Cell adhesion protein fibulin-7 and its C-terminal fragment negatively regulate monocyte and macrophage migration and functions in vitro and in vivo.

Fibulin-7 (Fbln7) has been identified as the latest member of the fibulin family of secreted glycoproteins in developing teeth, functioning as a cell adhesion molecule and interacting with other matrix proteins, receptors, and growth factors. More recently, we have shown that the C-terminal Fbln7 fragment (Fbln7-C) has antiangiogenic activity in vitro. Fbln7 is also expressed in immune-privileged tissues, such as eye and placenta, but its functional significance is unknown. In the current study, we show that human monocytes adhere to both full-length Fbln7 (Fbln7-FL) and Fbln7-C, in part, via integrins α5ß1 and α2ß1. Morphologic studies and surface expression analyses of CD14, mannose receptor (CD206), major histocompatibility complex II, and CD11b receptors revealed that both Fbln7-FL and Fbln7-C inhibit M-CSF-induced monocyte differentiation. Fbln7-C had significantly greater negative effects on cell spreading and stress fiber formation, including the production of IL-6 and metalloproteinase-1/-9 compared with Fbln7-FL. Furthermore, in an LPS-induced systemic inflammation model, Fbln7-C and Fbln7-FL reduced the infiltration of immune cells, such as neutrophils and macrophages, to the inflamed peritoneum. Thus, these results suggest that Fbln7 and Fbln7-C could modulate the activity of immune cells and have therapeutic potential for inflammatory diseases.-Sarangi, P. P., Chakraborty, P., Dash, S. P., Ikeuchi, T., de Vega, S., Ambatipudi, K., Wahl, L., Yamada, Y. Cell adhesion protein fibulin-7 and its C-terminal fragment negatively regulate monocyte and macrophage migration and functions in vitro and in vivo.

Heme oxygenase-1 induction alters chemokine regulation and ameliorates human immunodeficiency virus-type-1 infection in lipopolysaccharide-stimulated macrophages.

We have elucidated a putative mechanism for the host resistance against HIV-1 infection of primary human monocyte-derived macrophages (MDM) stimulated with lipopolysaccharide (LPS). We show that LPS-activated MDM both inhibited HIV-1 entry into the cells and were refractory to post-entry productive viral replication. LPS-treated cells were virtually negative for mature virions as revealed by transmission electron microscopy. LPS activation of MDM markedly enhanced the expression of heme oxygenase-1 (HO-1), a potent inducible cytoprotective enzyme. Increased HO-1 expression was accompanied by elevated production of macrophage inflammatory chemokines (MIP1α and MIP1ß) by LPS-activated MDM, significantly decreased surface chemokine receptor-5 (CCR-5) expression, and substantially reduced virus replication. Treatment of cells with HO-1 inhibitor SnPP IX (tin protoporphyrin IX) attenuated the LPS-mediated responses, HIV-1 replication and secretion of MIP1α, MIP1ß, and LD78ß chemokines with little change in surface CCR-5 expression. These results identify a novel role for HO-1 in the modulation of host immune response against HIV infection of MDM.

Endotoxin tolerance impairs IL-1 receptor-associated kinase (IRAK) 4 and TGF-beta-activated kinase 1 activation, K63-linked polyubiquitination and assembly of IRAK1, TNF receptor-associated factor 6, and IkappaB kinase gamma and increases A20 expression.

