Showing papers in "Journal of Leukocyte Biology in 2005"

Myeloperoxidase: friend and foe

Abstract: Neutrophilic polymorphonuclear leukocytes (neutrophils) are highly specialized for their primary function, the phagocytosis and destruction of microorganisms. When coated with opsonins (generally complement and/or antibody), microorganisms bind to specific receptors on the surface of the phagocyte and invagination of the cell membrane occurs with the incorporation of the microorganism into an intracellular phagosome. There follows a burst of oxygen consumption, and much, if not all, of the extra oxygen consumed is converted to highly reactive oxygen species. In addition, the cytoplasmic granules discharge their contents into the phagosome, and death of the ingested microorganism soon follows. Among the antimicrobial systems formed in the phagosome is one consisting of myeloperoxidase (MPO), released into the phagosome during the degranulation process, hydrogen peroxide (H2O2), formed by the respiratory burst and a halide, particularly chloride. The initial product of the MPO-H2O2-chloride system is hypochlorous acid, and subsequent formation of chlorine, chloramines, hydroxyl radicals, singlet oxygen, and ozone has been proposed. These same toxic agents can be released to the outside of the cell, where they may attack normal tissue and thus contribute to the pathogenesis of disease. This review will consider the potential sources of H2O2 for the MPO-H2O2-halide system; the toxic products of the MPO system; the evidence for MPO involvement in the microbicidal activity of neutrophils; the involvement of MPO-independent antimicrobial systems; and the role of the MPO system in tissue injury. It is concluded that the MPO system plays an important role in the microbicidal activity of phagocytes.

The cytokine activity of HMGB1.

Abstract: High mobility group box 1 (HMGB1) is a highly conserved, ubiquitous protein present in the nuclei and cytoplasm of nearly all cell types. We recently discovered that HMGB1 is secreted into the extracellular milieu and acts as a proinflammatory cytokine. Administration of HMGB1 to normal animals causes inflammatory responses, including fever, weight loss and anorexia, acute lung injury, epithelial barrier dysfunction, arthritis, and death. Anti-HMGB1 treatment, with antibodies or specific antagonists, rescues mice from lethal endotoxemia or sepsis and ameliorates the severity of collagen-induced arthritis and endotoxin-induced lung injury. Here, we give an abridged review of the cytokine activity of HMGB1, its secretion and release into the extracellular milieu, the putative signal transduction pathways, including interaction with cell-surface receptors and intracellular signaling, and its role in several inflammatory diseases. Finally, the therapeutic potential of blocking HMGB1 in the treatment of inflammatory diseases is discussed.

Nanotechnology for the biologist.

Abstract: Nanotechnology refers to research and technology development at the atomic, molecular, and macromolecular scale, leading to the controlled manipulation and study of structures and devices with length scales in the 1- to 100-nanometers range. Objects at this scale, such as "nanoparticles," take on novel properties and functions that differ markedly from those seen in the bulk scale. The small size, surface tailorability, improved solubility, and multifunctionality of nanoparticles open many new research avenues for biologists. The novel properties of nanomaterials offer the ability to interact with complex biological functions in new ways-operating at the very scale of biomolecules. This rapidly growing field allows cross-disciplinary researchers the opportunity to design and develop multifunctional nanoparticles that can target, diagnose, and treat diseases such as cancer. This article presents an overview of nanotechnology for the biologist and discusses "nanotech" strategies and constructs that have already demonstrated in vitro and in vivo efficacy.

Physical activity and modulation of systemic low‐level inflammation

Abstract: It has been recognized for some time that cardiovascular disease and type 2 diabetes are, to a major extent, inflammatory disorders associated with an environment characterized by a sedentary lifestyle together with abundant intakes of calories. Systemic low-level inflammation is suggested to be a cause as well as consequence of pathological processes with local tumor necrosis factor alpha production as an important biological driver. It is hypothesized that physical inactivity contributes to an enhanced proinflammatory burden independently of obesity, as regular muscle contractions mediate signals with myokines/cytokines as important messengers, which suppress proinflammatory activity at distant sites as well as within skeletal muscle. Muscle-derived interleukin (IL)-6 is considered to possess a central role in anti-inflammatory activities and health beneficial effects in relation to physical exercise. It is discussed how this fits the consistent observation that enhanced plasma levels of IL-6 represent a strong risk marker in chronic disorders associated with systemic low-level inflammation and all-cause mortality.

Interleukin-10 and the immune response against cancer: a counterpoint.

Abstract: Although interleukin-10 (IL-10) is commonly regarded as an anti-inflammatory, immunosuppressive cytokine that favors tumor escape from immune surveillance, a wealth of evidence is accumulating that IL-10 also possesses some immunostimulating properties. In fact, IL-10 has the pleiotropic ability of influencing positively and negatively the function of innate and adaptive immunity in different experimental models, which makes it questionable to merely categorize this cytokine as a target of anti-immune escape therapeutic strategies or rather, as an immunological adjuvant in the fight against cancer. Here, we review available data about the immunostimulating anticancer properties of IL-10, and in particular, we focus on the hypothesis that in contrast to what occurs in secondary lymphoid organs, IL-10 overexpression within the tumor microenvironment may catalyze cancer immune rejection.

