 12% o f the unfixed p e r i p h e r al l y m p h o c y t e s s h o w e d w e a k C D 4 p o s i t i v e F I T C reactions. H o w e v e r , it is u n d e t e c t a b l e o n the fixed cell samples. Since C D 8 ÷ T-cells were n o t s t a i n e d for C D 4 m o l e cules, a n d 5 0 % o f the m i x e d C D 4 ÷ a n d C D 8 ÷ T-cells showed positive FITC and peroxidase immunostaining ( M i c r o g r a p h s n o t shown), C D 4 ÷ T-cell s u b c l o n i n g was successful. I m m u n o l a b e l l i n g o f T-cell surface p o l y p e p tides at 4 ° C successfully localize these m o l e c u l e s in situ ( W a n g et al. 1990). W h e n u n f i x e d C D 4 ÷ T-cells were immunolabeled with antibody-peroxidase (Ab-PO) complexes at 4 ° C a n d t h e n r e i n c u b a t e d a t 37 ° C f o r 45 min, p a t c h e s o f C D 4 - A b - P O c o m p l e x e s on the cell surface a n d i n t e r n a l i z a t i o n were o b s e r v e d in m o s t cells (Fig. 2). T h e i n t e r n a l i z e d C D 4 - A b - P O c o m p l e x e s were seen in vesicles a n d v a c u o l e s n e a r the cell m e m b r a n e a n d also d e e p in the cell center close to the nucleus (Fig. 2). T h e G o l g i a p p a r a t u s in the cell center d i d n o t c o n t a i n C D 4 . W h e n the cells were fixed first a n d t h e n i m m u n o l a b e l e d , p o s i t i v e C D 4 - A b - P O d e p o s i t s were seen h o m o g e n e o u s l y d i s t r i b u t e d o n the cell surface. T h e c y t o p l a s m was u n l a -

Figs. 2-13 were not stained with uranyl acetate and lead citrate Fig. 1 A, B. Non-fixed CD4 ÷ T-cells were positively immunostained by fluorescence isothiocyanate (FITC)-antibody complexes. Patches of fluorescence illuminations were observed on the cell surfaces (A). The same field of cells under the fluorescence microscope showed the presence of both stained and nonstained cells (arrowhead) (B). × 400. Bar=25 Ixm

Fig. 2. Non-fixed CD4 + T-cells were immunostained at 4 ° C and then reincubated at 37°C for 45 min before fixation. CD4-Ab-

peroxidase deposits were seen on the surface. Patches and internalizations were also shown in most cells. Small vesicles were seen in vacuoles. Note the Golgi saccuoles were negative of bindings and the vacuole located between the nucleus (N) and the Golgi apparatus (G). x 16500. Bar=0.5 gm

Fig. 3. Fixed CD4 ÷ T-cells were immunolabeled and showed homogeneous distribution of CD4-Ab-PO deposits on the cell surfaces (arrowheads). Note there are no deposits found in the cytoplasm, x 16500. Bar=0.5 gm

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Figs. 4-7. Negative control groups for immunocytochemistry Fig. 4. The splenic mononuclear cell were isolated by Ficoll-Hypaque and fixed in glutaraldehyde before immunostaining. Negative CD4 bindings were shown on the cell surface (arrowhead). x 15600. Bar=0.5 gm Fig. 5. The freshly prepared h u m a n monocyte/macrophages were fixed and then immunostained for CD4 antigen. Note the negative deposites were found on the surfaces of cells (arrowhead) and platelets (p). x 57500. Bar=0.5 gm Fig. 6. Cells of histiocyte lymphoma showed negative CD4 bindings on the cell surface (arrowhead). Note the endogeneous peroxidase

activity in the cytoplasmic granules. A red blood cell also showed negative of binding, x 16500. Bar=0.5 gm Fig. 7. Negative CD4 bindings were seen on the surface (arrowhead) and in the cytoplasm of a CD4 + T-cells when immunolabeled with secondary antibody only and reincubated at 37 ° C for 45 min before fixation. Note the Golgi saccules (G) were also negative of CD4 bindings. N: nucleus. × J8000. B a r = 0 . 5 gm Fig. 8. Positive CD4 bindings were seen on the cell membrane (arrowhead) and in the vacuoles near the nuclear envelope (N) similarly treated as that in Fig. 7, whereas, with primary antibody incubation. Note the similar background stainings. × 24000. Bar=0.5 Ixm

