Williams Hematology



Dendritic Cells
Motility and Chemotaxis
Energetics and Endocytosis


Receptors Involved in Phagocytosis

Scavenger Receptors
Resistance to Infection
Antimicrobial Mechanisms

Reactive Oxygen Intermediates


Nitric Oxide
Cytostatic and Cytocidal Activity

Antibody-Dependent Cellular Cytotoxicity

Antibody-Independent Tumor Cytolysis


Cytotoxic Mechanisms

Cytotoxic Proteins and Peptides
Chapter References

Mononuclear phagocytes play central roles in resistance to many infectious diseases, including tuberculosis, leishmaniasis, typhoid fever, and systemic mycoses. Highly specialized mononuclear cells called dendritic cells excel in presenting antigens to T cells, a critical step in initiating the adaptive immune response. Unlike short-lived neutrophils, macrophages can survive within tissues for weeks and even months. They exhibit a prodigious capacity for macromolecular synthesis, secrete numerous bioactive molecules, and are highly responsive to the internal milieu. Macrophages possess receptors for cytokines, including interferon gamma and tumor necrosis factor alpha, allowing their functional state to be modulated by such molecules. Additional surface receptors enhance their phagocytic properties by recognizing various host-derived factors, including immunoglobulins, complement, and integrins. Macrophage receptors have been identified that recognize molecular motifs characteristic of microbial membranes and cell walls, including lipopolysaccharide, mannans, and (lipo)teichoic acids. The antimicrobial mechanisms of macrophages are mediated largely, but not exclusively, by various oxidants produced by their NADPH oxidase and/or inducible nitric oxide synthase (iNOS) systems. Macrophages also exhibit cytotoxic and cytostatic properties in vitro, although attempts to harness these activities have not yet been successfully applied to humans.

Acronyms and abbreviations that appear in this chapter include: ADCC, antibody-dependent cellular cytotoxicity; BPI, bactericidal/permeability enhancing factor; CGD, chronic granulomatous disease; CSF-1, colony-stimulating factor 1; FGF, fibroblast growth factor; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-monocyte colony-stimulating factor; ICAMs, intercellular adhesion molecules; iNOS or NOS2, inducible nitric oxide synthase; IRF-1, interferon regulatory factor 1; LBP, lipopolysaccharide binding protein; LDL, low-density lipoprotein; LPS, lipopolysaccharide; M-CSF, macrophage colony-stimulating factor; MHC, major histocompatibility complex; MPO, myeloperoxidase; NADPH, nicotinamide adenine dinucleotide phosphate (reduced form); NO, nitric oxide; Nramp, natural resistance-associated macrophage protein; O2–, superoxide; ONOO–, peroxynitrite; PDGF, platelet-derived growth factor; RANTES, regulated upon activation, normal T-cell expressed and presumably secreted; SR-A, type A scavenger receptor; TGF-b, transforming growth factor b.

