Signaling pathways triggered by HIV-1 Tat in human monocytes to induce TNF-alpha

In this study we investigated the signaling pathways triggered by Tat in human monocyte to induce TNF-alpha. In monocytes, the calcium, the PKA, and the PKC pathways are highly implicated in the expression of cytokine genes. Thus, these three major signaling pathways were investigated. Our data show that (i) PKC and calcium pathways are required for TNF-alpha production, whereas the PKA pathway seems to be not involved; (ii) downstream from PKC, activation of NFkappaB is essential while ERK1/2 MAP kinases, even though activated by Tat, are not directly involved in the pathway signaling leading to TNF-alpha production.     

Localized adhesion of monocytes to human atherosclerotic plaques demonstrated in vitro: implications for atherogenesis.

Blood-derived macrophages in the arterial intima are a characteristic feature of active atherosclerotic plaques. Adherent monocytes on the luminal surface and increased adhesion molecules on the endothelium have suggested that specific molecular mechanisms are involved in monocyte/macrophage traffic into the arterial wall. Adhesion of human monocytes and related cell lines was therefore studied in vitro to histological sections of human plaques. At 37 degrees C, these cells bound selectively to the plaques. Binding to the endothelium occurred and was also present extensively in the diseased intima. Inhibition studies showed that the endothelial and general intimal binding had largely similar molecular properties. Strong inhibition was produced by antibodies to the monocyte-specific adhesion molecule CD14, to beta2 integrins, and to ICAM-1. Likewise, a peptide containing the Arg-Gly-Asp sequence was strongly inhibitory, suggesting that binding of leukocyte integrins to arterial extracellular matrix was synergistic with cell-cell interactions. A P-selectin antibody was exceptional in giving selective inhibition of endothelial adhesion, which correlates with the specific endothelial localization of this adhesion molecule. These results show that monocytes adhere to atherosclerotic plaques through the focal activation of multiple arterial wall adhesion molecules, confirming the adhesion hypothesis. A positive feedback theory for the pathogenesis of atherosclerosis can be suggested, based on the ability of macrophages in the wall to activate the endothelium, induce adhesion molecules, and facilitate additional monocyte entry. The adhesion assay provides a means for the identification of adhesion inhibitors with therapeutic potential.     

Benoxaprofen inhibits the adhesion of human monocytes to cultured vascular endothelium

Pretreatment of human monocytes with benoxaprofen for at least 2 h produced a dose-dependent abrogation of their adhesion to monolayers of cultured porcine endothelium with 0.05 g/ml and 50.0 g/ml of the drug inducing a mean 33% and 83% inhibition of adhesion respectively. When the endothelium was treated with the drug there was no modification of monocyte adhesion. In contrast, pretreatment of endothelium with 5.0 and 50.0 g/ml benoxaprofen for at least 6 h, resulted in a mean 35% and 31% inhibition of polymorphonuclear cell (PMN) adhesion in 6/11 experiments. This inhibitory effect was not seen when drug-treated PMNs were added to endothelium. An impairment of monocyte chemotactic migration was only apparent with high concentrations of the drug (50 g/ml). These results suggest that an important anti-inflammatory property of benoxaprofen is the inhibition of monocyte adhesion to vascular endothelium.     

Pentoxifylline in vivo and in vitro down-regulates the expression of the intercellular adhesion molecule-1 in monocytes.