Endotoxin tolerance reprograms Toll-like receptor 4 responses by impairing LPS-elicited production of pro-inflammatory cytokines without inhibiting expression of anti-inflammatory or anti-microbial mediators. In septic patients, Toll-like receptor tolerance is thought to underlie decreased pro-inflammatory cytokine expression in response to LPS and increased incidence of microbial infections. The impact of endotoxin tolerance on recruitment, post-translational modifications and signalosome assembly of IL-1 receptor-associated kinase (IRAK) 4, IRAK1, TNF receptor-associated factor (TRAF) 6, TGF-ß-activated kinase (TAK) 1, and IκB kinase (IKK) γ is largely unknown. We report that endotoxin tolerization of THP1 cells and human monocytes impairs LPS-mediated receptor recruitment and activation of IRAK4, ablates K63-linked polyubiquitination of IRAK1 and TRAF6, compromises assembly of IRAK1-TRAF6 and IRAK1-IKKγ platforms, and inhibits TAK1 activation. Deficiencies in these signaling events in LPS-tolerant cells coincided with increased expression of A20, an essential deubiquitination enzyme, and sustained A20-IRAK1 associations. Overexpression of A20 inhibited LPS-induced activation of NF-κB and ablated NF-κB reporter activation driven by ectopic expression of MyD88, IRAK1, IRAK2, TRAF6, and TAK1/TAB1, while not affecting the responses induced by IKKß and p65. A20 shRNA knockdown abolished LPS tolerization of THP1 cells, mechanistically linking A20 and endotoxin tolerance. Thus, deficient LPS-induced activation of IRAK4 and TAK1, K63-linked polyubiquitination of IRAK1 and TRAF6, and disrupted IRAK1-TRAF6 and IRAK1-IKKγ assembly associated with increased A20 expression and A20-IRAK1 interactions are new determinants of endotoxin tolerance.

Cranberry juice constituents impair lymphoma growth and augment the generation of antilymphoma antibodies in syngeneic mice.

In addition to its nutritional value, cranberry juice has been effective in treating urinary tract infections. Various reports have also demonstrated its potential for inhibiting in vitro growth of transformed cell lines. Here we show that a fraction [nondialyzable material (NDM) of a molecular weight range 12,000-30,000 (NDM 12-30K)] derived from cranberry juice impairs in vitro growth and invasion through extracellular matrix of Rev-2-T-6 murine lymphoma cells. Furthermore, intraperitoneal injection of this fraction at nontoxic doses both inhibits the growth of Rev-2-T-6 tumors in vivo and enhances the generation of antilymphoma antibodies. These findings demonstrate the in vivo efficacy of cranberry components against malignant lymphoma in immune competent hosts.

A monocyte chemoattractant protein-1 gene polymorphism is associated with occult ischemia in a high-risk asymptomatic population.

Monocyte chemoattractant protein-1 (MCP-1) recruits monocytes into atherosclerotic plaques. A single nucleotide polymorphism in the MCP-1 gene promoter (-2578A>G) results in greater production of MCP-1 protein. We examined the association of this polymorphism with occult coronary artery disease (CAD) and its interaction with CAD risk factor burden, as assessed by the Framingham risk score (FRS) for hard events. We genotyped 679 apparently healthy 24-59-year-old siblings (SIBS) of people with premature CAD, tested for occult ischemia with exercise treadmill tests and thallium-201 single photon emission computed tomography, and assessed CAD risk factors to calculate the FRS. Occult ischemia occurred in 18% of SIBS and overall was somewhat more prevalent in those with the G allele (20.6%) compared to those without (15.6%), p=0.095. In SIBS at higher risk (highest quartile of FRS, >or=6.8%), occult ischemia occurred significantly more frequently in those with the G allele (44.4% versus 26.1%, p=0.017), while there was no significant difference in SIBS with lower FRS. After adjusting for individual risk factors included in the FRS, multivariate logistic regression modeling demonstrated that the G allele independently predicted occult ischemia in the entire study population (p=0.014, OR=1.86, 95% CI=1.14-3.04). This study demonstrates for the first time that the MCP-1 gene -2578A>G polymorphism is associated with an excess risk of coronary atherosclerosis in an asymptomatic population and demonstrates an apparent interaction with CAD risk factor burden.

Synergistic enhancement of cytokine-induced human monocyte matrix metalloproteinase-1 by C-reactive protein and oxidized LDL through differential regulation of monocyte chemotactic protein-1 and prostaglandin E2.

