Down-regulation of CXCR-4 and CCR-5 expression by interferon-gamma is associated with inhibition of chemotaxis and human immunodeficiency virus (HIV) replication but not HIV entry into human monocytes

Alterations in the expression of CXCR4 and CCR5, the co-receptors for HIV entry, may be associated with susceptibility of monocytic cells to HIV infection. Interferon (IFN)-gamma has been shown to inhibit HIV replication in monocytic cells, but the molecular mechanism involved is not well understood. To determine if IFN-gamma regulates HIV replication by altering CXCR-4/CCR-5 expression and hence virus entry into monocytic cells, we investigated the effects of IFN-gamma on CXCR-4 and CCR-5 expression and its biological implications with respect to HIV entry, replication and chemotaxis towards the CXCR-4 and CCR-5 ligands SDF-1 and MIP-1alpha, respectively. IFN-gamma decreased CXCR-4 and CCR-5 expression on monocytes derived from HIV-negative adults, HIV-positive adults and HIV-negative cord blood. This down-regulation of chemokine receptor expression did not result in a corresponding change in mRNA expression but was associated with elevated levels of the endogenously produced chemokines SDF-1 and RANTES. Furthermore, IFN-gamma inhibited chemotaxis in response to SDF-1 and MIP-1alpha, inhibited HIV replication, but failed to inhibit virus entry in monocytic cells. These results suggest that although IFN-gamma-induced down-regulation of CXCR-4 and CCR-5 expression is associated with an inhibition of SDF-1-/MIP-1alpha-mediated chemotaxis, IFN-gamma-induced inhibition of HIV replication may be mediated at levels subsequent to the virus entry.     

Inhibition of thymopoiesis of CD34+ cell maturation by HIV-1 in an in vitro CD34+ cell and thymic epithelial organ culture model

The mechanisms by which HIV-1 affects thymopoiesis were determined by preincubating CD34+ cells or cultured thymic epithelial (CTE) cells with lymphotropic (T-) and monotropic (M-) strains of HIV-1 in an in vitro CTE organ and CD34+ cell coculture model that allows for analysis of development of thymocytes and mature T cells. When purified CD34+ cells were precultured with either T- or M-tropic strains of HIV-1, thymopoiesis was impaired in a two-week coculture manifested by decreased cell number of thymocytes generated. However, the percentages of thymocyte subpopulations were comparable to control uninfected cocultures. Furthermore, HIV infection of thymocytes was predominantly observed in the CD44+CD3- population. However, in a four-week coculture experiment, HIV infection and depletion of more mature thymocytes were also observed. When CTE cells were preincubated with T- and M-tropic strains of HIV before addition of CD34+ cells, the number of thymocytes and subpopulations of thymocytes at early and later stages of maturation were markedly decreased. Furthermore, CD34+ and CD44+CD3- cells become HIV-infected. In summary, HIV-1 infection inhibited thymocyte maturation at early stages of thymocyte maturation CD44+CD25-CD3-. In addition, HIV also depleted later stages of CD4+ thymocyte subpopulations.     