Since pentoxifylline (PTX) was recently recognized as a substance with antiinflammatory capacities, we studied the in vivo and in vitro effect of PTX on the expression of the intercellular adhesion molecule-1 (ICAM-1) on human monocytes. For this purpose four healthy volunteers were treated with PTX (5 x 400 mg/day) for 2 days. Monocytes were isolated before and after PTX treatment and ICAM-1 expression was investigated. As shown by fluorescence-activated cell sorter (FACS) analysis, cultured monocytes isolated after oral application of PTX expressed significantly decreased amounts of ICAM-1 when compared with monocytes collected prior to oral PTX application. Northern blot analysis revealed reduced amounts of ICAM-1 mRNA in monocytes derived from volunteers after oral PTX treatment in comparison with monocytes isolated before oral PTX administration. Similarly, in monocytes treated with PTX (200 micrograms/ml) in vitro ICAM-1 was found decreased both at the protein and mRNA level in comparison with untreated cells. The inhibitory effect of PTX on ICAM-1 expression in monocytes could be reversed by the addition of exogenous tumour necrosis factor-alpha (TNF-alpha; 200 U/ml) suggesting that ICAM-1 down-regulation is mediated secondary to TNF-alpha suppression by PTX. The specific role of TNF-alpha in mediating ICAM-1 expression in cultured monocytes could be confirmed by the finding that a neutralizing anti-TNF-alpha antibody partially down-regulated ICAM-1 expression. The observed suppressive in vivo and in vitro effects of PTX on ICAM-1 expression in monocytes may contribute to the recently described antiinflammatory effects of PTX, e.g. in sepsis or allergic contact dermatitis.     

Reservoirs of HIV-1 in vivo: implications for antiretroviral therapy

The eradication of HIV-1 from infected individuals remains the ultimate goal of all anti-HIV therapeutics. Although highly active antiretroviral therapy (HAART) has led to a profound decrease in morbidity and mortality in infected people by suppressing HIV replication, the virus continues to evolve slowly during therapy even when patients achieve below detectable levels of HIV in plasma. HIV-1 persists in latently infected memory CD4+ T cells and there is minimal decay of HIV in this compartment despite prolonged HAART. Various other reservoirs and sanctuary sites harboring HIV are also established in vivo during antiretroviral therapy. Collectively these sites represent a major impediment to the eradication of HIV-1. This review presents a detailed overview of various reservoir sites in vivo, and discusses their impact on the success and failure of HAART for HIV patients. In addition, it addresses the effect of sub-optimal drug concentrations on reservoir establishment and outlines future therapeutic strategies to counteract these reservoirs and sanctuaries.     

Antibodies to the HIV-1 Tat protein correlated with nonprogression to AIDS: a rationale for the use of Tat toxoid as an HIV-1 vaccine

OBJECTIVES: To investigate which immune parameters, such as antibodies against HIV-1 specificities, or viral parameters, such as p24 antigenemia, are predictive of disease progression. STUDY DESIGN: We performed studies on serum collected from individuals exhibiting two extremes of disease evolution--67 fast progressors (FP) and 182 nonprogressors (NP)--at their enrollment. After a 1- to 2-year clinical follow-up of 104 nonprogressors after their enrollment, we could determine the best serologic predictors for disease progression. METHODS: We investigated levels of antibodies to tetanus toxoid and to HIV antigens including Env, Gag, Nef, and Tat proteins, as well as p24 antigenemia, viremia, CD4 cell count, and interferon-alpha (IFN-alpha) titers in FPs and NPs, and we correlated these data with clinical and biologic signs of progression. RESULTS: p24 Antigenemia, a marker of viral replication, and anti-Tat antibodies were highly and inversely correlated in both groups (P < .001). Furthermore, anti-p24 antibodies and low serum IFN-alpha levels were correlated to the NP versus the FP cohort. Finally, among NPs, only antibodies to Tat and not to the other HIV specificities (Env, Nef, Gag) were significantly predictive of clinical stability during their follow-up. CONCLUSION: Antibodies toward HIV-1 Tat, which are inversely correlated to p24 antigenemia, appear as a critical marker for a lack of disease progression. This study strongly suggests that rising anti-Tat antibodies through active immunization may be beneficial in AIDS vaccine development to control viral replication.     