Impact of LL-37 on anti-infective immunity.

Abstract: Host defense peptides (often called cationic antimicrobial peptides) have pleiotropic immunomodulatory functions. The human host defense peptide LL-37 is up-regulated at sites of infection and has little or no antimicrobial activity in tissue-culture media but under the same conditions, demonstrates immunomodulatory effects on epithelial cells, monocytes, and dendritic cells (DC). These effects include the induction of chemokine production in a mitogen-activated protein kinase-dependent manner in epithelial cell lines and monocytes and profound alterations of DC differentiation, resulting in the capacity to enhance a T helper cell type 1 response. Although the exact mechanisms of interaction between LL-37 and these cell types have not been elucidated, there is evidence for specific (i.e., receptor-mediated) and nonspecific interactions. The relative significance of the direct antimicrobial activities and immunomodulatory properties of LL-37 and other cationic host defense peptides in host defense remains unresolved. To demonstrate that antimicrobial activity was not necessarily required for protection in vivo, model peptides were synthesized and tested for antimicrobial and immunomodulatory activities. A peptide with no antimicrobial activity was found to be protective in animal models of Staphylococcus aureus and Salmonella infection, implying that a host defense peptide can protect by exerting immunomodulatory properties.

Structural organization of the neutrophil NADPH oxidase: phosphorylation and translocation during priming and activation

Abstract: The reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is part of the microbicidal arsenal used by human polymor- phonuclear neutrophils (PMNs) to eradicate invad- ing pathogens. The production of a superoxide anion (O2 - ) into the phagolysosome is the precur- sor for the generation of more potent products, such as hydrogen peroxide and hypochlorite. However, this production of O2 - is dependent on translocation of the oxidase subunits, including gp91 phox , p22 phox , p47 phox , p67 phox , p40 phox , and Rac2 from the cytosol or specific granules to the plasma membrane. In response to an external stim- uli, PMNs change from a resting, nonadhesive state to a primed, adherent phenotype, which allows for margination from the vasculature into the tissue and chemotaxis to the site of infection upon acti- vation. Depending on the stimuli, primed PMNs display altered structural organization of the NADPH oxidase, in that there is phosphorylation of the oxidase subunits and/or translocation from the cytosol to the plasma or granular membrane, but there is not the complete assembly required for O2 - generation. Activation of PMNs is the complete assembly of the membrane-linked and cytosolic NADPH oxidase components on a PMN mem- brane, the plasma or granular membrane. This review will discuss the individual components asso- ciated with the NADPH oxidase complex and the function of each of these units in each physiologic stage of the PMN: rested, primed, and activated. J. Leukoc. Biol. 78: 1025-1042; 2005.

Inflammation-mediated dysfunction and apoptosis in pancreatic islet transplantation: implications for intrahepatic grafts

Abstract: Recent advances in clinical protocols have improved the outcomes of pancreatic islet transplantation (PIT), yet PIT recipients typically require pancreatic islet grafts derived from multiple donors to achieve insulin independence. This along with experimental models of syngeneic PIT, showing that up to 60% of pancreatic islet tissue undergoes apoptosis within the first several days post-transplantation, strongly suggest the involvement of nonalloantigen-specific, inflammatory events in partial destruction of the graft following PIT. Interleukin-1beta appears to be among the most important inflammatory mediators, causing pancreatic islet dysfunction and apoptosis through the up-regulation of inducible nitric oxide (NO) synthase and cyclooxygenase-2. Kupffer cells secrete many molecules, including cytokines, NO, and free radicals, which are known to be directly toxic to the pancreatic islets, and depletion or inhibition of Kupffer cells improves outcomes following experimental PIT. Immediately after transplantation, the pancreatic islets are perfused only by portal vein blood until the process of angiogenesis restores arterial blood flow some 7-10 days later. This delayed vascularization may have implications for the expression of leukocyte adhesion molecules, the effects of free radicals, and the role of ischemia-reperfusion injury. Finally, in the immediate post-transplant period, hepatocytes may contribute to pancreatic islet injury through the production of NO. This paper reviews literature regarding the inflammatory events that follow PIT as well as the pathogenesis of diabetes and the pathophysiology of hepatic ischemia-reperfusion and their relation to the survival and function of intrahepatic pancreatic islet grafts.

Leukocyte apoptosis and its significance in sepsis and shock.

