PECAM-1 ligation negatively regulates TLR4 signaling in macrophages

Uncontrolled TLR4 signaling may induce excessive production of proinflammatory cytokines and lead to harmful inflammation; therefore, negative regulation of TLR4 signaling attracts much attention now. PECAM-1, a member of Ig-ITIM family, can mediate inhibitory signals in T cells and B cells. However, the role and the mechanisms of PECAM-1 in the regulation of TLR4-mediated LPS response in macrophages remain unclear. In this study, we demonstrate that PECAM-1 ligation with CD38-Fc fusion protein negatively regulates LPS-induced proinflammatory cytokine TNF-alpha, IL-6, and IFN-beta production by inhibiting JNK, NF-kappaB, and IFN regulatory factor 3 activation in macrophages. In addition, PECAM-1 ligation-recruited Src homology region 2 domain-containing phosphatase 1 (SHP-1) and Src homology region 2 domain-containing phosphatase 2 (SHP-2) may be involved in the inhibitory effect of PECAM-1 on TLR4 signaling. Consistently, silencing of PECAM-1 enhances the macrophage response to LPS stimulation. Taken together with the data that PECAM-1 is constitutively expressed in macrophages and its expression is up-regulated by LPS stimulation, PECAM-1 might function as a feedback negative regulator of LPS inflammatory response in macrophages. This study may provide a potential target for intervention of inflammatory diseases.     

The phosphatase SRC homology region 2 domain-containing phosphatase-1 is an intrinsic central regulator of dendritic cell function

Dendritic cells (DCs) initiate proinflammatory or regulatory T cell responses, depending on their activation state. Despite extensive knowledge of DC-activating signals, the understanding of DC inhibitory signals is relatively limited. We show that Src homology region 2 domain-containing phosphatase-1 (SHP-1) is an important inhibitor of DC signaling, targeting multiple activation pathways. Downstream of TLR4, SHP-1 showed increased interaction with several proteins including IL-1R-associated kinase-4, and modulated LPS signaling by inhibiting NF-κB, AP-1, ERK, and JNK activity, while enhancing p38 activity. In addition, SHP-1 inhibited prosurvival signaling through AKT activation. Furthermore, SHP-1 inhibited CCR7 protein expression. Inhibiting SHP-1 in DCs enhanced proinflammatory cytokines, IL-6, IL-12, and IL-1β production, promoted survival, and increased DC migration to draining lymph nodes. Administration of SHP-1-inhibited DCs in vivo induced expansion of Ag-specific cytotoxic T cells and inhibited Foxp3(+) regulatory T cell induction, resulting in an enhanced immune response against pre-established mouse melanoma and prostate tumors. Taken together, these data demonstrate that SHP-1 is an intrinsic global regulator of DC function, controlling many facets of T cell-mediated immune responses.     

The IREM-1 (CD300f) inhibitory receptor associates with the p85alpha subunit of phosphoinositide 3-kinase

The immune receptor expressed by myeloid cell 1 (IREM-1) (CD300f) inhibitory receptor displays five cytoplasmic tyrosine residues, two of them (Y205 and Y249) fit with ITIMs, whereas Y236 and Y263 constitute putative binding sites for PI3K. In the present study, immunoprecipitation analysis revealed that both the p85alpha subunit of PI3K and Src homology region 2 domain-containing phosphatase-1 could be recruited by IREM-1 in transfected cells as well as in the U937 monocytic leukemia cells, which constitutively express the receptor. By assaying the ability of different IREM-1 mutants to regulate the secretion of beta-hexosaminidase induced via FcRepsilonI in rat basophilic leukemia cells, both Y205 and Y249 appeared crucial for IREM-1-mediated inhibition. Remarkably, engagement of an IREM-1 mutant (Y(205,249,284)F), which did not recruit Src homology region 2 domain-containing phosphatase-1 and lost its inhibitory function, induced rat basophilic leukemia cell degranulation. This effect was dependent on the recruitment of PI3K, requiring the integrity of Y236 and Y263, and was blocked by PI3K inhibitors (i.e., wortmannin and LY-294002). Altogether, these data reveal a putative functional duality of the IREM-1 myeloid cell receptor.     