HIV-1 does not alter in vitro and in vivo IL-10 production by human monocytes and macrophages

The present study analyses the ability of HIV-1 to modulate IL-10 production in cells of monocyte-macrophage lineage cultured in the presence of macrophage colony-stimulating factor (M-CSF). Both monocytes and macrophages spontaneously produced low amount of IL-10. Lipopolysaccharide (LPS) induced a strong IL-10 response in fresh monocytes and in M-CSF-treated macrophages. In contrast, macrophages cultured in the absence of M-CSF exhibited a marked decrease in their susceptibility to LPS stimulation. M-CSF increased the IL-10 response of macrophages to LPS by enhancing both the expression of membrane-bound CD14, the protein that serves as LPS receptor, and the sensibility of CD14-expressing cells to LPS stimulation. Neither spontaneous nor LPS-induced expression of IL-10 was modulated in monocytes and macrophages by infection with eight monocytotropic strains, as demonstrated by ELISA and cytofluorimetric analysis. In contrast, all the HIV-1 strains primed macrophages for an increased IL-6 response to LPS stimulation. To determine whether IL-10 production was associated with in vivo infection, monocytes from AIDS individuals were analysed for IL-10 production. We found that neither spontaneous nor LPS-induced IL-10 production were different between healthy controls and HIV-infected patients. Taken together, these data strongly suggest that HIV-1 infection of monocytes-macrophages does not play a significant role in the regulation of IL-10 in infected patients. This study also emphasizes the role of M-CSF activation in the regulation of the cytokine response in macrophages.     

Interleukin-1 increases tumor cell adhesion to endothelial cells through an RGD dependent mechanism: in vitro and in vivo studies

The effects of human recombinant interleukin-1α and β (rIL-1α; rIL-1β) on the adhesion of human A549 lung carcinoma cells and M6 melanoma cells (TC) to human endothelial cells (HECs) in vitro were studied, and on TC/lung entrapment in vivo. In vitro, there was a significant increase in TC/HEC adhesion to HECs pretreated for 4 h with rIL-1α or rIL-1β. The effects of rIL-1α and β on TC/HEC adhesion were time dependent and reached a plateau within 4–6h. TC/HEC adhesion was not blocked when measured in the presence of antibodies to either fibronectin, glycoprotein IIb/IIIa, anti-ICAM, or anti-LFA. However, enhanced TC/HEC adhesion was completely blocked in the presence of the peptide, GRGDS. In vivo, pretreatment of nude mice for 4 h with rIL-1α (given i.p. before ix. injection of TCs) enhanced TC retention in the lung 24 h later. Our data demonstrate that IL-1 enhances TC adhesion to the vascular surface both in vitro and in vivo, suggesting that IL-1 can facilitate the metastatic process.     

HIV-1 infection of in vitro cultured human monocytes: early events and influence of anti HIV-1 antibodies

Summary To characterize the role of the humoral immune response on HIV-1 infection of monocytes and macrophages (Ms) we examined the susceptibility of in vitro cultured monocyte/Ms to various HIV-1 isolates and the influence of heterologous and particularly autologous anti HIV-1 sera on this infection. Depending on the period of in vitro cultivation and the virus isolate used different patterns of susceptibility were detected. One week old monocyte/Ms were highly susceptible to HIV-1 infection, in contrast to monocyte/Ms cultured 4 weeks. The infection by virus isolated immediately after seroconversion lead to persistent infection with high level of antigen production in contrast to infection by homologous virus isolated later. MAb against the V3-IIIB loop and sCD4 inhibited the infection of monocyte/Ms in a dose dependent manner, indicating that infection requires binding to CD4 and that post binding events may be common to the infection of lymphocytes. Anti HIV-1 sera showed neutralizing activity against heterologous and even autologous escape virus. This finding, together with the observation that monocytes and Ms are infected in vivo, suggests that protection against HIV-1 infection of monocytes and Ms in vivo may not be obtainable by the humoral immune response alone.     