Mononuclear phagocytes (monocytes and macrophages) are relatively large phagocytic cells with abundant cytoplasm and a round to reniform nucleus (see Chap. 73). Macrophages that depart from this appearance can bear eponyms, such as Gaucher or Kupffer cells, or have pseudonyms, such as foam (lipid-laden) or epithelioid cells. Mononuclear phagocytes combine prodigious biosynthetic and secretory abilities with an ability to vary their output in response to local conditions and chemical mediators. Although cells of the monocyte-macrophage lineage may undergo malignant transformation or exuberant proliferation (Chap. 78), most often their routine duties—host defense, antigen presentation, and removal of detritus—are performed away from the spotlight of disease.
Although human blood monocytes can be obtained readily, most other human macrophage populations are less accessible. Consequently, much of our information about macrophages is derived from in vivo experiments on mice or from in vitro experiments with cultured blood monocytes or cell lines. This chapter will review selected aspects of the biology of mononuclear phagocytes, including their endocytic and phagocytic behavior, receptors, secretory properties, and microbicidal and cytotoxic mechanisms. Production, distribution, and fate of monocytes and macrophages are discussed in Chap. 75.
Dendritic cells are widely distributed, HLA-DR/DQ–positive, migratory marrow–derived cells that are specialized for antigen capture and T-cell stimulation, rather than for phagocytosis and direct host defense.1 Antigen-presenting cells are important because, rather than recognizing intact protein antigens directly, T-cell receptors recognize peptide fragments that are bound to cell surface, major histocompatibility complex (MHC) molecules. Class I MHC molecules normally bind peptides, such as viral coat components, that are derived from intracellular proteins. MHC class II molecules typically present peptides derived from extracellular antigens.
Unlike macrophages, dendritic cells lack receptors for immunoglobulin, complement, and colony-stimulating factor 1 (CSF-1).2 They are only weakly phagocytic and take up exogenous antigens principally by fluid-phase endocytosis and adsorptive pinocytosis. After these internalized antigens are partially degraded and bound to MHC class II molecules, the antigens return to the dendritic cell’s surface as a MHC II peptide complex. Recognition of this complex by antigen receptors on CD4-positive T-helper cells generates one signal for these cells. Full T-cell activation requires a second, costimulatory signal from the antigen-presenting cell. Although the mechanisms that convey this second signal are incompletely known, dendritic cells excel in providing it, perhaps aided by specific chemokines.3 In addition to their MHC class II molecules, dendritic cells express multiple surface adhesion molecules, including LFA-1 (CD11a/CD18), LFA-3 (CD58), ICAM-1(CD54), and ICAM-2. By promoting close associations with T lymphocytes, such adhesion molecules can enhance the dendritic cell’s ability to signal T-cell receptors.
Dendritic cells constitute approximately 0.1 to 1 percent of the blood’s mononuclear cell.2 Their dynamic, veil-like or branching cytoplasmic processes provide a large surface area for interactions with T cells. GM-CSF and certain interleukins, including IL-4 or IL-13, promote in vitro differentiation of functional dendritic cells from blood monocytes4 or CD 34+ bone marrow or blood progenitor cells.5,6 and 7 Other dendritic cell populations include migratory veiled cells in afferent lymph, interdigitating cells in thymic medulla, and interstitial cells in the lung and heart. The Langerhans cells of skin are dendritic cells that express MHC class II antigens constitutively. Dendritic cells can pick up local antigens or haptens and migrate via the lymphatics to enter T-dependent, paracortical areas of regional lymph nodes.
Many mononuclear phagocytes are present in the lamina propria of organs regularly exposed to microbes, including the intestinal and genitourinary tracts, skin, and lungs. Local tissue populations of macrophages are rapidly augmented by entry of blood monocytes responding to various signals that arise during infection and inflammation. Macrophage motility depends on the contractile properties of actin and myosin, regulated by many additional proteins, including profilin, gelsolin, acumentin, tropomyosin, actin-binding protein, and calmodulin.8
Chemotaxis refers to the ability of cells to orient in and move along a chemical gradient. Many molecules that are generated during infection or injury are recognized by the surface receptors of monocytes and macrophages and trigger chemotactic responses. Such substances include N-formylated peptides produced by bacteria,9,10 complement component C5a,11 leukotriene B4 and other eicosanoids,12,13 collagen and elastin fragments,14,15 thrombin,16 platelet factor 4, platelet-derived growth factor (PDGF),17 and at least two neutrophil proteins, cathepsin G and azurocidin.18
Chemokines (i.e., chemotactic cytokines) are important mediators of chemotactic and migratory behavior. These 8- to 10-kDa molecules contain four conserved cysteines that are linked by disulfide bonds. They have been divided into two groups, based on their homology and the spacing of their first two cysteine residues. CXC chemokines, also called a-chemokines, have an amino acid interposed between these cysteines. In CC chemokines, also called b-chemokines, these cysteines are adjacent. The genes for CXC and CC chemokines are clustered on human chromosomes 4 and 17, respectively.18 Whereas CXC chemokines such as IL-8 act primarily on neutrophils, CC chemokines such as MCP 1–4, MIP-1a, MIP-b, and RANTES are potent activators of monocytes and T lymphocytes.18,19
Multiple, structurally related receptors for chemokines have been identified. Typically, these receptors have seven transmembrane domains and signal through heterotrimeric GTP-binding proteins. Chemokines and their receptors vary with respect to binding specificity. Many chemokines bind to more than one receptor, and most chemokine receptors bind more than one chemokine. Expression of chemokine receptors is regulated by the ambient cytokine environment, thereby allowing complex and graded responses.
Certain chemokine receptors have been subverted by pathogens in ways detrimental to the host. For example, CCR5, the macrophage receptor for RANTES, MIP-1a, and MIP-1b, is used by monocyte/macrophage-tropic strains of HIV-1 as a coreceptor for intracellular entry.20 Additionally, many members of the poxvirus, herpesvirus, and retrovirus families have captured genes encoding cytokine or chemokine receptors and modified them in ways that enhance viral pathogenicity.21,22 and 23 For example, molluscum contagiosum virus secretes a modified CC chemokine that interferes with the chemotactic response of human leukocytes to multiple CC and CXC chemokines, thereby blunting the in vivo inflammatory response to the virus.24 Human herpesvirus 8, a Kaposi’s sarcoma–associated herpesvirus, appears to use similar strategies to deliver signals that initiate inappropriate growth or transformation.25
Mononuclear phagocytes derive most of their metabolic energy from glycolytic metabolism. In alveolar macrophages, this is augmented substantially by oxidative phosphorylation. Macrophages imbibe extracellular fluid continually by a process known as pinocytosis (literally, “cell drinking”). Their fluid uptake occurs in several types of vesicles, including macropinosomes that are larger than 0.2 µm in diameter, clathrin-coated vesicles, and small uncoated vesicles. Receptor-mediated endocytosis takes place principally via clathrin-coated vesicles.26 Exposure of macrophages to M-CSF promptly induces active cell ruffling and enhanced macropinosome formation.27
The content of degradative enzymes in macrophages increases after they take up digestible substances by endocytosis or phagocytosis. Although proteins retained within the lysosomal apparatus emerge only after extensive degradation, endocytic mechanisms specialized for antigen presentation allow partially degraded antigens to be displayed on the macrophage cell membrane, bound to MHC molecules.28
Certain secretory products of macrophages, such as lysozyme, are produced regularly and in large amounts. Most others are produced and released in a highly controlled fashion, determined by the functional state of the cell and its exposure to regulatory stimuli. In addition to many cytokines (e.g., IL-1a and b, IL-6, TNF-a, and interferons a, b, and g) and chemokines, macrophages produce numerous growth factors, including G-CSF, GM-CSF, erythroid colony potentiating factor, transforming growth factor (TGF)-b, PDGF, and fibroblast growth factor (FGF).29 By secreting classical and alternative pathway complement factors,30 macrophages can augment local tissue concentrations of these host-defense molecules. Macrophages release various enzymes (e.g., plasminogen activator, elastase, collagenases, and acid hydrolases) that participate in tissue remodeling and wound healing. They also produce matrix proteins such as fibronectin, thrombospondin, proteoglycans, and diverse lipid mediators, including prostaglandins (PGE2, PGF2a), prostacyclin, and various lipoxygenase products.31
Receptors allow mononuclear phagocytes to recognize and respond to other cells. They also permit macrophages to adhere to extracellular matrix, bind and ingest microorganisms, and respond to various cytokines and growth factors. Expression of membrane receptors varies according to the macrophage’s functional state and reflects its prior exposure to cytokines.31 When receptor-ligand binding events occur, this is communicated to the intracellular machinery by transduction pathways that ultimately impinge on molecules that regulate transcription. These include NF-kB, NF-IL6, PU.1, interferon regulatory factor 1 (IRF-1), Egr-1, and Stat-1.
