Gram-positive and gram-negative bacteria do not trigger monocytic cytokine production through similar intracellular pathways.

Toll-like receptors (TLRs) are involved in human monocyte activation by lipopolysaccharide (LPS) and Staphylococcus aureus Cowan (SAC), suggesting that gram-positive and gram-negative bacteria may trigger similar intracellular events. Treatment with specific kinase inhibitors prior to cell stimulation dramatically decreased LPS-induced cytokine production. Blocking of the p38 pathway prior to LPS stimulation decreased interleukin-1alpha (IL-1alpha), IL-1ra, and tumor necrosis factor alpha (TNF-alpha) production, whereas blocking of the ERK1/2 pathways inhibited IL-1alpha, IL-1beta, and IL-1ra but not TNF-alpha production. When cells were stimulated by SAC, inhibition of the p38 pathway did not affect cytokine production, whereas only IL-1alpha production was decreased in the presence of ERK kinase inhibitor. We also demonstrated that although LPS and SAC have been shown to bind to CD14 before transmitting signals to TLR4 and TLR2, respectively, internalization of CD14 occurred only in monocytes triggered by LPS. Pretreatment of the cells with SB203580, U0126, or a mixture of both inhibitors did not affect internalization of CD14. Altogether, these results suggest that TLR2 signaling does not involve p38 mitogen-activated protein kinase signaling pathways, indicating that divergent pathways are triggered by gram-positive and gram-negative bacteria, thereby inducing cytokine production.

Interaction of human immunodeficiency virus type 1 envelope glycoprotein V3 loop with CCR5 and CD4 at the membrane of human primary macrophages.

We show that infection of primary monocyte-derived macrophages (MDMs) and blood lymphocytes (PBLs) by human immunodeficiency virus type 1 (HIV-1) R5 strains, but not that of PBLs by X4 strain HIV-1LAI, is inhibited by beta-chemokines RANTES and MIP-1alpha. A biotinylated disulfide-bridged peptide mimicking the complete loop of clade B consensus V3 domain of gp120 (V3Cs), but not a biotinylated V3LAI peptide or a control beta-endorphin peptide of approximately the same molecular weight (MW), was found to bind specifically to MDM membrane proteins, in particular two proteins of 42 and 62 kDa migrating as sharp bands after electroblotting onto Immobilon, and this was specifically inhibited by anti-V3 antibodies. When biotinylated V3Cs was incubated with intact MDMs, which were then washed and lysed, and the resulting material was incubated with streptavidin-agarose beads and electroblotted onto Immobilon, fresh V3Cs also bound to proteins of the same molecular weight recovered in the V3Cs-interacting material. This binding was inhibited by anti-V3 antibodies, and no binding occurred with the control peptides. V3Cs also bound to soluble recombinant CD4, and CD4 monoclonal antibody Q4120 specifically recognized the V3Cs-interacting 62-kDa protein, which should thus correspond to CD4. Recombinant radiolabeled RANTES, MIP-1alpha, and MIP-1beta, but not IL-8, also bound to a 42-kDa protein on the membrane of MDMs as well as to the V3Cs-interacting 42-kDa protein, and excess unlabeled V3Cs inhibited such binding. This protein was also recognized by antibodies to CCR5, the RANTES/MIP-1alpha/MIP-1beta receptor. These data show that V3Cs binds to MDM membrane proteins that comprise CD4 and CCR5, and that multimolecular complexes involving at least gp120 V3, CD4, and CCR5 are formed on the surface of MDMs as part of V3-mediated postbinding events occurring during HIV-1 infection.

The inhibitory effect of RANTES on the infection of primary macrophages by R5 human immunodeficiency virus type-1 depends on the macrophage activation state.

We investigated whether culture conditions could affect the RANTES antiviral effect on human immunodeficiency virus type 1 (HIV-1) infection of primary macrophages. Monocyte-derived macrophages (MDM) were obtained either as (1) the adherent cells of 5-day cultures of blood mononuclear cells (PBMC), followed by 2 days without nonadherent PBMC or added cytokines (MDM-5d), or (2) as the adherent cells recovered from 1-h incubation of PBMC, which were cultured for 7 days with macrophage colony-stimulating factor (M-CSF; MDM-MCSF). Infection of MDM-5d from different donors with HIV-1 R5 strains was reproducibly inhibited by RANTES (IC50 < or = 10 nM), but infection of MDM-MCSF was not inhibited by > or = 100 nM RANTES, even when added at initiation of cultures, although it was still inhibited by a CD4 antibody. RANTES had no antiviral effect when MDM-5d were treated with physiological concentrations of M-CSF or GM-CSF before infection. CCR5 and CXCR4 expression as well as that of other cell surface molecules, including adhesion molecules, was not affected by the cytokines. MDM-MCSF from delta 32CCR5 homozygous individuals did not render them permissive to HIV-1, suggesting that it is unlikely that the virus uses another coreceptor. RANTES binding to MDM was chondroitin sulfate, but not heparan sulfate, dependent, and RANTES bound more efficiently to MDM-5d than to MDM-MCSF. Chondroitin sulfate removal partially offset the RANTES antiviral effect for MDM-5d. Thus RANTES anti-HIV-1 activity for primary macrophages depends on culture conditions and their consequent activation status, which may lead to differences in proteoglycan surface expression. These data may be relevant for the development of chemokine-based therapy for HIV-1 infection.