Syk protein tyrosine kinase involves PECAM-1 signaling through tandem immunotyrosine inhibitory motifs in human THP-1 macrophages

Although recent evidence supports a functional relationship between platelet endothelial cell adhesion molecule (PECAM-1) and Syk tyrosine kinase, little is known about the interaction of Syk with PECAM-1. We report that down-regulation of Syk inhibits the spreading of human THP-1 macrophage cells. Moreover, our data indicate that Syk binds PECAM-1 through its immune tyrosine-based inhibitory motif (ITIM), and dual phosphorylation of the ITIM domain of PECAM-1 leads to activation of Syk. Our results indicate that the distance between the phosphotyrosines could be up to 22 amino acids in length, depending on the conformational flexibility, and that the dual ITIM tyrosine motifs of PECAM-1 facilitate immunoreceptor tyrosine-based activation motif-like signaling. The preferential binding of PECAM-1 to Src homology region 2 domain-containing phosphatase-2 or Syk may depend on their relative affinities, and could provide a mechanism by which signal transduction from PECAM-1 is internally regulated by both positive and negative signaling enzymes.     

FcR-like 2 Inhibition of B cell receptor-mediated activation of B cells

FcR-like (FCRL) 2 is a transmembrane protein with immunomodulatory potential that is preferentially expressed by memory B cells in humans. It has two consensus ITIMs in addition to a putative ITAM sequence in its cytoplasmic domain. We have confirmed the cellular distribution of FCRL2 and analyzed its functional potential to show that coligation with the BCR leads to tyrosine phosphorylation of its ITIM motifs and subsequent Src homology region 2 domain-containing phosphatase-1 recruitment to facilitate inhibition of BCR signaling. Mutational analysis indicates that the tyrosine residues in both inhibitory motifs of FCRL2 are required for complete inhibition of BCR signaling, whereas tyrosines in the putative activation motif are dispensable for signal modulation. These findings suggest a negative immunomodulatory function for FCRL2 in the regulation of memory B cells.     

TGF-β1 re-programs TLR4 signaling in L. donovani infection: enhancement of SHP-1 and ubiquitin-editing enzyme A20

Visceral leishmaniasis (VL), caused by Leishmania donovani, is a major health concern in India. It represents T-helper type 2 (Th2) bias of cytokines in active state and Th1 bias at cure. However, the role of the parasite in regulating Toll-like receptor (TLR)-mediated macrophage activation in VL patients remains elusive. In this report, we demonstrated that later stages of L. donovani infection rendered tolerance to macrophages, leading to incapability for the production of inflammatory cytokines like tumor necrosis factor (TNF)-α and interleukin (IL)-1β in response to TLR stimulation. Overexpression of transforming growth factor (TGF)-β(1), but not IL-10, resulted in suppressed lipopolysaccharide (LPS)-induced production of TNF-α and downregulation of TLR4 expression in L. donovani-infected macrophages. Recombinant human (rh)TGF-β(1) markedly enhanced tyrosine phosphatase (Src homology region 2 domain-containing phosphatase-1) activity, but inhibited IL-1 receptor-activated kinase (IRAK)-1 activation. Addition of neutralizing TGF-β(1) antibody reversed these effects, and thus suggesting the pivotal role of TGF-β(1) in promoting refractoriness for LPS in macrophages. Surprisingly, the use of a tyrosine phosphatase inhibitor (sodium orthovanadate, Na(3)VO(4)) promoted IRAK-1 activation, confirming the negative inhibitory role of tyrosine phosphatase in macrophage activation. Furthermore, rhTGF-β(1) induced tolerance in infected macrophages by reducing inhibitory protein (IκBα) degradation in a time-dependent manner. In addition, short interfering RNA studies proved that overexpression of A20 ubiquitin-editing protein complex induced inhibitory activity of TGF-β(1) on LPS-mediated nuclear factor-κB activation. Thus, these findings suggest that TGF-β(1) promotes overexpression of A20 through tyrosine phosphatase activity that ensures transient activation of inflammatory signaling pathways in macrophages in active L. donovani infection.     

