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Morphology and phenotype of dendritic cells from peripheral blood and their productive and ... whilst type 1 DC did not support virus growth. Examination of ...

Immunology 1991 72 361-367

ADONIS 0019280591000624

Morphology and phenotype of dendritic cells from peripheral blood and their productive and non-productive infection with human immunodeficiency virus type 1 S. PATTERSON, J. GROSS, P. BEDFORD & S. C. KNIGHT Division of Immunological Medicine, Clinical Research Centre, Harrow Acceptedfor publication 3 December 1990

SUMMARY Immununoelectron microscopy of human peripheral blood mononuclear cells enriched for the presence of antigen-presenting dendritic cells (DC) has revealed two morphologically distinct cell types both expressing DR and DQ major histocompatibility complex (MHC) class II antigens but lacking T, B, natural killer (NK) and monocyte/macrophage markers. The first (type 1) has an irregular surface with numerous projections and shows cytoplasmic vacuoles. The second (type 2) has a paler nucleus showing only a thin rim of dense heterochromatin, large expanses of cytoplasm devoid of organelles, fewer vacuoles and a smooth cell boundary with few processes. In addition a few cells with a morphology similar to veiled cells of the afferent lymphatics (type 3 DC) were observed. Cells with a morphology intermediate between these three types were observed, suggesting that they may represent stages of the veiled cell differentiation pathway. Type 2 and 3 DC were shown by electron microscopy to be susceptible to productive infection with human immunodeficiency virus (HIV), whilst type 1 DC did not support virus growth. Examination of infected DC preparations by in situ hybridization revealed a higher number of DC positive for viral DNA and RNA than for RNA alone. Thus, in addition to productively infected DC, there may be some that are latently infected, contain defective virus genome or replicate virus at a very low level.

INTRODUCTION Skin Langerhans' cells, blood dendritic cells, afferent lymph veiled cells and interdigitating dendritic cells (DC) in the Tdependent area of the lymph node are thought to belong to a single family of cells that have their origin in the bone marrow 1-6 although there are conflicting views on their precise lineage.78 These cells are highly efficient presenters of antigen in the context of major histocompatibility complex (MHC) class II glycoproteins. They differ from other MHC class TI-bearing cells in that they are potent stimulators of resting and memory as well as recently primed lymphocytes.9"0 The inter-relationship between these cells is illustrated by Langerhans' cells, which are able to acquire antigen in the skin, migrate as veiled cells via the afferent lymphatics to the T-dependent region of the lymph node, and there to differentiate into antigen-presenting interdigitating DC.2'5 DC of the afferent lymphatics are heterogenous with respect to morphology and phenotype. For example, in the rat these cells are heterogenous in morphology3 with some cells exhibiting surface folds or veils whilst others are characterized by short blunt pseudopodia. More recently these cells have been shown

to be heterogenous with respect to the expression of rat Thy- . 1 antigen and interleukin-2 (IL-2) receptors.""2 Human peripheral blood DC are susceptible to human immunodeficiency virus (HIV) infection in vitro'3 and HIV can

also be detected in the DC from HIV-infected individuals.'4 DC infected in vitro' with HIV and DC from HIV-infected individuals'4 are functionally impaired and this may contribute to the development of immunosuppression in acquired immunodeficiency syndrome (AIDS). These findings have prompted us to conduct a more detailed morphological study of peripheral blood DC in relation to HIV infection. DC with distinct morphologies were identified which differed in their permissiveness for productive HIV infection in vitro. Furthermore, studies employing in situ hybridization to detect viral DNA and RNA suggested that DC may become latently infected with HIV and thus provide a site where the virus could persist and evade the host immune system.

MATERIALS AND METHODS Cells DC-enriched preparations were prepared from peripheral blood as previously described.'6 Briefly, mononuclear cells from the blood of normal human volunteers were separated by centrifugation over Ficoll. They were then incubated on plastic overnight and the non-adherent low density cells isolated by

Correspondence: Dr S. Patterson, Division of Immunological Medicine, Clinical Research Centre, Watford Road, Harrow HAl 3UJ, U.K.

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centrifugation over metrizimide (13.7% v/w). These preparations contained 20-40% DC, the remaining cells being mainly monocytes; less than 3% were lymphocytes.