Expression of functional chemokine receptors of human placental cells

PROBLEM: Chemokine receptors of placental trophoblasts possibly act as co-receptors or alternative receptors of maternal fetal infection by HIV. To clarify their possible expression and the physiological roles of chemokines on human placentae, we studied chemokine chemokine receptor expression and the effects of exogenous chemokines on choriocarcinoma cell lines. MATERIALS AND METHODS: Placental samples were obtained from 13 placentae of various gestational ages. Villous tissue was mechanically dissected from samples. Trophoblasts were enriched by anti-human chorionic gonadotropin (hCG)-coated magnetic beads. Human choriocarcinoma cell lines (JAR, BeWo, JEG-3) were maintained in RPMI 1640 media supplemented with 10% FCS. Expression of chemokine receptors was studied by RT-PCR. The effects of MIP-1alpha, RANTES, MCP-1 on hCG production were estimated by EIA. Effects of chemokines on proliferation of choriocarcinoma cell lines were examined by MTT assay. RESULTS: We observed mRNA expression of CCR-1, 2, 3, 4, 5 and CXCR-1, 2, 4 in 1st trimester placental villi, CCR-I, 2, 4 and CXCR-1, 2. 4 in 2nd trimester placental villi, CCR-1, 2, 4 and CXCR-4 in 3rd trimester placental villi. Using MACS enriched trophoblasts, we observed identical results. A choriocarcinoma cell line BeWo expressed CCR-1, 3, 4 and CXCR-1, 2, 4 while JEG-3 and JAR expressed CCR-1, 3, 4, 5 and CXCR-1, 2, 4. Expression of the CCR-5 and CXCR-4 protein in choriocarcinoma cell lines and MACS-enriched trophoblats were confirmed by flow cytometry. Chemokine MCP-3, MIP-1alpha, RANTES mRNA were expressed by the 1st, 2nd and 3rd trimester placental samples and the three choriocarcinoma cell lines examined. MCP-1 was expressed by 1st and 2nd trimester placental villi. Administration of chemokines up-regulated proliferation (10(-1) - 10 ng/mL) and hCG production (10(-1) - 10(-2)ng/ mL) of the three choriocarcinoma cell lines examined. CONCLUSIONS: Our results suggest possible roles of chemokines/chemokine receptors on placental physiology and their involvement in HIV transmission as alternative receptors.     

Optimization of culture and storage conditions for an in vitro system to evaluate thymocyte phenotype and function

Studies on thymopoiesis are critical to the understanding of T-cell homeostasis as well as the host response to T-cell depletion. Various in vitro culture systems have been used in the study of thymocyte development; however it is unclear if current co-culture methods have been fully optimized. In this study in vitro suspension cultures have been re-evaluated and the optimal storage conditions for thymocytes have been established by evaluating various methods of storing/isolating thymic tissue and isolated thymocytes as well as the source of thymic epithelial cells (TEC). It was determined that thymocytes must be freshly isolated from whole thymic tissue and ideally stored at 4 degrees C prior to co-culture. Co-culture with either autologous or allogeneic TEC results in similar thymocyte subset distribution as well as interleukin-7 receptor-alpha (CD127) expression on these subsets. To evaluate the influence of the source of TEC on one aspect of thymocyte function the effect of IL-7 stimulation on the expression of CD127 was evaluated. IL-7 stimulation resulted in a downregulation of the expression of CD127 on all thymic subsets similar to that observed in circulating CD8+ T-cells. The effect of this was the same whether TEC were autologous or allogeneic. Optimizing culture techniques and facilitating the study of individual thymocyte subsets will lead to a better understanding of thymic function and development. It could also lead to therapeutic approaches that enhance immune recovery after T-cell depletion in HIV infection, bone marrow transplantation or following chemotherapy.     

The HIV-2 genotype and the HIV-1 syncytium-inducing phenotype are associated with a lower virus replication in dendritic cells

During sexual transmission, HIV infects the mucosal dendritic cells and is transferred to CD4 T cells. Whether HIV variants of a particular genetic (sub)type or phenotype selectively infect dendritic cells (DC) or are preferentially transferred to T cells remains highly controversial. To avoid the cumbersome use of primary dendritic cells, in vitro dendritic cell models were generated from precursors, either hematopoietic progenitor cells (HPC) or monocytes (MO). Productive infection in the dendritic cells and transfer of the virus to T cells was assessed for a range of HIV variants. HPC-derived dendritic cells (HPC-DC) were more susceptible to HIV-1 than to HIV-2 isolates. The HIV-1 group O strains were more productive in HPC-DC than group M, but amongst the latter, no subtype-related difference was observed. Both non-syncytium-inducing (NSI) and SI HIV isolates and lab strains could productively infect HPC-DC, albeit with a different efficiency. Adding blocking antibodies confirmed that both CCR-5 and CXCR-4 co-receptors were functional. Biological HIV-1 clones of the NSI/R5 phenotype infected more readily HPC-DC than SI/X4 clones. MO-derived dendritic cells were, however, more exclusive in their preference for NSI/R5 clones. Some HIV variants, that did not grow readily in HPC-DC alone, could be rescued by adding resting or pre-activated T cells. The present data show that HIV-2 isolates and SI clones replicate less in model-DC, but no preference for a particular HIV-1 subtype was evident. Co-culture with T cells could "correct" a limited growth in dendritic cells. Clearly, both intrinsic dendritic cell susceptibility and enhancement by T cells are explained only partly by HIV genotype and phenotype. The in vitro dendritic cell models seem useful tools to further unravel interactions between HIV, DC, and T cells.     

