A feedback regulatory pathway between LDL and alpha-1 proteinase inhibitor in chronic inflammation and infection.

Dietary lipids are transported via lymph to the liver and transformed to lipoproteins which bind to members of the low density lipoprotein receptor family (LDL-RFMs). Certain LDL-RFMs, e.g., very low density lipoprotein receptor (VLDLR), are also bound by inactivated proteinase inhibitors, the most abundant being α1proteinase inhibitor (α1PI, α1antitrypsin). Inflammation/infection, including HIV-1 infection, is accompanied by low levels of CD4+ T cells and active α1PI and high levels of inactivated α1PI. By inducing LDL-RFMs-mediated cellular locomotion, active α1PI regulates the number of CD4+ T cells. We sought to investigate whether CD4+ T cells and α1PI directly impact lipoprotein levels. At the cellular level, we show that active α1PI is required for VLDLR-mediated uptake of receptor-associated cargo, specifically CD4-bound HIV-1. We show that active α1PI levels linearly correlate with LDL levels in HIV-1 infected individuals (P<0.001) and that therapeutic, weekly infusions of active α1PI elevate the number of CD4+ T cells and HDL levels while lowering LDL levels in patients on antiretroviral therapy with controlled HIV-1. Based on the unusual combination of lipodystrophy and low levels of α1PI and CD4+ T cells in HIV-1 disease, we reveal that LDL and α1PI participate in a feedback regulatory pathway. We demonstrate integral roles for sequentially acting active and inactive α1PI in the uptake and recycling of receptors and cargo aggregated with VLDLR including CD4 and chemokine receptors. Evidence supports a role for α1PI as a primary sentinel to deploy the immune system as a consequence of its role in lipoprotein transport.

NF-kappaB signaling, elastase localization, and phagocytosis differ in HIV-1 permissive and nonpermissive U937 clones.

To identify positive or negative factors for HIV-1 infectivity, clones from the U937 promonocytic cell line that express similar levels of CD4 and CXCR4, but differ in HIV-1 susceptibility, were compared. In contrast to HIV-1 permissive clone 10 (plus), nonpermissive clone 17 (minus) was adherent to coverslips coated with chemokines, was phagocytic, killed bacteria, and expressed human leukocyte elastase (HLE) in a granule-like compartment (HLEG) that was never detected at the cell surface (HLECS). In contrast to the minus clone, the plus clone expressed HLE on the cell surface and was adherent to coverslips coated with the HLECS ligands alpha1proteinase inhibitor (alpha1PI, alpha1antitrypsin) and the HIV-1 fusion peptide. The phosphorylation status of several important signaling proteins was studied at the single cell level. Tumor suppressor p53, NF-kappaB p65, and Akt were constitutively phosphorylated in the plus clone, but not in the minus clone. Surprisingly, both alpha1PI and LPS induced phosphorylation of NF-kappaB p65 Ser-536 in both clones, but induced dephosphorylation of Ser-529 in the plus clone only. HIV-1 permissivity was conferred to the minus clone in a manner that required stimulation by both alpha1PI and LPS and was coincident to NF-kappaB p65 phosphorylation/dephosphorylation events as well as translocation of HLE to the cell surface. Even when stimulated, the minus clone exhibited greater reverse transcriptase activity, but less p24, than the plus clone. Results presented suggest that HIV-1 uptake and production efficiency are influenced by signaling profiles, receptor distribution, and the phagocytic capacity specific to the stage of differentiation of the CD4+ target cell.

p56lck, LFA-1 and PI3K but not SHP-2 interact with GM1- or GM3-enriched microdomains in a CD4-p56lck association-dependent manner.

We previously showed that the association of CD4 and G(M3) ganglioside induced by CD4 ligand binding was required for the down-regulation of adhesion and that aggregation of ganglioside-enriched domains was accompanied by transient co-localization of LFA-1 (lymphocyte function-associated antigen-1), PI3K (phosphoinositide 3-kinase) and CD4. We also showed that these proteins co-localized with the G(M1) ganglioside that partially co-localized with G(M3) in these domains. In the present study, we show that CD4-p56(lck) association in CD4 signalling is required for the redistribution of p56(lck), PI3K and LFA-1 in ganglioside-enriched domains, since ganglioside aggregation and recruitment of these proteins were not observed in a T-cell line (A201) expressing the mutant form of CD4 that does not bind p56(lck). In addition, we show that although these proteins associated in different ways with G(M1) and G(M3), all of the associations were dependent on CD4-p56(lck) association. Gangliosides could associate with these proteins that differ in affinity binding and could be modified following CD4 signalling. Our results suggest that through these associations, gangliosides transiently sequestrate these proteins and consequently inhibit LFA-1-dependent adhesion. Furthermore, while structural diversity of gangliosides may allow association with distinct proteins, we show that the tyrosine phosphatase SHP-2 (Src homology 2 domain-containing protein tyrosine phosphatase 2), also required for the down-regulation of LFA-1-dependent adhesion, transiently and partially co-localized with PI3K and p56(lck) in detergent-insoluble membranes without association with G(M1) or G(M3). We propose that CD4 ligation and binding with p56(lck) and their interaction with G(M3) and/or G(M1) gangliosides induce recruitment of distinct proteins important for CD4 signalling to form a multimolecular signalling complex.