SHP-1 and SHP-2 associate with immunoreceptor tyrosine-based switch motif of programmed death 1 upon primary human T cell stimulation, but only receptor ligation prevents T cell activation

To study the cis- and trans-acting factors that mediate programmed death 1 (PD-1) signaling in primary human CD4 T cells, we constructed a chimeric molecule consisting of the murine CD28 extracellular domain and human PD-1 cytoplasmic tail. When introduced into CD4 T cells, this construct mimics the activity of endogenous PD-1 in terms of its ability to suppress T cell expansion and cytokine production. The cytoplasmic tail of PD-1 contains two structural motifs, an ITIM and an immunoreceptor tyrosine-based switch motif (ITSM). Mutation of the ITIM had little effect on PD-1 signaling or functional activity. In contrast, mutation of the ITSM abrogated the ability of PD-1 to block cytokine synthesis and to limit T cell expansion. Further biochemical analyses revealed that the ability of PD-1 to block T cell activation correlated with recruitment of Src homology region 2 domain-containing phosphatase-1 (SHP-1) and SHP-2, and not the adaptor Src homology 2 domain-containing molecule 1A, to the ITSM domain. In TCR-stimulated T cells, SHP-2 associated with PD-1, even in the absence of PD-1 engagement. Despite this interaction, the ability of PD-1 to block T cell activation required receptor ligation, suggesting that colocalization of PD-1 with CD3 and/or CD28 may be necessary for inhibition of T cell activation.     

Characterization of sparstolonin B, a Chinese herb-derived compound, as a selective Toll-like receptor antagonist with potent anti-inflammatory properties

Blockade of excessive Toll-like receptor (TLR) signaling is a therapeutic approach being actively pursued for many inflammatory diseases. Here we report a Chinese herb-derived compound, sparstolonin B (SsnB), which selectively blocks TLR2- and TLR4-mediated inflammatory signaling. SsnB was isolated from a Chinese herb, Spaganium stoloniferum; its structure was determined by NMR spectroscopy and x-ray crystallography. SsnB effectively inhibited inflammatory cytokine expression in mouse macrophages induced by lipopolysaccharide (LPS, a TLR4 ligand), Pam3CSK4 (a TLR1/TLR2 ligand), and Fsl-1 (a TLR2/TLR6 ligand) but not that by poly(I:C) (a TLR3 ligand) or ODN1668 (a TLR9 ligand). It suppressed LPS-induced cytokine secretion from macrophages and diminished phosphorylation of Erk1/2, p38a, IκBα, and JNK in these cells. In THP-1 cells expressing a chimeric receptor CD4-TLR4, which triggers constitutive NF-κB activation, SsnB effectively blunted the NF-κB activity. Co-immunoprecipitation showed that SsnB reduced the association of MyD88 with TLR4 and TLR2, but not that with TLR9, in HEK293T cells and THP-1 cells overexpressing MyD88 and TLRs. Furthermore, administration of SsnB suppressed splenocyte inflammatory cytokine expression in mice challenged with LPS. These results demonstrate that SsnB acts as a selective TLR2 and TLR4 antagonist by blocking the early intracellular events in the TLR2 and TLR4 signaling. Thus, SssB may serve as a promising lead for the development of selective TLR antagonistic agents for inflammatory diseases.     

Cutting edge: human 2B4, an activating NK cell receptor, recruits the protein tyrosine phosphatase SHP-2 and the adaptor signaling protein SAP.

The genetic defect in X-linked lymphoproliferative syndrome (XLP) is the Src homology 2 domain-containing protein SAP. SAP constitutively associates with the cell surface molecule, signaling lymphocytic activation molecule (SLAM), and competes with SH2-domain containing protein tyrosine phosphatase-2 (SHP-2) for recruitment to SLAM. SLAM exhibits homology with the mouse cell surface receptor 2B4. The human homologue of 2B4 has now been identified. It is recognized by the c1.7 mAb, a mAb capable of activating human NK cells. Human 2B4 became tyrosine phosphorylated following pervanadate-treatment of transfected cells and recruited SHP-2. SAP was also recruited to 2B4 in activated cells. Importantly, the 2B4-SAP interaction prevented the association between 2B4 and SHP-2. These results suggest that the phenotype of XLP may result from perturbed signaling not only through SLAM, but also other cell surface molecules that utilize SAP as a signaling adaptor protein.     