Abstract: Sepsis and multiple organ failure continue to be significant problems among trauma, burn, and the critically ill patient population. Thus, a number of laboratories have focused on understanding the role of altered apoptotic cell death in contributing to immune and organ dysfunction seen in sepsis and shock. Immune cells that undergo altered apoptotic changes include neutrophils, macrophages, dendritic cells, as well as various lymphocyte populations. Evidence of epithelial as well as endothelial cell apoptotic changes has also been reported. Although mediators such as steroids, tumor necrosis factor, nitric oxide, C5a, and Fas ligand (FasL) appear to contribute to the apoptotic changes, their effects are tissue- and cell population-selective. As inhibiting Fas-FasL signaling (e.g., gene deficiency, Fas fusion protein, or Fas short interfering RNA administration), caspase inhibition (caspase mimetic peptides), and/or the overexpression of downstream antiapoptotic molecules (e.g., Bcl-2, Akt) improve survival of septic mice, it not only demonstrates the pathological significance of this process but points to novel targets for the treatment of sepsis.

New insights into the molecular mechanism of interleukin-10-mediated immunosuppression.

Abstract: Interleukin-10 (IL-10) is an important immunomodulatory cytokine, which has attracted much attention because of its anti-inflammatory properties. It reduces antigen presentation and inhibits T cell activation. IL-10-treated myeloid cells lose their ability to respond toward the endotoxin lipopolysaccharide (LPS) with the production of several proinflammatory mediators. Thereby, IL-10 limits excessive inflammatory reactions in response to endotoxin as it occurs in colitis or endotoxin shock. Mice can be tolerized toward endotoxin shock when pretreated with a sublethal dose of LPS. This can be mimicked in vitro as LPS desensitization, resulting in a similar LPS hyporesponsiveness as observed with IL-10 pretreatment. However, an early block in LPS signaling characterizes LPS desensitization, whereas IL-10 seems to target late events. Controversial reports have been published where IL-10 would interfere with the induction of proinflammatory mediators, and little is known about the molecular mechanisms behind the anti-inflammatory activities of IL-10. Some recent publications have tried to gain more insight into the molecular mechanism of IL-10 by gene-expression profiling and functional studies in myeloid-derived cells. These results are reviewed here and compared with the progress that has been made to understand the induction of endotoxin tolerance by LPS itself.

Active participation of endothelial cells in inflammation

Abstract: Leukocyte migration from the blood into tissues is vital for immune surveillance and inflammation. During this diapedesis of leukocytes, the leukocytes bind to endothelial cell adhesion molecules and then migrate across the vascular endothelium. Endothelial cell adhesion molecules and their counter-receptors on leukocytes generate intracellular signals. This review focuses on the active function of endothelial cells during leukocyte-endothelial cell interactions. We include a discussion of the "outside-in" signals in endothelial cells, which are stimulated by antibody cross-linking or leukocyte binding to platelet-endothelial cell adhesion molecule-1, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1. Some of these signals in endothelial cells have been demonstrated to actively participate in leukocyte migration. We suggest that some of the adhesion molecule signals, which have not been assigned a function, are consistent with signals that stimulate retraction of lateral junctions, stimulate endothelial cell basal surface adhesion, or induce gene expression.

Cytokines link osteoblasts and inflammation : microarray analysis of interleukin-17- and TNF-α-induced genes in bone cells

Abstract: The novel cytokine interleukin (IL)-17 has been implicated in many infectious and autoimmune settings, especially rheumatoid arthritis. Consistent with its proinflammatory effects on bone, osteoblast cells are highly responsive to IL-17, particularly in combination with other inflammatory cytokines. To better understand the spectrum of activities controlled by IL-17, we globally profiled genes regulated by IL-17 and tumor necrosis factor alpha (TNF-alpha) in the preosteoblast cell line MC3T3-E1. Using Affymetrix microarrays, 80-90 genes were up-regulated, and 19-50 genes were down-regulated with IL-17 and TNF-alpha as compared with TNF-alpha alone. These included proinflammatory chemokines and cytokines, inflammatory genes, transcriptional regulators, bone-remodeling genes, signal transducers, cytoskeletal genes, genes involved in apoptosis, and several unknown or unclassified genes. The CXC family chemokines were most dramatically induced by IL-17 and TNF-alpha, confirming the role of IL-17 as a potent mediator of inflammation and neutrophil recruitment. Several transcription factor-related genes involved in inflammatory gene expression were also enhanced, including molecule possessing ankyrin repeats induced by lipopolysaccharide/inhibitor of kappaBzeta (MAIL/kappaBzeta), CCAAT/enhancer-binding protein delta (C/EBPdelta), and C/EBPbeta. We also identified the acute-phase gene lipocalin-2 (LCN2/24p3) as a novel IL-17 target, which is regulated synergistically by TNF-alpha and IL-17 at the level of its promoter. A similar but not identical pattern of genes was induced by IL-17 and TNF-alpha in ST2 bone marrow stromal cells and murine embryonic fibroblasts. This study provides a profile of genes regulated by IL-17 and TNF-alpha in osteoblasts and suggests that in bone, the major function of IL-17 is to cooperate and/or synergize with other cytokines to amplify inflammation.

