Peter M. Henson

Neutrophil-mediated injury to endothelial cells. Enhancement by endotoxin and essential role of neutrophil elastase.

The neutrophil has been implicated as an important mediator of vascular injury, especially after endotoxemia. This study examines neutrophil-mediated injury to human microvascular endothelial cells in vitro. We found that neutrophils stimulated by formyl-methionyl-leucyl-phenylalanine (FMLP), the complement fragment C5a, or lipopolysaccharide (LPS) (1-1,000 ng/ml) alone produced minimal endothelial injury over a 4-h assay. In contrast, neutrophils incubated with endothelial cells in the presence of low concentrations of LPS (1-10 ng/ml) could then be stimulated by FMLP or C5a to produce marked endothelial injury. Injury was maximal at concentrations of 100 ng/ml LPS and 10(-7) M FMLP. Pretreatment of neutrophils with LPS resulted in a similar degree of injury, suggesting that LPS effects were largely on the neutrophil. Endothelial cell injury produced by LPS-exposed, FMLP-stimulated neutrophils had a time course similar to that induced by the addition of purified human neutrophil elastase, and different from that induced by hydrogen peroxide (H2O2). Further, neutrophil-mediated injury was not inhibited by scavengers of a variety of oxygen radical species, and occurred with neutrophils from a patient with chronic granulomatous disease, which produced no H2O2. In contrast, the specific serine elastase inhibitor methoxy-succinyl-alanyl-alanyl-prolyl-valyl-chloromethyl ketone inhibited 63% of the neutrophil-mediated injury and 64% of the neutrophil elastase-induced injury. However, neutrophil-mediated injury was not inhibited significantly by 50% serum, 50% plasma, or purified alpha 1 proteinase inhibitor. These results suggest that, in this system, chemotactic factor-stimulated human neutrophil injury of microvascular endothelial cells is enhanced by small amounts of LPS and may be mediated in large part by the action of neutrophil elastase.

Reactive Oxygen Species Regulate Activation-Induced T Cell Apoptosis

Reactive oxygen species (ROS) mediate apoptosis in a number of cell types. We studied the role that ROS play in activated T cell apoptosis by activating T cells in vivo and then culturing them for a short time. Activated T cells died independently of Fas and TNF alpha. Their death was characterized by rapid loss of mitochondrial transmembrane potential (delta psi(m)), caspase-dependent DNA fragmentation, and superoxide generation. A superoxide dismutase mimetic, Mn (III) tetrakis (5, 10, 15, 20-benzoic acid) porphyrin (MnTBAP), protected T cells from superoxide generation, caspase-dependent DNA loss, loss of delta psi(m), and cell death. These results indicate that ROS can regulate signals involved in caspase activation and apoptosis and may contribute to peripheral T cell deletion.

Minimal Differentiation of Classical Monocytes as They Survey Steady-State Tissues and Transport Antigen to Lymph Nodes

It is thought that monocytes rapidly differentiate to macrophages or dendritic cells (DCs) upon leaving blood. Here we have shown that Ly-6C⁺ monocytes constitutively trafficked into skin, lung, and lymph nodes (LNs). Entry was unaffected in gnotobiotic mice. Monocytes in resting lung and LN had similar gene expression profiles to blood monocytes but elevated transcripts of a limited number of genes including cyclo-oxygenase-2 (COX-2) and major histocompatibility complex class II (MHCII), induced by monocyte interaction with endothelium. Parabiosis, bromodoxyuridine (BrdU) pulse-chase analysis, and intranasal instillation of tracers indicated that instead of contributing to resident macrophages in the lung, recruited endogenous monocytes acquired antigen for carriage to draining LNs, a function redundant with DCs though differentiation to DCs did not occur. Thus, monocytes can enter steady-state nonlymphoid organs and recirculate to LNs without differentiation to macrophages or DCs, revising a long-held view that monocytes become tissue-resident macrophages by default.

