Joseph M. Petty

Pulmonary stromal-derived factor-1 expression and effect on neutrophil recruitment during acute lung injury

The severe and protracted inflammation that characterizes acute lung injury (ALI) is driven by the ongoing recruitment of neutrophils to the lung. Although much of the cytokine signaling responsible for the initial phase of ALI has been elaborated, relatively little is known about the mechanisms governing the recruitment of neutrophils from the bone marrow to the lung in the later period of this disease. Given its previously described chemoattractant effects on marrow neutrophils, we investigated whether stromal-derived factor-1 (SDF-1) (CXCL12) might participate in this later phase of recruitment. Using immunohistochemistry to examine both banked human lung specimens from patients with ALI and lungs from mice with LPS-induced pneumonitis, we found that pulmonary SDF-1 expression increases during ALI. We further determined that both lung SDF-1 protein expression and mRNA expression rise in a delayed but sustained pattern in this mouse model and that the major source of the increase in expression appears to be the lung epithelium. Lastly, we found that expression of the SDF-1 receptor CXCR4 rises in a similar temporal pattern on neutrophils in both the blood and airspace of LPS-injured mice and that Ab-mediated SDF-1 blockade significantly attenuates late but not early pulmonary neutrophilia in this model. These results implicate SDF-1 in neutrophil recruitment to the lung in the later period of acute lung injury and suggest a novel role for this cytokine in coordinating the transition from the inflammatory response to the initiation of tissue repair.

Obesity Is Associated with Neutrophil Dysfunction and Attenuation of Murine Acute Lung Injury

Although obesity is implicated in numerous health complications leading to increased mortality, the relationship between obesity and outcomes for critically ill patients appears paradoxical. Recent studies have reported better outcomes and lower levels of inflammatory cytokines in obese patients with acute lung injury (ALI)/acute respiratory distress syndrome, suggesting that obesity may ameliorate the effects of this disease. We investigated the effects of obesity in leptin-resistant db/db obese and diet-induced obese mice using an inhaled LPS model of ALI. Obesity-associated effects on neutrophil chemoattractant response were examined in bone marrow neutrophils using chemotaxis and adoptive transfer; neutrophil surface levels of chemokine receptor CXCR2 were determined by flow cytometry. Airspace neutrophilia, capillary leak, and plasma IL-6 were all decreased in obese relative to lean mice in established lung injury (24 h). No difference in airspace inflammatory cytokine levels was found between obese and lean mice in both obesity models during the early phase of neutrophil recruitment (2-6 h), but early airspace neutrophilia was reduced in db/db obese mice. Neutrophils from uninjured obese mice demonstrated diminished chemotaxis to the chemokine keratinocyte cytokine compared with lean control mice, and adoptive transfer of obese mouse neutrophils into injured lean mice revealed a defect in airspace migration of these cells. Possibly contributing to this defect, neutrophil CXCR2 expression was significantly lower in obese db/db mice, and a similar but nonsignificant decrease was seen in diet-induced obese mice. ALI is attenuated in obese mice, and this blunted response is in part attributable to an obesity-associated abnormal neutrophil chemoattractant response.

Crosstalk between CXCR4/Stromal Derived Factor-1 and VLA-4/VCAM-1 Pathways Regulates Neutrophil Retention in the Bone Marrow

Neutrophil retention in and release from the bone marrow is a critical process that remains incompletely understood. Previous work has implicated the CXCR4/stromal derived factor-1 (SDF-1) chemokine axis in the marrow retention of neutrophils, yet the adhesion pathways responsible for this retention are unknown. Because α4β1 integrin (VLA-4) and its ligand VCAM-1 play a central role in the interactions of hematopoietic stem cells, lymphocytes, and developing neutrophils in the marrow, we investigated whether this integrin might be involved in marrow neutrophil retention and release. In this study, we show that VLA-4 is expressed on murine marrow neutrophils and decreases with maturation, whereas blockade of this integrin leads to the release of marrow neutrophils. Marrow neutrophils adhere via VLA-4 to VCAM-1, which is expressed on marrow endothelium and stroma, and inhibition of VCAM-1 causes release of marrow neutrophils. Furthermore, SDF-1 (CXCL12) signaling through neutrophil CXCR4 augments VLA-4 adhesion to VCAM-1 in vitro, an effect that is blocked by preincubation with pertussis toxin. In vivo blockade of both CXCR4 and α4 causes synergistic release of marrow neutrophils, showing that cross-talk between CXCR4 and VLA-4 modulates marrow retention of these cells. Taken together, these results indicate that the VLA-4/VCAM adhesion pathway is critical in the retention and maturation-controlled release of neutrophils from the marrow, while providing an important link between the CXCR4/SDF-1 signaling axis and the adhesion events that govern this process.