C-reactive protein (CRP) and oxidized LDL (ox-LDL) are associated with inflammatory lesions, such as coronary artery disease, in which monocytes and matrix metalloproteinases (MMPs) may play a major role in the rupture of atherosclerotic plaques. Monocytes are recruited to inflammation sites by monocyte chemoattractant protein-1 (MCP-1), which may also participate in the activation of monocytes. The objective of this study was to compare the individual and combined effect of CRP and ox-LDL on human monocyte MMP-1 and the role of MCP-1 in this effect. Although CRP or ox-LDL failed to induce MMP-1 in control monocytes, these molecules enhanced MMP-1 production induced by tumor necrosis factor alpha (TNF-alpha) and granulocyte macrophage-colony stimulating factor (GM-CSF) with a synergistic increase in MMP-1 occurring in the presence of both mediators. Enhancement of MMP-1 by CRP and ox-LDL was attributable to a differential increase in MCP-1 and prostaglandin E2(PGE2). CRP, at physiological concentrations, induced high levels of MCP-1 and relatively low levels of PGE2, whereas ox-LDL caused a significant enhancement of PGE2 with little affect on MCP-1. Accordingly, CRP- and ox-LDL-induced MMP-1 production by monocytes was inhibited by anti-MCP-1 antibodies and indomethacin, respectively. Moreover, addition of exogenous MCP-1 or PGE2 enhanced MMP-1 production by TNF-alpha- and GM-CSF-stimulated monocytes. These results show that the combination of CRP and ox-LDL can cause a synergistic enhancement of the role of monocytes in inflammation, first, by increasing MCP-1, which attracts more monocytes and directly enhances MMP-1 production by activated monocytes, and second, by elevating PGE2 production, which also leads to higher levels of MMP-1.

An adenoviral vector for probing promoter activity in primary immune cells.

Functional analysis of the DNA regulatory regions that control gene expression has largely been performed through transient transfection of promoter-reporter constructs into transformed cells. However, transformed cells are often poor models of primary cells. To directly analyze DNA regulatory regions in primary cells, we generated a novel adenoviral luciferase reporter vector, pShuttle-luciferase-GFP (pSLUG) that contains a promoterless luciferase cassette (with an upstream cloning site) for probing promoter activity, and a GFP expression cassette that allows for the identification of transduced cells. Recombinant adenoviruses generated from this vector can transduce a wide range of primary immune cells with high efficiency, including human macrophages, dendritic cells and T cells; and mouse T cells transgenic for the coxsackie and adenoviral receptor (CAR). In primary T cells, we show inducible nuclear factor of activated T cells (NF-AT) activity using a recombinant pSLUG adenovirus containing a consensus NF-AT promoter. We further show inducible IL-12/23 p40 promoter activity in primary macrophages and dendritic cells using a recombinant pSLUG adenovirus containing the proximal human IL-12/23 p40 promoter. The pSLUG system promises to be a powerful tool for the analysis of DNA regulatory regions in diverse types of primary immune cells.

Selective side-chain modification of cysteine and arginine residues blocks pathogenic activity of HIV-1-Tat functional peptides.

Extracellular Tat protein of HIV-1 activates virus replication in HIV-infected cells and induces a variety of host factors in the uninfected cells, some of which play a critical role in the progression of HIV infection. The cysteine-rich and arginine-rich basic domains represent key components of the HIV-Tat protein for pathogenic effects of the full-length Tat protein and, therefore, could be ideal candidates for the development of a therapeutic AIDS vaccine. The present study describes selective modifications of the side-chain functional groups of cysteine and arginine amino acids of these HIV-Tat peptides to minimize the pathogenic effects of these peptides while maintaining natural peptide linkages. Modification of cysteine by introducing either a methyl or t-butyl group in the free sulfhydryl group and replacing the guanidine group with a urea linkage in the side chain of arginine in the cysteine-rich and arginine-rich Tat peptide sequences completely blocked the ability of these peptides to induce HIV replication, chemokine receptor CCR-5 expression, and NF-kappaB activity in monocytes. Such modifications also inhibited angiogenesis and migration of Kaposi's sarcoma cells normally induced by Tat peptides. Such chemical modifications of the cysteine-rich and arginine-rich peptides did not affect their reactivity with antibodies against the full-length Tat protein. With an estimated 40 million HIV-positive individuals worldwide and approximately 4 million new infections emerging every year, a synthetic subunit HIV-Tat vaccine comprised of functionally inactive Tat domains could provide a safe, effective, and economical therapeutic vaccine to reduce the progression of HIV disease.

