Coexpression of TLR2 or TLR4 with HLA-DR potentiates the superantigenic activities of Mycoplasma arthritidis-derived mitogen.

Mycoplasma arthritidis-derived mitogen (MAM) is a member of the superantigen family that structurally differs from other members while still capable of initiating cognate APC/T cell interaction. In addition to the critical role of MHC class II molecules, it has been suggested that TLR2 and TLR4 may cooperate with MHC class II during MAM-induced responses. In this study, we investigated the direct involvement of TLR2 and TLR4 in MAM binding and presentation to T cells. Our results showed that MAM fails to bind to TLR2- and TLR4-transfected cells. However, coexpression of TLR2 or TLR4 with HLA-DR significantly increases MAM binding and the subsequent T cell activation compared with cells expressing HLA-DR alone. The upregulated MAM binding and activity in HLA-DR/TLR-transfected cells is abrogated by an anti-HLA-DR Ab. Interestingly, we also found that MAM complexed with soluble HLA-DR is capable of binding to both TLR2 and TLR4. The enhancing effect of TLR2 or TLR4 on MAM-induced T cell proliferation was not due to TLR ligand contamination in the MAM preparation. Taken together, these results strongly suggest that binding of MAM to HLA-DR leads to a conformational change in MAM structure allowing its interaction with TLR2 and TLR4 and a better recognition by T cells.

Functional interaction of CD154 protein with α5ß1 integrin is totally independent from its binding to αIIbß3 integrin and CD40 molecules.

In addition to its classical CD40 receptor, CD154 also binds to αIIbß3, α5ß1, and αMß2 integrins. Binding of CD154 to these receptors seems to play a key role in the pathogenic processes of chronic inflammation. This investigation was aimed at analyzing the functional interaction of CD154 with CD40, αIIbß3, and α5ß1 receptors. We found that the binding affinity of CD154 for αIIbß3 is ∼4-fold higher than for α5ß1. We also describe the generation of sCD154 mutants that lost their ability to bind CD40 or αIIbß3 and show that CD154 residues involved in its binding to CD40 or αIIbß3 are distinct from those implicated in its interaction to α5ß1, suggesting that sCD154 may bind simultaneously to different receptors. Indeed, sCD154 can bind simultaneously to CD40 and α5ß1 and biologically activate human monocytic U937 cells expressing both receptors. The simultaneous engagement of CD40 and α5ß1 activates the mitogen-activated protein kinases, p38, and extracellular signal-related kinases 1/2 and synergizes in the release of inflammatory mediators MMP-2 and -9, suggesting a cross-talk between these receptors.

Critical role of lipid rafts in CD154-mediated T cell signaling.

Although signal pathways triggered via the CD40 molecule are well characterized, those induced via CD154 are less known. This study demonstrates that engagement of CD154 in Jurkat D1.1 cells with soluble CD40 leads to PKC alpha and delta activation, calcium mobilization, and phosphorylation of the Map kinases ERK1/2 and p38. Such response is accompanied by significant recruitment of CD154 into lipid rafts. Disruption of lipid rafts integrity with nystatin or methyl beta-cyclodextrin abrogated PKCalpha PKCdelta and p38 phosphorylation, but had no effect on ERK1/2 phosphorylation. Inhibition of PKC activation completely abolished p38 phosphorylation but had no effect on ERK1/2 phosphorylation, suggesting that localization of CD154 within lipid rafts is an absolute requirement for CD154-induced PKCalpha- and PKCdelta-dependent p38 phosphorylation. Furthermore, CD154 acts as co-stimulator for the production of IL-2 in an APC-superantigen-T-cell activation model. The results obtained demonstrate for the first time, that lipid rafts are of immunological relevance for CD154-triggered signals, and reinforce the importance of CD154 in T-cell activation.

CD40 ligand binds to alpha5beta1 integrin and triggers cell signaling.

