Isolated Pneumocystis carinii cell wall glucan provokes lower respiratory tract inflammatory responses

Macrophage-induced lung inflammation contributes substantially to respiratory failure during Pneumocystis carinii pneumonia. We isolated a P. carinii cell wall fraction rich in glucan carbohydrate, which potently induces TNF-alpha and macrophage-inflammatory protein-2 generation from alveolar macrophages. Instillation of this purified P. carinii carbohydrate cell wall fraction into healthy rodents is accompanied by substantial increases in whole lung TNF-alpha generation and is associated with neutrophilic infiltration of the lungs. Digestion of the P. carinii cell wall isolate with zymolyase, a preparation containing predominantly beta-1,3 glucanase, substantially reduces the ability of this P. carinii cell wall fraction to activate alveolar macrophages, thus suggesting that beta-glucan components of the P. carinii cell wall largely mediate TNF-alpha release. Furthermore, the soluble carbohydrate beta-glucan receptor antagonists laminariheptaose and laminarin also substantially reduce the ability of the P. carinii cell wall isolate to stimulate macrophage-inflammatory activation. In contrast, soluble alpha-mannan, a preparation that antagonizes macrophage mannose receptors, had minimal effect on TNF-alpha release induced by the P. carinii cell wall fraction. P. carinii beta-glucan-induced TNF-alpha release from alveolar macrophages was also inhibited by both dexamethasone and pentoxifylline, two pharmacological agents with potential activity in controlling P. carinii-induced lung inflammation. These data demonstrate that P. carinii beta-glucan cell wall components can directly stimulate alveolar macrophages to release proinflammatory cytokines mainly through interaction with cognate beta-glucan receptors on the phagocyte.     

BCL10 mediates lipopolysaccharide/toll-like receptor-4 signaling through interaction with Pellino2

Toll-like receptor (TLR) pathways signal through microbial components stimulation to induce innate immune responses. Herein, we demonstrate that BCL10, a critical molecule that signals between the T cell receptor and IkappaB kinase complexes, is involved in the innate immune system and is required for appropriate TLR4 pathway and nuclear factor-kappaB (NF-kappaB) activation. In response to lipopolysaccharide (LPS) stimulation, BCL10 was recruited to TLR4 signaling complexes and associated with Pellino2, an essential component down-stream of BCL10 in the TLR4 pathway. In a BCL10-deficient macrophage cell line, LPS-induced NF-kappaB activation was consistently defective, whereas activator protein-1 and Elk-1 signaling was intact. In addition, we found that BCL10 was targeted by SOCS3 for negative regulation in LPS signaling. The recruitment of BCL10 to TLR4 signaling complexes was attenuated by induced expression of SOCS3 in a feedback loop. Furthermore, ectopic SOCS3 expression blocked the interaction between BCL10 and Pellino2 together with BCL10-generated NF-kappaB activation and inducible nitric-oxide synthase expression. Together, these data define an important role of BCL10 in the innate immune system.     

Participation of Rip2 in lipopolysaccharide signaling is independent of its kinase activity

Rip2 (Rick, Cardiak, CCK2, and CARD3) is a serine/threonine kinase containing a caspase recruitment domain (CARD) at the C terminus. Previous reports have shown that Rip2 is involved in multiple receptor signaling pathways that are important for innate and adaptive immune responses. However, it is not known whether Rip2 kinase activity is required for its function. Here we confirm that Rip2 participates in lipopolysaccharide (LPS)/Toll-like receptor (TLR4) signaling and demonstrate that its kinase activity is not required. Upon LPS stimulation, Rip2 was transiently recruited to the TLR4 receptor complex and associated with key TLR signaling mediators IRAK1 and TRAF6. Furthermore, Rip2 kinase activity was induced by LPS treatment. These data indicate that Rip2 is directly involved in the LPS/TLR4 signaling. Whereas macrophages from Rip2-deficient mice showed impaired NF-kappaB and p38 mitogen-activated protein kinase activation and reduced cytokine production in response to LPS stimulation, LPS signaling was intact in macrophages from mice that express Rip2 kinase-dead mutant. These results demonstrate that Rip2-mediated LPS signaling is independent of its kinase activity. Our findings strongly suggest that Rip2 functions as an adaptor molecule in transducing signals from immune receptors.     

