The Fas/FasL system and T cell apoptosis in HIV-1-infected lymphoid tissue during highly active antiretroviral therapy.

Apoptosis has been proposed as a mechanism responsible for T cell depletion in HIV-1 infection. In the present study we have phenotyped apoptotic T cells in tonsillar lymphoid tissue from 11 HIV-1-infected patients by flow cytometry light-scatter characteristics during 48 weeks of highly active antiretroviral therapy (HAART). We found that the decline in tonsillar viral load was associated with a decrease in the proportion of apoptotic CD4+ and CD8+ T cells. CD4 cell apoptosis was predominantly seen within the memory CD28+ Fas+ FasL+ population. The increased level of apoptotic CD8+ T cells was found among activated Fas+ memory cells irrespective of CD28 and FasL expression. These T cell subsets were expanded in untreated infection, but normalized with therapy. We conclude that HIV-1 triggers FasL-mediated apoptosis of uninfected CD4+ T cells, whereas CD8+ T cell apoptosis is driven by chronic immune activation. Virus suppression reverses both of these mechanisms, contributing to immune reconstitution during HAART.

Isolation of human tonsillar dendritic cells.

Tonsillectomy remains a frequently performed operation in developed countries ensuring that tonsils are the most readily available source of human lymphoid tissue and an easily accessible source of dendritic cells (DC). Tonsil lymphoid tissue also provides a source of the different DC that are resident within the B- and T-cell microenvironments. Although an alternative model for follicular dendritic cell (FDC) ontogeny has been proposed (1) the FDC within tonsil B cell areas probably develop in situ from mesenchymal precursors (2). Whatever their origin, the phenotype and function of FDC (3) seem to be unrelated to the bone-marrow-derived DC that are the subject of these protocols. The precise relationship between the distinct sub-populations of the bone-marrow-derived DC within the tonsil is still not clear (see ref. 4 for review).

Deregulation of the CD95/CD95L system in lymphocytes from patients with primary acute HIV infection.

OBJECTIVE: To analyze the role of CD95/CD95 ligand (CD95L) expression and functionality in peripheral blood lymphocytes (PBL) during primary, acute HIV syndrome (AHS) and in the subsequent period. PATIENTS: Twelve patients were studied during the acute phase of the viral infection and most were followed for some months. METHODS: Cell culture and cytotoxicity assays based upon 51Cr release and flow cytometry were used to evaluate cell killing via CD95 molecule, flow cytometry to assess surface antigens, enzyme-linked immunosorbent assay (ELISA) for the determination of soluble CD95 and CD95L plasma levels, quantitative competitive (QC) reverse transcription polymerase chain reaction (RT-PCR) with an original RNA competitor for the analysis of CD95L mRNA expression and QC RT-PCR for determining plasma viral load. RESULTS: The analysis of PBL during this phase revealed that almost all cells, including CD8 T cells with a virgin phenotype, B lymphocytes and natural killer cells displayed CD95 molecules on the plasma membrane. Activation of CD95 on the surface of isolated lymphocytes by anti-CD95 monoclonal antibodies or binding to CD95L induced rapid apoptosis. However, CD95L mRNA was not expressed in PBL from these patients and was poorly inducible. Soluble CD95 was found in the plasma of all patients, but only in a few at high levels, even some months after seroconversion. In contrast, soluble CD95L was detected in only one patient, this occurring after the symptomatic period. For 10 of the 12 patients, expression of CD95 on the cell membrane or in the plasma did not correlate with the plasma viral load, which varied widely from patient to patient. Further, plasma levels of soluble CD95 were not altered by decreased lymphocyte activation or by efficient antiretroviral therapy. CONCLUSIONS: In patients experiencing an acute, primary HIV infection, a prolonged deregulation of the CD95/CD95L system may exist, which is probably not entirely related to virus production but may contribute to the pathogenesis of the disease. The hypothesis can be put forward that a complex balance exists between proapoptotic events (increase in CD95 expression), probably triggered by the host as a method to limit viral production, and antiapoptotic events (decrease in CD95L expression) probably triggered by the virus as a way to increase its production and survival.

Correlates of apoptosis of CD4+ and CD8+ T cells in tonsillar tissue in HIV type 1 infection.