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Fig. 9 A - H . Capping and internalization of CD4-Ab-PO complexes in the CD4 ÷ T-cells after reincubations at 37 ° C for 5 to 30 min. x 33 000. Bar = 0.5 gm. A Homogeneous and various concentrated patches and capping (arrowhead) of CD4-Ab-PO complexes on the cell surface were observed. B A concentrated CD4-Ab-PO deposit in an endocytotic vesicles (arrowhead) was observed at the base of the microvilli. C A tadpole-shaped endocytotic vacuoles containing small vesicles with a tail-like tubule (arrowhead) was found near the cell membrane. D A vacuole containing swelling vesicles and two tails (arrowheads). Note the size of this vacuole is larger than that in Fig. 8 C. E CD4-Ab-PO deposits appeared in a vacuole (diameter of 600 nm) and in small vesicles and tubules (diameter of 60 nm) nearby. Small vesicles near the hilium of the

vacuole and a tubule at the opposite side were fusing with the vacuole (arrowheads). F Vesicles of positive or negative CD4-AbPO bindings were seen nearby a vacuole (arrowhead) close to the cell surface. G The CD4-Ab-PO deposits were seen in the vacuole (arrowhead) moving deeper into the cytoplasm. Tubules with positive bindings were seen close to the vacuole. H CD4-Ab-PO positive vacuoles and tubules were observed extended from the cell membrane to the deep cytoplasm of the cell center. Note the tubules (arrowheads) and small vesicles at opposite sides of the vacuole. The non-fixed CD4 ÷ T-cells were immunolabeled with CD4-AbPO complexes, and reincubated for 30min (Figs. 10-11) and 45 min (Figs. 12-13) at 37 ° C

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The non-fixed CD4 ÷ T-cells were immunolabeled with CD4-AbPO complexes, and reincubated for 30 min (Figs. 10-11) and 45 min (Figs. 12-13) at 37 ° C. Fig. 10. Positive CD4-Ab-PO deposites were observed on the surface and internalized in vacuoles near the surface, in tubules close to the Golgi apparatus (G) and near the nucleus (iV). Note the negative bindings in the Golgi saccuoles were observed, x 30000. Bar = 0.5 g m

Fig. 11. The deposits were seen on the surface and in the tubules and vacuoles in the cytoplasm. Note the typical Golgi apparatus

(G) which showed negative CD4 bindings. N: nucleus, x 19800. B a r = 0 . 5 gm

Fig. 12. Large vacuoles with dense CD4-Ab-PO deposits were observed near the cell membrane and deep amoung the Golgi saccules (G). Note the Golgi apparatus is still negative of CD4 bindings. N: nucleus, x 18000. B a r = 0 . 5 gm Fig. 13. Positive and negative vacuoles were seen arround the Golgi apparatus (G) near the cell surface as well as near the nucleus (N). Note the Golgi apparatus is still negative of CD4 bindings. × 25000. B a r = 0 . 5 [xm

57 beled even without blocking the endogenous peroxidase activity (Fig. 3). Fresh lymphocytes isolated from human splenic mononuclear cells were CD4 negative (Fig. 4). Monocyte/macrophages were also freshly prepared from different healthy individuals and were CD4 negative (Fig. 5). The bone marrow cells from normal individuals and a patient of histiocyte lymphoma showed negative CD4 bindings on the surface, however, the endogenous reactions of peroxidase activity were shown in the cytoplasmic granules (Fig. 6). When primary antibody was omitted or preimmunized serum was applied prior to the secondary antibody-peroxidase incubation, no binding was observed on the cell surface or cytoplasm after 45 rain of reincubation (Fig. 7). Cell sample with primary antibody and similar secondary antibody-peroxidase reincubation showed positive deposites on the cell membrane and in vacuoles close to the nucleus (Fig. 8). No binding was present on monocyte cell surfaces after Dengue-2 MAb and then secondary antibody incubations (micrographs not shown). Capping and internalization of CD4-Ab-PO complexes from 5 to 30 min of reincubation were observed (Fig. 9A-H). Homogeneous distribution was observed first, and various densities of concentrated CD4-Ab-PO capping on the surface and at the root of microvilli appeared subsequently after 5 rain of reincubation (Fig. 9A, B). Diverse vacuoles of different sizes and shapes were formed after internalization (Fig. 9C-H). A vacuole with tail-like tubule formed under the cell membrane (Fig. 9 C). Coated vesicles were not identified. Small vesicles were found near and within the vacuoles (Fig. 9 C-D). Some vesicles attached to the inner surface of the vacuole (Fig. 9 E-HI). Tubules of various shapes with positive or negative CD4-Ab-PO deposits were seen near or extended from the surface of the vacuoiles (Fig. 9 E). The tubules are thought to connect the vacuoles to the cell membrane during their formation (Fig. 9F). They could also be a transferring channel from the apical surface towards the inner cell center (Figs. 9G-H). The maximum diameter of the tubules and vacuoles were 60 nm and 600 nm respectively. The network of tubules and vacuoles indicates the possibility of recetor-mediated endocytosis. After 30 rain of reincubation at 37° C , CD4-positive vesicles with diameter range from 60 600 nm were found near the cell surface and also in the cell center close to the nucleus and Golgi apparatus (Figs. 10-11). After 45 rain of 37° C reincubation, large vacuoles accumulated with dense CD4-AbPO deposites accumulated between the nucleus and the Golgi apparatus (Figs. 12 13). The Golgi saccuoles were negative of CD4 bindings (Figs. 10-13). Discussion