Alcohol-induced oxidative stress in brain endothelial cells causes blood-brain barrier dysfunction

Abstract: Brain microvascular endothelial cells (BMVEC) connected by tight junctions (TJ) form a tight monolayer at the blood-brain barrier (BBB). We investigated the idea that BBB dysfunction seen in alcohol abuse is associated with oxidative stress stemming from ethanol (EtOH) metabolism in BMVEC. Exposure to EtOH induced catalytic activity/expression of EtOH-metabolizing enzymes, which paralleled enhanced generation of reactive oxygen species (ROS). EtOH-mediated oxidative stress led to activation of myosin light chain (MLC) kinase, phosphorylation of MLC and TJ proteins, decreased BBB integrity, and enhanced monocyte migration across BBB. Acetaldehyde or ROS donors mimicked changes induced by EtOH in BMVEC. Thus, oxidative stress resulting from alcohol metabolism in BMVEC can lead to BBB breakdown in alcohol abuse, serving as an aggravating factor in neuroinflammatory disorders.

Macrophage galactose-type C-type lectins as novel markers for alternatively activated macrophages elicited by parasitic infections and allergic airway inflammation.

Abstract: Molecular markers, especially surface markers associated with type II, cytokine-dependent, alternatively activated macrophages (aaMF), remain scarce. Besides the earlier documented markers, macrophage mannose receptor and arginase 1, we demonstrated recently that murine aaMF are characterized by increased expression of found in inflammatory zone 1 (FIZZ1) and the secretory lectin Ym. We now document that expression of the two members of the mouse macrophage galactose-type C-type lectin gene family (mMGL1 and mMGL2) is induced in diverse populations of aaMF, including peritoneal macrophages elicited during infection with the protozoan Trypanosoma brucei brucei or the Helminth Taenia crassiceps and alveolar macrophages elicited in a mouse model of allergic asthma. In addition, we demonstrate that in vitro, interleukin-4 (IL-4) and IL-13 up-regulate mMGL1 and mMGL2 expression and that in vivo, induction of mMGL1 and mMGL2 is dependent on IL-4 receptor signaling. Moreover, we show that expression of MGL on human monocytes is also up-regulated by IL-4. Hence, macrophage galactose-type C-type lectins represent novel surface markers for murine and human aaMF.

Psoriatic scales : a promising source for the isolation of human skin-derived antimicrobial proteins

Abstract: Patients with psoriasis, a chronic, hyperproliferative and noninfectious skin disease, suffer surprisingly fewer cutaneous infections than would be expected. This observation led us to the hypothesis that a local "chemical shield" in the form of antimicrobial proteins provides psoriatic skin with resistance against infection. We subsequently began a systematic analysis of in vitro antimicrobially active proteins in psoriatic-scale extracts. A biochemical approach with rigorous purification and characterization combined with antimicrobial testing identified a number of mostly new human antibiotic peptides and proteins. In this review, we will focus on the most prominent antimicrobial proteins in psoriatic-scale extracts, which we identified as the S100-protein psoriasin, human beta-defensin 2 (hBD-2), RNase 7, lysozyme, and human neutrophil defensin 1-3. Apart from these cutaneous, antimicrobial proteins, only a few others, including hBD-3, have been characterized. A great number of minor antimicrobial proteins await further structural characterization.

Involvement of CC chemokine ligand 18 (CCL18) in normal and pathological processes.

Abstract: CC chemokine ligand 18 (CCL18) was originally discovered as pulmonary and activation-regulated chemokine (PARC), dendritic cell (DC)-chemokine 1 (DC-CK1), alternative macrophage activation-associated CC chemokine-1 (AMAC-1), and macrophage inflammatory protein-4 (MIP-4). CCL18 primarily targets lymphocytes and immature DC, although its agonistic receptor remains unknown so far. CCL18 is mainly expressed by a broad range of monocytes/macrophages and DC. A more profound understanding of the various activation programs and functional phenotypes of these producer cells might give a better insight in the proinflammatory versus anti-inflammatory role of this CC chemokine. It is interesting that CCL18 is constitutively present at high levels in human plasma and likely contributes to the physiological homing of lymphocytes and DC and to the generation of primary immune responses. Furthermore, enhanced CCL18 production has been demonstrated in several diseases, including various malignancies and inflammatory joint, lung, and skin diseases. The lack of a rodent counterpart for human CCL18 sets all hope on primate animal models to further elucidate the importance of CCL18 in vivo. This review will address these different aspects in more detail.