Enhanced replication of M-tropic HIV-1 strains in Herpesvirus saimiri immortalised T-cells which express CCR5.

A better characterisation of mononuclear cell-tropic (M-tropic) HIV-1 is central to disease control as these viruses predominate in disease transmission. M-tropic viruses do not replicate in conventional T-cell lines, and virus titres obtained in peripheral blood mononuclear cells (PBMC) are low. Human T-lymphocytes which have been immortalised by Herpesvirus saimiri strain C488 (HVS T-cells) are highly permissive to the replication of T-cell tropic strains of HIV. This study aimed to determine if HVS T-cells support replication of M-tropic HIV isolates that have not been adapted to conventional T-cell lines. A panel of PBMC low passage/primary field isolates and their molecular clones was used. Results show that infection in HVS T-cells was longer lived than in PBMC. In terms of peak virus titre and duration of productive infection, the two HVS T-cell lines studied were superior to PBMC, and one supported enhanced replication of all M-tropic isolates. This is important for generating M-tropic virus pools of sufficient titre for further biological studies such as virus neutralisation, co receptor usage and testing of antivirals. Phenotypic analysis showed that HVS T-cells are CD4+-activated memory cells expressing both CXCR-4 and CCR5 co receptors. Thus, HVS immortalisation appears to select for the T-cell subset targeted by HIV-1 in vivo.     

c-myc gene expression and activation of human thymocytes

The relationship between c-myc expression and thymocyte activation was studied in freshly isolated human thymocytes and in thymocytes activated with various inducing agents. In freshly isolated thymocytes c-myc mRNA is expressed at low levels, while thymocytes activated with Concanavalin A (Con A), the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), Con A in combination with TPA or interleukin 2 (IL-2) are induced to express higher levels of c-myc mRNA. The expression of c-myc is increased within 3 h of stimulation with these inducing agents; the amount of c-myc mRNA which is accumulated is not correlated with the rate of thymocyte proliferation. Dexamethasone and Cyclosporin A (CsA) which inhibit early events of T cell activation and the expression of the interleukin-2 (IL-2) and gamma-interferon (gamma-IFN) genes also markedly suppress the expression of c-myc mRNA in Con A, and Con A + TPA-activated thymocytes. We conclude that activation of c-myc gene expression is an early event observed in activated human thymocytes. The level of c-myc expression is dependent on the mode of thymocyte activation rather than on the rate of thymocyte proliferation. Since freshly isolated thymocytes express low levels of c-myc mRNA it is possible that IL-2 induces c-myc expression at least in a responsive subpopulation of thymocytes during ontogeny.     

Impaired migration of NOD mouse thymocytes: a fibronectin receptor-related defect

We previously showed intrathymic alterations in non-obese diabetic (NOD) mice, including the appearance of giant perivascular spaces, filled with mature thymocytes, intermingled with an extracellular matrix network. This raised the hypothesis of a defect in thymocyte migration with partial arrest of exiting thymocytes in the perivascular spaces. Herein, we investigated the expression of receptors for fibronectin [very late antigen (VLA)-4 and VLA-5] and laminin (VLA-6), known to play a role in thymocyte migration. When compared with two normal and one other autoimmune mouse strains, a decrease of VLA-5 expression in NOD thymocytes was noticed, being firstly observed in late CD4/CD8 double-negative cells, and more pronounced in mature CD4(+) and CD8(+) thymocytes. Functionally, thymocyte exit from the lymphoepithelial complexes, the thymic nurse cells, was reduced. Moreover, NOD thymocyte adhesion to thymic epithelial cells as well as to fibronectin was diminished, and so was the migration of NOD thymocytes through fibronectin-containing transwell chambers. In situ, intra-perivascular space thymocytes were VLA-5-negative, suggesting a correlation between the thymocyte arrest within these structures and loss of VLA-5 expression. Overall, our data reveal impairment in NOD thymocyte migration, and correspond to the first demonstration of a functional fibronectin receptor defect in the immune system.     