Induction of cross-tolerance by lipopolysaccharide and highly purified lipoteichoic acid via different Toll-like receptors independent of paracrine mediators

Exposure of macrophages to LPS induces a state of hyporesponsiveness to subsequent stimulation with LPS termed LPS desensitization or tolerance. To date, it is not known whether similar mechanisms of macrophage refractoriness are induced on contact with components of Gram-positive bacteria. In the present study, we demonstrate that pretreatment with highly purified lipoteichoic acid (LTA) results in suppression of cytokine release on restimulation with LTA in vitro and in vivo in both C3H/HeN and C3H/HeJ mice, but not in macrophages from Toll-like receptor (TLR)-2-deficient mice. Furthermore, desensitization in response to LPS or LTA exposure also inhibits responses to the other stimulus ("cross-tolerance"), suggesting that signaling pathways shared by TLR2 and TLR4 are impaired during tolerance. Finally, we show that LPS- or LTA-induced cross-tolerance is not transferred to hyporesponsive cells cocultured with LPS/LTA-responsive macrophages, showing that soluble mediators do not suffice for tolerance induction in neighboring cells.     

Phosphorylation of TPL-2 on serine 400 is essential for lipopolysaccharide activation of extracellular signal-regulated kinase in macrophages

Tumor progression locus 2 (TPL-2) kinase is essential for Toll-like receptor 4 activation of the mitogen-activated protein kinase extracellular signal-regulated kinase (ERK) and for upregulation of the inflammatory cytokine tumor necrosis factor (TNF) in lipopolysaccharide (LPS)-stimulated macrophages. LPS activation of ERK requires TPL-2 release from associated NF-kappaB1 p105, which blocks TPL-2 access to its substrate, the ERK kinase MEK. Here we demonstrate that TPL-2 activity is also regulated independently of p105, since LPS stimulation was still needed for TPL-2-dependent activation of ERK in Nfkb1(-/-) macrophages. In wild-type macrophages, LPS induced the rapid phosphorylation of serine (S) 400 in the TPL-2 C-terminal tail. Mutation of this conserved residue to alanine (A) blocked the ability of retrovirally expressed TPL-2 to induce the activation of ERK in LPS-stimulated Nfkb1(-/-) macrophages. TPL-2(S400A) expression also failed to reconstitute LPS activation of ERK and induction of TNF in Map3k8(-/-) macrophages, which lack endogenous TPL-2. Consistently, the S400A mutation was found to block LPS stimulation of TPL-2 MEK kinase activity. Thus, induction of TPL-2 MEK kinase activity by LPS stimulation of macrophages requires TPL-2 phosphorylation on S400, in addition to its release from NF-kappaB1 p105. Oncogenic C-terminal truncations of TPL-2 that remove S400 could promote its transforming potential by eliminating this critical control step.     

Cross-tolerization between Nod1 and Nod2 signaling results in reduced refractoriness to bacterial infection in Nod2-deficient macrophages

Nod2 is an intracellular innate immune receptor that plays a role in host defense and susceptibility to inflammatory disease. We show in this study that macrophages rendered refractory to TLR4 and Nod2 signaling by exposure to LPS and muramyl dipeptide (MDP) exhibit impaired TNF-alpha and IL-6 production in response to pathogenic Listeria monocytogenes and Yersinia pseudotuberculosis as well as commensal bacteria including Escherichia coli and Bacteroides fragilis. Surprisingly, Nod2 deficiency was associated with impaired tolerization in response to pathogenic and commensal bacteria. Mechanistically, reduced tolerization of Nod2-null macrophages was mediated by recognition of bacteria through Nod1 because it was abolished in macrophages deficient in Nod1 and Nod2. Consistently, Nod2-null macrophages tolerant to LPS and MDP showed enhanced production of TNF-alpha and IL-6 as well as increased NF-kappaB and MAPK activation in response to the dipeptide KF1B, the Nod1 agonist. Furthermore, reduced tolerization of Nod2-deficient macrophages in response to bacteria was abolished when mutant macrophages were also rendered tolerant to the Nod1 ligand. Finally, MDP stimulation induced refractoriness not only to MDP, but also to iE-DAP stimulation, providing a mechanism to explain the reduced tolerization of Nod2-deficient macrophages infected with bacteria. These results demonstrate that cross-tolerization between Nod1 and Nod2 leads to increase recognition of both pathogenic and commensal bacteria in Nod2-deficient macrophages pre-exposed to microbial ligands.     

Cutting edge: purinergic signaling regulates radical-mediated bacterial killing mechanisms in macrophages through a P2X7-independent mechanism.

Signaling by extracellular nucleotides through P2 purinergic receptors affects diverse macrophage functions; however, its role in regulating antimicrobial radicals during bacterial infection has not been investigated. Mycobacterium tuberculosis-infected macrophages released ATP in a dose-dependent manner, which correlated with nitrite accumulation. P2 receptor inhibitors, including oxidized ATP, blocked NO synthase (NOSII) up-regulation and NO production induced by infection with M. tuberculosis or bacille Calmette-Guérin, or treatment with LPS or TNF-alpha. Oxidized ATP also inhibited oxygen radical production and activation of NF-kappaB and AP-1 in response to infection and inhibited NO-dependent killing of bacille Calmette-Guérin by macrophages. Experiments using macrophages derived from P2X7 gene-disrupted mice ruled out an essential role for P2X7 in NOSII regulation. These data demonstrate that P2 receptors regulate macrophage activation in response to bacteria and proinflammatory stimuli, and suggest that extracellular nucleotides released from infected macrophages may enhance production of oxygen radicals and NO at sites of infection.     

