Human Toll-like receptor (TLR) 4 and TLR2 receptors recognize LPS or lipoteichoic acid (LTA), respectively. Prolonged exposure of human macrophages/monocytes to bacterial LPS induces a state of adaptation/tolerance to subsequent LPS challenge. Inflammatory gene expressions such as IL-1β and TNF-α are selectively repressed, while certain anti-inflammatory genes such as secretory IL-1R antagonist are still induced in LPS-adapted/tolerant cells. In this report, we demonstrate that LPS-tolerized human promonocytic THP-1 cells develop cross-tolerance and no longer respond to LTA-induced IL-1β/TNF-α production, indicating that disruption of common intracellular signaling is responsible for the decreased IL-1β/TNF-α production. We observe that down-regulation of IL-1R-associated kinase (IRAK) protein level and kinase activity closely correlates with the development of cross-tolerance. IRAK protein levels and kinase activities in LPS-tolerized cells remain low and hyporesponsive to subsequent LPS or LTA challenges. We also demonstrate that THP-1 cells with prolonged LTA treatment develop LTA tolerance and do not express IL-1β/TNF-α upon further LTA challenge. Strikingly, cells tolerized with LTA are only refractory to subsequent LTA challenge and can still respond to LPS stimulation. Correspondingly, stimulation of TLR2 by LTA, although activating IRAK, does not cause IRAK degradation. IRAK from LTA-tolerized cells can be subsequently activated and degraded by further LPS challenge, but not LTA treatment. Our studies reveal that LTA-induced tolerance is distinct compared with that of LPS tolerance, and is likely due to disruption of unique TLR2 signaling components upstream of MyD88/IRAK.

IL-1R-associated kinase (IRAK) plays a pivotal role in IL-1R/Toll-like receptor (TLR)-mediated signaling and NF-κB activation. IRAK from leukocytes undergoes rapid activation and inactivation/degradation following IL-1 or LPS stimulation. The rapid degradation of IRAK may serve as a negative feedback mechanism of down-regulating IL-1R/TLR-mediated signaling and cytokine gene transcription. Although IL-1/IL-1R-triggered IRAK degradation has been studied in detail, the mechanism of LPS-induced IRAK activation and degradation is not clearly defined. In this study, we demonstrate that the IRAK N-terminal 186-aa region is required for LPS-induced degradation. The N-terminally truncated IRAK protein expressed in human monocytic THP-1 cells remains stable upon LPS challenge. In comparison, IRAK as well as the IRAK mutant with C-terminal truncation undergo degradation with LPS stimulation. We demonstrate that pretreatment with protein kinase C inhibitor calphostin inhibits LPS-induced IRAK degradation. Furthermore, we observe coimmunoprecipitation of endogenous IRAK and protein kinase C-ζ protein. We show that functional TLR4 is required for LPS-mediated IRAK degradation. IRAK protein in the murine GG2EE cells harboring a mutated TLR4 gene does not undergo degradation upon LPS treatment. In sharp contrast, we observe that the IRAK homolog, IRAK2, does not undergo degradation upon prolonged LPS treatment, suggesting complex regulation of the innate immunity network upon microbial challenge.

Hydrolysis of surfactant-associated phospholipids by secretory phospholipases A2 is an important potential mechanism for surfactant dysfunction in inflammatory lung diseases. In these conditions, airway secretory phospholipase A2(sPLA2) activity is increased, but the type of sPLA2 and its impact on surfactant function are not well understood. We examined in vitro the effect of multiple secretory phospholipases A2 on surfactant, including their ability to 1) release free fatty acids, 2) release lysophospholipids, and 3) increase the minimum surface tension (γmin) on a pulsating bubble surfactometer. Natural porcine surfactant and Survanta were exposed to mammalian group I (recombinant porcine pancreatic) and group II (recombinant human) secretory phospholipases A2. Our results demonstrate that mammalian group I sPLA2 hydrolyzes phosphatidylcholine (PC), producing free fatty acids and lysophosphatidylcholine, and increases γmin. In contrast, mammalian group II sPLA2 demonstrates limited hydrolysis of PC and does not increase γmin. Group I and group II secretory phospholipases A2 from snake venom hydrolyze PC and inhibit surfactant function. In summary, mammalian secretory phospholipases A2 from groups I and II differ significantly from each other and from snake venom in their ability to hydrolyze surfactant-associated PC.

Secretory A(2) phospholipases (sPLA(2)) hydrolyze surfactant phospholipids cause surfactant dysfunction and are elevated in lung inflammation. Phospholipase-mediated surfactant hydrolysis may disrupt surfactant function by generation of lysophospholipids and free fatty acids and/or depletion of native phospholipids. In this study, we quantitatively assessed multiple mechanisms of sPLA(2)-mediated surfactant dysfunction using non-enzymatic models including supplementation of surfactants with exogenous lysophospholipids and free fatty acids. Our data demonstrated lysophospholipids at levels >or=10 mol% of total phospholipid (i.e., >or=10% hydrolysis) led to a significant increase in minimum surface tension and increased the time to achieve a normal minimum surface tension. Lysophospholipid inhibition of surfactant function was independent of the lysophospholipid head group or total phospholipid concentration. Free fatty acids (palmitic acid, oleic acid) alone had little effect on minimum surface tension, but did increase the maximum surface tension and the time to achieve normal minimum surface tension. The combined effect of equimolar free fatty acids and lysophospholipids was not different from the effect of lysophospholipids alone for any measurement of surfactant function. Surfactant proteins did not change the percent lysophospholipids required to increase minimum surface tension. As a mechanism that causes surfactant dysfunction, depletion of native phospholipids required much greater change (equivalent to >80% hydrolysis) than generation of lysophospholipids. In summary, generation of lysophospholipids is the principal mechanism of phospholipase-mediated surfactant injury in our non-enzymatic models. These models and findings will assist in understanding more complex in vitro and in vivo studies of phospholipase-mediated surfactant injury.