Thomas S. Wilkinson

IL-6 and Its Soluble Receptor Orchestrate a Temporal Switch in the Pattern of Leukocyte Recruitment Seen during Acute Inflammation

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.

Interplay between IFN-γ and IL-6 signaling governs neutrophil trafficking and apoptosis during acute inflammation

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.

Differential Regulation of Neutrophil-Activating Chemokines by IL-6 and Its Soluble Receptor Isoforms

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 (GROα) 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-1β-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.

The human cationic host defense peptide LL‐37 mediates contrasting effects on apoptotic pathways in different primary cells of the innate immune system

The human cathelicidin LL‐37 is a cationic host defense peptide (antimicrobial peptide) expressed primarily by neutrophils and epithelial cells. This peptide, up‐regulated under conditions of inflammation, has immunomodulatory and antimicrobial functions. We demonstrate that LL‐37 is a potent inhibitor of human neutrophil apoptosis, signaling through P2X7 receptors and G‐protein‐coupled receptors other than the formyl peptide receptor‐like‐1 molecule. This process involved modulation of Mcl‐1 expression, inhibition of BID and procaspase‐3 cleavage, and the activation of phosphatidylinositol‐3 kinase but not the extracellular signal‐regulated kinase 1/2 mitogen‐activated protein kinase pathway. In contrast to the inhibition of neutrophil apoptosis, LL‐37 induced apoptosis in primary airway epithelial cells, demonstrating alternate consequences of LL‐37‐mediated modulation of apoptotic pathways in different human primary cells. We propose that these novel immunomodulatory properties of LL‐37 contribute to peptide‐mediated enhancement of innate host defenses against acute infection and are of considerable significance in the development of such peptides and their synthetic analogs as potential therapeutics for use against multiple antibiotic‐resistant infectious diseases.

Galectin-3 reduces the severity of pneumococcal pneumonia by augmenting neutrophil function

The Gram-positive Streptococcus pneumoniae is the leading cause of community-acquired pneumonia worldwide, resulting in high mortality. Our in vivo studies show that galectin-3(-/-) mice develop more severe pneumonia after infection with S. pneumoniae, as demonstrated by increased bacteremia and lung damage compared to wild-type mice and that galectin-3 reduces the severity of pneumococcal pneumonia in part by augmenting neutrophil function. Specifically, we show that 1) galectin-3 directly acts as a neutrophil-activating agent and potentiates the effect of fMLP, 2) exogenous galectin-3 augments neutrophil phagocytosis of bacteria and delays neutrophil apoptosis, 3) phagocytosis of apoptotic neutrophils by galectin-3(-/-) macrophages is less efficient compared to wild type, and 4) galectin-3 demonstrates bacteriostatic properties against S. pneumoniae in vitro. Furthermore, ad-back of recombinant galectin-3 in vivo protects galectin-3-deficient mice from developing severe pneumonia. Together, these results demonstrate that galectin-3 is a key molecule in the host defense against pneumococcal infection. Therapeutic strategies designed to augment galectin-3 activity may both enhance inflammatory cell function (by directly affecting neutrophil responsiveness and prolonging neutrophil longevity) and have direct bacteriostatic activity, improving clinical outcomes after severe pneumococcal infection.

The Human Cathelicidin LL-37 Preferentially Promotes Apoptosis of Infected Airway Epithelium