Replication of HIV-1 in vivo and in vitro

A complex relationship exists between HIV and its cellular targets. The lethal effect of HIV on circulating CD4⁺ helper T lymphocytes parallels the degree of the infected individual's immunodeficiency and ultimately the transition to AIDS and death. However, as with other members of the Lentivirus family of retroviruses, the ubiquitous, mobile macrophage is also a prime target for HIV infection, and apparently, in most instances, is the initial infected cell, since most people are infected with a CCR5 chemokine-tropic virus. Unlike the lymphocyte, the macrophage is apparently a more stable viral host, capable of a long infected life as an HIV reservoir and a chronic source of infectious virus. Published in vitro studies have indicated that whereas lymphocytes replicate HIV solely on their plasma membrane, macrophages have been envisaged to predominantly replicate HIV within cytoplasmic vacuoles, and thus have been likened to a “Trojan horse,” when it comes to the immune system. Recent studies have revealed an ingenious way by which the cultured monocyte-derived macrophage (MDM) replicates HIV and releases it into the medium. The key macrophage organelle appears to be what is alternatively referred to as the “late endosome” (LE) or the “multivesicular body” (MVB), which have a short and a long history, respectively. Proof of the association is that chemically, LE/MVB and their vesicles possess several pathopneumonic membrane markers (e.g., CD63) that are found on released HIV particles. The hypothesis is that HIV usurps this vesicle-forming mechanism and employs it for its own replication. Release of the intravacuolar virus from the cell is hypothesized to occur by a process referred to as exocytosis, resulting from the fusion of virus-laden LE/MVB with the plasma membrane of the macrophage. Interestingly, LE/MVB are also involved in the infection stage of MDM by HIV. Close review of the literature reveals that along with the Golgi, which contributes to the formation of LE/MVB, the MVB was first identified as a site of HIV replication by macrophages many years ago, but the full implication of this observation was not appreciated at the time. As in many other areas of HIV research, what has been totally lacking is an in vivo confirmation of the in vitro phenomenon. Herein, the ultrastructure of HIV interaction with cells in vitro and in vivo is explored. It is shown that while HIV is regularly found in LE/MVB in vitro, it is infrequently the case in vivo. Therefore, the results challenge the “Trojan horse” concept.     

ICAM-1-mediated adhesion of peripheral blood monocytes to the maternal surface of placental syncytiotrophoblasts: implications for placental villitis.

Accumulation of maternal monocytes in the villous/intervillous space (villitis) is associated with increased risk of perinatal morbidity and mortality and may initiate in utero transmission of cell-associated infectious agents such cytomegalovirus and HIV-1. We have developed an in vitro model of trophoblast syncytialization and have investigated the adhesive interactions between this tissue and peripheral blood monocytes. We show that monocytes strongly adhere to cultured syncytiotrophoblasts (STs) and that treatment with the inflammatory cytokines interferon-gamma, tumor necrosis factor-alpha, and interleukin-1 alpha greatly increase the number bound. Pretreatment of STs with these cytokines upregulated apical expression of intercellular cell adhesion molecule (ICAM)-1 but not E-or L-selection, ICAM-2 or -3, or various integrins. ICAM-1 expression was cytokine concentration dependent, significantly increased within 6 hours of treatment, peaked after 24 hours, and remained undiminished for 48 hours after cytokine removal from the cultures. Adhesion of monocytes to STs was inhibited > 80% by antibody to ICAM-1 or its cognate ligand LFA-1. ICAM-1 was detected immunohistochemically only in rare foci on intact term placental villi. These results suggest that villous trophoblast expression of ICAM-1 occurs only during an immune inflammatory reaction and that aberrant expression of this molecule may be an important pathological feature in those immunoinflammatory disorders of the placenta characterized by an excessive accumulation of leukocytes in the intervillous/villous space such as spontaneous abortion, perinatal hematogenous infections, and villitis of unknown etiology.     