Control mechanisms of virus replication in naturally SIVsmm infected mangabeys and experimentally infected macaques

As a close cousin to Asian macaques, the African sooty mangabey monkey, a species naturally infected with SIV without ever developing disease, represents an intriguing and thought provoking model for the study of lentiviral infection and disease development. Pursuant to our previous findings that documented the presence of high frequencies of CD8+ T-cells capable of inhibiting lentiviral replication in vitro via a soluble factor, the potential contribution of beta-chemokines and their receptor was evaluated in samples from sooty mangabeys and disease susceptible macaques. Both mangabey and Rhesus macaque PBMC were found to secrete comparable levels of MIP-1alpha, MIP-1beta and RANTES after stimulation in vitro. Constitutive expression of CCR-5 appeared lower in mangabey PBMC but the vast majority of T-cells from mangabeys or macaques were found to express CCR-5 after in vitro activation. Sequence analysis of macaque and mangabey MIP-1alpha and RANTES showed complete homology to the human counterpart. MIP-1beta on the other hand while highly conserved among both monkey species, showed only 93% homology to human MIP-1beta. In addition, CCR-5, CCR-2b and CXCR-4 presented no consistent differences between rhesus and mangabey sequences. Thus, based on current data, differences in disease susceptibility between macaques and mangabeys do not appear to rely on differences in chemokine pathways. However, analyses of the ontogeny of CD8+ T-cell-mediated inhibition of SIV replication showed that macaque PBMC acquire this function shortly after infection, and show a progressive loss thereafter, correlated with progression towards disease. Mangabeys, on the other hand, appear to acquire the CD8+ T-cell inhibitory function long before any evidence of seroconversion to SIV and maintain this function for the lifetime of the animal. In vitro analyses of induction of the CD8+ inhibitory function showed that rhesus CD8+ T-cells have the potential to secrete the inhibitory factor(s) prior to SIV infection.     

CXCR-4 AND CCR-5 expression in normal term human placenta

The maternal-fetal interphase has an active Immunitary System (IS) whose mediators -cells, cytokines and chemokines- coordinately act to favour pregnancy normal development. It is not known exactly which of those mediators are present in each placental cellular stratus and what the physiological or potentially pathologic consequences derived from their presence can be. It is known that chemokines recruit cells with regulatory activity towards the deciduous and some of their receptors are coreceptors to infectious agents like HIV, making research of chemokines expression and their receptors in the maternal-fetal interphase of great interest in recent years. In the present study, the CXCR-4 and CCR-5 expression was investigated in 8 samples of normal human placenta obtained from term pregnancies, with low obstetric risk, by using Immunocitochemical techniques (Biotin-Avidin-Peroxidase). The most relevant finding in this study was the demonstration that CXCR-4 and CCR-5 differential expression in trophoblast, stroma and endothelium represents, as far as we know, the first report of the presence of these receptors in all layers of placental tissue. These results help to broaden the knowledge about the expression of chemokines receptors -that act as main coreceptors in the HIV infection- in the maternal-fetal interphase, and this can be a contribution to be taken into account in the vertical transmission study of this infectious agent.     