Myxoma virus lacking the pyrin-like protein M013 is sensed in human myeloid cells by both NLRP3 and multiple Toll-like receptors, which independently activate the inflammasome and NF-κB innate response pathways

The myxoma virus (MYXV)-encoded pyrin domain-containing protein M013 coregulates inflammatory responses mediated by both the inflammasome and the NF-κB pathways. Infection of human THP-1 monocytic cells with a MYXV construct deleted for the M013 gene (vMyxM013-KO), but not the parental MYXV, activates both the inflammasome and NF-κB pathways and induces a spectrum of proinflammatory cytokines and chemokines, like interleukin-1β (IL-1β), tumor necrosis factor (TNF), IL-6, and monocyte chemoattractant protein 1. Here, we report that vMyxM013-KO virus-mediated activation of inflammasomes and secretion of IL-1β are dependent on the adaptor protein ASC, caspase-1, and NLRP3 receptor. However, vMyxM013-KO virus-mediated activation of NF-κB signaling, which induces TNF secretion, was independent of ASC, caspase-1, and either the NLRP3 or AIM2 inflammasome receptors. We also report that early synthesis of pro-IL-1β in response to vMyxM013-KO infection is dependent upon the components of the inflammasome complex. Activation of the NLRP3 inflammasome and secretion of IL-1β was also dependent on the release of cathepsin B and production of reactive oxygen species (ROS). By using small interfering RNA screening, we further demonstrated that, among the RIG-I-like receptors (RLRs) and Toll-like receptors (TLRs), only TLR2, TLR6, TLR7, and TLR9 contribute to the NF-κB-dependent secretion of TNF and the inflammasome-dependent secretion of IL-1β in response to vMyxM013-KO virus infection. Additionally, we demonstrate that early triggering of the mitogen-activated protein kinase pathway by vMyxM013-KO virus infection of THP-1 cells plays a critical common upstream role in the coordinate induction of both NF-κB and inflammasome pathways. We conclude that an additional cellular sensor(s)/receptor(s) in addition to the known RLRs/TLRs plays a role in the M013 knockout virus-induced activation of NF-κB pathway signaling, but the activation of inflammasomes entirely depends on sensing by the NLRP3 receptor in response to vMyxM013-KO infection of human myeloid cells.     

Spontaneously diabetic Ins2(+/Akita):apoE-deficient mice exhibit exaggerated hypercholesterolemia and atherosclerosis

Type 2 diabetes (T2DM) is characterized by hyperglycemia, dyslipidemia, and increased inflammation. Previously, we showed that high glucose (HG) induces Toll-like receptor (TLR) expression, activity, and inflammation via NF-κB followed by cytokine release in vitro and in vivo. Here, we determined how HG-induced inflammation is affected by free fatty acids (FFA) in human monocytes. THP-1 monocytic cells, CD14(+) human monocytes, and transiently transfected HEK293 cells were exposed to various FFA (0-500 μM) and glucose (5-20 mM) for evaluation of TLR2, TLR4, NF-κB, IL-1β, monocyte chemoattractant protein-1 (MCP-1), and superoxide release. In THP-1 cells, palmitate increased cellular TLR2 and TLR4 expression, generated reactive oxygen species (ROS), and increased NF-κB activity, IL-1β, and MCP-1 release in a dose- and time-dependent manner. Similar data were observed with stearate and FFA mixture but not with oleate. Conversely, NADPH oxidase inhibitor treatment repressed glucose- and palmitate-stimulated ROS generation and NF-κB activity and decreased IL-1β and MCP-1 expression. Silencing TLR2, TLR4, and p47phox with small inhibitory RNAs (siRNAs) significantly reduced superoxide release, NF-κB activity, IL-1β, and MCP-1 secretion in HG and palmitate-treated THP-1 cells. Moreover, data from transient transfection experiments suggest that TLR6 is required for TLR2 and MD2 for TLR4 to augment inflammation in FFA- and glucose-exposed cells. These findings were confirmed with human monocytes. We conclude that FFA exacerbates HG-induced TLR expression and activity in monocytic cells with excess superoxide release, enhanced NF-κB activity, and induced proinflammatory factor release.     

Death receptors bind SHP-1 and block cytokine-induced anti-apoptotic signaling in neutrophils.

