New T-lymphocytic cell lines for studying cell infectability by human immunodeficiency virus.

We identified five human T-lymphoid cell lines (PB-1, Sez-4, C19PL, HUT 102B and ATL-2) which highly express CD4 in addition to CXCR4 and CCR5. In order to evaluate if these cells are infectabile by human immunodeficiency virus (HIV) and could be employed as a model in HIV research we exposed these cell lines to X4 (T-cell tropic) and R5 (macrophage tropic) and subsequently tried to correlate their infectability with (i) level of chemokine coreceptor (CXCR4 and CCR5) expression, (ii) coreceptor functionality (calcium flux, chemotaxis and phosphorylation of MAPK p42/44 and AKT) and (iii) endogenous expression and secretion of HIV-related chemokines which compete with the virus for binding to CXCR4 (SDF-1/CXCL12) or CCR5 (MIP-1beta/CCL4, MIP-1alpha/CCL3, RANTES/CCL5, MCP-2/CCL8, MCP-3/CCL7 and MCP-4/CCL13). We demonstrated that while PB-1 cells are infectable by both X4 and R5 HIV, Sez-4, C91PL, HUT 102B and ATL-2 cells were infected by X4 HIV only. Moreover, we noticed that the susceptibility of these cells to HIV did not correspond either with the level of surface expression or with the functionality of CXCR4 or CCR5; however, it was modulated to some degree by the endogenously secreted HIV-related chemokines. Thus all five mature T-cell lines described here may provide useful new models for studying various aspects of HIV infection. In addition we demonstrate that the infectability of cells by HIV is modulated by so far unidentified intrinsic factors as well as some already known endogenously secreted chemokines. The identification of these factors may be important for developing new strategies to protect cells from HIV infection.

Biological significance of the expression of HIV-related chemokine coreceptors (CCR5 and CXCR4) and their ligands by human hematopoietic cell lines.

The aim of this study was to learn more about the role of the HIV-related chemokine-chemokine receptor axes in human hematopoiesis. To address this issue we phenotyped 35 selected hematopoietic cell lines for the expression of CD4, CXCR4 and CCR5. We next evaluated the functionality of these chemokine receptors by calcium flux and chemotaxis assays, and by the ability of SDF-1, MIP-1alpha, MIP-1beta and RANTES to influence the growth of the cells expressing CXCR4 and/or CCR5. Lastly, we examined whether human hematopoietic cell lines may secrete some HIV-related chemokines, and whether endogenously secreted chemokines might interfere with the infectability. of hematopoietic cells by X4 and R5 HIV strains. These results demonstrate that: (1) HIV-related receptors are widely expressed on human hematopoietic cell lines; (2) stimulation of CXCR4 by SDF-1 induces calcium flux and chemotaxis in several hematopoietic cell lines more efficiently than stimulation of CCR5 by receptor-specific beta-chemokines; (3) chemokines do not regulate proliferation of the hematopoietic cells; and finally (4) infectability of the hematopoietic cells by HIV-1 may be auto-modulated by endogenously secreted chemokines. These data shed more light on the role of HIV-related chemokine-chemokine receptors axes in human hematopoiesis and interaction of hematopoietic cells with HIV.

The limited infectability by R5 HIV of CD34(+) cells from thymus, cord, and peripheral blood and bone marrow is explained by their ability to produce beta-chemokines.