Macrophages can adhere reversibly to various surfaces, including endothelial cells and extracellular matrix proteins. This property allows them to migrate on such surfaces and is imparted by adhesive plasma membrane glycoprotein receptors called adhesins. At least three families of adhesins participate in these processes—selectins, integrins, and intercellular adhesion molecules (ICAMs). The integrin superfamily is composed of heterodimeric molecules with noncovalently associated a and b chains. Several b2-integrins are prominent in macrophages, including LFA-1 (CD11a/CD18), MAC-1 (CD11b/CD18), and p150/95 (CD 11c/CD18). The ligands (often called counterreceptors) of these adhesins include ICAMS-1, -2, and -3 (LFA-1); fibrinogen and fibronectin (MAC-1); and iC3b (p150,95). Several b1-integrins (VLA-4, –5, and –6) expressed by monocytes are fibronectin receptors that may promote recruitment of monocytes to inflammatory foci.32 Leukocyte adhesion deficiency—a complex inherited disorder that results from a marked deficiency of b2-integrins33—is associated with frequent and severe infections, poor wound healing, and diminished accrual of leukocytes at sites of infection.
Mononuclear phagocytes and other leukocytes contain “homing receptors” called L-selectins or LECAMs. These adhesive, lectinlike molecules mediate an initial, low-affinity rolling type of adhesion between leukocytes and their counterreceptors on vascular endothelium, before stronger connections are made via integrins.34
The surface membranes of mononuclear phagocytes contain specific receptors for immunoglobulins, including IgG1 and other IgG subtypes, IgA, and IgE. IgG binds organisms via its Fab sites and binds the macrophage’s Ig receptors via its Fc portion. IgG receptors promote both attachment and ingestion of immunoglobulin-coated particles. Macrophages also display receptors for several complement components, including C3b, C3bi, C3a, and C5a. The C3b receptor (also called CR1 and CD35) is also found on neutrophils. It recognizes opsonized particles and accelerates C3b breakdown by factor I. CR1 mediates attachment without ingestion unless small amounts of IgG are present35 or the macrophages are otherwise stimulated.36 The C3bi receptor (also called CR3, Mac1, and CD 11b/CD18) recognizes an Arg-Gly-Asp (RGD) triplet in its ligand, C3bi,37 as well as in fibrinogen and fibrin. CR3 binds many other ligands, including molecules found on bacteria, fungi, and protozoans as well as ICAM-1 and other ligands of endothelial cells.38 The contact sites of the C3bi receptor for several such ligands have been shown to partially overlap.39 IgG and C3b receptors allow macrophages to recognize microorganisms opsonized (tagged for phagocytosis) by the deposition of immunoglobulin and/or complement on their surface. In vivo administration of interferon-g to normal subjects significantly increases the expression of Ig receptors (Fc gammaRI, Fc gammaRII, Fc gammaRIII), integrins (CD11a/CD18, CD11b/CD18), and HLA-DR by monocytes.40
Certain macrophage receptors recognize molecules or molecular arrays that are typically found on microbes and can be classified as “pattern recognition receptors.”41 A well-studied example is the macrophage mannose receptor—a 180-kDa transmembrane protein with eight tandem carbohydrate recognition domains.42 Two of these domains interact with linear or branched-chain mannosyl and fucosyl residues, allowing the mannose receptor to recognize a wide variety of bacteria, mycobacteria, yeasts, and parasites and initiate phagocytic, endocytic, or antigen capture responses. Mannose receptors are also expressed on dendritic cells.43
Several macrophage receptors can recognize lipopolysaccharide (LPS), an abundant glycolipid that occupies most of a gram-negative bacterium’s outer membrane surface.44 These receptors include CD 14, a GPI-anchored glycoprotein; the CD11/18 family of b2-integrins; and the macrophage’s type A scavenger receptor (SR-A). Binding of LPS to CD14 is enhanced by lipopolysaccharide binding protein (LBP), a 65-kDa plasma protein homologous to bactericidal/permeability enhancing factor (BPI). CD14-deficient mice are markedly resistant to shock induced by LPS or gram-negative bacterial challenge.45 After exposure to LPS, normal monocytes produce and release many inflammatory mediators, including reactive oxygen and nitrogen intermediates, prostaglandins, and various proinflammatory cytokines, including TNF-a, IL-1b, IL-6, and IL-8.
Macrophages contain several receptors that recognize various negatively charged macromolecules. Among these are the trimeric, macrophage type I and type II scavenger receptors, which bind oxidized and acetylated low-density lipoproteins (LDLs) and mediate their endocytic uptake. These receptors contribute to cholesterol deposition in atherosclerotic foam cells in humans and mice.46,47 Types I and II scavenger receptors have a collagenlike domain, and the type I receptor also has a cysteine-rich domain common to several other receptors. Type I and type II scavenger receptors also mediate adhesion and bind lipopolysaccharide,48 lipoteichoic acids from gram-positive bacteria,49 and advanced glycation protein end products. Additional macrophage scavenger receptors include CD36 and CD68 (macrosialin). CD36 can bind apoptotic cells, Plasmodium falciparum-infected erythrocytes, long-chain fatty acids, and oxidized LDL.50
Other macrophage receptors allow these cells to recognize regulatory molecules such as macrophage colony-stimulating factor (M-CSF), hormones, leukotrienes, other eicosanoids, coagulation factors, transport proteins, antiproteases, and many other bioactive molecules.51
By subjecting inbred mice differing in resistance to infection by different microbes to detailed genetic analysis, Nramp1 (natural resistance-associated macrophage protein)—a macrophage protein that contributes substantially to innate resistance to intracellular infections—was identified. Nramp1 is a hydrophobic, integral membrane protein that is encoded by the Lsh/Ity/Bcg gene, which regulates resistance to Leishmania, Salmonella and Mycobacteria. The expression of Nramp1 in mice is restricted to macrophages and is enhanced by treatment with interferon-g and lipopolysaccharide. Nramp1 is also found in late endosomal and lysosomal vesicular compartments of the macrophage. The protein may enhance vacuolar acidification and endosome-phagosome fusion52 or act to influence transport of iron or other trace metals needed for microbial growth.53 Intracellular pathogens, such as mycobacteria, can interfere with phagosome-lysosome fusion, thereby preventing vacuolar acidification and reducing the delivery of Nramp1 to phagosomes.54
Experimental studies of murine listeriosis were instrumental in developing the concept of “activated macrophages.”55 In this model, bacterial numbers increased logarithmically in the liver and spleen for 3 days after intravenous inoculation of L. monocytogenes. Thereafter, net bacterial growth ceased, and viable bacteria declined sharply in numbers, disappearing by the next week. These beneficial changes were accompanied by the appearance of delayed hypersensitivity. An altered phenotype was evident in the peritoneal macrophages, which enlarged, became more phagocytic, and more effectively resisted in vitro challenge by L. monocytogenes. Similar changes were noted after mice were infected with Brucella abortus, Salmonella typhimurium or Mycobacterium bovis.55 The antimicrobial efficacy of these macrophages was nonspecific, since infection by any one of these intracellular pathogens engendered macrophages with an enhanced ability to inhibit intracellular replication by all of them. Although such activated macrophages reverted to their basal state after 1 to 2 weeks, the phenotypic changes recurred within 24 h after a challenge by the same organism that had initiated the original infection. In vivo resistance was not transferred from immune to naive mice by serum, but the transfer of splenic T lymphocytes conferred protection.
During the past decade, much has been learned about the events responsible for these phenomena. Mononuclear phagocytes possess multiple mechanisms that allow them to kill ingested microorganisms or restrict their replication. Ingested microbes are sequestered within membrane-bounded compartments, called phagocytic vacuoles or phagosomes, which can be acidified to a pH of approximately 4.5. Within such phagosomes, the microbes are exposed to a mixture of lysolipids, macrophage-derived enzymes and proteins, and various oxidants. Moreover, microbial access to micronutrients, especially iron, that are essential for their growth is limited. Although entrapment within phagolysosomes is a lethal event for most microbes, successful pathogens have developed stratagems that allow them to survive and even thrive in this environment. Some, such as Listeria monocytogenes, escape from phagosomes and enter the cytoplasmic compartment,56 where they co-opt the host cell’s actin and use it to propel themselves into adjacent cells.57,58 Other pathogens modulate their phagosomal microenvironments by inhibiting vacuolar acidification59 or phagolysosomal fusion60 or by undergoing phenotypic changes that enhance their resistance.61,62