Hyaluronate activation of CD44 induces insulin-like growth factor-1 expression by a tumor necrosis factor-alpha-dependent mechanism in murine macrophages.

Macrophages participate in inflammatory and repair processes in part through the selective release of cytokines that contribute to tissue remodeling. Extracellular matrix components generated at inflammatory sites may influence tissue remodeling by effects on leukocyte adherence and local cytokine production. In murine bone marrow-derived macrophages, we found that soluble hyaluronic acid stimulated IL-1 beta, TNF alpha, and insulin-like growth factor-1 (IGF-1) mRNA transcript expression as well as IGF-1 protein synthesis. Monoclonal antibodies to the hyaluronic acid receptor CD44 blocked the effects of hyaluronic acid on IL-1 beta, TNF alpha, and IGF-1 expression. TNF alpha and IL-1 beta mRNA expression preceded IGF-1 protein synthesis, and TNF alpha, but not IL-1 beta, was found to directly stimulate IGF-1. Furthermore, IGF-1 induction was dependent on endogenous TNF alpha production since IGF-1 protein synthesis was inhibited in the presence of anti-TNF alpha antiserum. In addition, IL-1 beta was found to exert a regulatory role on IGF-1 production by enhancing the TNF alpha effect. IL-1 beta and TNF alpha mRNA transcript expression as well as IGF-1 protein synthesis were also stimulated by chrysotile asbestos. Anti-CD44 antibodies had no effect whereas anti-TNF alpha antiserum blocked asbestos-stimulated IGF-1 production. These results indicate that hyaluronate activation of CD44 induces cytokine expression and macrophage-derived IGF-1 production is dependent on TNF alpha expression.

Selective activation and functional significance of p38α mitogen-activated protein kinase in lipopolysaccharide-stimulated neutrophils

Activation of leukocytes by proinflammatory stimuli selectively initiates intracellular signal transduction via sequential phosphorylation of kinases. Lipopolysaccharide (LPS) stimulation of human neutrophils is known to result in activation of p38 mitogen-activated protein kinase (MAPk); however, the upstream activator(s) of p38 MAPk is unknown, and consequences of p38 MAPk activation remain largely undefined. We investigated the MAPk kinase (MKK) that activates p38 MAPk in response to LPS, the p38 MAPk isoforms that are activated as part of this pathway, and the functional responses affected by p38 MAPk activation. Although MKK3, MKK4, and MKK6 all activated p38 MAPk in experimental models, only MKK3 was found to activate recombinant p38 MAPk in LPS-treated neutrophils. Of p38 MAPk isoforms studied, only p38alpha and p38delta were detected in neutrophils. LPS stimulation selectively activated p38alpha. Specific inhibitors of p38alpha MAPk blocked LPS-induced adhesion, nuclear factor-kappa B (NF-kappaB) activation, and synthesis of tumor necrosis factor-alpha (TNF-alpha). Inhibition of p38alpha MAPk resulted in a transient decrease in TNF-alpha mRNA accumulation but persistent loss of TNF-alpha synthesis. These findings support a pathway by which LPS stimulation of neutrophils results in activation of MKK3, which in turn activates p38alpha MAPk, ultimately regulating adhesion, NF-kappaB activation, enhanced gene expression of TNF-alpha, and regulation of TNF-alpha synthesis.

Phagocytosis of aged human neutrophils by macrophages is mediated by a novel "charge-sensitive" recognition mechanism.