Morphological and immunolabelling studies Electron microscopy. Cells were pelleted by centrifugation, fixed in 3% glutaraldehyde in 0 1 M phosphate buffer, pH 7-2, and processed for thin section electron microscopy. For immunogold labelling studies 20 nm colloidal gold was prepared by the method of Frens'7 and then conjugated to staphylococcal protein A.'8 DC preparations were labelled with monoclonal antibodies directed against MHC class II DR (Becton-Dickinson, Mountain View, CA) and DQ (Leu-10; Becton Dickinson) determinants or with anti-CD14 (Leu-M3; Becton-Dickinson), CDl9 (Dako, Glostrup, Denmark), CD16 (Leu-l B; BectonDickinson) and CD3 (Leu-4; Becton-Dickinson), which are specific markers for macrophages/monocytes B, natural killer (NK) and T cells, respectively. All immunolabelling was performed on ice in the presence of 10 mm sodium azide. Cells were washed once in phosphate-buffered saline (PBS), then incubated for 45 min in appropriately diluted antibody followed by two washes and labelling for 45 min with protein A gold. After washing twice they were fixed in glutaraldehyde. Some preparations were incubated to detect endogenous peroxidase. '9

Table 1. Detection of HIV DNA and RNA by in situ hybridization in DC infected in vitro % cells labelling for

DNA

RNA

Donor I 5 days after infection 10 days after infection

62 46

57 29

Donor 2 5 days after infection 10 days after infection

65 46

40 27

Control HIV infected cells H9 cells persistently infected with the ITIB strain of HIV

89

89

In earlier unpublished experiments prior to the development of the combined immunolabelling and in situ hybridization, which allows unequivocal identification of DC, we also noted a greater proportion of cells labelling for HIV DNA than for RNA after in vitro infection of DC preparations.

Infection with HIV Between 5 x 105 and 106 DC enriched cells in 1 ml bicarbonatebuffered RPMI-1640 medium supplemented with 10% foetal calf serum (FCS), penicillin/streptomycin (100 IU/ml) and Lglutamine (2 mM) were infected with approximately 104 TCID50 units of the IIIB strain of HIV. They were cultured for 5 or 10 days before processing for electron microscopy or in situ hybridization. Cells to be examined by in situ hybridization were first labelled with a mixture of antibodies (CD 14, CD3, CD 19, CD1 6), which are specific for monocytes/macrophages, T, B and NK cells. After washing they were adsorbed onto poly-Llysine-coated glass slides and fixed for 45 min in 4% paraformaldehyde in 0-1 M phosphate buffer, pH 7-2. Antibody labelling was detected by the alkaline phosphatase/anti-alkaline phosphatase (APAAP) staining technique20 and HIV nucleic acid sequences were detected by in situ hybridization using an HIV BH1O lamda DNA probe2' labelled with 35S ATP and CTP by nick translation.22 The cell permeabilization, hybridization and washing procedures were as previously described.23 To label RNA and DNA simultaneously, hybridization mixture was added to the slide which was then heated to 950 for 6 min to denature the DNA, whilst to detect RNA alone the hybridization mixture was boiled for 5 min, rapidly cooled on ice and then added to the specimen. Infected cells binding radiolabelled DNA probe were detected by autoradiography and DC were identified by morphology and the absence of APAAP staining. H9 cells persistently infected with HIV or uninfected were processed in parallel with DC preparations. RESULTS

Morphology and immunogold labelling of uninfected DC preparations By electron microscopy the DC showed a range of morphologies; at one end of-the spectrum were cells that had moderately

Figure 1. Type 1 peripheral blood DC immunogold labelled for DR. This cell can be differentiated from type 2 DC by its numerous short pseudopodia, more frequent vacuoles and a nucleus which is more electron dense overall and has more electron dense heterochromatin at its margins. Bar represents 2 p.

electron dense nuclei, contained occasional vacuoles and had numerous short pseudopodia (Figs 1 and 2). These cells were similar to the human DC described by Van Voorhis et al.24 At the opposite end of the spectrum was a second major group of DC that had a smoother cell boundary with fewer processes, a nucleus containing mainly the less dense euchromatin with the dense heterochromatin being restricted to a thin rim adjacent to the nuclear membrane and expanses of cytoplasm relatively devoid of organelles (Figs 3-6). We have termed the former major cell population type 1 DC and the latter type 2 DC. Cells

Morphology and phenotype of DC infection with HIV-J

Figure 2. Type 1 peripheral blood DC immunogold labelled for DQ. Bar represents 1 p.