Slam Haplotypes Modulate the Response to Lipopolysaccharide In Vivo through Control of NKT Cell Number and Function

CD1d-restricted NKT cells make up an innate-like T cell subset that plays a role in amplifying the response of innate immune leukocytes to TLR ligands. The Slam locus contains genes that have been implicated in innate and adaptive immune responses. In this study, we demonstrate that divergent Slam locus haplotypes modulate the response of macrophages to the TLR4 ligand LPS through their control of NKT cell number and function. In response to LPS challenge in vivo, macrophage TNF production in Slam haplotype-2+ 129S1/SvImJ and 129X1/SvJ mice was significantly impaired in comparison with macrophage TNF production in Slam haplotype-1+ C57BL/6J mice. Although no cell-intrinsic differences in macrophage responses to LPS were observed between strains, 129 mice were found to be deficient in liver NKT cell number, in NKT cell cytokine production in response to the CD1d ligand α-galactosylceramide, and in NKT cell IFN-γ production after LPS challenge in vivo. Using B6.129c1 congenic mice and adoptive transfer, we found that divergent Slam haplotypes controlled the response to LPS in vivo, as well as the diminished NKT cell number and function, and that these phenotypes were associated with differential expression of signaling lymphocytic activation molecule family receptors on NKT cells. These data suggest that the polymorphisms that distinguish two Slam haplotypes significantly modulate the innate immune response in vivo through their effect on NKT cells.

Plasma granulocyte colony-stimulating factor levels correlate with clinical outcomes in patients with acute lung injury

Objectives: To evaluate the association between plasma granulocyte colony-stimulating factor (G-CSF) levels and clinical outcomes including mortality in patients with acute lung injury (ALI), and to determine whether lower tidal volume ventilation was associated with a more rapid decrease in plasma G-CSF over time in patients with ALI. Design: Retrospective measurement of G-CSF levels in plasma samples that were collected prospectively as part of a large multicenter clinical trial. Setting: Intensive care units in ten university centers. Patients: The study included 645 patients enrolled in the National Heart, Lung, and Blood Institute Acute Respiratory Distress Syndrome Clinical Network trial of lower tidal volumes compared with traditional tidal volumes for ALI. Measurements and Main Results: Baseline plasma levels of G-CSF were associated with an increased risk of death and a decrease in ventilator-free days and organ failure-free days in multivariate analyses controlling for ventilation strategy, age, and sex (Odds ratio death 1.2/log10 increment G-CSF, 95% confidence interval 1.01 to 1.4). Stratification of G-CSF levels into quartiles revealed a strong association between the highest levels of G-CSF and an increased risk of death and decreased ventilator-free days and organ failure-free days in multivariate analyses controlling for ventilation strategy, Acute Physiology and Chronic Health Evaluation III score, Pao2/Fio2 ratio, creatinine, and platelet count (p < 0.05). Subgroup multivariate analysis of patients with sepsis as their risk factor for ALI revealed a U-shaped association between mortality and G-CSF levels such that risk increased linearly from the second through fourth (highest) quartiles, yet also increased in the first (lowest) quartile. G-CSF levels decreased over time in both tidal volume groups, and there was no statistical difference in the extent of decrease between ventilator strategies. Conclusions: In patients with ALI, plasma G-CSF levels are associated with morbidity and mortality, but these levels are not influenced by tidal volume strategy. In patients with sepsis-related ALI, a bimodal association between baseline plasma G-CSF levels and subsequent morbidity and mortality from this disease was found.