The removal of neutrophils and their histotoxic contents from the inflamed site is a prerequisite for resolution of tissue injury, and a point at which factors critical to the pathogenesis of chronic inflammation may act. Engulfment of intact, senescent neutrophils by macrophages represents an important neutrophil disposal process. In this study the mechanism by which human monocyte-derived macrophages (M phi) recognized and ingested human neutrophils that had been aged in culture was studied using an in vitro phagocytic assay. Inhibition of M phi receptors for Ig Fc and the opsonic complement fragments C3b and iC3b with MAbs to M phi FcR, CR1, CR3, and CR4 had no effect on recognition, and the pattern of inhibition observed when polyanions were included in the medium at 1 mg/ml was different from that reported for the M phi receptor for protein advanced glycosylation end products (AGE), indicating a recognition mechanism different from those proposed for M phi phagocytosis of senescent erythrocytes. Furthermore, although aging neutrophils undergo programmed cell death (or apoptosis), which is directly related to recognition by M phi, the pattern of inhibition observed with monosaccharides was different from that reported to inhibit the binding of apoptotic mouse thymocytes to isologous M phi. By contrast, evidence was obtained for a novel recognition mechanism inhibitable by cationic sugars and amino acids in a charge-dependent fashion, and directly modulated by pH but not affected by inhibitors of the mannose-6-phosphate, sheep erythrocyte, mannosyl-fucosyl, asialoglycoprotein, and scavenger receptors of the macrophage. These observations suggest that hydrogen ions and charged molecules may modulate M phi uptake of senescent neutrophils at inflamed sites, and that recognition itself may involve charged structures on the cells.

Muc5b is required for airway defence

Respiratory surfaces are exposed to billions of particulates and pathogens daily. A protective mucus barrier traps and eliminates them through mucociliary clearance (MCC). However, excessive mucus contributes to transient respiratory infections and to the pathogenesis of numerous respiratory diseases. MUC5AC and MUC5B are evolutionarily conserved genes that encode structurally related mucin glycoproteins, the principal macromolecules in airway mucus. Genetic variants are linked to diverse lung diseases, but specific roles for MUC5AC and MUC5B in MCC, and the lasting effects of their inhibition, are unknown. Here we show that mouse Muc5b (but not Muc5ac) is required for MCC, for controlling infections in the airways and middle ear, and for maintaining immune homeostasis in mouse lungs, whereas Muc5ac is dispensable. Muc5b deficiency caused materials to accumulate in upper and lower airways. This defect led to chronic infection by multiple bacterial species, including Staphylococcus aureus, and to inflammation that failed to resolve normally. Apoptotic macrophages accumulated, phagocytosis was impaired, and interleukin-23 (IL-23) production was reduced in Muc5b−/− mice. By contrast, in mice that transgenically overexpress Muc5b, macrophage functions improved. Existing dogma defines mucous phenotypes in asthma and chronic obstructive pulmonary disease (COPD) as driven by increased MUC5AC, with MUC5B levels either unaffected or increased in expectorated sputum. However, in many patients, MUC5B production at airway surfaces decreases by as much as 90%. By distinguishing a specific role for Muc5b in MCC, and by determining its impact on bacterial infections and inflammation in mice, our results provide a refined framework for designing targeted therapies to control mucin secretion and restore MCC.

Adherence of neutrophils to cultured human microvascular endothelial cells. Stimulation by chemotactic peptides and lipid mediators and dependence upon the Mac-1, LFA-1, p150,95 glycoprotein family.