Production of matrix metalloproteinase-9 by activated human monocytes involves a phosphatidylinositol-3 kinase/Akt/IKKalpha/NF-kappaB pathway.

Matrix metalloproteinase-9 (MMP-9) is considered to be an important component in the progression of inflammation. Monocytes/macrophages are prominent at inflammation sites, and activation of these cells by stimulants, such as lipopolysaccharide (LPS) or tumor necrosis factor alpha and granulocyte macrophage-colony stimulating factor, leads to the production of significant amounts of MMP-9. Here, we show that LPS stimulation of monocytes results in MMP-9 production through a phosphatidylinositol-3 kinase (PI-3K)/Akt/inhibitor of kappaB (IkappaB) kinase-alpha (IKKalpha)/nuclear factor (NF)-kappaB pathway. This new role for Akt in signaling leading to MMP-9 production was demonstrated by inhibitor and immunoprecipitation studies. LY294002 or wortmannin, inhibitors of PI-3K, suppressed LPS-induced Akt activity and MMP-9 production. Evidence for the participation of Akt in monocyte MMP-9 synthesis was demonstrated by the inhibition of MMP-9 by SH-5, a specific inhibitor of Akt. The mechanism by which Akt regulates MMP-9 is through the activation of NF-kappaB, as shown by coimmunoprecipitation of the phosphorylated form of IKKalpha and Akt as well as the SH-5 suppression of the dissociation of IkappaB from NF-kappaB and the activation of NF-kappaB p65. The role of NF-kappaB in regulation of MMP-9 was demonstrated further by the inhibition of MMP-9 production by proteasome inhibitors, lactacystin and MG-132, which prevented the ubiquitination and dissociation of IkappaB from NF-kappaB. This is the first demonstration that Akt is involved in the signaling pathway leading to the production of monocyte MMP-9 and provides an additional approach in the regulation of this enzyme in human primary monocytes.

Angiotensin II increases human monocyte matrix metalloproteinase-1 through the AT2 receptor and prostaglandin E2: implications for atherosclerotic plaque rupture.

Angiotensin II (Ang II)-mediated hypertension increases the risk for acute coronary syndrome, a consequence of atherosclerotic plaque rupture, which may be caused by matrix metalloproteinases (MMPs). Here, we show that human primary monocytes stimulated with tumor necrosis factor alpha (TNF-alpha) and granulocyte macrophage-colony stimulating factor (GM-CSF) release Ang II, which is an integral component of the signal transduction pathway that leads to MMP-1 production. An Ang II-mediated increase in MMP-1 synthesis occurred only in conjunction with cytokine stimulation. Moreover, Ang II mediated its effect through the Ang II type 2 (AT(2)) receptor, as demonstrated by enhancement of MMP-1 production by an AT(2) agonist, CGP-42112A, and inhibition of MMP-1 production by PD1233319, an AT(2) antagonist. Additionally, exogenous Ang II caused a significant enhancement in MMP-1 production by cytokine-stimulated monocytes, and the most effective enhancement occurrred when Ang II was added 6 h after stimulation. Furthermore, Ang II and the AT(2) agonist increased prostaglandin E(2) (PGE(2)), which in turn mediated the increase in MMP-1, as shown by the inhibition of MMP-1 by indomethacin or aspirin. In contrast, the AT(2) antagonist inhibited the PGE(2) production induced by TNF-alpha and GM-CSF. Ang II, through its interaction with the AT(2) receptor, has a central role in mediating the PGE(2)-dependent production of MMP-1 by monocytes stimulated with TNF-alpha and GM-CSF. These observations provide insight into the association between hypertension and acute coronary syndrome and a possible mechanism by which Ang-converting enzyme inhibitor and aspirin may reduce the risk for heart attacks.

Oxidized low-density and high-density lipoproteins regulate the production of matrix metalloproteinase-1 and -9 by activated monocytes.