Involvement of the HIV-1 external envelope glycoprotein 120 (gp120) C2 region in gp120 oligomerization.

A synthetic peptide resembling the C2 region of human immunodeficiency virus type 1 (HIV-1) gp120 (C2-Lai: amino acids (aa) 273-288), inhibited (C50 = 200 microM) gp120 calcium-dependent binding of N-acetyl-beta-D-glucosaminyl and mannosyl residues exposed on natural glycoprotein bovine fetuin whereas a peptide derived from an aa sequence downstream of C2-Lai (C2-SC19) had no such effect (C50 > 1000 microM). No calcium-carbohydrate-specific binding of C2-Lai to fetuin was detected. In addition, C2-Lai was also found to inhibit the calcium-dependent oligomerization of gp120: while recombinant gp120 (rgp120) was recovered mainly as oligomers (78%) in 10 mM CaCl2, in contrast to 100% monomers in 1mM CaCl2, mostly monomers (67%) were found in 10 mM CaCl2 in the presence of C2-Lai. Peptide C2-SC19 and carbohydrate structures such as fetuin, fucoidin, dextran or mannan did not significantly affect gp120 oligomerization. Electrophoresis and gel filtration analysis also showed that C2-Lai aggregated in the form of 20 kDa compounds, which is compatible with association of 10 molecules. Taken together, these results demonstrate that the C2 domain is involved in gp120 oligomerization and suggest that gp120 oligomers but not monomers have specific carbohydrate binding properties.

Effect of mannosylated derivatives on HIV-1 infection of macrophages and lymphocytes.

We previously demonstrated that gp120/160 (Env) of HIV-1 interact in a carbohydrate-specific manner with mannosyl/N-acetylglucosaminyl derivatives and that HIV-1LAI infection of monocytic U937 and lymphoid CEM cells was inhibited by CD4-free Concanavalin A-reactive glycopeptides from U937 cells. We report here that the natural glycoproteins bovine fetuin and asialofetuin, human orosomucoid and alpha-fetoprotein, and mannan, which all specifically interact with Env, inhibited infection of primary macrophages by macrophage-tropic HIV-1 strains, whereas dextran had no such effect. This activity was conserved if fetuin, asialofetuin, or orosomucoid were heat-treated, which rules out the role of their three-dimensional structure. Orosomucoid and mannan partially inhibited Env binding to macrophages but not to U937 or CEM cells. This indicates that Env does not bind in the same manner to primary macrophages and to immortalized CD4+ cells, and that orosomucoid and mannan act at CD4-independent stages of virus binding to macrophages. Mannan also inhibited Env binding to surface glycopeptides obtained after trypsin treatment of macrophages. Furthermore, orosomucoid and fetuin interacted with, and they inhibited the binding of a V3 loop B clade consensus peptide to several macrophage membrane proteins, including two 36 and 42 kDa proteins. These data indicate that these glycoproteins interfere with post-binding events during HIV-1 infection of primary macrophages. In contrast, the compounds did not affect infection of U937 or CEM cells by T-cell tropic HIV-1LAI nor infection of peripheral blood lymphocytes by HIV-1LAI or HIV-1(Ba-L). Thus, carbohydrate-specific inhibition of HIV infection depends on the cell type more than on the viral strain, and differences in the glycan structure of cell-type-specific cofactors may be important for HIV entry into cells.

alpha 1-Acid glycoprotein binds human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein via N-linked glycans.

In the present study, we demonstrate a specific low-affinity interaction between recombinant precursor gp160 (rgp160) or surface unit gp120 (rgp120) of human immunodeficiency virus type 1 (HIV-1) and alpha 1-acid glycoprotein (AGP), a human glycoprotein displaying complex type N-glycans. Binding of rgp160/rgp120 to agarose-coupled AGP was dose-dependent, saturable, calcium-, pH- and temperature-dependent. Binding was inhibited by soluble AGP, asialo-AGP, fetuin, beta-D-GlcNAc47-BSA, alpha-D-Man20-BSA, mannan, complex-type asialo-agalacto-tetraanternary precursor oligosaccharide from human AGP and oligomannose 9 from porcine thyroglobulin; fully deglycosylated AGP was not inhibitory. The three AGP glycoforms separated on immobilized ConA bound rgp160 to the same extent as did unfractionated AGP. These findings extend our previous results on the carbohydrate-binding properties of HIV-1 envelope (Env) glycoprotein in that they demonstrate the involvement of AGP glycan moieties in the binding to rgp160/rgp120. Preincubation of rgp160 with AGP or mannan significantly reduced its binding to monocyte-derived macrophages (MDM), suggesting that AGP may play a role in preventing binding of soluble or virus-bound Env glycoprotein to CD4+ monocytic cells.