In order to successfully demonstrate CD4 molecules on T-cells, high levels of CD4 expression are necessary (Chen et al. 1986; Hoxie et al. 1986; Stein et al. 1987). The down-regulation and re-expression of CD4 molecules on T lymphocytes is controled by diverse mechanisms including phorbol ester (Solbach et al. 1982), interleukin-1 (IL-1) (Tvede et al. 1988) and bryostatin-1

(Boto et al. 1991). Previous work indicates that development and proliferation of bovine mature T cells needs autocrine growth stimulation by IL-2 (Takamatsu et al. 1990). High levels of CD4 molecules were expressed on the T-cell surface after the activation of T lymphocytes by treatment with IL-2 (Toribio et al. 1989; Takamatsu et al. 1990). Human T-cell preparations in vitro also requires co-incubation with IL-2 (Chen et al. 1986) to prepare T-cell subclones. Fresh lymphocytes isolated from Ficoll-Hypaque density gradient centrifugation without IL-2 incubation have been used to detect the capping of CD4 molecules on cell surfaces (Pavan et al. 1990). However, the CD4 molecules expressed on our unfixed fresh cell samples were weakly demonstrated on only 12% of cells. After fixation, the CD4 epitopes were probably destroyed and barely detected by the monoclonal antibody used in this experiment, and then eluded the immunocytochemical detection. When the unfixed cell samples were immunostained with FITC and then reincubated at 37° C, it is difficult to recognize the internalization of CD4-Ab-FITC complexes. Using electron microscopy and rewarmed HIVinfected cells by incubation at 37°C for 60 rain, Stein et al. (1987) suggested that HIV fused with CD4 + T-cell membranes and the HIV core was then internalized into the T-cell. It seems that the receptor-mediated HIV endocytosis is not essential for the internalization of HIV receptor, CD4 (Sattentau and Weiss 1988). However, McClure et al. (1988) suggested that after HIV fuses with the cell membrane at neutral pH, the receptor-mediated endocytosis for the entry of HIV is followed. In addition, ligand-uptake via receptors is 1000-fold more faster than random endocytosis in antigen presenting cells (Singer and Lindermann 1990). The receptor-mediated internalization of membrane polypeptides have been identified similar to the pathways of ligand bindings, e.g. lectin or antibody, in our previous works (Wang et al. 1989, 1990) and in others (Goldstein et al. 1985; Tvede et al. 1988; Dunn et al. 1989). Various vesicles and vacuoles containing CD4-Ab-PO complexes near the Golgi areas were observed, similar to the accumulation of T-cell growth factor, IL-4, and other membrane polypeptides in cultured T-cells (Wang et al. 1989). In parallel with results obtained by fracture-flip immunogold technique (Pavan et al. 1990), homogeneous distribution of CD4 molecules was observed on cell surfaces after fixation. While diverse densities of patches were revealed after various reincubation time. Independent experiments of FITC-stained and peroxidasestained T cells before and after fixation showed homogeneous and diverse distributions of capping CD4-Ab complexes indicating that movement of CD4 molecules were induced by ligand binding. Entry of HIV by endocytosis and fusion with the plasma membrane are CD4 dependent and part of the endocytosed material may exposed through endosomes (Dunn et al. 1988; Stoorvogal et al. 1991) to the lysosomal compartment (Habeshaw et al. 1989) which could be one of the pathway of signal transduction. Wheather or not the CD4 molecules internalize together with HIV particles is still unclear. However, the