Induction of Apoptosis and p53 Expression in Immature Thymocytes by Direct Interaction with Thymic Epithelial Cells

Apoptosis of normal thymocytes was shown to be triggered by several mechanisms (e.g. glucocorticoids, γ-irradiation). In the present study the authors report on thymocyte apoptosis that is induced by thymic epithelial cells. The thymocytes undergo a massive apoptotic death within 24 h of cocultivation with thymic epithelial cell monolayers derived from primary cultures (PTEC) or from a thymic epithelial cell line (TEC). Non-thymic monolayers were inactive. Apoptosis induction in this experimental model requires direct contact between the thymocytes and the thymic epithelial monolayer and can be blocked by anti-CD2 and anti-LFA-1 antibodies. The immature CD3^−/+dull CD4⁺CD8⁺ thymocytes were the cells which undergo apoptosis. The fact that the authors are dealing with a massive apoptotic process of immature cells in the absence of exogenous antigen suggests that it involves the nonselected thymocytes. The apoptotic pathway selected by thymocytes following their culturing on TEC involves p53 expression. Indeed it was found that TEC-induced apoptosis, led to the accumulation of p53 protein that preceded the step of DNA fragmentation in freshly isolated thymocytes as well as in a glucocorticoid resistant thymoma cell line. Since glucocorticoid-induced thymocyte apoptosis is p53-independent, glucocorticoids are conceivably not involved in TEC-induced thymocyte death. The in vitro experimental model presented here may reflect the physiological sequence of events leading to thymocyte death in the thymus.     

Induction of interleukin 2 receptors on immature human thymocytes and co-expression of T3 and T6 antigens.

We have recently demonstrated that human thymocytes can be induced to express interleukin 2 (IL-2) receptors and to synthesize IL-2. The present study shows that relatively immature T6+ human thymocytes as well as the more mature T3+ thymocytes could be induced to express functional IL-2 receptors when activated with either Concanavalin A (Con A), Con A and 12-O-tetradecanoylphorbol 13-acetate (TPA) or IL-2 in combination with Con A or TPA. The phenotype of the common, immature thymocyte was identified by the binding of either fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated monoclonal anti-T6 (OKT 6) antibody and of mature thymocytes by the binding of monoclonal anti-T3 (OKT 3) antibody. We also observed that the expression of the T3 antigen on thymocytes, freshly isolated from thymic specimens obtained in the course of cardiac surgery of infants and children, was greater than 40% in 14 of 18 donors and that thymocytes co-expressed the T3 and the T6 antigen as determined by dual colour cytofluorometry. In thymocytes activated in vitro the expression of IL-2 receptors, determined by dual colour cytofluorometry with the PE-conjugated monoclonal anti-human IL-2 receptor antibody (PE anti-IL-2 R), was detected by the second day of induction in both immature T6+ and mature T3+ thymocytes. T6+ thymocytes proliferated in response to IL-2 and persisted in cultures for the duration of the study (18 days) and continued to express IL-2 receptors.     

Replication of M-tropic HIV-1 in activated human intestinal lamina propria lymphocytes is the main reason for increased virus load in the intestinal mucosa

The gastrointestinal tract is the site of early abundant HIV replication and associated marked CD4 T-cell depletion. The aim of this study was to characterize the basis for the increased HIV replication in this compartment. Isolated mononuclear cells of the peripheral blood (PBMCs), the intestinal lamina propria (LPMCs), and purified gut lamina propria CD4 T-cell subpopulations (LP T cells) were isolated, phenotypically characterized, and infected in vitro with 2 different HIV-1 strains. T-cell subpopulations were analyzed by fluorescence-activated cell sorter. HIV-1 core protein p24 was determined in supernatants after in vitro infection. Furthermore the effect of T-cell stimulation on the replication of M- and T-tropic HIV strains was studied. In vitro replication of HIV-1 was significantly increased in CD69 compared with CD69 CD4 LP T cells, while there was no difference between CD103 and CD103 CD4 LP T cells. Experimental stimulation of LPMCs, which mimics activation by intestinal pathogens frequently present in the bowel of HIV-infected patients, further dramatically enhances HIV replication (24.5-fold) compared with nonstimulated LPMCs. M-tropic HIV-1 showed a preferential replication in LPMCs, while T-tropic HIV-1 strain showed a preferential replication in PBMCs. Thus, the elevated activation state of target cells in the intestine and not the expression of the homing marker CD103 is directly linked to massive HIV production.     