Endotoxin-induced maturation of MyD88-deficient dendritic cells

LPS, a major component of the cell wall of Gram-negative bacteria, can induce a variety of biological responses including cytokine production from macrophages, B cell proliferation, and endotoxin shock. All of them were completely abolished in MyD88-deficient mice, indicating the essential role of MyD88 in LPS signaling. However, MyD88-deficient cells still show activation of NF-kappaB and mitogen-activated protein kinase cascades, although the biological significance of this activation is not clear. In this study, we have examined the effects of LPS on dendritic cells (DCs) from wild-type and several mutant mice. LPS-induced cytokine production from DCs was dependent on MyD88. However, LPS could induce functional maturation of MyD88-deficient DCs, including up-regulation of costimulatory molecules and enhancement of APC activity. MyD88-deficient DCs could not mature in response to bacterial DNA, the ligand for Toll-like receptor (TLR)9, indicating that MyD88 is differentially required for TLR family signaling. MyD88-dependent and -independent pathways originate at the intracytoplasmic region of TLR4, because both cytokine induction and functional maturation were abolished in DCs from C3H/HeJ mice carrying the point mutation in the region. Finally, in vivo analysis revealed that MyD88-, but not TLR4-, deficient splenic CD11c(+) DCs could up-regulate their costimulatory molecule expression in response to LPS. Collectively, the present study provides the first evidence that the MyD88-independent pathway downstream of TLR4 can lead to functional DC maturation, which is critical for a link between innate and adaptive immunity.     

Mechanisms of endotoxin tolerance in human intestinal microvascular endothelial cells

Lipopolysaccharide (endotoxin) tolerance is well described in monocytes and macrophages, but is less well characterized in endothelial cells. Because intestinal microvascular endothelial cells exhibit a strong immune response to LPS challenge and play a critical regulatory role in gut inflammation, we sought to characterize the activation response of these cells to repeated LPS exposure. Primary cultures of human intestinal microvascular endothelial cells (HIMEC) were stimulated with LPS over 6-60 h and activation was assessed using U937 leukocyte adhesion, expression of E-selectin, ICAM-1, VCAM-1, IL-6, IL-8, manganese superoxide dismutase, HLA-DR, and CD86. Effect of repeat LPS stimulation on HIMEC NF-kappaB and mitogen-activated protein kinase (MAPK) activation, generation of superoxide anion, and Toll-like receptor 4 expression was characterized. LPS pretreatment of HIMEC for 24-48 h significantly decreased leukocyte adhesion after subsequent LPS stimulation. LPS pretreatment inhibited expression of E-selectin, VCAM-1, IL-6, and CD86, while ICAM-1, IL-8, and HLA-DR were not altered. Manganese superoxide dismutase expression increased with repeated LPS stimulation, with a reduction in intracellular superoxide. NF-kappaB activation was transiently inhibited by LPS pretreatment for 6 h, but not at later time points. In contrast, p44/42 MAPK, p38 MAPK, and c-Jun N-terminal kinase activation demonstrated inhibition by LPS pretreatment 24 or 48 h prior. Toll-like receptor 4 expression on HIMEC was not altered by LPS. HIMEC exhibit endotoxin tolerance after repeat LPS exposure in vitro, characterized by diminished activation and intracellular superoxide anion concentration, and reduced leukocyte adhesion. HIMEC possess specific mechanisms of immunoregulatory hyporesponsiveness to repeated LPS exposure.     

Sphingosine kinase protects lipopolysaccharide-activated macrophages from apoptosis

Lipopolysaccharide (LPS) signaling is critical for the innate immune response to gram-negative bacteria. Here, evidence is presented for LPS stimulation of sphingosine kinase (SPK) in the RAW 264.7 murine macrophage cell line and rat primary hepatic macrophages (HMs). LPS treatment of RAW 264.7 cells resulted in a time- and dose-dependent activation of SPK and membrane translocation of SPK1. Further, LPS-induced SPK activation was blocked by SPK1-specific small interfering RNA (siRNA). Overexpression of Toll-like receptor 4 and MD2, the receptor and coreceptor of LPS, in HEK 293 cells activated SPK activity in the absence of LPS treatment. Inhibition of SPK by the pharmacological inhibitor N,N-dimethylsphingosine (DMS) or SPK1-specific siRNA blocked LPS stimulation of extracellular signal-regulated kinase 1/2 and p38 but enhanced LPS-induced c-Jun N-terminal kinase activation. The SPK inhibitor DMS and dominant-negative SPK1 also blocked LPS activation of Elk-1 and NF-kappaB reporters in RAW 264.7 cells. Inhibition of SPK sensitized RAW 264.7 cells and HMs to LPS-induced apoptosis. These data demonstrate the critical role of SPK1 in LPS signaling in macrophages and suggest that SPK1 is a potential therapeutic target to block hyperimmune responses induced by gram-negative bacteria.     

