BACKGROUNDNosocomial infection occurs commonly in intensive care units (ICUs). Although critical illness is associated with immune activation, the prevalence of nosocomial infections suggests concomitant immune suppression. This study examined the temporal occurrence of immune dysfunction across three immune cell types, and their relationship with the development of nosocomial infection.METHODSA prospective observational cohort study was undertaken in a teaching hospital general ICU. Critically ill patients were recruited and underwent serial examination of immune status, namely percentage regulatory T-cells (Tregs), monocyte deactivation (by expression) and neutrophil dysfunction (by CD88 expression). The occurrence of nosocomial infection was determined using pre-defined, objective criteria.RESULTSNinety-six patients were recruited, of whom 95 had data available for analysis. Relative to healthy controls, percentage Tregs were elevated 6-10 days after admission, while monocyte HLA-DR and neutrophil CD88 showed broader depression across time points measured. Thirty-three patients (35%) developed nosocomial infection, and patients developing nosocomial infection showed significantly greater immune dysfunction by the measures used. Tregs and neutrophil dysfunction remained significantly predictive of infection in a Cox hazards model correcting for time effects and clinical confounders {hazard ratio (HR) 2.4 [95% confidence interval (CI) 1.1-5.4] and 6.9 (95% CI 1.6-30), respectively, P=0.001}. Cumulative immune dysfunction resulted in a progressive risk of infection, rising from no cases in patients with no dysfunction to 75% of patients with dysfunction of all three cell types (P=0.0004).CONCLUSIONSDysfunctions of T-cells, monocytes, and neutrophils predict acquisition of nosocomial infection, and combine additively to stratify risk of nosocomial infection in the critically ill.

Introduction and Objectives The clinical diagnosis of ventilator-associated pneumonia (VAP) remains notoriously difficult, as several non-infective conditions mimic VAP. Microbiological confirmation of the diagnosis using conventional cultures typically takes 48–72 h. Identification of molecules measurable within a short time frame and closely associated with microbiologically confirmed VAP is therefore highly desirable. VAP is associated with significant influx of activated neutrophils into the alveolar space. We postulated that extracellular neutrophil proteases in bronchoalveolar lavage fluid (BALF) may reliably identify VAP in suspected cases. Methods Fifty-four intubated and mechanically ventilated patients in the intensive care unit developed clinically suspected VAP and were recruited. Bronchoalveolar lavage (BAL) was performed using a standardised protocol. An aliquot of BALF was sent to the diagnostic microbiology laboratory for quantitative culture, with confirmation of VAP defined as growth of a pathogen(s) at >104 colony forming units/ml. Remaining BALF was centrifuged. The following neutrophil-specific proteases were assayed in cell-free BALF supernatant—matrix metalloproteinase (MMP)-8 and MMP-9 by Luminex assay, and human neutrophil elastase (HNE) by enzyme-linked immunosorbent assay. Urea was simultaneously measured in serum and BALF, and used to correct for the dilution of epithelial lining induced by BAL. Receiver operating characteristic (ROC) curves were constructed and optimal specificity and sensitivity for each marker calculated. Results Eleven patients (20%) had confirmed VAP. For HNE (cut off 670ng/ml) the ROC area under curve (AUC) was 0.87 (p<0.0001), sensitivity 93%, specificity 79%. For MMP-8 (13 ng/ml), ROC AUC was 0.81 (p<0.005), sensitivity 91%, specificity 63%. For MMP-9 (22 ng/ml), ROC AUC was 0.79 (p<0.005), sensitivity 82%, specificity 63%. Conclusions Neutrophil proteases are strongly associated with confirmed infection in cases of suspected VAP. The values for HNE, in particular, compare extremely favourably with any previously published equivalent values. These data suggest that neutrophil protease concentrations in BALF deserve further attention as potentially diagnostic markers for VAP. They further suggest that neutrophil proteases, inappropriately released into the extracellular space, may contribute to the pathophysiology of VAP.