The resistance of human bone marrow (BM) CD34(+) cells to human immunodeficiency virus (HIV) infection is at this point not fully understood. Recently we reported that the chemokines MIP-1alpha, MIP-1beta, and RANTES secreted by BM-derived CD34(+) cells may compete with the macrophagotropic HIV (R5 HIV) strain for the CCR5 coreceptor.In this study we extended our previous observations and examined various lympho-hematopoietic CD34(+) cells isolated from thymus (Th), cord blood (CB), mobilized peripheral blood (mPB), and BM for the expression of beta-chemokines binding to CCR5, i.e., MIP-1alpha, MIP-1beta, RANTES, MCP-2, MCP-3, and MCP-4, and the alpha chemokine SDF-1 (binding to CXCR4) as these chemokines may compete with the R5 and X4 HIV strains, respectively, for entry into cells. We found that Th-, CB-, mPB-, and BM-derived CD34(+) cells express mRNA transcripts for all the beta-chemokines tested but not for SDF-1. Using sensitive ELISA assays we found that although MIP-1alpha and MIP-1beta proteins were secreted by all the lympho-hematopoietic CD34(+) cells tested, RANTES was detectable only in media conditioned by BM- and CB-derived CD34(+) cells and not Th-derived cells. However, media conditioned by BM-, mPB- and Th-derived CD34(+) cells protected the T lymphocytic cell line (PB-1) from infection by the R5 but not the X4 HIV strain. Hence this study demonstrates that beta-chemokines are secreted by lympho-hematopoietic CD34(+) cells originating from various sources and that these endogenously secreted chemokines may limit entry of the R5 HIV strain into the cells by competing for the CCR5 coreceptor.

Bone marrow CD34(+) cells and megakaryoblasts secrete beta-chemokines that block infection of hematopoietic cells by M-tropic R5 HIV.

CD34(+) cells are nonpermissive to infection by HIV strains X4 and R5, despite the fact that many CD34(+) cells express high levels of the viral receptor protein CD4 and the coreceptor CXCR4 on their surface. In these cells, the co-receptor CCR5 protein, which, like CXCR4, is a chemokine receptor, is detected mainly intracellularly. We hypothesized that CD34(+) cells secrete CCR5-binding chemokines and that these factors interfere with HIV R5 interactions with these cells, possibly by binding CCR5 or by inducing its internalization. We found that human CD34(+) cells and CD34(+)KIT(+) cells, which are enriched in myeloid progenitor cells, expressed and secreted the CCR5 ligands RANTES, MIP-1alpha, and MIP-1beta and that IFN-gamma stimulated expression of these chemokines. In contrast, SDF-1, a CXCR4 ligand, was not detectable in the CD34(+)KIT(+) cells, even by RT-PCR. Conditioned media from CD34(+) cell culture significantly protected the T lymphocyte cell line PB-1 from infection by R5 but not X4 strains of HIV. Interestingly, the secretion of endogenous chemokines decreased with the maturation of CD34(+) cells, although ex vivo, expanded megakaryoblasts still secreted a significant amount of RANTES. Synthesis of CCR5-binding chemokines by human CD34(+) cells and megakaryoblasts therefore largely determines the susceptibility of these cells to infection by R5 HIV strains. We postulate that therapeutic agents that induce the endogenous synthesis of chemokines in human hematopoietic cells may protect these cells from HIV infection.

Induction of syncytia by neuropathogenic murine leukemia viruses depends on receptor density, host cell determinants, and the intrinsic fusion potential of envelope protein.

Infection by the neuropathogenic murine leukemia virus (MLV) TR1.3 results in hemorrhagic disease that correlates directly to in vivo syncytium formation of brain capillary endothelial cells (BCEC). This phenotype maps to amino acid 102 in the envelope (Env) protein of TR1.3. Substitution of glycine (G) for tryptophan (W) at this position (W102G Env) in the nonpathogenic MLV FB29 induces both syncytium formation and neurologic disease in vivo. Using an in vitro gene reporter cell fusion assay, we showed that fusion either with murine NIH 3T3 cells or with nonmurine target cells that expressed receptors at or below endogenous murine levels mirrored that seen in BCEC in vivo. In these instances only TR1.3 and W102G Env induced cell fusion. In contrast, when receptor levels on nonmurine cells were raised above endogenous murine levels, FB29 Env was as fusogenic as the neuropathogenic TR1.3 and W102G Env. These results indicate that TR1.3 Env and W102G Env are intrinsically more fusogenic than FB29 Env, that the induction of fusion requires a threshold number of receptors that is greater for FB29 Env than for TR1.3 or W102G Env, and that receptor density on murine NIH 3T3 cells and BCEC is below the threshold for FB29-dependent fusion. Surprisingly, receptor density on NIH 3T3 cells could not be increased by stable expression of exogenous receptors, and FB29-dependent fusion was not observed in NIH 3T3 cells that transiently expressed elevated receptor numbers. These results suggest that an additional undefined host cell factor(s) may limit both receptor expression and fusion potential in murine cells.