Mammalian defensins: structures and mechanism of antibiotic activity

Abstract: Antibiotic peptides are important effector molecules in host-parasite interactions throughout the living world. In vertebrates, they function in first-line host defense by antagonizing a wide range of microbes including bacteria, fungi, and enveloped viruses. The antibiotic activity is thought to be based on their cationic, amphipathic nature, which enables the peptides to impair vital membrane functions. Molecular details for such activities have been elaborated with model membranes; however, there is increasing evidence that these models may not reflect the complex processes involved in the killing of microbes. For example, the overall killing activity of the bacterial peptide antibiotic nisin is composed of independent activities such as the formation of target-mediated pores, inhibition of cell-wall biosynthesis, formation of nontargeted pores, and induction of autolysis. We studied the molecular modes of action of human defense peptides and tried to determine whether they impair membrane functions primarily and whether additional antibiotic activities may be found. We compared killing kinetics, solute efflux kinetics, membrane-depolarization assays, and macromolecular biosynthesis assays and used several strains of Gram-positive cocci as test strains. We found that membrane depolarization contributes to rapid killing of a significant fraction of target cells within a bacterial culture. However, substantial subpopulations appear to survive the primary effects on the membrane. Depending on individual strains and species and peptide concentrations, such subpopulations may resume growth or be killed through additional activities of the peptides. Such activities can include the activation of cell-wall lytic enzymes, which appears of particular importance for killing of staphylococcal strains.

Cannabinoid receptors in microglia of the central nervous system: immune functional relevance

Abstract: Microglia, resident macrophages of the brain, function as immune effector and accessory cells. Paradoxically, they not only play a role in host defense and tissue repair but also have been implicated in a variety of neuropathological processes. Microglia, in addition to exhibiting phenotypic markers for macrophages, express CB1 and CB2 cannabinoid receptors. Recent studies suggest the existence of a third, yet-to-be cloned, non-CB1, non-CB2 cannabinoid receptor. These receptors appear to be functionally relevant within defined windows of microglial activation state and have been implicated as linked to cannabinoid modulation of chemokine and cytokine expression. The recognition that microglia express cannabinoid receptors and that their activation results in modulation of select cellular activities suggests that they may be amenable to therapeutic manipulation for ablating untoward inflammatory responses in the central nervous system.

Maturation requirements for dendritic cells in T cell stimulation leading to tolerance versus immunity

Abstract: The model that dendritic cell (DC) "maturation" describes the change from an immature, antigen-capturing cell to a mature, antigen-presenting cell is well-established. Classification of DCs in terms of function has been problematic previously. It is therefore proposed that mature and not immature DCs are responsible for antigen presentation and stimulation of T cells. Furthermore, DC antigen presentation to T cells can have two outcomes: tolerance or immunity. The particular outcomes appear to be determined by the activation state of the mature DC. DCs can be activated by a range of environmental stimuli or "danger signals". Here, the hypothesis is advanced that activated, mature DCs induce T cell immunity, and resting, nonactivated but fully differentiated mature antigen-presenting DCs can induce tolerance. This proposal extends to conventional DCs and plasmacytoid DCs. The paper also concentrates on the spleen as a site for DC maturation, in light of evidence from this laboratory for differentiation of DCs from splenic precursors in long-term, stroma-dependent cultures. The hypothesis advanced here serves to simplify many current issues regarding DC maturation and function.

Targeting leukocyte integrins in human diseases

Abstract: As our understanding of integrins as multifunctional adhesion and signaling molecules has grown, so has their recognition as potential therapeutic targets in human diseases. Leukocyte integrins are of particular interest in this regard, as they are key molecules in immune-mediated and inflammatory processes and are thus critically involved in diverse clinical disorders, ranging from asthma to atherosclerosis. Antagonists that interfere with integrin-dependent leukocyte trafficking and/or post-trafficking events have shown efficacy in multiple preclinical models, but these have not always predicted success in subsequent clinical trials (e.g., ischemia-reperfusion disorders and transplantation). However, recent successes of integrin antagonists in psoriasis, inflammatory bowel disease, and multiple sclerosis demonstrate the tremendous potential of antiadhesion therapy directed at leukocyte integrins. This article will review the role of the leukocyte integrins in the inflammatory process, approaches to targeting leukocyte integrins and their ligands, and the results of completed clinical trials.

The low-toxicity versions of LPS, MPL adjuvant and RC529, are efficient adjuvants for CD4+ T cells.

Abstract: Lipopolysaccharide (LPS) has long been known to enhance innate and adaptive immune responses; however, its extreme toxicity precludes its use in clinical settings. The combined toxicity and adjuvanticity of LPS have contributed to the view that immunological adjuvants need to be highly inflammatory to be maximally effective. Here, we compared the effects of LPS with its less-toxic derivatives, monophosphoryl lipid A (MPL) and a chemical mimetic, RC529, on CD4+ T cell clonal expansion, long-term survival, and T helper cell type 1 (Th1) differentiation. We found that LPS, MPL, and RC529 had similar effects on CD4+ T cell clonal expansion, cell division, and ex vivo survival. Analysis of the ability of activated CD4+ T cells to produce interferon-gamma following a 21-day immunization and challenge protocol with LPS and MPL resulted in similar Th1 differentiation. In contrast, we found that LPS was more effective in promoting long-term CD4+ T cell responses, as we recovered nearly sixfold more cells following immunization/challenge as compared with treatment with MPL. Our results indicate that low-inflammation adjuvants, such as MPL and RC529, are capable of enhancing short-term CD4+ T cell clonal expansion and Th1 differentiation, but inflammatory signaling aids in the long-term retention of antigen-specific T cells.