Introduction and objectives The pathogens most commonly implicated in ventilator-associated pneumonia (VAP) are Pseudomonas aeruginosa and Staphylococcus aureus. Although a florid inflammatory response characteristically occurs in the alveolar space in VAP, the underlying mechanisms remain unclear, partly owing to a lack of adequate models of alveolar injury. We therefore sought to characterise the response of primary human type II alveolar epithelial (ATII) cells to virulence factors from these pathogens. Methods Primary ATII cells were derived from seven patients undergoing surgical resection for lung cancer. Lung tissue was refrigerated overnight; flushed with saline; incubated with trypsin; diced; incubated with DNAse I; and strained/filtered. Macrophages and fibroblasts were removed by adherence. The resulting cell population was centrifuged, washed, resuspended and plated onto tissue culture plates pre-coated with type I bovine collagen at 2×106 cells/ml. When cells achieved confluence medium was replenished and the following were added for 24 h: 100 ng/ml P aeruginosa lipopolysaccharide (LPS); 10 ug/ml S aureus lipoteichoic acid (LTA); 10 ug/ml S aureus peptidoglycan (PGN); 10 ng/ml human recombinant tumour necrosis factor alpha (TNFα); or control medium. Supernatant was aspirated at 24 h and cytokines were measured by cytometric bead array. Results Interleukin (IL)-1β, IL-6, IL-8 IL-10, IL-12p70 and TNFα were all detectable in control medium at 24 h. None of the measured cytokines were significantly altered by application of LPS or LTA. In contrast, PGN induced a significant rise in concentrations of IL-1β, IL-6, IL-8 IL-10 and TNFα. Addition of TNFa induced a significant increase in IL-6, IL-8 and IL-10. The only cytokine to be uniformly uninfluenced by stimulation was IL-12p70. Conclusions In our hands primary ATII cells appeared to be unresponsive to P aeruginosa LPS or to S aureus LTA. By contrast, S aureus PGN provoked a brisk and significant inflammatory response simultaneously affecting a range of cytokines. These data suggest that ATII cells have strikingly different responses to individual bacterial virulence factors. They further suggest that PGN (but not LTA) contributes, at least in part, to the florid inflammatory response seen in Staphylococcal pneumonia.

BACKGROUND:The optimal method for diagnosing ventilator-associated pneumonia (VAP) is controversial and its effect on reported incidence uncertain. This study aimed to model the impact of using either endotracheal aspirate or bronchoalveolar lavage on the reported incidence of pneumonia and then to test effects suggested from theoretical modelling in clinical practice.METHODS:A three-part single-centre study was undertaken. First, diagnostic performance of aspirate and lavage were compared using paired samples from 53 patients with suspected VAP. Secondly, infection surveillance data were used to model the potential effect on pneumonia incidence and antibiotic use of using exclusively aspirate or lavage to investigate suspected pneumonia (643 patients; 110 clinically suspected pneumonia episodes). Thirdly, a practice change initiative was undertaken to increase lavage use; pneumonia incidence and antibiotic use were compared for the 12 months before and after the change.RESULTS:Aspirate overdiagnosed VAP compared with lavage (89% vs 21% of clinically suspected cases, p<0.0001). Modelling suggested that changing from exclusive aspirate to lavage diagnosis would decrease reported pneumonia incidence by 76% (95% CI 67% to 87%) and antibiotic use by 30% (95% CI 20% to 42%). After the practice change initiative, lavage use increased from 37% to 58%. Although clinically suspected pneumonia incidence was unchanged, microbiologically confirmed VAP decreased from 18 to 9 cases per 1000 ventilator days (p = 0.001; relative risk reduction 0.61 (95% CI 0.46 to 0.82)), and mean antibiotic use fell from 9.1 to 7.2 antibiotic days (21% decrease, p = 0.08).CONCLUSIONS:Diagnostic technique impacts significantly on reported VAP incidence and potentially on antibiotic use.

Hyaluronan (HA) accumulates in vascular disease but its functional role is not fully understood. To investigate the impact of HA enriched extracellular matrices (ECM) on cell phenotype, arterial smooth muscle cells (ASMCs) were transduced with retroviral constructs (LXSN) encoding murine has-1, has-2, and has-3. HA synthesis was significantly elevated in has transduced ASMCs. Metabolically labeled HA from has-1 and has-2 transduced cells was present mostly in high molecular weight (HWA) fractions (2-10x10(6) Da), whereas HA produced by has-3 and control cells was present in lower molecular weight fractions (approximately 2x10(6) Da). Both has-1 and has-3 transduced ASMCs accumulated more pericellular HA than has-2 transduced ASMCs. All has transduced ASMCs had attenuated growth and migration rates, and a decreased detachment response. Affinity histochemistry revealed that has-1 transduced ASMCs accumulated the greatest amount of HA containing ECM than the other transduced ASMCs. This ECM was hyaluronidase sensitive and bound a significantly greater number of monocytes than the ECM generated by has-2 or has-3 transduced ASMCs. Confocal microscopy showed CD44 positive monocytes bound to hyaluronidase sensitive ECM in has-1 transduced ASMCs. These data implicate specific has isoforms in the formation of HA enriched pro-inflammatory ECMs.