Viral pathogenesis and immunity within the thymus.

Replication of viruses within the thymic microenvironment may have a unique impact on viral persistence and pathology. The author's laboratory has studied thymic infection by both human and murine retroviruses. For human lentiviruses, such as HIV-1, the consequences of persistent thymic replication are frequently a severe disruption of the normal processes of thymopoiesis and potentially of progression to AIDS. Murine retroviruses, such as Gross murine leukemia virus, establish persistent infection with less cytopathic, but no less devastating effects. These include the alteration of immune recognition to retroviral antigens by the peripheral immune response, the thymic persistence of virus, and the establishment of viral-induced thymic leukemia. This article summarizes the analysis of both the common and distinctive means of pathology induced by these two retroviral families with particular attention on the influence and impact of the thymus as a unique site of virus replication.

The role of the immune response in MULV-induced lymphomagenesis.

Although the exact mechanisms of murine leukemia virus (MuLV)-induced lymphomagenesis have yet to be elucidated, it is clear that the immune reponse to viral proteins plays a critical role in this disease process. The parameters for lymphomagenesis are governed by an inverse relationship between viral persistence and immune responsiveness. MuLV have evolved ways to avoid immune detection either by altering their own genome or by altering the host environment. In addition, the intrathymic replication of MuLV during thymocyte maturation and immune selection plays an important role in T cell repertoire development and immune inhibition. These viruses have served as a highly effective experimental model in understanding the many pathways by which MuLV have overcome immune detection and thereby led to lymphomagenesis.

Manipulation of the immune response by foreign gene expression in the thymus.

Retroviral gene transfer vectors have been developed for optimal in vivo gene therapy. Ideally, these vectors should target gene expression specifically to selected tissues or organs. Our studies focus on the development of retroviral vectors for gene delivery to the thymus. The goal of these studies is to utilize thymic expression of exogenous genes to manipulate the immune repertoire. We have characterized the selective thymic tropism of a molecular clone of Gross murine leukemia virus, GD-17, to thymic medullary epithelial cells using immunohistochemical staining and confocal microscopy. Specific expression of viral antigens in the thymus lead to the induction of immunologic tolerance to GMuLV proteins. This tissue specific vector may thus be used to study the requirements of epithelial mediated tolerance induction, and provide a more efficient tool for gene therapy.

A point mutation in the env gene of a murine leukemia virus induces syncytium formation and neurologic disease.

TR1.3 is a Friend-related murine leukemia virus that has been shown to cause intracerebral hemorrhages and neurologic disease due to infection and subsequent cytopathology of cerebral vessel endothelium. A striking feature of this pathology is the formation of endothelial cell syncytia. The pathogenesis of this disease has now been mapped to a single amino acid substitution of tryptophan to glycine in the variable region of the envelope protein. This same mutation enabled TR1.3 to form syncytia and retard cell proliferation in vitro in the SC-1 mouse embryoblast line but did not affect the pH dependence of viral entry. These results demonstrate that subtle molecular changes in retroviral env genes can induce both syncytium formation and overt clinical disease.

Intracerebral hemorrhages and infarction induced by a murine leukemia virus is influenced by host determinants within endothelial cells.

The strain and developmental parameters that control susceptibility to murine leukemia virus (MuLV)-induced intracerebral hemorrhages and infarction were studied using the endothelial cell tropic MuLV TR1.3. Inoculated animals displayed an absolute age dependence on the development of intracerebral vascular disease; however, other genetic determinants affected the timing and magnitude of susceptibility to neurologic disease. BALB/c mice were susceptible to neurologic disease only when inoculated prior to Day 4 postpartum. In contrast, Swiss/NIH and C3H/HeN mice consistently showed a less virulent phenotype and were only susceptible when infected prior to Day 3 postpartum. These studies demonstrate that susceptibility to TR1.3 murine leukemia virus-induced neurologic disease is regulated by age- and strain-dependent factors encoded within cerebral endothelial cells.