The expression and roles of Toll-like receptors in the biology of the human neutrophil

Abstract: Neutrophils are amongst the first im- mune cells to arrive at sites of infection, where they initiate antimicrobial and proinflammatory func- tions, which serve to contain infection. Sensing and defeating microbial infections are daunting tasks as a result of their molecular heterogeneity; however, Toll-like receptors (TLRs) have emerged as key components of the innate-immune system, activat- ing multiple steps in the inflammatory reaction, eliminating invading pathogens, and coordinating systemic defenses. Activated neutrophils limit in- fection via the phagocytosis of pathogens and by releasing antimicrobial peptides and proinflamma- tory cytokines and generating reactive oxygen in- termediates. Through the production of chemo- kines, they additionally recruit and activate other immune cells to aid the clearance of the microbes and infected cells and ultimately, mount an adap- tive immune response. In acute inflammation, in- flux of neutrophils from the circulation leads to extremely high cell numbers within tissues, which is exacerbated by their delayed, constitutive apopto- sis caused by local inflammatory mediators, poten- tially including TLR agonists. Neutrophil apoptosis and safe removal by phagocytic cells limit tissue damage caused by release of neutrophil cytotoxic granule contents. This review addresses what is currently known about the function of TLRs in the biology of the human neutrophil, including the reg- ulation of TLR expression, their roles in cellular recruitment and activation, and their ability to de- lay apoptotic cell death. J. Leukoc. Biol. 77: 886-892; 2005.

Disruption of granulocyte macrophage-colony stimulating factor production in the lungs severely affects the ability of mice to control Mycobacterium tuberculosis infection.

Abstract: Mice lacking expression of granulocyte macrophage-colony stimulating factor (GM-CSF KO) are unable to contain Mycobacterium tuber- culosis (M. tuberculosis) growth and succumb to infection by 35 days following pulmonary chal- lenge. GM-CSF KO mice do not express normal levels of the inflammatory cytokine tumor necro- sis factor (TNF-) nor the chemokines, regu- lated on activation, normal T expressed and se- creted (RANTES), macrophage-inflammatory pro- tein-1 (MIP-1), MIP-1, and lymphotactin, which are required for recruitment of lymphocytes and expression of a T helper cell type 1 (TH1) response within the lungs. In contrast, transgenic mice overexpressing GM-CSF in the lungs but with a lack of GM-CSF in other organs (GM) are able to recruit lymphocytes and to express a TH1 re- sponse with production of TNF- and interferon- in the lungs. However, GM mice succumb to in- fection between 60 and 90 days post-challenge, as they are unable to develop a normal granuloma- tous response. Although GM mice are able to express the chemokine RANTES, they lack the ability to express other inflammatory chemokines such as lymphotactin and MIP-1. We conclude that GM-CSF is essential to the recruitment of lym- phocytes and expression of a TH1 response in the lung, to the generation of a normal mononuclear granuloma, and most importantly, to the contain- ment of M. tuberculosis bacterial growth. J. Leu- koc. Biol. 77: 914-922; 2005.

Regulation of matrix metalloproteinase-9 release from IL-8-stimulated human neutrophils

Abstract: Matrix metalloproteinase-9 (MMP-9) is present in the tertiary granules of neutrophils and can be released following stimulation. We examined the signaling mechanisms that regulate interleukin-8 (IL-8)-mediated MMP-9 release from neutrophils. IL-8 activates neutrophils by interacting with two receptors: CXC chemokine receptor 1 (CXCR1) and CXCR2. Blocking CXCR1 had no effect on IL-8-mediated MMP-9 release, whereas blocking CXCR2 significantly reduced MMP-9 release. We also found that stimulating CXCR2 alone was sufficient to induce MMP-9 release. This process was independent of changes in the intracellular calcium concentration. Src-family kinases and protein kinase C (PKC) were involved in two mutually exclusive pathways regulating IL-8-mediated MMP-9 release. Inhibition of extracellular signal-regulated kinase (ERK)1/2 blocked IL-8-mediated MMP-9 release; however, inhibition of p38 mitogen-activated protein kinase had no effect on MMP-9 release. We found ERK1/2 was activated downstream of PKC, but not Src-family kinases, in this system. These data suggest that IL-8-induced MMP-9 release from neutrophils is mediated through CXCR2 and involves two distinct pathways, one involving PKC and ERK1/2 and the other involving Src-family kinases. Furthermore, our data show that the mechanisms that regulate MMP-9 release from tertiary granules are different from those that regulate primary granule release.