It was originally thought that the critical role of the CD40 ligand (CD40L) in normal and inflammatory immune responses was mainly mediated through its interaction with the classic receptor, CD40. However, data from CD40L(-/-) and CD40(-/-) mice suggest that the CD40L-induced inflammatory immune response involves at least one other receptor. This hypothesis is supported by the fact that CD40L stabilizes arterial thrombi through an alphaIIbbeta3-dependent mechanism. Here we provide evidence that soluble CD40L (sCD40L) binds to cells of the undifferentiated human monocytic U937 cell line in a CD40- and alphaIIbbeta3-independent manner. Binding of sCD40L to U937 cells was inhibited by anti-CD40L monoclonal antibody 5C8, anti-alpha5beta1 monoclonal antibody P1D6, and soluble alpha5beta1. The direct binding of sCD40L to purified alpha5beta1 was confirmed in a solid phase binding assay. Binding of sCD40L to alpha5beta1 was modulated by the form of alpha5beta1 expressed on the cell surface as the activation of alpha5beta1 by Mn(2+) or dithiothreitol resulted in the loss of sCD40L binding. Moreover, sCD40L induced the translocation of alpha5beta1 to the Triton X-100-insoluble fraction of U937 cells, the rapid activation of the MAPK pathways ERK1/2, and interleukin-8 gene expression. The binding of sCD40L to CD40 on BJAB cells, an alpha5beta1-negative B cell line, and the resulting activation of ERK1/2 was not inhibited by soluble alpha5beta1, suggesting that sCD40L can bind concomitantly to both receptors. These results document the existence of novel CD40L-dependent pathways of physiological relevance for cells expressing multiple receptors (CD40, alpha5beta1, and alphaIIbbeta3) for CD40L.

Delineation of the HLA-DR region and the residues involved in the association with the cytoskeleton.

Whereas the association of major histocompatibility complex (MHC) class II molecules with the cytoskeleton and their recruitment into lipid rafts play a critical role during cognate T/antigen-presenting cell interactions, MHC class II-induced signals, regions, and residues involved in their association and recruitment have not yet been fully deciphered. In this study, we show that oligomerization of HLA-DR molecules induces their association with the cytoskeleton and their recruitment into lipid rafts. The association of oligomerized HLA-DR molecules with the cytoskeleton and their recruitment into lipid rafts occur independently. Furthermore, the association with the cytoskeleton is HLA-DR-specific, since oligomerization of HLA-DP triggers its recruitment only into lipid rafts. HLA-DR molecules devoid of both alpha and beta cytoplasmic tails did not associate with the cytoskeleton, but their recruitment into lipid rafts was unimpeded. Deletion of either the alpha or beta cytoplasmic tail did not affect the association of HLA-DR with the cytoskeleton and/or recruitment into lipid rafts. HLA-DR molecules that were devoid of the alpha cytoplasmic chain and that had their beta cytoplasmic chain replaced with the HLA-DP beta chain or with a beta chain in which the residues at positions Gly(226)-His(227)-Ser(228) were substituted by alanine no longer associated with the cytoskeleton. They were, however, still recruited into lipid rafts. Together, these results support the involvement of different regions of the cytoplasmic tails in the association and the recruitment of HLA-DR into different compartments. The differential behavior of HLA-DP and -DR with respect to their association with the cytoskeleton may explain the previously described difference in their transduced signals.

Zinc-binding sites in the N terminus of Mycoplasma arthritidis-derived mitogen permit the dimer formation required for high affinity binding to HLA-DR and for T cell activation.