Cationic host defense peptides are key, evolutionarily conserved components of the innate immune system. The human cathelicidin LL-37 is an important cationic host defense peptide up-regulated in infection and inflammation, specifically in the human lung, and was shown to enhance the pulmonary clearance of the opportunistic pathogen Pseudomonas aeruginosa in vivo by as yet undefined mechanisms. In addition to its direct microbicidal potential, LL-37 can modulate inflammation and immune mechanisms in host defense against infection, including the capacity to modulate cell death pathways. We demonstrate that at physiologically relevant concentrations of LL-37, this peptide preferentially promoted the apoptosis of infected airway epithelium, via enhanced LL-37-induced mitochondrial membrane depolarization and release of cytochrome c, with activation of caspase-9 and caspase-3 and induction of apoptosis, which only occurred in the presence of both peptide and bacteria, but not with either stimulus alone. This synergistic induction of apoptosis in infected cells was caspase-dependent, contrasting with the caspase-independent cell death induced by supraphysiologic levels of peptide alone. We demonstrate that the synergistic induction of apoptosis by LL-37 and Pseudomonas aeruginosa required specific bacteria-epithelial cell interactions with whole, live bacteria, and bacterial invasion of the epithelial cell. We propose that the LL-37-mediated apoptosis of infected, compromised airway epithelial cells may represent a novel inflammomodulatory role for this peptide in innate host defense, promoting the clearance of respiratory pathogens.

Overexpression of hyaluronan synthases alters vascular smooth muscle cell phenotype and promotes monocyte adhesion

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 (2–10 × 106 Da), whereas HA produced by has‐3 and control cells was present in lower molecular weight fractions (∼2 × 106 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. J. Cell. Physiol. 206: 378–385, 2006.

Differential regulation of chemokine production in human peritoneal mesothelial cells: IFN-gamma controls neutrophil migration across the mesothelium in vitro and in vivo.

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.

NO-loaded Zn2+-exchanged zeolite materials: A potential bifunctional anti-bacterial strategy

Nitric oxide (NO) is important for the regulation of a number of diverse biological processes, including vascular tone, neurotransmission, inflammatory cell responsiveness, defence against invading pathogens and wound healing. Transition metal exchanged zeolites are nanoporous materials with high-capacity storage properties for gases such as NO. The NO stores are liberated upon contact with aqueous environments, thereby making them ideal candidates for use in biological and clinical settings. Here, we demonstrate the NO release capacity and powerful bactericidal properties of a novel NO-storing Zn(2+)-exchanged zeolite material at a 50 wt.% composition in a polytetrafluoroethylene polymer. Further to our published data showing the anti-thrombotic effects of a similar NO-loaded zeolite, this study demonstrates the anti-bacterial properties of NO-releasing zeolites against clinically relevant strains of bacteria, namely Gram-negative Pseudomonas aeruginosa and Gram-positive methicillin-sensitive and methicillin-resistant Staphylococcus aureus and Clostridium difficile. Thus our study highlights the potential of NO-loaded zeolites as biocompatible medical device coatings with anti-infective properties.

Monocytes Control Second-Phase Neutrophil Emigration in Established Lipopolysaccharide-induced Murine Lung Injury

RATIONALE Acute lung injury (ALI) is an important cause of morbidity and mortality, with no currently effective pharmacological therapies. Neutrophils have been specifically implicated in the pathogenesis of ALI, and there has been significant research into the mechanisms of early neutrophil recruitment, but those controlling the later phases of neutrophil emigration that characterize disease are poorly understood. OBJECTIVES To determine the influence of peripheral blood monocytes (PBMs) in established ALI. METHODS In a murine model of LPS-induced ALI, three separate models of conditional monocyte ablation were used: systemic liposomal clodronate (sLC), inducible depletion using CD11b diphtheria toxin receptor (CD11b DTR) transgenic mice, and antibody-dependent ablation of CCR2(hi) monocytes. MEASUREMENTS AND MAIN RESULTS PBMs play a critical role in regulating neutrophil emigration in established murine LPS-induced lung injury. Gr1(hi) and Gr1(lo) PBM subpopulations contribute to this process. PBM depletion is associated with a significant reduction in measures of lung injury. The specificity of PBM depletion was demonstrated by replenishment studies in which the effects were reversed by systemic PBM infusion but not by systemic or local pulmonary infusion of mature macrophages or lymphocytes. CONCLUSIONS These results suggest that PBMs, or the mechanisms by which they influence pulmonary neutrophil emigration, could represent therapeutic targets in established ALI.