WASP deficiency leads to global defects of directed leukocyte migration in vitro and in vivo

Abstract: Intact cellular migration is critically important for the induction and regulation of the immune response. The Wiskott-Aldrich syndrome protein (WASP) regulates surface receptor signaling to the actin cytoskeleton in hematopoietic cells and thus plays a pivotal role in cellular locomotion. WASP deficiency causes the Wiskott-Aldrich syndrome (WAS), characterized by immunodeficiency, thrombocytopenia, and eczema. Cell migration defects may contribute to the pathophysiology of WAS. In this study, we used a variety of in vitro and in vivo assays to comprehensively analyze migration properties of lymphocytes, dendritic cells (DC), and neutrophils from WASP-deficient mice. We provide evidence that WASP-deficient lymphocytes show a marked reduction in tethering in an in vitro flow chamber assay as well as decreased migration of T cells in response to the CC chemokine ligand 19 (CCL19). In vivo, compared with wild-type lymphocytes, WASP-deficient lymphocytes showed significantly impaired homing to Peyer's patches upon adoptive transfer into recipient mice. In addition, bone marrow-derived DC migrated less efficiently in response to CCL19. In vivo studies showed decreased migration of DC from skin to draining lymph nodes in WASP-deficient animals. Finally, we also document decreased neutrophil migration in vitro and in vivo. In summary, our studies suggest that WASP plays an important role in the locomotion of lymphocytes, DC, and granulocytes in vitro and in vivo and thus, reveal a crucial role of WASP in physiological trafficking of various hematopoietic cell lineages. These results further delineate immunological abnormalities in WASP-deficient mice, which will be useful to assess preclinical gene therapy studies.

Leukocyte-versus microparticle-mediated tissue factor transfer during arteriolar thrombus development

Abstract: Circulating tissue factor accumulates in the developing thrombus and contributes to fibrin clot formation. To determine whether tissue factor derived from hematopoietic cells is delivered to the thrombus via tissue factor-bearing microparticles or circulating leukocytes expressing tissue factor on the plasma membrane, we compared the kinetics of tissue factor accumulation in the developing arteriolar thrombus with the time course of leukocyte-thrombus interaction and microparticle-thrombus interaction in the microcirculation of a living mouse using intravital high-speed widefield and confocal microscopy. Tissue factor rapidly accumulated in the developing thrombus, appearing immediately following vessel wall injury, reaching a first peak in approximately 100 s. In contrast, leukocyte-thrombus interaction was not observed until after 2-3 min following vessel wall injury. Maximal leukocyte rolling and firm leukocyte adherence on thrombi in wild-type mice were observed after approximately 8 min and were dependent on P-selectin and P-selectin glycoprotein ligand-1. This delay in P-selectin-dependent leukocyte rolling is a result of time-dependent platelet activation and P-selectin expression on the luminal surface of the thrombus. In contrast, microparticle accumulation in the developing arteriolar thrombus was rapid, and peak accumulation was within 60 s. The accumulation of hematopoietic cell-derived tissue factor in the developing thrombus correlates to the kinetics of microparticle accumulation and does not correlate temporally with leukocyte-thrombus interaction. These results indicate that tissue factor derived from hematopoietic cells is delivered by microparticles during the initial phase of thrombus development in vivo.

Spontaneous neutrophil apoptosis and regulation of cell survival by granulocyte macrophage-colony stimulating factor

Abstract: Polymorphonuclear leukocytes (PMNs or neutrophils) are the most prominent cellular component of the innate immune system in humans and produce an array of potent cytotoxic molecules. It is important that neutrophils undergo constitutive (spontaneous) apoptosis as a mechanism to facilitate normal cell turnover and immune system homeostasis. Conversely, several proinflammatory cytokines, including granulocyte macrophage-colony stimulating factor (GM-CSF), prolong neutrophil survival. The molecular mechanisms that regulate PMN apoptosis or survival remain incompletely defined. To that end, we compared global gene expression in human neutrophils during spontaneous apoptosis with that in cells cultured with human GM-CSF. Genes encoding proteins that inhibit apoptosis, such as myeloid cell leukemia sequence 1, caspase 8 and Fas-associated via death domain-like apoptosis regulator (CFLAR), B cell chronic lymphocytic leukemia/lymphoma 2 (BCL2)/adenovirus E1B 19 kDa-interacting protein 2 (BNIP2), and serum/glucocorticoid-regulated kinase (SGK), were down-regulated coincident with neutrophil apoptosis. In contrast, those encoding apoptosis inhibitor 5, BCL2-like 1, BNIP2, CFLAR, SGK, and tumor necrosis factor alpha-induced protein 8 were up-regulated in PMNs cultured with GM-CSF. Correspondingly, GM-CSF delayed PMN apoptosis (P<0.03), increased cell viability (P<0.03), and prolonged neutrophil phagocytic capacity (P<0.05). Prolonged functional capacity was paralleled by striking up-regulation of proinflammatory genes and proteins, including CD14, CD24, CD66, and human leukocyte antigen-DR. In addition, expression of SGK protein diminished during PMN apoptosis but was restored by culture with GM-CSF, suggesting SGK is involved in leukocyte survival. These studies provide a global view of the molecular events that regulate neutrophil survival and apoptosis.

Defensin deficiency, intestinal microbes, and the clinical phenotypes of Crohn's disease.