Interleukin-6 signaling via its soluble receptor (sIL-6R) differentially regulates inflammatory chemokine expression and leukocyte apoptosis to coordinate transition from neutrophil to mononuclear cell infiltration. sIL-6R activities may, however, be influenced in vivo by the occurrence of two sIL-6R isoforms that are released as a consequence of differential mRNA splicing (DS) or proteolytic cleavage (PC) of the cognate IL-6R (termed DS- and PC-sIL-6R). Using human peritoneal mesothelial cells and a murine model of peritoneal inflammation, studies described in this work have compared the ability of both isoforms to regulate neutrophil recruitment. In this respect, DS- and PC-sIL-6R were comparable in their activities; however, these studies emphasized that IL-6 trans signaling differentially controls neutrophil-activating CXC chemokine expression. In vitro, stimulation of mesothelial cells with IL-6 in combination with either DS-sIL-6R or PC-sIL-6R showed no induction of CXC chemokine ligand (CXCL)1 (GRO alpha) and CXCL8 (IL-8), whereas both isoforms enhanced CXCL5 (ENA-78) and CXCL6 (granulocyte chemotactic protein-2) expression. Moreover, when complexed with IL-6, both isoforms specifically inhibited the IL-1 beta-induced secretion of CXCL8. These findings were paralleled in vivo, in which induction of peritoneal inflammation in IL-6-deficient (IL-6(-/-)) mice resulted in enhanced keratinocyte-derived chemokine and macrophage-inflammatory protein-2 (the murine equivalent of CXCL1 and CXCL8) levels, but reduced LPS-induced CXC chemokine (the murine equivalent of CXCL5) expression. Reconstitution of IL-6 signaling in IL-6(-/-) mice with IL-6 and its soluble receptor isoforms corrected this chemokine imbalance and suppressed overall neutrophil infiltration. These data confirm that sIL-6R-mediated signaling primarily limits neutrophil influx; however, induction of CXCL5 and CXCL6 may regulate other neutrophil responses.

Hyaluronan (HA) is an important constituent of the extracellular matrix and accumulates during inflammatory lung diseases like asthma. Little is known about the factors that regulate HA synthesis by lung cells. Accordingly, we investigated the effect of T-helper 1 (TH1) and 2 (TH2) cytokines and the anti-inflammatory agents fluticasone and salmeterol on HA synthesis in human lung fibroblasts. Interleukin-1beta (IL-1beta) and tumor necrosis factor (TNF)-alpha were the most potent stimulators of HA synthesis and when combined, caused synergistic increases in HA accumulation. Time-course analysis of HA accumulation and [3H]-glucosamine incorporation into HA demonstrated continued synthesis over the 24 h of stimulation. Peak synthesis at 6-12 h coincided with an increased proportion of high molecular weight HA. Reverse transcriptase polymerase chain reaction (RT-PCR) revealed that IL-1beta and TNF-alpha induced HA synthase-2 messenger RNA (mRNA) 3 h following stimulation and remained elevated throughout the 24-h stimulation period. Fluticasone inhibited IL-1beta and TNF-alpha induced HA synthesis (44.5%) whereas salmeterol had no effect. When combined, fluticasone and salmeterol inhibited HA synthesis to a greater extent (85.2%). Further, fluticasone attenuated IL-1beta and TNF-alpha stimulated hyaluronan synthase-2 messenger RNA (mRNA), and the addition of salmeterol cooperatively enhanced this inhibition. These results indicate that enhanced synthesis of HA by the proinflammatory cytokines IL-1beta and TNF-alpha can be abrogated by specific corticosteroid and beta2 blocker combinations shown to be effective in the treatment of asthma.

RATIONALE: RV-induced PBMC production of IFN 7 correlates with clinical outcomes of rhinovirus infections, but whether this occurs via an IL-12-dependent mechanism has yet to be determined.METHODS: PBMCs from six allergic asthmatic volunteers were incubated 48 hours with RV 16 or media along with monoclonal blocking antibodie s to IL-12p40, IL-12p35, IL-15, IL-18, ICAM-I (the RVI6 receptor), or an lgGl isotype control.IFN 7 in culture supernates was measured by ELISA.In a second experiment, PBMCs were incubated with RV16 for 24 hours, and then IL-12p40 subunit and active IL-12p70 were assessed by ELISA. RESULTS:The mean PBMC production of IFN'/was significantly higher in RV 16-incubated cells than in media controls ( 194 vs. 4 pg/ml, n = 6, p < 0.001), and blocking ICAM-1 significantly decreased IFN'[ (19 pg/ml, n = 6, p<0.001).Blocking IL-12p40 or IL-12p35 caused a small decrease in IFN 7 (132 or 157 pg/ml, respectively), but this was not significantly different from lgG1 control (147 pg/ml, n = 6).Blocking IL-15 or IL-18 had no significant effect on IFN 7.Only small amounts of IL-12p40 (20 pg/ml) or IL-12p70 (2 pg/ml) were detected.CONCLUSIONS: Although IL-12, IL-15, and IL-18 can induce IFN 7, these cytokines do not significantly contribute to RV-induced IFN,/production by PBMCs, which may occur through a different mechanism.