Potentiation of caspase-1 activation by the P2X7 receptor is dependent on TLR signals and requires NF-kappaB-driven protein synthesis

The proinflammatory cytokines IL-1beta and IL-18 are inactive until cleaved by the enzyme caspase-1. Stimulation of the P2X7 receptor (P2X7R), an ATP-gated ion channel, triggers rapid activation of caspase-1. In this study we demonstrate that pretreatment of primary and Bac1 murine macrophages with TLR agonists is required for caspase-1 activation by P2X7R but it is not required for activation of the receptor itself. Caspase-1 activation by nigericin, a K+/H+ ionophore, similarly requires LPS priming. This priming by LPS is dependent on protein synthesis, given that cyclohexamide blocks the ability of LPS to prime macrophages for activation of caspase-1 by the P2X7R. This protein synthesis is likely mediated by NF-kappaB, as pretreatment of cells with the proteasome inhibitor MG132, or the IkappaB kinase inhibitor Bay 11-7085 before LPS stimulation blocks the ability of LPS to potentiate the activation of caspase-1 by the P2X7R. Thus, caspase-1 regulation in macrophages requires inflammatory stimuli that signal through the TLRs to up-regulate gene products required for activation of the caspase-1 processing machinery in response to K+-releasing stimuli such as ATP.     

An essential role of the NF-kappa B/Toll-like receptor pathway in induction of inflammatory and tissue-repair gene expression by necrotic cells

Tissue damage induced by infection or injury can result in necrosis, a mode of cell death characterized by induction of an inflammatory response. In contrast, cells dying by apoptosis do not induce inflammation. However, the reasons for underlying differences between these two modes of cell death in inducing inflammation are not known. Here we show that necrotic cells, but not apoptotic cells, activate NF-kappaB and induce expression of genes involved in inflammatory and tissue-repair responses, including neutrophil-specific chemokine genes KC and macrophage-inflammatory protein-2, in viable fibroblasts and macrophages. Intriguingly, NF-kappaB activation by necrotic cells was dependent on Toll-like receptor 2, a signaling pathway that induces inflammation in response to microbial agents. These results have identified a novel mechanism by which cell necrosis, but not apoptosis, can induce expression of genes involved in inflammation and tissue-repair responses. Furthermore, these results also demonstrate that the NF-kappaB/Toll-like receptor 2 pathway can be activated both by exogenous microbial agents and endogenous inflammatory stimuli.     

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.     

Induction of in vitro reprogramming by Toll-like receptor (TLR)2 and TLR4 agonists in murine macrophages: effects of TLR "homotolerance" versus "heterotolerance" on NF-kappa B signaling pathway components

In this study, tolerance induction by preexposure of murine macrophages to Toll-like receptor (TLR)2 and TLR4 agonists was revisited, focusing on the major signaling components associated with NF-kappaB activation. Pretreatment of macrophages with a pure TLR4 agonist (protein-free Escherichia coli (Ec) LPS) or with TLR2 agonists (Porphyromonas gingivalis LPS or synthetic lipoprotein Pam3Cys) led to suppression of TNF-alpha secretion, IL-1R-associated kinase-1, and IkappaB kinase (IKK) kinase activities, c-jun N-terminal kinase, and extracellular signal-regulated kinase phosphorylation, and to suppression of NF-kappaB DNA binding and transactivation upon challenge with the same agonist (TLR4 or TLR2 "homotolerance," respectively). Despite inhibited NF-kappaB DNA binding, increased levels of nuclear NF-kappaB were detected in agonist-pretreated macrophages. For all the intermediate signaling elements, heterotolerance was weaker than TLR4 or TLR2 homotolerance with the exception of IKK kinase activity. IKK kinase activity was unperturbed in heterotolerance. TNF-alpha secretion was also suppressed in P. gingivalis LPS-pretreated, Ec LPS-challenged cells, but not vice versa, while Pam3Cys and Ec LPS did not induce a state of cross-tolerance at the level of TNF-alpha. Experiments designed to elucidate novel mechanisms of NF-kappaB inhibition in tolerized cells revealed the potential contribution of IkappaBepsilon and IkappaBxi inhibitory proteins and the necessity of TLR4 engagement for induction of tolerance to Toll receptor-IL-1R domain-containing adapter protein/MyD88-adapter-like-dependent gene expression. Collectively, these data demonstrate that induction of homotolerance affects a broader spectrum of signaling components than in heterotolerance, with selective modulation of specific elements within the NF-kappaB signaling pathway.