Abstract: Crohn's disease is a chronic, inflammatory disease of the intestinal mucosa. Although intestinal bacteria are implicated in disease pathogenesis, the etiology is still unclear. The main location of disease is the small intestine (ileum) and the colon. Ileal disease has been linked to a mutation in the NOD2 gene. Defensins are antimicrobial peptides and in the ileum, are mainly expressed in Paneth cells, epithelial cells that also express NOD2. In the colon, defensins are expressed by enterocytes or metaplastic Paneth cells. Crohn's disease patients with ileal involvement, compared with controls or Crohn's patients without ileal involvement, have diminished expression of ileal Paneth cell defensins. This decrease is even more pronounced in Crohn's patients displaying a NOD2 mutation. In contrast, Crohn's disease of the colon is characterized by an impaired induction of beta-defensins in enterocytes. The colonic expression of the constitutive beta-defensin 1 is also decreased in the inflamed colonic mucosa, but this decrease is less specific to Crohn's disease, as it can also be found in ulcerative colitis patients. In conclusion, the regional localizations of Crohn's disease, ileal or colonic disease, can be linked to different defensin profiles. Crohn's disease of the ileum is associated with diminished defensin expression in Paneth cells. Crohn's disease of the colon is associated with diminished beta-defensin expression in enterocytes. Thus, it can be speculated that decreased defensin levels lead to a weakened intestinal barrier function to intestinal microbes and might be crucial in the pathophysiology of Crohn's disease.

Enhanced superoxide release and elevated protein kinase C activity in neutrophils from diabetic patients: association with periodontitis

Abstract: Inflammation and oxidative stress are important factors in the pathogenesis of diabetes and contribute to the pathogenesis of diabetic complications. Periodontitis is an inflammatory disease that is characterized by increased oxidative stress, and the risk for periodontitis is increased significantly in diabetic subjects. In this study, we examined the superoxide (O(2)(-))-generating reduced nicotinamide adenine dinucleotide phosphate-oxidase complex and protein kinase C (PKC) activity in neutrophils. Fifty diabetic patients were grouped according to glycemic control and the severity of periodontitis. Neutrophils from diabetic patients with moderate [amount of glycated hemoglobin (HbA(1c)) between 7.0% and 8.0%] or poor (HbA(1c) >8.0%) glycemic control released significantly more O(2)(-) than neutrophils from diabetic patients with good glycemic control (HbA(1c) <7.0%) and neutrophils from nondiabetic, healthy individuals upon stimulation with 4beta-phorbol 12-myristate 13-acetate or N-formyl-Met-Leu-Phe. Depending on glycemic status, neutrophils from these patients also exhibited increased activity of the soluble- and membrane-bound forms of PKC, elevated amounts of diglyceride, and enhanced phosphorylation of p47-phox during cell stimulation. In addition, we report a significant correlation between glycemic control (HbA(1c) levels) and the severity of periodontitis in diabetic patients, suggesting that enhanced oxidative stress and increased inflammation exacerbate both diseases. Thus, hyperglycemia can lead to a novel form of neutrophil priming, where elevated PKC activity results in increased phosphorylation of p47-phox and O(2)(-) release.

Polysaccharide purified from Ganoderma lucidum induced activation and maturation of human monocyte-derived dendritic cells by the NF-κB and p38 mitogen-activated protein kinase pathways

Abstract: Ganoderma lucidum, a fungus native to China, has been widely used to promote health and longevity in the Chinese. The polysaccharide component with a branched (136)--D-glucan moiety of G. lucidum (PS-G) has been reported to exert anti-tumor activity and activation of natu- ral killer cells. In this study, we investigated the effects of PS-G on human monocyte-derived den- dritic cells (DC). Treatment of DC with PS-G resulted in the enhanced cell-surface expression of CD80, CD86, CD83, CD40, CD54, and hu- man leukocyte antigen (HLA)-DR, as well as the enhanced production of interleukin (IL)-12p70, p40, and IL-10 and also IL-12p35, p40, and IL-10 mRNA expression, and the capacity for endocytosis was suppressed in DC. In addition, treatment of DC with PS-G resulted in enhanced T cell-stimulatory capacity and increased T cell secretion of interferon- and IL-10. Neutraliza- tion with antibodies against Toll-like receptor (TLR)-4 inhibited the PS-G-induced production of IL-12 p40 and IL-10, suggesting a vital role for TLR-4 in signaling DC upon incubation with PS-G. Further study showed that PS-G was able to augment inhibitor of B( IB) kinase and nuclear factor (NF)-B activity and also IB and p38 mitogen-activated protein kinase (MAPK) phosphorylation. Further, inhibition of NF-B by helenalin and p38 MAPK by SB98059 prevented the effects of PS-G in the expression of CD80, CD86, CD83, CD40, CD54, and HLA-DR and production of IL-12p70, p40, and IL-10 in various degrees. Taken together, our data demonstrate that PS-G can effectively pro- mote the activation and maturation of immature DC, suggesting that PS-G may possess a potential in regulating immune responses. J. Leukoc. Biol. 78: 533-543; 2005.