Safety and immunogenicity of HIV-1 Tat toxoid in immunocompromised HIV-1-infected patients

OBJECTIVES: To antagonize the deleterious effects of the HIV-1 toxin extracellular Tat on uninfected immune cells, we developed a new strategy of anti-HIV-1 vaccine using an inactivated but immunogenic Tat (Tat toxoid). Tat toxoid has been assayed for safety and immunogenicity in seropositive patients. METHOD: The phase I vaccine clinical trial testing Tat toxoid preparation in Seppic Isa 51 oil adjuvant was performed on 14 HIV-1-infected asymptomatic although biologically immunocompromised individuals (500-200 CD4+ cells/mm3). RESULTS: Following as many as 8 injections, no clinical defects were observed. All patients exhibited an antibody (Ab) response to Tat, and some had cell-mediated immunity (CMI) as evaluated by skin test in vivo and T-cell proliferation in vitro. CONCLUSION: These results provide initial evidence of safety and potency of Tat toxoid vaccination in HIV-1-infected individuals.     

Antibody to HIV-1 Tat protein inhibits the replication of virus in culture

Summary The HIV-1 transactivator protein Tat is essential for viral replication. Tat is released from infected cells and can be taken up and transactivate HIV-LTR in LTR-CAT transfected cell lines. The present study shows that the addition of monoclonal antibody to Tat in IIIB and MN-infected cultures reduces the HIV antigen production in a concentration dependent manner. These data suggest that external Tat might be important in the replication of HIV, exerting the effect in a paracrine fashion. Using 1 µg/ml of anti-Tat antibody resulted in a decline of HIV antigen production to 33% and 45% of controls in IIIB and MN infected H9 cells, respectively. A time course experiment showed progressively increased inhibition of replication during 7 days of exposure to anti-Tat antibody, which could be due to increasing Tat concentration. The inhibitory effect of anti-Tat antibodies on the replication of HIV could play an important regulatory role during infection in vivo.     

Fibrinogen binding to intercellular adhesion molecule 1: implications for Plasmodium falciparum adhesion

Intercellular adhesion molecule 1 (ICAM-1) is an endothelial cell adhesion molecule implicated in cerebral malaria. We investigated whether fibrinogen affects Plasmodium falciparum binding to ICAM-1, as the ICAM-1 binding sites of P. falciparum and fibrinogen overlap. We show that fibrinogen dramatically reduces P. falciparum adhesion to ICAM-1 under flow conditions.     

Role of the LFA-1 adhesion glycoprotein in neutrophil adhesion to endothelium and plastic surfaces.

Neutrophil adherence to endothelium is known to be mediated, at least in part, by adhesion molecules such as LFA-1. Deficiency of these adhesion molecules leads to recurrent infection and early death from infection. As screening for defects of these adhesion glycoproteins is often performed by the ability of neutrophils to adhere to plastic plates, in this study a comparison of neutrophil adherence by the CD18/CD11a (LFA-1) mechanism to endothelium and plastic surfaces was examined. Baseline neutrophil adherence was two-fold higher to plastic than to endothelium (17% +/- 9 for plastic, 8% +/- 5 for endothelium). Baseline adherence to endothelium was partially inhibitable by anti-LFA-1 antibodies, whereas no inhibition of adherence occurred on plastic. Neutrophil stimulants increased adherence to both surfaces, although only on endothelium was this increase attributable to the LFA-1 mechanism. IL-1 increased adherence to endothelium, but had no effect on plastic. We conclude that adherence of neutrophils to plastic surfaces probably represents overall activation status through undefined mechanisms, is not by LFA-1 receptor ligand interactions, and is therefore a non-physiological phenomenon. Endothelial receptors are pivotal in neutrophil adherence. It would be more appropriate to screen leucocytes for leucocyte adhesion deficiency by assaying for specific receptor occupancy with monoclonal antibodies, rather than an assay such as adhesion to plastic where the adhesion ligand is non specific.