Macrophages with enhanced antimicrobial or cytotoxic activity generally show an increased production of reactive oxygen intermediates.63 To generate these oxidants, phagocytes, including monocytes and macrophages, contain multiple protein components that, when assembled and activated, form an NADPH oxidase complex that transfers electrons to molecular oxygen from intracellular NADPH. Activation of NADPH oxidase is triggered by protein kinases64 and involves translocation to the plasma membrane of several cytosolic components, including p67phox, p47phox, p40phox. The fully active NADPH oxidase complex also includes several small GTP-binding proteins.65 In the plasma membrane, the several cytosolic components interact via proline-rich and SH3 domains with a flavo-hemoprotein, cytochrome b558, that is composed of large and small subunits called gp91phox and p22phox, respectively. Superoxide (O2–) anions generated by NADPH oxidase are unstable and undergo various reactions, including dismutation to form hydrogen peroxide (H2O2) and oxygen.
Chronic granulomatous disease (CGD) refers to a group of uncommon disorders associated with defective activation and assembly of NADPH oxidase by neutrophils and mononuclear phagocytes. The neutrophils and monocytes of children with CGD fail to produce superoxide and H2O2 and show markedly impaired antimicrobial activity against many bacteria and fungi in vitro. The most common variant of CGD is transmitted with X-linked inheritance and affects male children only.66 It results from the absence or abnormality of gp91phox, whose gene is located on the X chromosome, at Xp21.1. Defects in p47phox (chromosome 7q11.23) are transmitted autosomally and account for about 30 percent of total CGD cases, affecting males and females equally. Primary genetic defects involving p22phox, p67phox, and gp91 have also been described and account for the remaining cases of CGD. Children affected by CGD sustain repeated infections, most often caused by Staphylococcus aureus but caused also by bacteria and fungi of limited pathogenic potential (e.g., Serratia marcescens, Burkholderia cepacia, and Aspergillus fumigatus). Prophylactic administration of antibiotics, such as trimethoprim-sulfamethoxazole, and of interferon-g,67 decreases the frequency and severity of infections in patients with all forms of CGD (See Chap. 72).
Like neutrophils, blood monocytes contain myeloperoxidase (MPO) and use it to convert H2O2 into microbicidal oxidants.68 Their MPO is lost when monocytes differentiate into macrophages. Hereditary deficiency of MPO is relatively common, perhaps affecting as many as 1 in 2000 individuals.69 Neutrophils and monocytes of affected individuals lack MPO and are unable to convert the H2O2 produced by the dismutation of superoxide into more potent oxidants such as hypochlorite or chloramines. Neutrophils and monocytes from subjects with hereditary MPO deficiency show selectively impaired microbicidal activity in vitro, and several such patients (typically with additional predisposing factors, such as diabetes mellitus) have developed disseminated C. albicans infections. Although other abnormalities can also cause MPO-deficiency, R569W missense mutations occur in the MPO genes of many affected subjects, often associated with some other abnormality of the allelic gene.70,71
Nitric oxide (NO) and other reactive nitrogen intermediates play important roles in restricting the growth of many pathogenic organisms in mice and in murine macrophages.72,73 NO is both diffusible and unstable. It reacts with oxygen and water to yield equimolar amounts of nitrite and nitrate and reacts with other molecules to form S-nitrosothiols. NO and superoxide (O2–) can interact to form peroxynitrite (ONOO–), a potent oxidant that may also mediate antimicrobial activity.
Distinct NO synthase enzymes are responsible for the constitutive and inducible production of nitric oxide.74 In murine macrophages, stimulation by lipopolysaccharide or cytokines such as interferon g leads to the expression of an inducible nitric oxide synthase, called NOS2 or iNOS. This heme-containing enzyme converts L-arginine to citrulline + NO, using NADPH and oxygen as additional substrates and FAD, FMN, calmodulin, and tetrahydrobiopterin as cofactors.74 iNOS itself may mediate production of superoxide in L-arginine-depleted murine macrophages.75
Pathogens whose susceptibility to rodent macrophages can be attributed to NO production include Cryptococcus neoformans, Francisella tularensis, Leishmania major, and Schistosoma mansoni73,76; iNOS knockout mice show increased susceptibility to acute infection by many, but not all, organisms.77,78 For example, macrophages from knockout mice deficient in interferon regulatory factor 1 (IRF-1) efficiently controlled Listeria monocytogenes despite failing to produce iNOS and NO,79 and mice deficient in NF-IL6 were highly susceptible to Listeria despite a normal iNOS response.80 The role of NO in human host defense is less certain but is under intense study.81 Human lung macrophages from patients with tuberculosis express iNOS,82 and inflammatory human macrophages have been induced to express iNOS in vitro.83
Activation of macrophage cytostatic or cytocidal activity usually requires the delivery of two signals, one that “primes” and the other that “triggers.” Priming signals often derive from T cells and can include molecules such as interferon g, IL-2, IL-4, G-CSF, and GM-CSF or combinations thereof.84 The cytostatic properties of activated macrophages85 arise from both oxygen-independent and oxygen-dependent mechanisms. The former are mediated by cytotoxic molecules, including TNF-a and enzymes (e.g., cytolytic proteases or arginase). The latter are mediated by reactive chemical intermediates formed by NADPH oxidase or inducible nitric oxide synthase.
Macrophage-mediated cellular cytotoxicity has been divided into four basic categories: rapid antibody-dependent cellular cytotoxicity (ADCC), slow ADCC, antibody-independent tumor cytolysis, and cytostasis.86 All require macrophage activation, and all but cytostasis required intimate physical contact between the macrophage and its target cell. Just as many pathogenic microbes can subvert the microbicidal actions of macrophages, tumors may also release cytokines and other regulatory factors that suppress macrophage-mediated cytotoxicity.87
ADCC can occur rapidly (within 4 to 6 h) or slowly (over 24 to 48 h) after macrophage–target cell contact occurs. Such contact can be induced by antibody and is promoted by adhesive glycoproteins of the integrin/selectin group. IgG1, IgG2a, IgG2b, or IgG3 isotype antibodies impart immunologic specificity to the process—with IgG2a perhaps being most efficient.88 ADCC against antibody-coated erythrocytes can contribute to erythrocyte destruction in autoimmune hemolytic anemias, and ADCC directed toward surface-bound gliadin may contribute to the pathogenesis of celiac disease.89
This process, reportedly specific for neoplastic or transformed cells, lyses a wide range of syngeneic, allogeneic, and xenogeneic targets, while sparing their normal counterparts. The macrophage delivers the fatal blow at close quarters, using molecular weaponry that includes TNF-a, reactive oxygen intermediates, and nitric oxide.31,90
Unlike ADCC, macrophage-mediated cytostasis is neither target specific nor contact-dependent. It affects proliferation of a broad range of normal and neoplastic targets and results from release of various macrophage-derived molecules, including prostaglandins, arginase, nitric oxide, and cytokines, including interferons, TNF-a or IL-1a, and –1b.31
Production of reactive oxygen intermediates by NADPH oxidase, discussed above with reference to antimicrobial activity, also contributes to the ability of macrophages to destroy tumor cells91,92 and to lyse antibody-coated erythrocyte targets.93
Cytostasis caused by NO or peroxynitrite (OONO) results from oxidation or nitrosylation of cytoplasmic enzymes, such as ribonucleotide reductase,94 and mitochondrial enzymes with iron-sulfur centers, such as aconitase and succinate-ubiquinone oxidoreductase.95 Damage of these vital enzymes inhibits the target cell’s energy metabolism and its production of deoxyribonucleotides.96 Expression of iNOS has been demonstrated in tumor-associated macrophages that infiltrate human breast and gastric cancers97; iNOS production in human macrophages is regulated by an interplay between membrane CD23, a low-affinity receptor for IgE FCe, and the cytokine IL-10.98
Although activated murine macrophages secrete a cytotoxic serine protease,99 and rabbit alveolar macrophages contain potentially cytotoxic peptide defensins,100 a contribution by human homologues of these molecules to macrophage-mediated cytotoxicity has not been shown. The cytotoxicity of cytolytic protease99 and defensins101 is enhanced by otherwise nontoxic concentrations of hydrogen peroxide.
TNF-a was originally identified in the circulation of LPS-treated animals and received its alternative original name, “cachexin,” from its ability to inhibit lipoprotein lipase and induce wasting in mice.102,103 A 26-kDa TNF-a precursor is displayed on the plasma membrane,104 and proteolytic processing trims this to a monomeric, 17-kDa form, which can form trimers.105 The cytotoxic properties of TNF-a are enhanced by concomitant production of nitric oxide by macrophages.106 Lymphocytes also produce TNF-a, as well as a closely related lymphotoxin molecule called TNF-b.107
TNF-a mediates most of its varied effects on cells via a specific 75-kDa receptor108 which also circulates in a soluble form. In addition to killing certain tumor cells in vitro and in vivo, TNF-a causes fever, leukopenia followed by leukocytosis, neutrophil priming and activation, T-cell activation, increased serum levels of IL-6, and an enhanced procoagulatory state.109 These effects resemble those of IL-1 and include many responses seen typically with acute inflammation and infection.