The immunological effects of interleukin 2 therapy in HIV+ patients

Human immunodeficiency virus (HIV) infection produces a profound impairment of immune functions that antiretroviral therapy is unable to restore. Because of its immuno-enhancing properties, interleukin 2 (IL-2) has been used as a therapeutic tool in HIV+ subjects. IL-2 produces an increase of CD4 and CD8 lymphocyte absolute counts that is preferentially due to the expansion of the "naive" cells. In addition, IL-2 increases cytokine production from the cells of the immune system and is able to up-regulate the expression of cytokine receptors, such as the chemokine receptors CCR-5 and CXCR-4. Less informations on the IL-2 activity on the CD8 subset are available at the moment. The advent of highly active antiretroviral therapy has changed this scenario, making the IL-2 effects less clear-cut than previously hypothesized. We suggest that the ongoing studies will define the precise role of IL-2 in the therapy of HIV infection.     

TLR在小鼠胸腺中的表达及其在胸腺细胞发育中的可能作用

检测体外培养和体内发育过程中,胎鼠胸腺处于不同发育阶段时Toll样受体(TLR)的表达,阐明TLR表达量与胸腺细胞发育相关性,为TLR和胸腺细胞发育分化相关研究提供基础数据。无菌取15d胎龄胎鼠胸腺进行体外培养(FTOC),在培养不同时间点(2d,4d,6d),检测处于不同发育期胸腺TLR的表达;同时在孕期不同天数(15~19d),分别取胎鼠胸腺,检测在体内发育过程中胸腺TLR的表达;在FTOC中加入二脱氧鸟苷培养6d以制备胸腺基质细胞,检测基质细胞与胸腺细胞TLR表达情况。结果:小鼠胸腺中检测到多种TLR。FTOC培养中:培养第2天(F2)开始检测到各种TLR,到培养第6天(F6),TLR1,TLR3,TLR6,TLR7,TLR8明显上调,而TLR4,TLR5保持低水平,TLR4在培养第6天又下降;体内发育过程中:TLR6表达量随胎龄增加有较明显上调,TLR1,TLR3-8保持低水平表达;TLR2,TLR9体内体外都未检测到明显表达。在对胸腺细胞与基质细胞TLR表达比较中发现TLR1,TLR5,TLR6,TLR7高表达于胸腺细胞。胎鼠胸腺表达某些TLR,并且在发育不同阶段表达量有所改变,提示TLR可能参与胸腺细胞的发育过程。     

Functional HIV CXCR4 coreceptor on human epithelial Langerhans cells and infection by HIV strain X4

HIV can cross the intact epithelium of genital mucosae via Langerhans cells. Fresh Langerhans cells are known to express CD4 and CCR5. The presence of CXCR4 on the surface of cultured but not freshly isolated Langerhans cells has been described. In the present study, we demonstrate that CXCR4 was expressed by fresh Langerhans cells isolated and purified from epidermis. However, the percentage of Langerhans cells expressing CXCR4 or CCR5 increased during maturation of the cells in culture, especially in the presence of exogenous granulocyte-macrophage colony-stimulating factor. To determine whether CXCR4 was functional, freshly isolated Langerhans cells were infected with HIV LAI, a T-cell-tropic strain, and p24 protein production was measured in culture supernatants. p24 production was observed when infected Langerhans cells were cocultured with SupT1 cells. However, the presence of HIV provirus DNA was evidenced within the infected Langerhans cells by nested PCR. Ultrastructural studies confirmed the formation of syncytia when Langerhans cells were cocultured with SupT1 cells. Preincubation of Langerhans cells with azidothymidine or SDF-1-alpha, a natural ligand for CXCR4, prevented infection. These data demonstrated that CXCR4 is present on the surface of Langerhans cells freshly isolated from human skin epidermis and that this expression is functional.