The immunopathogenesis of human immunodeficiency virus type 1 (HIV-1) infection has been associated with increased death by apoptosis of T cell subsets. In the present study, we have examined correlates of apoptosis of CD4+, CD8S+CD28+, and CD8+CD28- T cells in tonsillar lymphoid tissue in persons with HIV-1. Single-cell suspensions of tonsillar lymphocytes were analyzed by flow cytometry to determine the fraction of cells showing typical characteristics of apoptosis as well as the expression of activation markers within the live and the apoptotic cell populations. The proportion of cells carrying infectious provirus was quantified by limiting dilution analysis. Compared with uninfected controls, apoptosis of both CD4+ and CD8+ T cells was enhanced in HIV-1 infection and was higher among CD8+ than among CD4+ T cells. Apoptosis of CD28-cells was more prevalent than apoptosis of CD28+ cells for both CD4+ and CD8+ T cells. Occurrence of apoptosis of CD4+ T cells correlated with provirus levels and proportional expression of the activation marker HLA-DR. Apoptosis of CD8+CD28+ cells correlated with expression of the activation markers CD69 and HLA-DR while apoptosis within CD8+CD28- cells did not correlate with any of the studied parameters. Although apoptosis was much more prevalent among CD8+ than CD4+ T cells, CD8+ T cells still accumulated in tonsillar lymphoid tissue in persons with HIV-1. Our data may be interpreted to suggest that apoptosis of CD4+, CD8+CD28+, and CD8+CD28- cells in tonsillar tissue is regulated by different mechanisms and the results are of importance to our understanding of the immunopathogenesis of HIV-1 infection.

Developmental cell lineage.

Studies of the role of cell lineage in development began in the 1870s, fell into decline in the first half of the 20th century, and were revived in the 1960s. This revival was attended by the introduction of new and powerful analytical techniques. Cell lineage can be inferred to have a causative role in developmental cell fate in embryos in which induced changes in cell division pattern lead to changes in cell fate. Such a causative role of cell lineage is suggested also by cases where homologous cell types characteristic of symmetrical and longitudinally metameric body plan arise via homologous cell lineages. The developmental pathways of commitment to particular cell fates proceed according to a mixed typologic and topographic hierarchy, which appears to reflect an evolutionary compromise between maximizing the ease of ordering the spatial distribution of determinants of commitment and minimizing the need for migration of differentially committed embryonic cells.

Effects of monocyte purification and culture on integrin expression.

Selective alterations in the surface expression of members of the LeuCAM (leukocyte cell adhesion molecule) family of integrins occur during in vitro culture of human monocytes. Such changes may relate in part to cellular maturation, but also to activation following purification and culture of monocytes. In this paper, we examined the effects of monocyte isolation, adherence during culture and endotoxin exposure on the expression of these molecules and the ligand for LFA-1, ICAM-1 (CD54). Expressions of CD11b, CD18 and CD54, but not CD11a or CD11c, were higher on monocytes freshly isolated by density gradient separation and plastic adherence as compared with cells labelled directly in whole blood. However, the surface expression of the LeuCAMs and CD54 on cultured monocytes was not affected by short-term adherence to plastic for 2 h, as determined by comparisons of their expression on adherence-isolated and elutriated monocytes. In contrast, prolonged adhesion of monocytes for up to 21 days in culture altered expression of CD11a without affecting that of the other LeuCAMs or CD54. Expression of CD11a decreased more rapidly on adherence-maintained cells as compared with suspension-cultured cells. Our results show that cellular manipulations required for in vitro studies of monocyte/macrophages may alter expression of the LeuCAMs.

Macrophage tropism: fact or fiction?

Primary HIV-1 isolates can be distinguished by phenotypic qualities such as the ability to productively infect cells of established CD4-positive lines and to induce syncytia in MT-2 cells. Such viral phenotypes have also been reported to confer host cell specificity. It is perceived that primary isolates with the syncytium-inducing phenotype (SI or rapid/high) are T cell tropic and are therefore unable to infect primary cells of the monocyte/macrophage lineage. However, we have consistently found that these isolates are as capable of establishing infection in monocyte-derived macrophages (MDM) as the monocytotropic, non-syncytium-inducing variants (NSI or slow/low). It is known that differentiation, activation, and proliferation of human monocytes are affected by both isolation methods and culture conditions. Therefore, to test whether our inability to discriminate macrophage tropic HIV-1 isolates could be explained by differences in culturing techniques, we isolated monocytes by elutriation or short-term adherence and allowed the cells to mature and differentiate in either the presence or absence of autologous lymphocytes. After removal of nonadherent cells, MDM were infected with a panel of SI and NSI primary HIV-1 isolates. MDM were susceptible to infection by the SI as well as the NSI isolates, regardless of whether or not the cells were allowed to mature in the presence of autologous lymphocytes. However, MDM matured in the presence of autologous lymphocytes replicated HIV-1 isolates (both NSI and SI) to a higher titre than MDM matured in the absence of lymphocytes. In light of these findings and recently published reports on HIV-1 phenotype and chemokine receptor usage we believe that the term macrophage-tropic strains of HIV-1 is no longer appropriate.