Kamperdijk EWA, Nieuwenhuis P, Hoefsmit ECM: Dendritic Cells in Fundamental and Clinical Immunology, p 1. Plenum Press, New York, 1993.

Freudenthal PS, Steinman RM: The distinct surface of human blood dendritic cells, as observed after an improved isolation method. Proc Natl Acad Sci USA 87:7698, 1990.

Adema GJ, Hartgers F, Verstraten R, et al: A dendritic-cell-derived C-C chemokine that preferentially attracts naive T cells. Nature 387:713, 1997.

Chapuis F, Rosenzwajg M, Yagello M, et al: Differentiation of human dendritic cells from monocytes in vitro. Eur J Immunol 27:431, 1997.

Szabolcs P, Ciocon DH, Moore MA, Young JW: Growth and differentiation of human dendritic cells from CD34+ progenitors. Adv Exp Med Biol 417:15, 1997.

Brossart P, Grunebach F, Stuhler G, et al: Generation of functional human dendritic cells from adherent peripheral blood monocytes by CD40 ligation in the absence of granulocyte-macrophage colony-stimulating factor. Blood 92:4238, 1998.

Rosenzwajg M, Camus S, Guigon M, Gluckman JC: The influence of interleukin (IL)-4, IL-13, and Flt3 ligand on human dendritic cell differentiation from cord blood CD34+ progenitor cells. Exp Hemat 26:63, 1998.

Stossel TP: On the crawling of animal cells. Science 260:1086, 1993.

Marasco WA, Phan SH, Krutzsch H: Purification and identification of formylmethionyl-leucyl-phenylalanine as the major peptide neutrophil chemotactic factor produced by Escherichia coli. J Biol Chem 259:5430, 1984.

Snyderman R, Pike MC, Edge S, Lane B: A chemoattractant receptor on macrophages exists in two affinity states regulated by guanine nucleotides. J Cell Biol 98:444, 1984.

Shang XZ, Issekutz AC: Beta 2 (CD18) and beta 1 (CD29) integrin mechanisms in migration of human polymorphonuclear leucocytes and monocytes through lung fibroblast barriers: shared and distinct mechanisms. Immunology 92:527, 1997.

Malmsten CL, Palmblad J, Uden AM, et al: A highly potent stereospecific factor stimulates migration of polymorphonuclear leukocytes. Acta Physiol Scand 110:449, 1980.

Sozzani S, Zhou D, Locati M, et al: Stimulating properties of 5-oxo-eicosanoids for human monocytes: synergism with monocyte chemotactic protein-1 and -3. J Immunol 157:4664, 1996.

Hunninghake GW, Davison JM, Rennard S, et al: Elastin fragments attract macrophage precursors to diseased sites in pulmonary emphysema. Science 212:925, 1981.

Uemura Y, Okamoto K: Elastin-derived peptide induces monocyte chemotaxis by increasing intracellular cyclic GMP level and activating cyclic GMP dependent protein kinase. Biochem Mol Biol Int 41:1085, 1997.