Monocytes/macrophages are prominent in atherosclerotic plaques where the vascular remodeling and plaque rupture may be influenced by the lipids and cytokines at these sites. Therefore, we evaluated the effects of factors found within the vascular wall, such as cytokines, oxidized low-density lipoprotein (ox-LDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL), on monocyte-derived matrix metalloproteinase-1 (MMP-1) and -9 (MMP-9) and tissue inhibitor of metalloproteinases-1 (TIMP-1). ox-LDL, LDL, and HDL alone had no effect on MMP-1, MMP-9, or TIMP-1 production. However, in the presence of tumor necrosis factor (TNF)-alpha and GM-CSF, ox-LDL enhanced MMP-1 significantly by two- to threefold, increased MMP-9 slightly, and had no effect on TIMP-1 production. In contrast, HDL suppressed the induction of MMP-1 by TNF-alpha and GM-CSF as well as the ox-LDL-mediated increase in MMP-1 production. The enhancement of MMP-1 production by ox-LDL occurred through, in part, a prostaglandin E2 (PGE2)-dependent pathway as indomethacin suppressed and PGE2 restored MMP-1 production. This conclusion was supported further by ox-LDL-mediated increases in PGE2 and cyclooxygenase-2 (COX-2) production. These data suggest that the interaction of primary monocytes with ox-LDL and proinflammatory cytokines may contribute to vascular remodeling and plaque rupture.

Cytokine-induced monocyte MMP-1 is negatively regulated by GSK-3 through a p38 MAPK-mediated decrease in ERK1/2 MAPK activation.

Elucidation of the signal transduction events leading to the production of MMPs by monocytes/macrophages may provide insights into the mechanisms involved in the destruction of connective tissue associated with chronic inflammatory lesions. Here, we show that GSK-3 is a negative regulator of cytokine-induced MMP-1 production by monocytes. Inhibition of monocyte GSK-3 pharmacologically with SB216763 or GSK-3ß siRNA caused a significant enhancement of MMP-1 by TNF-α- and GM-CSF-activated monocytes, indicating that induction of MMP-1 by TNF-α and GM-CSF involved phosphorylation/inactivation of GSK-3. TNF-α- and GM-CSF-induced phosphorylation of GSK-3 and subsequent MMP-1 production was blocked with the PKC inhibitor Gö6976 but not by the AKT1/2 inhibitor AKT VIII, showing that cytokine phosphorylation of GSK-3 occurs primarily through a PKC pathway. Inhibition of GSK-3 resulted in decreased phosphorylation of p38 MAPK with a corresponding increase in phosphorylation of ERK1/2 MAPK. Enhanced MMP-1 production by treatment with SB216763 was a result of increased ERK1/2 activation, as demonstrated by inhibition of MMP-1 by PD98059, a specific ERK1/2 inhibitor. Conversely, the p38 MAPK inhibitor SB203580 enhanced cytokine activation of ERK1/2 and the production of MMP-1 similar to that of SB216763. These findings demonstrate that the degree of cytokine-mediated phosphorylation/inhibition of GSK-3 determines the level of MMP-1 production through a mechanism involving decreased activation of p38 MAPK, a negative regulator of ERK1/2 required for cytokine-induced production of MMP-1 by monocytes.

Polyreactive antibodies plus complement enhance the phagocytosis of cells made apoptotic by UV-light or HIV.

Polyreactive antibodies are a major component of the natural antibody repertoire and are capable of binding a variety of structurally unrelated antigens. Many of the properties attributed to natural antibodies, in fact, are turning out to be due to polyreactive antibodies. In humans, each day, billions of cells undergo apoptosis. In the present experiments, we show by ImageStream technology that although polyreactive antibodies do not bind to live T cells they bind to both the plasma membrane and cytoplasm of late apoptotic cells, fix complement, generate the anaphylatoxin C5a and increase by as much as 5 fold complement-mediated phagocytosis by macrophages. Of particular importance, T cells undergoing apoptosis following infection with HIV also bind polyreactive antibodies and are phagocytosed. We conclude that the polyreactive antibodies in the natural antibody repertoire contribute in a major way to the clearance of cells made apoptotic by a variety of natural and infectious processes.