Regulated recruitment and clearance of neutrophils (PMN) is the hallmark of competent host defense and resolution of inflammation. We now report that IFN-gamma controls PMN infiltration and modulates IL-6 signaling through its soluble receptor (sIL-6R) to promote their apoptosis and clearance. Induction of peritoneal inflammation in IFN-gamma-deficient (IFN-gamma-/-) mice emphasized that the initial rate of PMN recruitment was impaired. This defect in PMN recruitment was also associated with the suppressed intraperitoneal expression of IL-1beta and IL-6. Reconstitution of IFN-gamma signaling restored the rate of PMN infiltration and IL-6 levels and was accompanied by normalization of PMN-activating CXC chemokine expression. To test whether local IL-6 signaling modulated PMN recruitment, inflammation was induced in IFN-gamma-/- and IL-6-/- mice and cytokine signaling adapted by intraperitoneal sIL-6R-IL-6 fusion protein (HYPER-IL-6) or IFN-gamma. Although HYPER-IL-6 attenuated PMN influx in IFN-gamma-/- mice, IFN-gamma had no effect on PMN infiltration in IL-6-/- mice. Examination of the leukocyte infiltrate from IFN-gamma-/-, IL-6-/-, and wild-type mice showed that apoptosis was aberrant in the absence of IFN-gamma and IL-6 as a result of impaired sIL-6R signaling. These data emphasize a pivotal role for IFN-gamma in regulating innate immunity through control of both the recruitment and clearance phases of PMN trafficking.

Leukocyte recruitment into the infected peritoneal cavity consists of an early, predominant polymorphonuclear leukocyte (PMN) influx and subsequent, prolonged mononuclear cell migration phase. Although chemokine secretion by resident peritoneal cells plays a primary role in mediating this migration, the mechanisms involved in controlling the switch in phenotype of cell infiltrate remain unclear. The present study investigates a potential role for the Th1-type cytokine IFN-γ in the process of leukocyte recruitment into the peritoneal cavity. Stimulation of cultured human peritoneal mesothelial cells with IFN-γ (1–100 U/ml) alone or in combination with IL-1β (100 pg/ml) or TNF-α (1000 pg/ml) resulted in significant up-regulation of monocyte chemoattractant protein-1 and RANTES protein secretion. In contrast, IFN-γ inhibited basal and IL-1β-, and TNF-α-induced production of IL-8. The modulating effects of IFN-γ on chemokine production occurred at the level of gene expression, and the degree of regulation observed was dependent on the doses of IL-1β and TNF-α used. Analysis of the functional effects of IFN-γ on IL-1β-induced transmesothelial PMN migration with an in vitro human transmigration system and an in vivo murine model of peritoneal inflammation demonstrated that IFN-γ was able to down-regulate PMN migration induced by optimal doses of IL-1β. These effects were mediated in vivo via down-regulation of CXC chemokine synthesis. These findings suggest that IFN-γ may play a role in controlling the phenotype of infiltrating leukocyte during the course of an inflammatory response, in part via regulation of resident cell chemokine synthesis.

During acute inflammation, leukocyte recruitment is characterized by an initial infiltration of neutrophils, which are later replaced by a more sustained population of mononuclear cells. Based on both clinical and experimental evidence, we present a role for IL-6 and its soluble receptor (sIL-6R) in controlling this pattern of leukocyte recruitment during peritoneal inflammation. Liberation of sIL-6R from the initial neutrophil infiltrate acts as a regulator of CXC and CC chemokine expression, which contributes to a suppression of neutrophil recruitment and the concurrent attraction of mononuclear leukocytes. Soluble IL-6R-mediated signaling is therefore an important intermediary in the resolution of inflammation and supports transition between the early predominantly neutrophilic stage of an infection and the more sustained mononuclear cell influx.