Effects of HIV-1 on the surface expression of LFA-1 on cultured monocytes.

CD11a, the alpha chain of LFA-1, which is a member of the LeuCAM family of integrins, has been implicated in the formation of HIV-induced syncytia and may contribute to the depletion of CD4-positive lymphocytes seen in patients with HIV infection. In this study, we examined the effects of HIV-1 infection on the expression of CD11a on cultured monocyte-derived macrophages (MDMs). Monocytes isolated from peripheral blood and maintained in suspension culture were infected in vitro with a monocytotropic variant of HIV-1 (Ba-L). Surface expression of CD11a, measured by indirect immunofluorescence and flow cytometry, was significantly higher on HIV-infected cells than on mock-infected cells from the same donor. Upregulation of CD11a expression was unaffected by the HIV reverse transcriptase inhibitor, zidovudine, indicating that it did not depend on reverse transcription. A step before reverse transcription, such as viral binding, appears sufficient to trigger an increase in CD11a expression. This hypothesis is supported by our findings of soluble recombinant CD4 inhibition of HIV-induced CD11a upregulation. It is possible that induction of a cytokine network by HIV underlies this effect, given our findings that exposure of uninfected MDMs to granulocyte-macrophage colony-stimulating factor (GM-CSF) specifically increased CD11a expression and that HIV-infected MDMs secreted more GM-CSF than mock-infected cells.

The kinetics of surface expression of CD11/CD18 integrins and CD54 on monocytes and macrophages.

Cells of the macrophage lineage mediate extremely important normal functions of the immune system. Such functions are in part related to interactions between cell-bound LeuCAMs and their ligands. MoAb staining and flow cytometric analysis were used to follow changes in surface expression of LeuCAMs and the LFA-1 ligand CD54 during maturation of peripheral blood monocytes (BM) in vitro. Surface expression of these molecules increased on BM following isolation, the greatest increase being in CD54 and CD11c. Following an initial increase, there was a reduction in CD11a expression after 2 weeks in culture, this being greater on adherent compared with suspension-maintained cells. Expression of CD11b remained high throughout the culture period. LeuCAM and CD54 expression was further compared on freshly isolated alveolar macrophages (AM) and BM paired donors. A reciprocal relationship was observed between CD11c and CD11b on AM and BM, in that BM expressed higher levels of CD11b than CD11c, whilst the converse was true for AM. CD54 expression was also higher on AM than on BM, whilst there was no significant difference in expression of CD11a on these cells. These data suggest that consistent changes occur in the surface expression of the LeuCAMs and CD54 as monocytes mature into macrophages, which may reflect the specific functions of these cells.

Acetylcholine-induced retraction of an identified axon in the developing leech embryo.

At early stages of embryonic development of the glossiphoniid leech, Theromyzon rude, a branch, termed MAC, of the axon of the segmentally iterated Retzius neuron extends into the anterior interganglionic connective nerve. At later stages, this branch disappears again in about 30% of the Retzius neurons in the standard midbody segments and in about 75% of the Retzius neurons in the two reproductive midbody segments. The frequency of disappearance of the MAC branch increases to about 85% in all Retzius neurons upon exposure of the embryos to culture media containing 1 mM acetylcholine (ACh) and 10 microM physostigmine during a sensitive period of axon outgrowth. This disappearance represents a retraction of the MAC branch to its point of origin, while other axon branches of the Retzius neuron remained unaffected. In later development, the retracted (medial) MAC branch was replaced by a new (lateral) branch termed LAC. The observations were made using confocal microscopy of fixed embryos stained with anti-5-HT antibody and confirmed by Lucifer yellow injection of individual Retzius neurons. The specific retraction of a single axon branch might be attributable to the local presence of extracellular matrix molecules in the ganglionic neuropil, which is contacted only by the MAC axon branch and could render this branch susceptible to growth-regulating signals. Since Retzius axon morphology in standard segments of ACh-treated embryos resembled that of reproductive segments in untreated embryos, it appears possible that ACh treatment may have simulated a process that contributes to the segmental differentiation of the Retzius neuron.