Naldini A, Sower L, Bocci V, et al: Thrombin receptor expression and responsiveness of human monocytic cells to thrombin is linked to interferon-induced cellular differentiation. J Cell Physiol 77:76, 1998.

Deuel TF, Senior RM, Huang JS, Griffin GL: Chemotaxis of monocytes and neutrophils to platelet-derived growth factor. J Clin Invest 69:1046, 1982.

Baggiolini M, deWald B, Moser B: Human chemokines: an update. Ann Rev Immunol 15:675, 1997.

Boulay F, Naik N, Giannini E, et al: Phagocyte chemoattractant receptors. Ann NY Acad Sci 832:69, 1997.

Dragic T, Litwin V, Allaway GP, et al: HIV-1 entry into CD4+ cells is mediated by the chemokine receptor CC-CKR-5. Nature 381:667, 1996.

Guo HG, Browning P, Nicholas J, et al: Characterization of a chemokine receptor-related gene in human herpesvirus 8 and its expression in Kaposi’s sarcoma. Virology 228:371, 1997.

McFadden G, Lalani A, Everett H, et al: Virus-encoded receptors for cytokines and chemokines. Semin Cell Dev Biol 9:359,1998.

Rucker J, Edinger AL, Sharron M, et al: Utilization of chemokine receptors, orphan receptors, and herpesvirus-encoded receptors by diverse human and simian immunodeficiency viruses. J Virol 71:8999, 1997.

Damon I, Murphy PM, Moss B: Broad spectrum chemokine antagonistic activity of a human poxvirus chemokine homolog. Proc Nat Acad Sci USA 95:6403, 1998.

Geras-Raaka E, Arvanitakis L, Bais C, et al: Inhibition of constitutive signaling of Kaposi’s sarcoma-associated herpesvirus G protein-coupled receptor by protein kinases in mammalian cells in culture. J Exp Med 187:801,1998.

Mukherjee S, Ghosh RN, Maxfield FR: Endocytosis. Physiol Rev 77: 759, 1997.

Racoosin EL, Swanson JA: M-CSF-induced macropinocytosis increases solute endocytosis but not receptor-mediated endocytosis in mouse macrophages. J Cell Science 102:867, 1992.

Unanue ER: Antigen presenting function of the macrophage. Annu Rev Immunol 2:395, 1984.

Nathan CF: Secretory products of macrophages. J Clin Invest 79:319, 1987.

Ezekowitz RAB, Sim RB, MacPherson GG, Gordon S: Interaction of human monocytes, macrophages, and polymorphonuclear leukocytes with zymosan in vitro. Role of type 3 complement receptors and macrophage-derived complement. J. Clin Invest 76:2368, 1985.

Adams DO, Hamilton TA: Macrophages as destructive cells in host defense, in Inflammation, Basic Principles and Clinical Correlates, 2d ed, edited by JI Gallin, IM Goldstein, R Snyderman, p 637. Raven, New York, 1992.

Hemler ME: VLA proteins in the integrin family: Structures, functions, and their role on leukocytes. Annu Rev Immunol 8:365, 1990.

Mazzone A, Ricevuti G: Leukocyte CD11/CD18 integrins: biological and clinical relevance. Haematologica 80:161, 1995.

VanAndvian U, Chambers J, McEvoy L, et al: Two-step model of leukocyte-endothelial cell interaction inflammation: distinct roles for LECAM-I and the leukocyte beta 2 integrins in vivo. Proc Natl Acad Sci USA 88:7538, 1991.

Ehlenberger AG, Nussenzweig V: The role of membrane receptors for C3b and C3d in phagocytosis. J Exp Med 145:357, 1977.

Griffin FMJ, Griffin JA: Augmentation of macrophage complement receptor function in vitro: II. Characterization of the effects of a unique lymphokine upon the phagocytic capabilities of macrophages. J lmmunol 125:844, 1980.

Wright SD, Reddy PA, Jong MTC, Erickson BW: C3bi receptor (complement receptor type 3) recognizes a region of complement protein C3 containing the sequence Arg-Gly-Asp. Proc Natl Acad Sci USA 84:1965, 1987.

Wright SD: Receptors for complement and the biology of phagocytosis, in Inflammation, Basic Principles and Clinical Correlates, 2d ed, edited by JI Gallin, IM Goldstein, R Snyderman, p 477. Raven, New York, 1992.

Zhang L, Plow EF: Overlapping, but not identical, sites are involved in the recognition of C3bi, neutrophil inhibitory factor, and adhesive ligands by the alpha M beta 2 integrin. J Biol Chem 271:18211, 1996.

Schiff DE, Rae J, Martin TR, et al: Increased phagocyte Fc gammaRI expression and improved Fc gamma-receptor-mediated phagocytosis after in vivo recombinant human interferon-gamma treatment of normal human subjects. Blood 90:3187, 1997.

Janeway CA: The immune system evolved to discriminate infectious nonself from noninfectious self. Immunol Today 13:11, 1992.

Stahl PD, Ezekowitz RAB: The mannose receptor is a pattern recognition receptor involved in host defense. Curr Opin Immunol 10:50, 1998.

Reis e Souza C, Stahl PD, Austyn JM: Phagocytosis of antigens by Langerhans cells in vitro. J Exp Med 178:509, 1993.

Fenton MJ, Golenbock DT: LPS-binding proteins and receptors. J Leuk Biol 64:25, 1998.

Haziot A, Ferrero E, Kontgen F, et al: Resistance to endotoxin shock and reduced dissemination of gram-negative bacteria in CD14-deficient mice. Immunity 4:407, 1996.

Brown MS, Goldstein JI: Lipoprotein metabolism in the macrophage: implications for cholesterol deposition in atherosclerosis. Annu Rev Biochem 52:223, 1983.

Yla-Herttuala S: Expression of lipoprotein receptors and related molecules in atherosclerotic lesions. Curr Opin Lipidol 7:292, 1996.

Hampton RY, Goklenbock DT, Penman M, et al: Recognition and plasma clearance of endotoxin by scavenger receptors. Nature 352: 342, 1991.

Dunne DW, Resnick D, Greenberg J, et al: The type I macrophage scavenger receptor binds to gram-positive bacteria and recognizes lipoteichoic acid. Proc Nat Acad Sci USA 91:1863, 1994.

Daviet L, McGregor JL: Vascular biology of CD36: roles of this new adhesion family molecule in different disease states. Thromb Haemostasis 78:65, 1997.