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Figure 4. Type 2 peripheral blood DC immunogold labelled for DQ. Bar represents 2 p.

Figure 3. Type 2 peripheral blood DC immunogold labelled for DR. This cell can be differentiated from type 1 DC by its smoother surface, expanses of cytoplasm devoid of organelles and by a nucleus that is generally paler and has only a thin rim of electron dense heterochromatin adjacent to the nuclear membrane. Bar represents 2 p.

with a morphology intermediate between types I and 2 were also observed (Fig. 7). In addition, a small number of cells were present that showed long thin processes (Fig. 8) which probably correspond to the folds or veils observed by scanning electron microscopy on veiled cells of the afferent lymphatics.3 The type 2 DC in Fig. 6 shows elongated processes which may represent veils and thus could represent a cell on the veiled cell differentiation pathway. Based on morphological criteria, a possible

Figure 5. (a) Type 2 peripheral blood DC 5 days after HIV infection. Bar represents 3 p. (b) Higher magnification of area indicated by arrows showing mature and budding (arrows) virus. Bar represents 300 nm.

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Figure 6. Type 2 peripheral blood DC 5 days after HIV infection. Arrows indicate virus particles. The long process may represent veils and thus provide a morphological link to the veiled cell. Bar represents 2 j.

Figure 7. Peripheral blood DC immunogold labelled with antibody to DR. This cell has a morphology intermediate to that of the type I and 2 DC. Bar represents 1 p.

differentiation pathway can therefore be traced from type 1 DC through type 2 DC to veiled cells. Both types of DC expressed MHC class II DR (Figs I and 3) and DQ antigens (Figs 2 and 4), and were negative for membrane markers specific for monocytes/macrophages, NK T and B cells. In addition, they lacked peroxidase-positive granules or endoplasmic reticulum which are characteristics of monocytes and macrophages, respectively. The ratio of type 1 to type 2 DC varied greatly in different preparations. In some experiments adherent cells were removed by incubating on plastic for 90 min prior to centrifugation over metrizamide. The DC in these preparations showed the same range of morphologies as those subjected to overnight culture. HIV infection of DC in vitro In a series of 12 experiments, seven out of the 12 showed evidence of HIV growth in DC by electron microscopy, with up to 20% of the DC infected. Virus was observed budding through

the cell membrane and mature virus was present on the cell surface (Fig. 5). However, virus replication was limited to type 2 DC and those cells resembling veiled cells (type 3 DC) (Figs 5, 6 and 8; see also Figs 1 and 2 from Patterson & Knight'3). HIV DNA and RNA in in vitro infected DC By in situ hybridization up to 60% of DC were found to be infected after 5 days in some experiments (Fig. 9). The percentage of infected cells was reduced after 10 days. Uninfected cultures contained 20-40% DC after 10-14 days, indicating there is not a preferential loss of DC during culture. When the numbers of cells positive for DNA and RNA or RNA alone were estimated, a greater number of cells were found to be positive for RNA and DNA than for RNA alone (Table 1).

DISCUSSION This work describes two morphologically distinct peripheral blood cells of DC phenotype and a cell resembling afferent

Morphology and phenotype of DC infection with HIV-J

Figure 8. (a) Peripheral blood DC with veiled cell morphology 5 days after HIV infection. Arrows indicate area with virus particles. Bar represents 2 u. (b) Higher magnification of the area indicated by the arrows. Bar represents 500 nm.

Figure 9. In situ hybridization to detect HIV DNA and RNA in a preparation of peripheral blood DC 5 days after HIV infection. Bar represents 20 M.