The process of neutrophil adhesion to and migration through the microvascular endothelium, an early event in the induction of the acute inflammatory response, has been attributed to the generation of extravascular chemoattractants. Although both chemotactic peptides and lipid mediators enhance neutrophil adherence in vitro and in vivo, the mechanism(s) involved in the interaction between circulating neutrophils and microvascular endothelial cells is still not completely understood. In a microtiter well adherence assay, the chemotactic peptides, FMLP and C5a, and the lipid mediators, leukotriene B4 (LTB4) and platelet activating factor (PAF), enhanced human neutrophil adherence to cultured human microvascular endothelial cells as well as to human umbilical vein endothelial cells in a dose-dependent manner with a rapid time course. This stimulated adhesive interaction between neutrophils and cultured human endothelial cells was dependent on the expression of the Mac-1, LFA-1, p150,95 glycoprotein family on the neutrophil surface since neutrophils from patients with leukocyte adhesion deficiency, lacking surface expression of the adhesive glycoproteins, exhibited markedly diminished adherence to human endothelial cells in response to stimulation with chemotactic factors compared to normal control neutrophils. All four mediators enhanced expression of the glycoprotein family on the surface of normal neutrophils as determined by flow cytofluorimetry using a monoclonal antibody (TS1/18) to the glycoprotein common beta subunit. In addition, TS1/18 inhibited up to 100% the adherence of normal neutrophils to endothelial cells stimulated by maximal concentrations of FMLP, C5a, LTB4, or PAF. Moreover, HL-60 cells, human promyelocytic leukemia cells, neither increased glycoprotein surface expression nor adherence in response to stimulation. Thus, peptide and lipid mediators of the acute inflammatory response appear to enhance adherence of circulating neutrophils to the microvascular endothelium by a mechanism dependent on expression of the Mac-1, LFA-1, p150,95 glycoprotein family on the neutrophil surface.

Fas Determines Differential Fates of Resident and Recruited Macrophages during Resolution of Acute Lung Injury

RATIONALE During acute lung injury (ALI) the macrophage pool expands markedly as inflammatory monocytes migrate from the circulation to the airspaces. As inflammation resolves, macrophage numbers return to preinjury levels and normal tissue structure and function are restored. OBJECTIVES To determine the fate of resident and recruited macrophages during the resolution of ALI in mice and to elucidate the mechanisms responsible for macrophage removal. METHODS ALI was induced in mice using influenza A (H1N1; PR8) infection and LPS instillation. Dye labeling techniques, bone marrow transplantation, and surface immunophenotyping were used to distinguish resident and recruited macrophages during inflammation and to study the role of Fas in determining macrophage fate during resolving ALI. MEASUREMENTS AND MAIN RESULTS During acute and resolving lung injury from influenza A and LPS, a high proportion of the original resident alveolar macrophages persisted. In contrast, recruited macrophages exhibited robust accumulation in early inflammation, followed by a progressive decline in their number. This decline was mediated by apoptosis with local phagocytic clearance. Recruited macrophages expressed high levels of the death receptor Fas and were rapidly depleted from the airspaces by Fas-activating antibodies. In contrast, macrophage depletion was inhibited in mice treated with Fas-blocking antibodies and in chimeras with Fas-deficient bone marrow. Caspase-8 inhibition prevented macrophage apoptosis and delayed the resolution of ALI. CONCLUSIONS These findings indicate that Fas-induced apoptosis of recruited macrophages is essential for complete resolution of ALI.

Modulation of macrophage efferocytosis in inflammation.

A critical function of macrophages within the inflammatory milieu is the removal of dying cells by a specialized phagocytic process called efferocytosis (“to carry to the grave”). Through specific receptor engagement and induction of downstream signaling, efferocytosing macrophages promote resolution of inflammation by (i) efficiently engulfing dying cells, thus avoiding cellular disruption and release of inflammatory contents, and (ii) producing anti-inflammatory mediators such as IL-10 and TGF-β that dampen pro-inflammatory responses. Evidence suggests that plasticity in macrophage programming, in response to changing environmental cues, modulates efferocytic capability. Essential to programming for enhanced efferocytosis is activation of the nuclear receptors PPARγ, PPARδ, LXR, and possibly RXRα. Additionally, a number of signals in the inflammatory milieu, including those from dying cells themselves, can influence efferocytic efficacy either by acting as immediate inhibitors/enhancers or by altering macrophage programming for longer-term effects. Importantly, sustained inflammatory programming of macrophages can lead to defective apoptotic cell clearance and is associated with development of autoimmunity and other chronic inflammatory disorders. This review summarizes the current knowledge of the multiple factors that modulate macrophage efferocytic ability and highlights emerging therapeutic targets with significant potential for limiting chronic inflammation.