Fraser I, Gordon S: An overview of receptors of MPS cells, in Blood Cell Biochemistry, vol 5, Macrophages and Related Cells, edited by MA Horton, p 1. Plenum, New York, 1993.

Hackam DJ, Rotstein OD, Zhang W, et al: Host resistance to intracellular infection: mutation of natural resistance-associated macrophage protein 1 (Nramp1) impairs phagosomal acidification. J Exp Med 188: 351, 1998.

Searle S, Bright NA, Roach TIA, et al: Localization of Nramp1 in macrophages: modulation with activation and infection. J Cell Sci 111:2855, 1998.

Sturgill-Koszycki S, Schaible U, Russell DG: Mycobacterium-containing phagosomes are accessible to early endosomes and reflect a transitional state in the normal phagosome biogenesis. EMBO J 15:6960, 1996.

Mackaness GB: Reflections on the history of the macrophage, in Mononuclear Phagocytes in Cell Biology, edited by G Lopez-Berestein, J Klostergaard, p 1. CRC, Boca Raton, FL, 1993.

Portnoy DA: Innate immunity to a facultative intracellular bacterial pathogen. Curr Opin Immunol 4:20, 1992.

Southwick FS, Purich DL: Dynamic remodeling of the actin cytoskeleton: lessons learned from Listeria locomotion. Bioessays 16:885, 1994.

Smith GA, Portnoy DA: How the Listeria monocytogenes ActA protein converts actin polymerization into a motile force. Trends Microbiol 5:272, 1997.

Sibley LD, Weidner E, Krahenbuhl JL: Phagosome acidification is blocked by intracellular Toxoplasma gondii. Nature 315:416, 1985.

Horwitz MA: The legionnaire’s disease bacterium (Legionella pneumophila) inhibits phagosome-lysosome fusion in human monocytes. J Exp Med 158:2108, 1983.

Alpuche A, Swanson JA, Loomis WP, Miller SI: Salmonella typhimurium activates virulence gene transcription within acidified macrophage phagosomes. Proc Natl Acad Sci USA 89:10079, 1992.

Rathman M, Sjaastad MD, Falkow S: Acidification of phagosomes containing Salmonella typhimurium in murine macrophages. Infect Immun 64:2765, 1996.

Nathan CF: Secretion of oxygen intermediates: Role in effector functions of activated macrophages. Fed Proc 41, 2206, 1982.

El Benna J, Faust RP, Johnson JL, Babior BM: Phosphorylation of the respiratory burst oxidase subunit p47phox as determined by two-dimensional phosphopeptide mapping. Phosphorylation by protein kinase C, protein kinase A, and a mitogen-activated protein kinase. J Biol Chem 271:6374, 1996.

Meischl C, Roos D: The molecular basis of chronic granulomatous disease. Springer Semin Immunopathol 19:417, 1998.

Roos D: X-CGDbase: a database of X-CGD-causing mutations. Immunol Today 17:517, 1996.

Gallin JI, Malech HL, Meinick DA: A controlled trial of interferon gamma to prevent infection in chronic granulomatous disease. The International Chronic Granulomatous Disease Study Group. New Engl J Med 324:509, 1991.

Klebanoff SJ: Oxygen metabolites from phagocytes, in Inflammation, Basic Principles and Clinical Correlates, 2d ed, edited by Jl Gallin, IM Goldstein, R Snyderman, p 541. Raven, New York, 1992.

Nauseef WM, Root RK, Malech HL: Biochemical and immunologic analysis of hereditary myeloperoxidase deficiency. J Clin Invest 71:1297, 1983.

DeLeo FR, Goedken M, McCormick SJ, Nauseef WM: A novel form of hereditary myeloperoxidase deficiency linked to endoplasmic reticulum/proteasome degradation. J Clin Invest 101:2900, 1998.

Nauseef WM, Cogley M, Bock S, Petrides PE: Pattern of inheritance in hereditary myeloperoxidase deficiency associated with the R569W missense mutation. J Leuk Biol 63:264, 1998.

James S: Role of nitric oxide in parasitic infections. Microb Rev 59:533, 1995.

Fang FC: Mechanisms of nitric oxide-related antimicrobial activity. J Clin Invest 99:2818, 1997.

Nathan C, Xie Q: Nitric oxide synthases: roles, tolls and controls. Cell 78:915, 1994.

Xia Y, Zweier JL: Superoxide and peroxynitrite generation from inducible nitric oxide synthase in macrophages. Proc Nat Acad Sci USA 94:6954, 1997.

Granger DL, Cameron ML, Lee-See K, Hibbs JB Jr: Role of macrophage-derived nitrogen oxides in antimicrobial function, in Mononuclear Phagocytes in Cell Biology, edited by G Lopez-Berestein, J Klostergaard, p 7, CRC, Boca Raton, FL, 1993.

MacMicking JD, Nathan C, Hom G, et al: Altered responses to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthase. Cell 81:641, 1995.

Sharton-Kersten T, Yap G, Magram J, Sher A: Inducible nitric oxide is essential for host control of persistent but not acute infection with the intracellular pathogen Toxoplasma gondii. J Exp Med 185:1261, 1997.

Fehr T, Schoedon G, Odermatt B, et al: Crucial role of interferon consensus sequence binding protein, but neither of interferon regulatory factor 1 nor of nitric oxide synthesis for protection against murine listeriosis. J Exp Med 185:921, 1997.

Tanaka T, Akira S, Yoshida K, et al: Targeted disruption of the NF-IL6 gene discloses its essential role in bacteria killing and tumor cytotoxicity by macrophages. Cell 80:353, 1995.

Nathan C: Inducible nitric oxide synthase: what difference does it make? J Clin Invest 100:2417, 1997.

Nicholson S, Bonecini-Almeida M Da G, Lapa e Silva JR, et al: Inducible nitric oxide synthase in pulmonary alveolar macrophages from patients with tuberculosis. J Exp Med 183:2293, 1996.

Nozaki Y, Hasegawa Y, Ichiyama S, et al: Mechanism of nitric-oxide dependent killing of Mycobacterium BCG in human alveolar macrophages. Infect Immun 65:3644, 1997.

Hamilton TA, Ohmori Y, Narumi S, Tannenbaum CS: Regulation of diversity of macrophage activation, in Mononuclear Phagocytes in Cell Biology, edited by G Lopez-Berestein, J Klostergaard, pp 48–70. CRC, Boca Raton, FL, 1993.

Krahenbuhl JL, Remington JS: The role of activated macrophages in specific and nonspecific cytostasis of tumor cells. J Immunol 113:507, 1974.