Glycosylated phosphatidylinositol molecules as second messengers.

Glycosylated phosphatidylinositol (GPI) lipids are a structurally and functionally diverse molecular family. One of the most interesting and controversial aspects of GPI function is their ability to participate in signaling mechanisms or to directly function as second messengers of biological receptors. For example, while there is little dispute that subsets of GPI molecules are hydrolyzed following receptor ligation, there is no consensus as to the subsequent roles of intact GPI molecules or their cleavage products. The importance of these observations is underscored by two facts; many GPI anchored proteins participate in the regulation of cell proliferation, and several hormones metabolize GPI forms that are not linked to proteins. The purpose of this review is to outline the major structural and biological features of GPI molecules as they relate to their role in cellular signaling.

Neonatal HIV-1 thymic infection.

The thymus is the primary site of T cell ontogeny and selection during fetal and neonatal development. Previous studies have established that the thymus is also a site of HIV-1 infection, as early as the first trimester of pregnancy. Alteration of the thymocyte maturation process by HIV-1 could impact on the peripheral T cell population and interfere with immune responses. A neonatal thymic organ culture system was established to study HIV-1 infection within the thymus. We have shown that this primary tissue isolate can support a productive HIV-1 infection. Infection occurred without detectable thymocyte cytopathology. The ability to infect the developing thymocyte within an intact micro environment will enable us to further establish the kinetics of acute HIV-1 thymic infection and its consequences on lymphocyte maturation.

Diminished tyrosine protein kinase activity in T cells unresponsive to TCR stimulation.

Tyrosine phosphorylation is thought to be one of the earliest steps in antigenic activation of T cells. Three nonreceptor tyrosine kinases, p56lck, p60fyn, and ZAP-70, are known to be involved in T cell receptor (TCR) signaling, albeit their functional roles appear to be different. Whereas p60fyn and ZAP-70 are functionally associated with the T cell antigen receptor, p56lck is essential for TCR signaling without being directly coupled to the TCR. We have studied a mutant variant of the Jurkat T cell line (J32-3.2), in which basal activities of p56lck and p60fyn are 2- to 2.5-fold reduced relative to those in its parental line (J32) while basal activity of ZAP-70 remains unchanged, and compared responses of J32-3.2 and J32 to TCR stimulation. We have demonstrated that tyrosine phosphorylation following CD3 cross-linking in J32-3.2 cells was extremely short-lived and thus insufficient for the induction of subsequent physiological responses. This was at least partially due to the diminished tyrosine kinase activity in these cells. A decrease in the activity of src-related kinases was caused primarily by their lower expression, whereas expression of ZAP-70 was unchanged but its response to CD3 cross-linking was diminished, correlating with the deficient tyrosine phosphorylation of the CD3 zeta-chain, recently observed in J32-3.2. These data are consistent with the idea that src-related kinases phosphorylate the zeta-chain, which in turn recruits ZAP-70 required to sustain the signal.

Human neonatal thymic organ culture: an ex vivo model of thymocyte ontogeny and HIV-1 infection.

An intact thymic microenvironment is required for the normal maturation and selection of thymocytes. This process is directed by the interaction of thymocyte progenitors with molecules on the surface of thymic stromal cells and with cytokines. The precise nature of these events is poorly understood in humans. We have developed a technique of human neonatal thymic organ culture (hNTOC) that enabled thymocyte development for up to 14 days of ex vivo culture. hNTOC supported the maturation of CD4+CD8+ double-positive cells into both CD4+CD8- and CD4-CD8+ single-positive thymocytes. hNTOC was also used to examine infection with HIV-1, as a means to address the thymic pathology of HIV-1. These results establish an experimental system for the analysis of human thymic ontogeny and for the experimental manipulation of these events by ex vivo administration of cytokines, drugs or infectious agents.

Pathogenesis of cerebral infarction and hemorrhage induced by a murine leukemia virus.