Involvement of TLR2 and TLR4 in cell responses to Rickettsia akari.

A better understanding of the pathogenesis of rickettsial disease requires elucidation of mechanisms governing host defense during infection. TLRs are primary sensors of microbial pathogens that activate innate immune cells, as well as initiate and orchestrate adaptive immune responses. However, the role of TLRs in rickettsia recognition and cell activation remains poorly understood. In this study, we examined the involvement of TLR2 and TLR4 in recognition of Rickettsia akari, a causative agent of rickettsialpox. Transfection-based complementation of TLR2/4-negative HEK293T cells with human TLR2 or TLR4 coexpressed with CD14 and MD-2 enabled IκB-α degradation, NF-κB reporter activation, and IL-8 expression in response to heat-killed (HK) R. akari. The presence of the R753Q TLR2 or D299G TLR4 polymorphisms significantly impaired the capacities of the respective TLRs to signal HK R. akari-mediated NF-κB reporter activation in HEK293T transfectants. Blocking Ab against TLR2 or TLR4 markedly inhibited TNF-α release from human monocytes stimulated with HK R. akari, and TNF-α secretion elicited by infection with live R. akari was reduced significantly only upon blocking of TLR2 and TLR4. Live and HK R. akari exerted phosphorylation of IRAK1 and p38 MAPK in 293/TLR4/MD-2 or 293/TLR2 stable cell lines, whereas only live bacteria elicited responses in TLR2/4-negative HEK293T cells. These data demonstrate that HK R. akari triggers cell activation via TLR2 or TLR4 and suggest use of additional TLRs and/or NLRs by live R. akari.

Endotoxin tolerance dysregulates MyD88- and Toll/IL-1R domain-containing adapter inducing IFN-beta-dependent pathways and increases expression of negative regulators of TLR signaling.

Endotoxin tolerance reprograms cell responses to LPS by repressing expression of proinflammatory cytokines, while not inhibiting production of anti-inflammatory cytokines and antimicrobial effectors. Molecular mechanisms of induction and maintenance of endotoxin tolerance are incompletely understood, particularly with regard to the impact of endotoxin tolerization on signalosome assembly, activation of adaptor-kinase modules, and expression of negative regulators of TLR signaling in human cells. In this study, we examined LPS-mediated activation of MyD88-dependent and Toll-IL-1R-containing adaptor inducing IFN-beta (TRIF)-dependent pathways emanating from TLR4 and expression of negative regulators of TLR signaling in control and endotoxin-tolerant human monocytes. Endotoxin tolerization suppressed LPS-inducible TLR4-TRIF and TRIF-TANK binding kinase (TBK)1 associations, induction of TBK1 kinase activity, activation of IFN regulatory factor (IRF)-3, and expression of RANTES and IFN-beta. Tolerance-mediated dysregulation of the TLR4-TRIF-TBK1 signaling module was accompanied by increased levels of suppressor of IkappaB kinase-epsilon (SIKE) and sterile alpha and Armadillo motif-containing molecule (SARM). LPS-tolerant cells showed increased expression of negative regulators Toll-interacting protein (Tollip), suppressor of cytokine signaling (SOCS)-1, IL-1R-associated kinase-M, and SHIP-1, which correlated with reduced p38 phosphorylation, IkappaB-alpha degradation, and inhibited expression of TNF-alpha, IL-6, and IL-8. To examine functional consequences of increased expression of Tollip in LPS-tolerized cells, we overexpressed Tollip in 293/TLR4/MD-2 transfectants and observed blunted LPS-inducible activation of NF-kappaB and RANTES, while TNF-alpha responses were not affected. These data demonstrate dysregulation of TLR4-triggered MyD88- and TRIF-dependent signaling pathways and increased expression of negative regulators of TLR signaling in endotoxin-tolerant human monocytes.

Stimulated stromal cells induce gamma-globin gene expression in erythroid cells via nitric oxide production.