Adams DO, Hamilton TA: Destruction of tumor cells by mononuclear phagocytes. Models for analyzing effector mechanisms and regulation of macrophage activation, in Mechanisms of Host Resistance to Infectious Agents, Tumors, and Allografts, edited by RM Steinman, RJ North, pp 185–204. Rockefeller University, New York, 1986.

Elgert KD, Alleva DG, Mullins DW: Tumor-induced immune dysfunction: the macrophage connection. J Leuk Biol 64:275, 1998.

Kipps TJ, Parham P, Punt J, Herzenberg AL: Importance of immunoglobulin isotype in human antibody-dependent cell-mediated cytotoxicity directed by murine monoclonal antibodies. J Exp Med 161:1, 1985.

Saalman R, Wold AE, Dahlgren UI, et al: Antibody-dependent cell-mediated cytotoxicity to gliadin-coated cells with sera from children with coeliac disease. Scand J Immunol, 47:37, 1998.

Keller R, Keist R, Wechsler A, et al: Mechanisms of macrophage-mediated tumor cell killing: a comparative analysis of the roles of reactive nitrogen intermediates and tumor necrosis factor. Int J Cancer 46:682, 1990.

Nathan CF: Reactive oxygen intermediates in lysis of antibody-coated tumor cells, in Macrophage-Mediated Antibody-Dependent Cellular Cytotoxicity, edited by HS Koren, pp 199–215. Marcel Dekker, New York, 1983.

Cohen MS, Taffet SM, Adams DO: The relationship between competence for secretion of H2O2 and completion of macrophage cytotoxicity by BCG-elicited murine macrophages. J Immunol 128:1781, 1982.

Fleer A, Roos D, von den Borne EG Jr, Engelfriet CP: Cytotoxic activity of human monocytes toward sensitized red cells is not dependent on the generation of reactive oxygen species. Blood 54:407, 1979.

Lepoivre M, Chenais B, Yapo A, et al: Alterations of ribonucleotide reductase activity following induction of the nitrite-generating pathway in adenocarcinoma cells. J Biol Chem 265:14143, 1990.

Darpier JC, Hibbs JB Jr: Murine cytotoxic activated macrophages inhibit aconitase in tumor cells: inhibition involves the iron-sulfur prosthetic group and is reversible. J Clin Invest 78:790, 1986.

Keller R, Geiges M, Keist R: Arginine dependent reactive nitrogen intermediates as mediators of tumor cell killing by activated macrophages. Cancer Res 50:1421, 1990.

Thomsen LT, Miles DW: Role of nitric oxide in tumour progression: lessons from human tumours. Cancer Metastasis Rev 17:107, 1998.

Dugas N, Palacios-Calender M, Dugas B, et al: Regulation by endogenous interleukin-10 of the expression of nitric oxide synthase induced after ligation of CD23 in human macrophages. Cytokine 10:680, 1998.

Adams DO, Hamilton TA: The cell biology of macrophage activation. Annu Rev Immunol 2:283, 1984.

Lehrer RI, Lichtenstein AK, Ganz T: Antimicrobial and cytotoxic peptides of mammalian cells. Annu Rev Immunol 11:105, 1993.

Lichtenstein AK, Ganz T, Selsted ME, Lehrer RI: Synergistic cytolysis mediated by hydrogen peroxide combined with peptide defensins. Cell Immunol 114:104, 1988.

Beutler B, Cerami A: Cachectin: more than a tumor necrosis factor. N Engl J Med 316:379, 1987.

Beutler B (ed): Tumor Necrosis Factors: The Molecules and Their Emerging Role in Medicine. Raven, New York, 1992.

Chaudhri G: Differential regulation of biosynthesis of cell surface and secreted TNF-alpha in LPS-stimulated murine macrophages. J Leuk Biol 62:249, 1997.

Jones EY, Stuart DI, Walker NPC: Structure of tumor necrosis factor. Nature 338:225, 1989.

Higuchi M, Higashi N, Taki H, Osawa T: Cytolytic mechanisms of activated macrophages. Tumor necrosis factor and L-arginine dependent mechanisms act synergistically as the major cytolytic mechanisms of activated macrophages. J Immunol 144:1425, 1990.

Nedwin GE, Naylor SI, Sakaguchi AY: Human lymphotoxin and tumor necrosis factor genes: structure, homology and chromosomal localization. Nucleic Acids Res 13:6361, 1985.

Dembric Z, Loetscher H, Gubler U: Two human TNF receptors have similar extracellular but distinct intracellular domain sequences. Cytokine 2:231, 1990.

Dinarello CA: Role of interleukin-1 and tumor necrosis factor in systemic responses to infection and inflammation, in Inflammation, Basic Principles and Clinical Correlates, 2d ed, edited by JI Gallin, IM Goldstein, R Snyderman, pp 211–232. Raven, New York, 1992.
Copyright © 2001 McGraw-Hill
Ernest Beutler, Marshall A. Lichtman, Barry S. Coller, Thomas J. Kipps, and Uri Seligsohn
Williams Hematology



  1. […] cancerAnti-Cancer Dietary Strategies: The Basics, Part 2Hybrid PET and MRI imaging on the horizonCHAPTER 74 BIOCHEMISTRY AND FUNCTION OF MONOCYTES AND MACROPHAGES body { background: […]

  2. hey there and thanks for your info ? I have definitely picked up anything new from right here. I did alternatively experience several technical issues using this web site, as I skilled to reload the website a lot of times prior to I may get it to load correctly. I have been wondering if your web host is OK? Not that I am complaining, however sluggish loading instances times will sometimes have an effect on your placement in google and can injury your high quality rating if ads and marketing with Adwords. Well I’m adding this RSS to my e-mail and can glance out for a lot extra of your respective fascinating content. Ensure that you replace this once more very soon..

  3. Valuable information. Lucky me I found your site unintentionally, and I am shocked why this twist of fate did not took place in advance! I bookmarked it.

  4. Execelent information and facts my friend, como ligar a las mujeres I just didn’t know what you published, excellent share. sistema del macho alfa

  5. Aw, this was a very nice post. In thought I want to put in writing like this moreover – taking time and precise effort to make an excellent article… but what can I say… I procrastinate alot and by no means seem to get something done.

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out / Change )

Twitter picture

You are commenting using your Twitter account. Log Out / Change )

Facebook photo

You are commenting using your Facebook account. Log Out / Change )

Google+ photo

You are commenting using your Google+ account. Log Out / Change )

Connecting to %s

%d bloggers like this: