The priming effect of C5a on monocytes is predominantly mediated by the p38 MAPK pathway.

The dysregulation of the inflammatory response after trauma leads to significant morbidity and mortality. Monocytes and macrophages play a central role in the orchestration of the inflammatory response after injury. Serum interleukin-6 (IL-6) concentration correlates with poor outcomes after injury. Tumor necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine that plays a crucial role in the pathogenesis of multiple organ dysfunction syndrome. Furthermore, in the presence of C5a, monocytes and macrophages have potentiated responses, but the mechanisms underlying this response remain largely unknown. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy volunteers and pretreated with C5a (100 ng/mL) for 1 h before adding lipopolysaccharide (LPS) (10 ng/mL) for up to 20 h. Inhibitors for the mitogen-activated protein kinases (MAPKs) were added 1 h before adding C5a. C5a primes monocytes for LPS-induced IL-6 and TNF-alpha production. Treatment of PBMCs with C5a leads to a rapid activation of the 3 MAPK pathways. SP600125 (inhibitor of c-Jun NH2-terminal kinase MAPK) and PD98059 (inhibitor of extracellular signal-regulated kinase MAPK) did not affect the C5a priming of the LPS-induced IL-6 and TNF-alpha production, whereas SB203580, a specific inhibitor of p38 MAPK, did suppress the C5a priming effect. These results demonstrate that C5a primes adherent PBMCs and modulates LPS-induced IL-6 and TNF-alpha production. Results from extracellular signal-regulated kinase and c-Jun NH2-terminal kinase MAPK blockade suggest that these signaling pathways have minimal or no role in reprogramming LPS-mediated IL-6 and TNF-alpha production. On the contrary, in PBMCs, C5a activates the p38 cascade, and this pathway plays a major role in the C5a enhancement of LPS-induced IL-6 and TNF-alpha production.

The influence of gender on human innate immunity.

BACKGROUND: Several experimental, clinical, and epidemiologic studies indicate a better prognosis in women after an infectious challenge. The monocyte/macrophage, as coordinators of the innate immune response to sepsis, secrete plasma inflammatory cytokines. Elevated plasma cytokine levels are inversely correlated with outcome. In addition, single-nucleotide polymorphisms related to these cytokine genes and in genes important for lipopolysaccharide (LPS) detection, particularly toll-like receptor-4, have been associated with variations in clinical outcome. We hypothesize that the gender differences in clinical outcome are due to measurable differences in cytokine responses and intracellular signaling, and these differences are independent of polymorphism carrier status. METHODS: Venous blood samples from healthy subjects (56 men, 23 women) were incubated with LPS, and supernatant cytokine levels were determined by enzyme-linked immunosorbent assay. In a randomly chosen subgroup, (8 men, 4 women), peripheral blood mononuclear cells were isolated, and LPS-mediated intracellular mitogen-activated protein kinase (MAPK) phosphorylation was assayed via Western blot analysis. Each subject was screened for the following SNPs: tumor necrosis factor alpha (TNF-alpha) -308G/A, interleukin (IL)-6 -174G/C, IL-1beta -31C/T, and toll-like receptor-4 (TLR4) +896A/G. RESULTS: Women produced significantly less LPS-induced TNF-alpha and IL-1beta but not IL-6. When the analysis was adjusted for the presence of each polymorphism, the differences in TNF-alpha and IL-1beta accumulation persisted. Female gender was associated with lower MAPK phosphorylation at each LPS concentration but was not statistically significant. CONCLUSIONS: Gender-specific differences in LPS-induced TNF-alpha and IL-1beta were observed, possibly attributed to alterations in MAPK phosphorylation. Furthermore, studies investigating the influence of genomic variation on the innate immune response should address potential gender-related differences.

Hypertonic saline modulates innate immunity in a model of systemic inflammation.

We sought to determine if hypertonic saline (HTS) impacted alveolar macrophage (AM) activation and intracellular inflammatory gene signaling in a model of systemic inflammation. Rats received an intravenous administration of 4 mL/kg of 7.5% HTS or L-lactate lactated Ringer's (L-LR). They were simultaneously treated with an intraperitoneal injection of zymosan, which induces noninfectious systemic inflammation. AM were harvested by bronchoalveolar lavage 24 h after treatment. AM activation was analyzed by measurement of baseline and lipopolysaccharide (LPS)-induced TNF-alpha production. Intracellular signaling was analyzed for activation of the mitogen-activated protein kinases (MAPKs): ERK1/2, JNK, and p38. AM from HTS-treated rats produced less TNF-alpha than from L-LR-treated rats (927 +/- 335 pg/mL [SEM] vs. 3628 +/- 783 pg/mL [SEM], P = 0.001) and were also less responsive to LPS (4444 +/- 86 pg/mL [SEM] vs. 6666 +/- 91 pg/mL [SEM], P = 0.058). However, there was no difference in MAPK activation. In vivo HTS prevents excessive AM activation during systemic inflammation. This suppression is mediated through alternate pathways and does not induce the classic MAPK signaling cascade.

Monocyte activation on polyelectrolyte multilayers.

The adherence and activation of primary human monocytes was investigated on a polyelectrolyte multilayer film containing hyaluronic acid (HA) and poly-L-lysine (PLL). The sequential layer-by-layer deposition of the multilayer film was characterized by surface plasmon resonance. Eight alternating bilayers displayed an effective thickness of 16.15 nm with a total polymer coverage of 2.10 microg/cm2. For cell studies, HA-PLL multilayers were constructed on tissue culture polystyrene (TCPS) substrates and characterized by time of flight second ion mass spectrometry (ToF-SIMS) analysis. Principal component analysis of the ToF-SIMS spectra resolved no significant difference in surface chemistry between PLL-terminated and HA-terminated multilayer surfaces. Monocyte adhesion on PLL- and HA-terminated surfaces was measured by the lactate dehydrogenase assay and showed a significant decrease in cell adhesion after 24 h incubation. Cell viability measured by Live/Dead fluorescent staining showed significant cell death in the adherent cell population over these 24 h. Tumor necrosis factor-alpha (TNF-alpha) production, a measure of monocyte activation, was quantified by ELISA and normalized to the number of adherent monocytes. The activation of monocytes on PLL-terminated and HA-terminated surfaces was nearly identical, and both surfaces had TNF-alpha levels that were 8-fold higher than TCPS. These results demonstrate that sufficient PLL had diffused into the surface layer to direct monocyte adherence and to induce cytokine activation and cell death on the HA-terminated multilayer films. The diffusion of the second multilayer component to the coating surface should, thus, be taken into account in the design of polyelectrolyte-based biomaterial coating strategies.

Calcium/calmodulin-dependent kinase II is required for platelet-activating factor priming.

Platelet-activating factor (PAF) primes the macrophage proinflammatory response to inflammatory stimuli, such as lipopolysaccharide (LPS). The cellular events responsible for this priming or reprogramming remain unresolved, but may occur through an increase in cytosolic calcium, inducing calcium/calmodulin-dependent kinase (CaMK) activation. To study this, differentiated THP-1 cells were used to study the effect of CaMK II and IV inhibition on PAF-induced reprogramming of TLR4-mediated events. LPS induced p38, ERK 1/2, and JNK/SAPK phosphorylation, NF-kappaB and AP-1 activation, and TNF-alpha and IL-10 production. PAF pretreatment selectively increased LPS-induced ERK 1/2, JNK/SAPK, NF-kappaB and AP-1 activation, and TNF-alpha production. Inhibition of CaMK II prevented PAF-induced priming of these events. Inhibition of CaMK IV prevented LPS-induced ERK 1/2, JNK/SAPK, NF-kappaB and AP-1 activation, and TNF-alpha production, but increased IL-10 production with or without PAF pretreatment. Neither CaMK II nor IV inhibition had any affect on p38 activity. These data suggest that the function of CaMK II is essential for PAF-induced macrophage priming. This priming event is mediated in part by modulation of ERK 1/2, JNK/SAPK, NF-kappaB, and AP-1 activation. CaMK IV, on the other hand, is not specific for priming by PAF and appears to have a direct link in TLR4-mediated events.

Anti-inflammatory drug delivery from hyaluronic acid hydrogels.

Two different types of hyaluronic acid (HA) hydrogels were synthesized by crosslinking HA with divinyl sulfone (DVS) and poly(ethylene glycol)-divinyl sulfone (VS-PEG-VS). Vitamin E succinate (VES), an anti-inflammatory drug, and bovine serum albumin (BSA), a model of anti-inflammatory protein drugs, were loaded into the gels and their release kinetics were measured in vitro. VES and BSA released with a burst from both HA hydrogels during the first few hours, and release continued gradually for several days. The rate of release from HA-VS-PEG-VS-HA hydrogels was faster than that from HA-DVS-HA hydrogels, presumably due to the lower crosslink density in the former. The anti-inflammatory action of released VES was tested by incubating peripheral blood mononuclear cells (PBMC) on HA hydrogels with and without VES in the gel. The number of cells adhering on HA hydrogels was very low compared to that on tissue culture polystyrene (TCPS), which might be one of the important advantages of using HA hydrogels for implant coatings or tissue engineering applications. ELISA test results showed that the tumor necrosis factor-alpha (TNF-alpha) concentration was very low in the supernatant of the wells containing the HA hydrogel with VES in contact with the activated macrophages compared to that without VES. This is probably the effect of the released VES reducing the production of anti-inflammatory cytokine, TNF-alpha. HA hydrogels containing anti-inflammatory drugs may have potential for use in tissue engineering and also as biocompatible coatings of implants.

Implications of proteasome inhibition: an enhanced macrophage phenotype.

The objective of this study was to elucidate the role of the cellular proteasome on endotoxin-mediated activation of the macrophage. To study this role, THP-1 cells were stimulated with lipopolysaccharide (LPS) with selective cells being pretreated with the proteasome inhibitor, lactacystin or MG-132. LPS stimulation led to the phosphorylation and degradation of IRAK, followed by activation of JNK/SAPK, ERK 1/2, and p38. Subsequently, LPS induced the degradation of IkappaB, and the nuclear activation of NF-kappaB and AP-1. Activation of these pathways was associated with the production of IL-6, IL-8, IL-10, and TNF-alpha. Proteasome inhibition with either lactacystin or MG-132 attenuated LPS-induced IRAK degradation, and enhanced activation of JNK/SAPK, ERK 1/2, and p38. Proteasome inhibition, also, led to increased LPS-induced AP-1 activation, and attenuated LPS-induced IkappaB degradation resulting in abolished NF-kappaB activation. Proteasome inhibition led to significant modulation of LPS-induced cytokine production; increased IL-10, no change in IL-6, and decreased IL-8, and TNF-alpha. Thus, this study demonstrates that cellular proteasome is critical to regulation of LPS-induced signaling within the macrophage, and inhibition of the proteasome results in a conversion to an anti-inflammatory phenotype.

Modulation of endotoxin-induced endothelial function by calcium/calmodulin-dependent protein kinase.

Endothelial cells facilitate sepsis-induced neutrophil adherence through the production of adhesion molecules and proinflammatory cytokines. The production of these factors requires coordinated intracellular inflammatory signaling. Recently, patients prone to sepsis-induced complications have been shown to have derangements in intracellular calcium and potentially calcium/calmodulin-dependent protein kinase (CaMK) activity, but the impact of these impairments is unknown. Human umbilical vein endothelial vein endothelial cells (HUVECs) were exposed to lipopolysaccharide (LPS) for various periods of time. Select HUVECs were pretreated with an inhibitor of CaMK II, KN62. Total cellular and nuclear proteins were extracted and analyzed for various components of the Toll-mediated signal cascade. Neutrophil adhesion was assayed fluorometrically using calcein-labeled neutrophils on treated HUVECs. LPS stimulation led to mitogen-activated protein kinase activation and translocation of activator protein-1 (AP-1) and nuclear factor (NF)-kappaB. CaMK blockade inhibited LPS induced ERK 1/2 and JNK but enhanced p38 activity. This selective MAPK inhibition was associated with a reduction in AP-1 activity, with no affect on NF-kappaB activity. Associated with this altered cell signaling was increased ICAM-1 production and enhanced neutrophil adhesion. Altered CaMK activity resulted in dysregulated mitogen-activated protein kinase signaling, demonstrated by reduced ERK 1/2 and JNK activity but enhanced p38 activity. This altered signaling is associated with reduced AP-1 activation and unaffected NF-kappaB activation. Neutrophil adhesion, however, is enhanced presumably through increased ICAM-1 production. Therefore, CaMK inhibition of endothelial cells, characteristic of sustained increases in intracellular calcium, appears to result in a dysregulated proadhesive phenotype.

Platelet-activating factor acetylhydrolase inhibits alveolar macrophage activation in vivo.

Platelet-activating factor (PAF) is an important proinflammatory mediator of septic shock. PAF is produced by activated macrophages and acts to perpetuate the response to endotoxin. PAF is metabolized by an endogenous PAF-acetylhydrolase (PAF-AH). Low circulating levels of PAF-AH have been associated with the development of postinjury multiple organ failure. We have previously shown that PAF-AH significantly inhibits macrophage activation by lipopolysaccharide (LPS) in vitro. The purpose of these studies was to determine whether this effect would translate to an in vivo model of remote lung injury. Wistar rats were administered a single intravenous dose of PAF-AH (5 mg/kg) or its carrier solution simultaneous with the induction of zymosan peritonitis. After 24 h, alveolar macrophages were obtained by bronchoalveolar lavage and stimulated in vitro with LPS (1 microg/mL). Supernatants were collected at 18 h for cytokine production and cellular and nuclear protein extractions were performed at 30 and 60 min to assess the activation of p38 and extracellular signal-regulated kinase (ERK) 1/2 kinases and the nuclear translocation of nuclear factor (NF)-kappaB. Administration of PAF-AH significantly inhibited LPS-induced tumor necrosis factor alpha and interleukin-1beta production by alveolar macrophages from zymosan-treated animals. This functional inhibition was associated with inhibition of ERK 1/2 kinase and NF-kappaB activation but not p38 kinase activation. Interleukin 6 production was depressed in the macrophages from zymosan-treated animals but no additional inhibition resulted from PAF-AH treatment. In conclusion, zymosan peritonitis leads to priming of alveolar macrophages such that their subsequent tumor necrosis factor alpha response to LPS is enhanced. In vivo administration of PAF-AH abrogates this response, suggesting that this priming may be PAF dependent. This effect of PAF-AH may be mediated by the inhibition of intracellular signaling via inhibition of ERK kinase and NF-kappaB activation.

Endotoxin-induced endothelial cell proinflammatory phenotypic differentiation requires stress fiber polymerization.

Endotoxin-induced intercellular adhesion molecule-1 (ICAM-1) and interleukin 8 (IL-8) production in endothelial cells, which is mediated by Toll-receptor signaling, is essential for optimal neutrophil recruitment and migration during sepsis. Endotoxin also causes stress fiber polymerization that has recently been shown to affect intracellular signaling. However, the role of this polymerization process on endothelial-induced neutrophil adhesion and migration is unknown. Human umbilical vein endothelial cells (HUVEC) were stimulated with lipopolysaccharide (LPS). Selected cells were pretreated with cytochalasin D (CD) or lactrunculin A (LA), agents that disrupt actin polymerization. Cellular protein was extracted and analyzed by Westem blot for the phosphorylated form of IL-1-associated kinase (IRAK) and production of ICAM-1. Extracted nuclear protein was analyzed by Western blot and electrophoretic mobility shift assay (EMSA) for nuclear translocation and activity of NF-kappaB. IL-8 production was determined by enzyme-linked immunoabsorbant assay (ELISA). Neutrophil adhesion was assayed fluorometrically using calcein-AM-labeled neutrophils on treated endothelial cells. LPS treatment led to phosphorylation of IRAK, and subsequent NF-kappaB translocation and activation. This cellular signaling was followed by ICAM-1 expression and IL-8 production. Pretreatment of cells with CD or LA led to a significant inhibition of IRAK phosphorylation, and NF-kappaB nuclear translocation and activation. Actin depolymerization also significantly inhibited LPS-induced ICAM-1 and IL-8 production. HUVEC pretreated with CD or LA demonstrated significant inhibition of LPS-induced neutrophil adhesion. Endotoxin-induced actin polymerization is essential for optimal intracellular signaling through IRAK and NF-kappaB. Failure of these signaling events is associated with a marked reduction in adhesion molecule production, IL-8 production, and neutrophil adhesion. These findings support the necessity of stress fiber polymerization for optimal recruitment of neutrophils during sepsis.

Modulation of endotoxin-induced endothelial activity by microtubule depolymerization.

BACKGROUND: Endotoxin not only activates the Toll-mediated signaling pathway within endothelial cells that leads to neutrophil migration but also causes the polymerization of microtubules. The potential role of this polymerization event, however, is unknown. METHODS: Human umbilical vein endothelial cells stimulated with endotoxin were pretreated with or without the microtubule depolymerizing agent colchicine. Toll-mediated signaling events and protein production were in turn investigated by Western blot, gel shift, and enzyme-linked immunosorbent assay. Finally, neutrophil adhesion was assayed fluorometrically under the various conditions. RESULTS: Endotoxin led to activation of the various Toll-mediated pathways, production of intercellular adhesion molecule-1 and interleukin-8, and subsequent neutrophil adhesion. Pretreatment with colchicine led to selective inhibition of anti-dual phosphorylated extracellular signal-regulated kinase-1/2, anti-dual phosphorylated c-jun N-terminal kinase, and adaptor protein-1; selective enhancement of p38; and no effect on nuclear factor-kappaB. This selective modulation of intracellular signaling resulted in attenuated intercellular adhesion molecule-1, interleukin-8 and prostaglandin E2 production, but enhanced cyclooxygenase-2 expression. As a result, microtubule disruption led to a significant reduction in neutrophil adhesion. CONCLUSION: Microtubule formation is essential to optimal endotoxin-induced intracellular signaling through anti-dual phosphorylated extracellular signal-regulated kinase-1/2, anti-dual phosphorylated c-jun N-terminal kinase, and adaptor protein-1. Failure of these signaling events is associated with a marked reduction in the formation of a proadhesive phenotype that may prove to be beneficial in modulating neutrophil recruitment during sepsis.

Platelet-activating factor priming of inflammatory cell activity requires cellular adherence.

BACKGROUND: Platelet-activating factor (PAF) primes tissue-fixed inflammatory cells, but has no effect on circulating cells. Adherence of inflammatory cells leads to cytoskeletal reorganization, which is essential for optimal inflammatory function. The purpose of this study was to investigate whether cellular adherence plays a role in PAF priming of inflammatory cells. METHODS: Differentiated THP-1 cells were maintained under adherent and nonadherent conditions. Selected cells were pretreated with PAF, followed by endotoxin stimulation. Cellular and nuclear proteins were analyzed by Western blot for components of the Toll-like receptor-mediated signaling cascade. Cytokine analysis was performed by enzyme-linked immunosorbent assay. RESULTS: Endotoxin led to activation of interleukin (IL)-1-associated kinase, extracellular signal-regulated kinase 1/2 and p38, and nuclear translocation of nuclear factor-kappaB, all of which were significantly enhanced by previous cellular adherence. PAF led to priming only under adherent conditions, demonstrated by increased IL-1-associated kinase and extracellular signal-regulated kinase 1/2 activity; nuclear factor-kappaB translocation; and IL-6, IL-8, and tumor necrosis factor-alpha production over non-PAF-treated cells. PAF had no significant effect on p38 activity or IL-10 production under any condition. CONCLUSIONS: PAF primes mononuclear cells by increasing Toll-mediated signaling only under adherent conditions. This, therefore, would limit PAF-induced priming in vivo to foci of stimulated adherent inflammatory cells with little effect systemically on circulating cells.

Slow channel calcium inhibition blocks proinflammatory gene signaling and reduces macrophage responsiveness.

BACKGROUND: This study investigates the possible intracellular mechanisms responsible for calcium antagonist protection in tissue-fixed macrophages, a central modulator of the proinflammatory phenotype. METHODS: Rabbit alveolar macrophages were exposed to lipopolysaccharide in the presence of different specific calcium antagonists. Cellular and nuclear protein were extracted and analyzed by Western blot for the phosphorylated forms of PYK2, ERK 1/2, and p38, and nuclear translocation of NF-kappaB and AP-1. Tumor necrosis factor-alpha (TNF-alpha) expression was measured by an L929 bioassay on cellular supernatants. Statistical analysis was performed by unpaired Student's t tests. RESULTS: Cells pretreated with 100 to 500 micromol/L of diltiazem or 50 to 100 micromol/L of verapamil, both slow channel calcium blockers, led to dose-dependent reductions in lipopolysaccharide-induced PYK2 and ERK 1/2 phosphorylation, and nuclear translocation of AP-1 when compared with controls (p < 0.05). Neither inhibitor had any significant effect on p38 or NF-kappaB translocation. EGTA an extracellular calcium chelator, had no significant effect on any intracellular process studied. A dose-dependent reduction in TNF-alpha production was demonstrated with diltiazem and verapamil (p < 0.05), with no effect induced by EGTA. CONCLUSION: Slow channel calcium influx is essential for optimal intracellular signaling through PYK2 and ERK 1/2. This reduced intracellular signaling correlated with reduced AP-1 translocation and TNF-alpha production. Extracellular calcium chelation had no significant effect on intracellular signaling or TNF-alpha production. This study further elucidates the protective mechanism of action of calcium channel blockade by diltiazem and verapamil by reducing intracellular calcium release and down-regulating the excessive proinflammatory phenotype.

The actin cytoskeleton: an essential component for enhanced TNFalpha production by adherent monocytes.

Monocyte adherence induces the formation of focal adhesions, the interaction sites of intracellular signaling molecules and cytoskeletal proteins such as actin. We previously demonstrated that adherence potentiates human monocyte LPS-induced TNFalpha production. Hence, we hypothesized that the actin cytoskeleton is integral to adherence-induced priming for enhanced LPS-induced TNFalpha production. In contrast to nonadherent cells, LPS induced significant transcription of TNFalpha mRNA and production of TNFalpha in adherent monocytes. Disrupting the actin cytoskeleton with cytochalasin D (CD) in adherent monocytes inhibited LPS-induced TNFalpha production by 55%, thereby abrogating adherence-induced priming. Moreover, CD pretreatment abrogated adherence-induced activation of Pyk2, a major focal adhesion kinase, and ERK 1/2, a component of the mitogen-activated protein kinase (MAPK) signaling pathway, and it completely inhibited LPS-induced ERK 1/2 activation. However, CD treatment of nonadherent monocytes failed to inhibit cytokine production. In conclusion, the actin cytoskeleton is integral in the reprogramming of the monocyte for enhanced cytokine production and in maintaining this "primed" state.

Hypertonic preconditioning inhibits macrophage responsiveness to endotoxin.

Hypertonic saline has been shown to modulate cell shape and the response of components of the innate immune response. However, the effect of hypertonic saline on the macrophage remains unknown. We hypothesized that hypertonic preconditioning would impair subsequent inflammatory mediator signaling through a reduction in stress fiber polymerization and mitogen-activated protein kinase activity after LPS stimulation. Rabbit alveolar macrophages were stimulated with 100 ng/ml of LPS. Selected cells were preconditioned with 40-100 mM of NaCl, mannitol, or urea for 4 h and returned to isotonic medium before LPS stimulation. Cellular protein was harvested and subjected to Western blot analysis for the dually phosphorylated active forms of p38 and extracellular signal-related kinase (ERK) 1/2. TNF production was determined by an L929 bioassay, and stress fiber polymerization was evaluated by confocal microscopy. Preconditioning of macrophages with NaCl or mannitol resulted in dose-dependent reduction in ERK 1/2 phosphorylation with no effect on p38 phosphorylation. Urea preconditioning had no effect on either mitogen-activated protein kinase. A dose-dependent attenuation of TNF production was seen with NaCl and mannitol preconditioning (p < 0.05), but not with urea. NaCl and mannitol preconditioning resulted in failure of LPS-induced stress fiber polymerization, whereas urea did not. Extracellular hypertonic conditions (i.e., NaCl and mannitol) have an immunomodulatory effect on macrophages, demonstrated through failure of optimal stress fiber polymerization, ERK 1/2 activity, and TNF production. Intracellular hypertonic conditions (i.e., urea) had no significant effect. Hypertonic saline or mannitol resuscitation, therefore, may help protect against multiple-organ dysfunction syndrome as a result of this reduced proinflammatory responsiveness.