Zinc-dependent superantigens can be divided into two subfamilies based on how they use zinc ions for interactions with major histocompatibility complex (MHC) class II molecules. Members of the first subfamily use zinc ions for interactions with histidine 81 on the beta-chain of MHC class II molecules, whereas members of the second subfamily use zinc ions for dimer formation. The zinc-binding motif is located in the C terminus of the molecule in both subfamilies. While our recent studies with Mycoplasma arthritidis-derived mitogen (MAM) have provided the first direct evidence demonstrating the binding to MHC class II molecules in a zinc-dependent manner, it still not known how zinc coordinates the interaction. Data presented here show that the zinc ion is mainly required to induce MAM/MAM dimer formation. Residues in the N terminus of MAM are involved in dimer formation and MHC class II binding, while histidine 14 and aspartic acid 31 of the MAM sequence are the major residues mediating MAM/MAM dimerization. Zinc-induced dimer formation is necessary for MAM binding, MHC class II-induced cell-cell adhesion, and efficient T cell activation. Together these results depict the unique mode of interaction of MAM in comparison with other superantigens.

Lipid raft-dependent and -independent signaling through HLA-DR molecules.

Lipid rafts are plasma membrane microdomains that are highly enriched in signaling molecules and that act as signal transduction platforms for many immune receptors. The involvement of these microdomains in HLA-DR-induced signaling is less well defined. We examined the constitutive presence of HLA-DR molecules in lipid rafts, their possible recruitment into these microdomains, and the role of these microdomains in HLA-DR-induced responses. We detected significant amounts of HLA-DR molecules in the lipid rafts of EBV(+) and EBV(-) B cell lines, monocytic cell lines, transfected HeLa cells, tonsillar B cells, and human monocytes. Localization of HLA-DR in these microdomains was unaffected by the deletion of the cytoplasmic domain of both the alpha and beta chains. Ligation of HLA-DR with a bivalent, but not a monovalent, ligand resulted in rapid tyrosine phosphorylation of many substrates, especially Lyn, and activation of ERK1/2 MAP kinase. However, the treatment failed to induce further recruitment of HLA-DR molecules into lipid rafts. The HLA-DR-induced signaling events were accompanied by the induction of cell-cell adhesion that could be inhibited by PTK and Lyn but not ERK1/2 inhibitors. Disruption of lipid rafts by methyl-beta-cyclodextrin (MbetaCD) resulted in the loss of membrane raft association with HLA-DR molecules, inhibition of HLA-DR-mediated protein tyrosine phosphorylation and cell-cell adhesion. MbetaCD did not affect the activation of ERK1/2, which was absent from lipid rafts. These results indicate that although all the HLA-DR-induced events studied are dependent on HLA-DR dimerization, some require the presence of HLA-DR molecules in lipid rafts, whereas others do not.

A proteomic approach to identify developmentally regulated proteins in Leishmania infantum.

We used comparative two-dimensional gel electrophoresis (2-DE) and mass spectrometry methodologies to highlight and identify proteins that are differentially expressed in the intracellular stage of the parasite Leishmania donovani infantum, a causative agent of visceral leishmaniasis. During its digenetic life cycle, Leishmania alternates between the alimentary tract of the sandfly vector as an extracellular promastigote and the acidic phagolysosomes of macrophage cells as an intracellular amastigote. Proteins differentially expressed in the intracellular form of the parasite are thought to be important for intracellular survival and pathogenesis. We used narrow pH range strips for isoelectric focusing to resolve soluble proteins of both developmental stages of L. infantum. More than 62 proteins differentially expressed in amastigotes were detected among approximately 2000 protein spots resolved by 2-DE. A quadrupole time-of-flight analysis of few selected protein spots, specifically expressed in the amastigote stage, permitted the identification of two proteins, part of the energetic metabolism pathways, the isocitrate dehydrogenase and the glycolytic enzyme triosephosphate isomerase. The kinetic parameters of these two enzymes were measured in both developmental stages of the parasite and their activity was indeed found to be higher in amastigotes. These findings bring a new insight in our understanding of metabolic and energy requirements of the intracellular form of Leishmania. Comparative analysis of the proteome of both developmental stages of the protozoan parasite Leishmania should permit the identification of protein candidates for the development of vaccines and new drugs.