Death domain-containing receptors of the tumor necrosis factor (TNF)/nerve growth factor (NGF) family can induce apoptosis upon activation in many cellular systems. We show here that a conserved phosphotyrosine-containing motif within the death domain of these receptors can mediate inhibitory functions. The Src homology domain 2 (SH2)-containing tyrosine phosphatase-1 (SHP-1), SHP-2 and SH2-containing inositol phosphatase (SHIP) bound to this motif in a caspase-independent but cell-dependent manner. We also found that stimulation of death receptors disrupted anti-apoptosis pathways initiated (at least under certain conditions) by survival factors in neutrophils. In these cells, activation of the tyrosine kinase Lyn, an important anti-apoptotic event, was prevented as a consequence of death-receptor stimulation, most likely through association of the receptor with activated SHP-1. Thus, we provide molecular and functional evidence for negative signaling by death receptors.     

Kir3.1 channel is functionally involved in TLR4-mediated signaling

We aimed to study the involvement of Kir3.1 channel in TLR4-mediated signaling. LPS stimulation induced the recruitment of TLR4 and Kir3.1 into the lipid raft in THP-1 cells. Treatment with Tertiapin-Q, an inhibitor of Kir3.1, markedly abolished the recruitment of TLR4 into the lipid raft and inhibited the LPS-induced NF-κB activation, resulting in decreased production of TNF-α, IL-1β, and IL-6. To verify the specific role of the Kir3.1 channel, we generated Kir3.1-knockdown THP-1 cells. The Kir3.1^KD THP-1 cells exhibited inhibition of NF-κB activation and production of these pro-inflammatory cytokines in response to TLR4 stimulation. Taken together, our results demonstrate that the Kir3.1 channel is involved in the TLR4-mediated signal at an early event by facilitating the recruitment of TLR4 into lipid raft.     

TLR2 activation enhances HIV nuclear import and infection through T cell activation-independent and -dependent pathways

TLR2 activation plays a crucial role in Neisseria gonorrheae-mediated enhancement of HIV infection of resting CD4(+) T cells. We examined signaling pathways involved in the HIV enhancing effect of TLR2. TLR2 but not IL-2 signals promoted HIV nuclear import; however, both signals were required for the maximal enhancing effect. Although TLR2 signaling could not activate T cells, it increased IL-2-induced T cell activation. Cyclosporin A and IkBα inhibitor blocked TLR2-mediated enhancement of HIV infection/nuclear import. PI3K inhibitor blocked HIV infection/nuclear import and T cell activation and exerted a moderate inhibitory effect on cell cycle progression in CD4(+) T cells activated by TLR2/IL-2. Blockade of p38 signaling suppressed TLR2-mediated enhancement of HIV nuclear import/infection. However, the p38 inhibitor did not have a significant effect on T cell activation or TCR/CD3-mediated enhancement of HIV infection/nuclear import. The cell cycle arresting reagent aphidicolin blocked TLR2- and TCR/CD3-induced HIV infection/nuclear import. Finally, cyclosporin A and IκBα and PI3K inhibitors but not the p38 inhibitor blocked TLR2-mediated IκBα phosphorylation. Our results suggest that TLR2 activation enhances HIV infection/nuclear import in resting CD4(+) T cells through both T cell activation-dependent and -independent mechanisms.     

CD72 negatively regulates signaling through the antigen receptor of B cells

The immunoreceptor tyrosine-based inhibition motif (ITIM) is found in various membrane molecules such as CD22 and the low-affinity Fc receptor for IgG in B cells and the killer cell-inhibitory receptor and Ly-49 in NK cells. Upon tyrosine phosphorylation at the ITIMs, these molecules recruit SH2 domain-containing phosphatases such as SH2-containing tyrosine phosphatase-1 and negatively regulate cell activity. The B cell surface molecule CD72 carries an ITIM and an ITIM-like sequence. We have previously shown that CD72 is phosphorylated and recruits SH2-containing tyrosine phosphatase-1 upon cross-linking of the Ag receptor of B cells (BCR). However, whether CD72 modulates BCR signaling has not yet been elucidated. In this paper we demonstrate that expression of CD72 down-modulates both extracellular signal-related kinase (ERK) activation and Ca2+ mobilization induced by BCR ligation in the mouse B lymphoma line K46micromlambda, whereas BCR-mediated ERK activation was not reduced by the ITIM-mutated form of CD72. Moreover, coligation with CD72 with BCR reduces BCR-mediated ERK activation in spleen B cells of normal mice. These results indicate that CD72 negatively regulates BCR signaling. CD72 may play a regulatory role in B cell activation, probably by setting a threshold for BCR signaling.