58 significance of CD4 internalization may indicate T cell activation (Blue et al. 1987) through the binding of tyrosine kinase p56 lck (Pinching and Nye 1990). After HIV binding, phosphorylation of CD4 molecules is induced (Acres et al. 1986; Fields et al. 1988). Down-regulation and internalization of CD4 molecules from the cell surface induced by phorbol esters has been suggested (Hoxie et al. 1986; Blue et al. 1987), however, CD8 molecules were not affected (Bedinger et al. 1988). The work of Fields et al. (1988) also indicated that a protein kinase C inhibitor, H7, can block phosphorylation of CD4 molecules induced by HIV binding, but cannot block the binding between HIV and CD4 receptor. When the intracellular domain of CD4 was mutated or replaced, CD4 molecules were not internalized after phorbol ester treatment, and domains beyound the H1V-binding region were not necessary for HIV infection (Bedinger et al. 1988). These results suggest that CD4-phosphorylation is not required for HIV infection, and it seems possible that internalization of CD4 molecules is not necessary for viral infection. However, internalization and rapid accumulation o f CD4/gpl20 complexes intracellularly have been demonstrated and could be the result of rapid decrease of surface CD4 antigens after HIV infection (Stevenson et al. 1988). Further experiments also indicated that there was a decrease of CD4 molecules on the HIV infected cell surfaces in vitro (Schnittman et al. 1989). It was suggested that internalization of CD4 was accompanied with HIV envelope proteins, and reduced amount of CD4 m R N A also occurred prior to the reduction of CD4 molecules on the surface (Hoxie et al. 1986). Our results suggest that non-HIV induced internalization of CD4 antigens may involve a receptor-mediated endocytosis to down-regulate the over-expressed CD4 molecules on the surface. A histiocyte lymphoma cell line, U937 cells, was reported to express a high level of CD4 molecules on the cell surface (Asjo et al. 1987). However, fresh histiocytes from bone marrow of a patient o f histiocyte lymphoma in our experiments were negative for CD4. Nonspecific PO reactions in the cytoplasmic granules of a histiocyte (Fig. 6) can be taken as a positive control for negative surface CD4-Ab-PO deposits. Normal human bone marrow progenitor cells have been reported to be the target cells of HIV-1 (Folks et al. 1988), however, CD4 molecules were not detected on fresh human bone marrow cells. The monocyte/macrophage cells were susceptible to HIV-1 infection (Ho et al. 1986; Kazazi et al. 1989; Valentin et al. 1990) and CD4 molecules may present in a variable degree on the surfaces of human monocytes and macrophages (Dalgeish et al. 1984; Klatzmann et al. 1984; McDougal et al. 1986; Kazazi et al. 1989). The mature monocyte population which expresses CD4 molecules have been shown to be infected by HTLV-III/ LAV (Ho et al. 1986; Nicholson et al. 1986; Westervelt et al. 1991). Weak CD4 binding have been demonstrated on about 12% of the unfixed peripheral lymphocytes, however, these receptors were not detectable on the fresh and fixed peripheral monocytes in our results. The less CD4 expression on monocyte/macrophages than CD4 +

T-helper cells and the method of fixation applied in this study may both reduce the detections of EM-immunocytochemistry. It has been suggested that CD4 molecules could not be needed for monocyte virus enhancement mechanism (Homsy et al. 1989) and neither for HIV infection to the adherent macrophage after 5 days of incubation (Kazazi et al. 1989). The absence of CD4 molecules in the Golgi apparatus after 45 rain of reincubation in this work parallels our previous findings with surface polypeptides and glycoproteins (Wang et al. 1989, 1990). The blocking of intracellular synthesis and transportation of HIV glycoproreins by CD4 molecules occurs in the endoplasmic reticulure but not in the Golgi saccules (Buonocore and Rose 1990). The Golgi apparatus is involved in the degradation and recycling of the receptor for epidermal growth factor (EGF-R) with or without ligand binding (Beguinot et al. 1985). However, recycling of CD4 did not occur in this experiment. Recycling and reexpression of CD4 molecules were found in phorbol ester-treated peripheral T-cells, but CD4 was not reexpressed in other lymphoidal cells (Hoxie et al. 1986), nor in our IL-2 treated CD4 + T-cells. We suggest that these CD4-containing vesicles of different shapes and sizes may indicate a signal transduction and an accumulation or possible a degradation pathway of CD4-Ab-PO complexes without the involvement of the Golgi saccules. Using immuno-electron microscopy, in situ hybridization or polymerase chain reaction to study the relationship between regulatory factors and latent viruses within the host cells will be important work. We suggest that CD4-enriched Tcells could be a good model for further studies of the infection, replication and assembly of HIV in human cells in vitro.

Acknowledgements. We thank the supports from the National Science Council of the Republic of China by grants of NSC-78-0412B016-68 and NSC-79-0412-B016-79. References

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