Expression of CD11/CD18 and ICAM-1 on monocytes and lymphocytes of HIV-1-infected individuals.

The role of the leukocyte integrins in the infection of cells by HIV-1 or in the progression of AIDS-related disease is of continuing interest. Using a dual-labeling flow cytometric method, we determined the level of expression of the leukocyte integrins and intercellular adhesion molecule 1 (ICAM-1) on monocytes and lymphocytes from HIV-1-infected and uninfected individuals. The mean fluorescence of CD18 on lymphocytes and CD11c on monocytes of HIV-1-infected subjects was significantly higher than for the control group (P = .008 and .014, respectively). There was a trend toward higher fluorescence of CD11a on lymphocytes from HIV-1-infected subjects compared with cells from the control group (P = .089). Integrin expression on lymphocytes or monocytes from HIV-infected individuals did not correlate with their CD4 lymphocyte number. However, the mean fluorescence associated with ICAM-1 on monocytes and CD11a and CD18 expression on lymphocytes was related to clinical stage of disease.

HIV-1 infection of monocyte-derived macrophages reduces Fc and complement receptor expression.

Fc receptor (FcR) and complement receptor (CR) expression on HIV-infected monocyte-derived macrophages may be an important determinant of immune function. We studied the effects of HIV-1 infection of macrophages in vitro on FcR and CR expression. Macrophages were infected with HIV-1DV 7 days following isolation, and the expression of Fc gamma RI-III and CR3 were measured at intervals thereafter by flow cytometry. We found a reduction in receptor expression with the percentage of cells expressing FcRI 14 days post infection declining from 77% to 13%, FcRII fell from 96% to 85%, FcRIII from 45% to 9%, and CR3 from 91% to 67% 14 days following infection. As these receptors are important for macrophage function, their down-modulation may contribute to the pathogenesis of HIV-related disease.

Developmental origin of segmental identity in the leech mesoderm.

Segmentation in the leech embryo is established by a stereotyped cell lineage. Each of the 32 segments arises from homologous, bilaterally symmetrical complements of mesodermal and ectodermal blast cell clones. Although segments are homologous, they are regionally differentiated along the longitudinal body axis. Various segments display idiosyncratic ensembles of features, which constitute discrete segmental identities. The differentiation of segment-specific features, such as the mesoderm-derived nephridia, genital primordia and identified Small Cardioactive Peptide immunoreactive neurons, reflects a diversification of the developmental fates of homologous blast cell clones. We have investigated whether segment-specific differentiation of homologous mesodermal blast cell clones depends on cell-intrinsic mechanisms (based on the cells' lineage history) or on cell-extrinsic mechanisms (based on the cells' interactions with their environment) in embryos of Theromyzon rude. For this purpose, we first mapped the segment-specific fates of individual mesodermal blast cell clones, and then induced mesodermal clones to take part in the formation of segments for which they are not normally destined. Two types of ectopic segmental position were produced: one in which a mesodermal blast cell clone was out of register with all other consegmental cells and one in which a mesodermal blast cell clone was out of register with its overlying ectoderm, but was in normal register with the mesoderm and ectoderm on the other side of the embryo. Mesodermal blast cell clones that developed in either type of ectopic segmental position gave rise to segment-specific features characteristic of their original segmental fates rather than their ectopic positions. Thus, the development of segmental identity in the leech mesoderm is attributable to a cell-intrinsic mechanism and, either before or soon after their birth, mesodermal blast cells are autonomously committed to segment-specific fates.

Cell position and developmental fate in leech embryogenesis.

The o and p blast cell bandlets of the leech Theromyzon rude, which normally produce two different sets of identifiable cells designated the "O" and "P" fates, respectively, form an equivalence group: in embryos experimentally deprived of their p bandlet, the blast cells of the adjacent o bandlet may "transfate" and take on the P fate. Loss of the p bandlet is not, however, a sufficient condition for transfating of the o bandlet. Rather, loss of the p bandlet allows the o bandlet to shift into ectopic positions, and it is the ultimate position of the o bandlet that mandates which fate--O or P--the blast cells will take on. Therefore, the choice of the pluripotent o blast cells to follow either the O or P developmental pathway depends on their perception of positional cues provided by cells outside the equivalence group rather than on a direct interaction with p blast cell equivalence group members.