CD4 ligation induces activation of protein kinase C zeta and phosphoinositide-dependent-protein kinase-1, two kinases required for down-regulation of LFA-1-mediated adhesion.

We previously showed that CD4 binding induced a down-regulation of LFA-1-dependent-antigen-independent adhesion of T and B lymphocytes in a phosphatidylinositol-3-kinase (PI3K)-dependent manner. We now show in A201-CD4 (+) T cell lines, that anti-CD4 Ab increases activation of phosphoinositide-dependent-protein-kinase 1 (PDK1) or PKC zeta, two main effectors down-stream from PI3K. CD4 binding also increases interactions between PI3K and activated PKCzeta and PDK1. Both events are dependent on CD4/p56Lck association, since they are not detected when p56Lck is unable to bind a truncated form of CD4 in transfected T cell lines. We also show using antisense oligonucleotides that both kinases are necessary for down-regulating LFA-1-dependent adhesion induced by CD4 signalling. We also suggest a role of PDK1 in the recruitment of the phosphatase SHP-2 in a multiprotein complex induced by anti-CD4 Ab. This study thus provides further insights into the mechanism underlying the CD4 triggered regulation of LFA-1-mediated adhesion.

CD4-induced down-regulation of T cell adhesion to B cells is associated with localization of phosphatidyl inositol 3-kinase and LFA-1 in distinct membrane domains.

We have previously shown that binding of anti-CD4 antibody inhibit LFA-1-dependent adhesion between CD4+ T cells and B cells in a p56(lck) and a PI3-kinase-dependent manner. In this work, we investigated with two different T cell lines (Jurkat and A201) whether CD4 binding could alter interactions of the proteins putatively involved in this adhesion regulatory pathway. Anti-CD4 binding was shown to induce a transient association between PI3-kinase and LFA-1, which took place in different regions of the plasma membrane. It was detected in detergent soluble membrane but also in detergent insoluble membrane consisting in raft microdomains, composed of GM1 and/or GM3 gangliosides. These results show that anti-CD4 Ab could modify the interaction between LFA-1 and signaling molecules, such as PI3-kinase and induce, in part, their recruitment in raft domains. By using specific inhibitors, raft integrity and CD4 association with GM3 were found necessary for observing the CD4-dependent inhibition of LFA-1-mediated adhesion. These results strongly suggest that these molecular rearrangements in the membrane are necessary to induce down-regulation of LFA-1-mediated adhesion.

Molecular events associated with CD4-mediated Down-regulation of LFA-1-dependent adhesion.

We have previously shown that CD4 ligand binding inhibits LFA-1-dependent adhesion between CD4+ T cells and B cells in a p56(lck)- and phosphatidylinositol 3-kinase (PI3-kinase)-dependent manner. In this work, downstream events associated with adhesion inhibition have been investigated. By using HUT78 T cell lines, CD4 ligands were shown to induce a dissociation of LFA-1 from cytohesin, a cytoplasmic protein known to bind LFA-1 and to enhance the affinity/avidity of LFA-1 for its ligand ICAM-1. A dissociation of PI3-kinase from cytohesin is also observed. In parallel, we have found that CD4 ligand binding induced a redistribution of PI3-kinase and of the tyrosine phosphatase SHP-2 to the membrane and induced a transient formation of protein interactions including PI3-kinase; an adaptor protein, Gab2; SHP-2; and a SH2 domain-containing inositol phosphatase, SHIP. By using antisense oligonucleotides or transfection of transdominant mutants, down-regulation of adhesion was shown to require the Gab2/PI3-kinase association and the expression of SHIP and SHP-2. We therefore propose that CD4 ligands, by inducing these molecular associations, lead to sustained local high levels of D-3 phospholipids and possibly regulate the cytohesin/LFA-1 association.