BACKGROUND: Inoculation of neonatal BALB/c mice with the Friend murine leukemia virus TR1.3 uniformly induces cerebral infarctions and hemorrhages within 18 days. The primary target of TR1.3 infection are endothelial cells of capillaries and small vessels. Preliminary post-mortem histologic analysis revealed multifocal endothelial cell pathology associated with the presence of thrombi and extravasation of red blood cells into the brain parenchyma. The consequences of viral infection on endothelial cell integrity and its relevance to hemorrhagic and ischemic lesions are unclear. EXPERIMENTAL DESIGN: Neonatal BALB/c mice were infected with TR1.3 murine leukemia virus and were monitored daily for symptoms of tremor, seizure and paralysis. Diseased mice were killed and the brains prepared for histopathologic analysis and electron microscopy studies. RESULTS: Hematoxylin and eosin-stained sections revealed widespread areas of infarction throughout white and grey matter with numerous scattered thrombi. Endothelial cell pathology was widespread and pronounced. This included enlarged cytoplasm, intracytoplasmic clefts, separation of tight junctions, swollen mitochondria, changes to the basal lamina and in many instances the formation of syncytia. Ultrastructural studies identified numerous viral particles within the endothelial cell cytoplasm and budding from the abluminal and luminal cell surfaces. CONCLUSIONS: These data confirm that TR1.3 virus replicates within endothelial cells and provides the first direct evidence of retrovirus-induced endothelial cell pathology. These results suggest that hemorrhage may be a direct consequence of this endothelial cell pathology, and that endothelial cell damage initiates the formation of thrombi and vessel occlusion that results in multiple cerebral infarctions.

Selective thymocyte depletion in neonatal HIV-1 thymic infection.

OBJECTIVE: To determine the impact of HIV-1 infection on thymocyte development, and the role of thymic infection on the pathogenesis of neonatal HIV-1 infection. DESIGN AND METHODS: The consequences of thymic infection by HIV-1 were examined by comparative histologic and molecular analyses of an asymptomatic, HIV-1-seropositive 3-day-old subject, versus age- and treatment-matched controls. The presence of replicating virus was established by in situ hybridization with specific molecular probes to HIV-1. The distribution of thymocyte subsets was determined by quantitative flow cytometry following staining with antibodies to CD4 and CD8 cell surface proteins. RESULTS: The results show clear evidence of severe thymic involution, HIV-1 infection of thymocytes, and selective depletion of thymocyte subpopulations. The consequences of HIV-1 infection were a marked depletion of CD3+CD4+ CD8hi and CD3+CD4+CD8- cells. The phenotype of the residual thymic lymphoid population was predominantly that of immature CD3-CD4-CD8- double negative and CD3+CD4+CD8lo cells. CONCLUSION: Changes in the distribution of thymocyte subsets suggests a role for thymic involvement in the pathogenesis of HIV-1 infection in neonates.

Multifunctional roles of glycosyl-phosphatidylinositol lipids.

The results presented here indicate that GPI lipids are a structurally and functionally diverse molecular family. Despite new detailed information on the structures of GPI-anchored proteins, there is relatively scant information on the structure of free-GPI. Thus, little is known of the relationships between GPI structures and the mechanism of their biological effects. For example, there is no distinction at the structural level between hormone-sensitive free-GPI and those that serve as precursors for protein-GPI. Nor is there precise biochemical data on the mechanism and importance of free-GPI in hormone signaling, or the signaling roles that GPI anchors play in protein function. The T-cell activation cascade is an ideal system for studying both forms of GPI and their derivatives. The study of GPI molecules in T lymphocytes offers the exciting possibility of addressing questions on the structure, function, genesis, and regulation of both free- and protein-GPI molecules in a single cell type. The detection of multiple protein-GPI and free-GPI forms, and of hormone-sensitive GPI, provides the first approach to these issues. For the moment, the potential for biochemical signaling by intact GPI or its metabolites is enormous. If significant progress is to be made, the structures of hormone sensitive free-GPI must be elucidated. Only then can we precisely define the roles of these molecules in the regulation of cell metabolism and proliferation.