OBJECTIVE: We have previously shown that nitric oxide (NO) is involved in the hydroxyurea-induced increase of gamma-globin gene expression in cultured human erythroid progenitor cells and that hydroxyurea increases NO production in endothelial cells via endothelial NO synthase (NOS). We have now expanded those studies to demonstrate that stimulation of gamma-globin gene expression is also mediated by NOS induction in stromal cells within the bone marrow microenvironment. MATERIALS AND METHODS: Using NO analyzer, we measured NO production in endothelial and macrophage cell cultures. In coculture studies of erythroid and stromal cells, we measured globin gene expression during stimulation by NO inducers. RESULTS: Hydroxyurea (30-100 microM) induced NOS-dependent production of NO in human macrophages (up to 1.2 microM). Coculture studies of human macrophages with erythroid progenitor cells also resulted in induction of gamma-globin mRNA expression (up to threefold) in the presence of hydroxyurea. NOS-dependent stimulation of NO by lipopolysaccharide (up to 0.6 microM) has been observed in human macrophages. We found that lipopolysaccharide and interferon-gamma together increased gamma-globin gene expression (up to twofold) in human macrophage/erythroid cell cocultures. Coculture of human bone marrow endothelial cells with erythroid progenitor cells also induced gamma-globin mRNA expression (2.4-fold) in the presence of hydroxyurea (40 microM). CONCLUSION: These results demonstrate an arrangement by which NO and fetal hemoglobin inducers may stimulate globin genes in erythroid cells via the common paracrine effect of bone marrow stromal cells.

Tyrosine phosphorylation of MyD88 adapter-like (Mal) is critical for signal transduction and blocked in endotoxin tolerance.

Toll-like receptor 4 (TLR4) recognition of lipopolysaccharide triggers signalosome assembly among TLR4, sorting (e.g. MyD88 adapter-like (Mal)) and signaling (e.g. MyD88) adapters, initiating recruitment and activation of kinases, activation of transcription factors, and production of inflammatory mediators. In this study we examined whether tyrosine phosphorylation of Mal regulates its interactions with TLR4, MyD88, interleukin-1 (IL-1) receptor-associated kinase (IRAK)-2, and tumor necrosis factor receptor-associated factor (TRAF)-6 and is important for signaling. Overexpression of wild-type Mal in human embryonic kidney 293T cells induced its constitutive tyrosine phosphorylation and led to activation of p38, NF-kappaB, and IL-8 gene expression. Mutagenesis of Tyr-86, Tyr-106, and Tyr-159 residues within the Toll-IL-1 receptor domain impaired Mal tyrosine phosphorylation, interactions with Bruton tyrosine kinase, phosphorylation of p38, and NF-kappaB activation. Lipopolysaccharide triggered tyrosine phosphorylation of endogenous Mal and initiated Mal-Bruton-tyrosine kinase interactions in 293/TLR4/MD-2 cells and human monocytes that were suppressed in endotoxin-tolerant cells. Compared with wild-type Mal, Y86A-, Y06A-, and Y159A-Mal variants exhibited higher interactions with TLR4 and MyD88, whereas associations with IRAK-2 and TRAF-6 were not affected. Overexpression of Y86A- and Y106A-Mal in 293/TLR4/MD-2 cells exerted dominant-negative effects on TLR4-inducible p38 phosphorylation and NF-kappaB reporter activation to the extent comparable with P125H-Mal-mediated suppression. In contrast, tyrosine-deficient Mal species did not affect NF-kappaB activation when signaling was initiated at the post-receptor level by overexpression of MyD88, IRAK-2, or TRAF-6. Thus, tyrosine phosphorylation of Mal is required for adapter signaling, regulates Mal interactions with TLR4 and receptor signaling, and is inhibited in endotoxin tolerance.

Urokinase-type plasminogen activator stimulation of monocyte matrix metalloproteinase-1 production is mediated by plasmin-dependent signaling through annexin A2 and inhibited by inactive plasmin.

Chronic inflammatory diseases are associated with connective tissue turnover that involves a series of proteases, which include the plasminogen activation system and the family of matrix metalloproteinases (MMPs). Urokinase-type plasminogen activator (uPA) and plasmin, in addition to their role in fibrinolysis and activation of pro-MMPs, have been shown to transduce intracellular signals through specific receptors. The potential for uPA and plasmin to also contribute to connective tissue turnover by directly regulating MMP production was examined in human monocytes. Both catalytically active high m.w. uPA, which binds to the uPAR, and low m.w. uPA, which does not, significantly enhanced MMP-1 synthesis by activated human monocytes. In contrast, the N-terminal fragment of uPA, which binds to uPAR, but lacks the catalytic site, failed to induce MMP-1 production, indicating that uPA-stimulated MMP-1 synthesis was plasmin dependent. Endogenous plasmin generated by the action of uPA or exogenous plasmin increased MMP-1 synthesis by signaling through annexin A2, as demonstrated by inhibition of MMP-1 production with Abs against annexin A2 and S100A10, a dimeric protein associated with annexin A2. Interaction of plasmin with annexin A2 resulted in the stimulation of ERK1/2 and p38 MAPK, cyclooxygenase-2, and PGE(2), leading to increased MMP-1 production. Furthermore, binding of inactive plasmin to annexin A2 inhibited plasmin induction of MMP-1, suggesting that inactive plasmin may be useful in suppressing inflammation.

The broad antibacterial activity of the natural antibody repertoire is due to polyreactive antibodies.

Polyreactive antibodies bind to a variety of structurally unrelated antigens. The function of these antibodies, however, has remained an enigma, and because of their low binding affinity their biological relevance has been questioned. Using a panel of monoclonal polyreactive antibodies, we showed that these antibodies can bind to both Gram-negative and Gram-positive bacteria and acting through the classical complement pathway can inhibit bacterial growth by lysis, generate anaphylatoxin C5a, enhance phagocytosis, and neutralize the functional activity of endotoxin. Polyreactive antibody-enriched, but not polyreactive antibody-reduced, IgM prepared from normal human serum displays antibacterial activity similar to that of monoclonal polyreactive IgM. We conclude that polyreactive antibodies are a major contributor to the broad antibacterial activity of the natural antibody repertoire.

Role of TLR4 tyrosine phosphorylation in signal transduction and endotoxin tolerance.

In this study, we examined whether tyrosine phosphorylation of the Toll-IL-1 resistance (TIR) domain of Toll-like receptor (TLR) 4 is required for signaling and blocked in endotoxin tolerance. Introduction of the P712H mutation, responsible for lipopolysaccharide (LPS) unresponsiveness of C3H/HeJ mice, into the TIR domain of constitutively active mouse DeltaTLR4 and mutation of the homologous P714 in human CD4-TLR4 rendered them signaling-incompetent and blocked TLR4 tyrosine phosphorylation. Mutations of tyrosine residues Y674A and Y680A within the TIR domains of CD4-TLR4 impaired its ability to elicit phosphorylation of p38 and JNK mitogen-activated protein kinases, IkappaB-alpha degradation, and activation of NF-kappaB and RANTES reporters. Likewise, full-length human TLR4 expressing Y674A or Y680A mutations showed suppressed capacities to mediate LPS-inducible cell activation. Signaling deficiencies of the Y674A and Y680A TLR4s correlated with altered MyD88-TLR4 interactions, increased associations with a short IRAK-1 isoform, and decreased amounts of activated IRAK-1 in complex with TLR4. Pretreatment of human embryonic kidney (HEK) 293/TLR4/MD-2 cells with protein tyrosine kinase or Src kinase inhibitors suppressed LPS-driven TLR4 tyrosine phosphorylation, p38 and NF-kappaB activation. TLR2 and TLR4 agonists induced TLR tyrosine phosphorylation in HEK293 cells overexpressing CD14, MD-2, and TLR4 or TLR2. Induction of endotoxin tolerance in HEK293/TLR4/MD-2 transfectants and in human monocytes markedly suppressed LPS-mediated TLR4 tyrosine phosphorylation and recruitment of Lyn kinase to TLR4, but did not affect TLR4-MD-2 interactions. Thus, our data demonstrate that TLR4 tyrosine phosphorylation is important for signaling and is impaired in endotoxin-tolerant cells, and suggest involvement of Lyn kinase in these processes.