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lymph veiled cells. The data suggest that they may form a developmental pathway, with the earliest stage being resistant to productive HIV infection whereas the later stages can be productively infected with virus. Cells termed type I DC have numerous short pseudopodia and are similar to those previously described.24 The type 2 DC, on the other hand, have fewer processes, nuclei with only a thin rim of dense heterochromatin, and are characterized by areas of cytoplasm relatively devoid of organelles. The expression of MHC class II antigens, DR and DQ, on both type 1 and 2 DC, and the absence of markers for other cell types of the peripheral blood suggests that they are both of the DC lineage. Previous work has suggested that DQ is found on peripheral blood DC but not on macrophages.25 Furthermore, the observation of cells with an intermediate morphology between type 1 and 2 cells indicates that they may represent different stages of the same differentiation pathway. A small number of cells morphologically similar to veiled cells was also observed and termed type 3 DC. Interestingly, some type 2 DC showed elongated processes which may represent the initial stages of the development of veils, although further evidence is needed for this to be substantiated. Some indications that human DC at different stages of development could be identified by electron microscopy were reported from the comparison between DC from peripheral blood and from the joints of patients with inflammatory arthritis; the DC in the joints were mainly of the 'more activated' appearance that we describe here as type 2 or type 3 DC.26 Interestingly, these type 2 DC from the joint were occasionally observed forming intimate contacts with lymphocytes (S. C. Knight, P. Fryer, S. Griffiths, B. Harding, J. Dixey and B. M. Ansell, unpublished observations), suggesting that they may be able to cluster lymphocytes, a property characteristic of DC.27 Differentiation could be driven by cytokines, such as granulocyte-macrophage colony-stimulating factor, as has been proposed for rat DC" and investigations to test this possibility are now in hand. Productive in vitro HIV infection of type 2 DC and of cells resembling veiled cells was observed by electron microscopy. This indicates that there are physiological or biochemical differences between type 1 and 2 DC despite the similarity in their membrane antigen profile. Susceptibility to infection does not seem to reflect differential expression of the virus receptor, as preliminary data suggest that both cell types can express low levels of CD4 (S. Patterson, J. Gross, P. Bedford and S. C. Knight, manuscript in preparation). Other investigations have shown that HIV replicates preferentially in activated or differentiated mature cells. For T lymphocytes productive infection only occurs in activated and not resting cells,2829 whilst visna, the sheep lentivirus, replicates in differentiated macrophages and not in the monocyte precursory The present findings of growth in type 2 and not in type I DC may indicate that type 2 DC are further along the differentiation pathway, whilst the observation of HIV replication in cells resembling veiled cells suggests that these cells are closer to type 2 than type 1 DC in the differentiation pathway. These conclusions correlate closely with the developmental pathway proposed on the basis of cell morphology. At present we have no evidence to suggest that HIV causes a change in DC morphology. In lymphocytes and macrophages the ability to replicate virus is thought to reflect the presence of cellular NF kappa Blike transcription factors that bind to regulatory enhancer sequences in the viral LTR.3'32 Whether similar factors are

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present in DC and determine latent versus productive viral infection is, as yet, unknown. Infected DC were readily identified by the combined immunolabelling in situ hybridization technique. These experiments showed a significantly greater number ofinfected cells than were observed by electron microscopy and may indicate the greater sensitivity of the technique. However, we have performed other experiments in which only a low percentage of DC were identified as being infected by in situ hybridization. The reason for this variation is not yet clear but probably reflects differences in different DC preparations, rather than in the virus, since H9 cells, a CD4-bearing T-cell line, showed consistently high rates of infection with the same preparation of virus. The reduction in the percentage of infected DC at 10 days compared with those at 5 days suggests that DC are killed by HIV infection. This finding correlates with our in vivo studies in which a marked reduction in DC numbers was observed as patients progressed to the AIDS stage of disease.'4 The finding that more DC were positive for viral DNA than for RNA may indicate that some DC are latently infected or transcribing very low amounts of RNA. From the percentage of cells with viral DNA (Table 1) it is possible that some type 1 DC may be latently infected. An alternative explanation is that some cells are infected with defective virus. Elucidation of this problem may be possible by employing reagents such as phorbol esters to stimulate virus replication in latently infected cells. Latent infection of DC could provide the virus with a site where it is able to evade immune surveillance and persist in the host.

ACKNOWLEDGMENTS The authors thank Dr G. L. Asherson, Clinical Research Centre, Harrow for critically reading the manuscript and Mrs J. Gilbert for preparing it for publication.

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