Mechanisms of host defence

The respiratory tract is situated at the interface between the environmental air and the internal tissues of the organism. During normal ventilation or as a result of aspiration, noxious materials, including infectious agents, are deposited on mucosal surfaces of the airways and penetrate into the depths of the lung parenchyma. Foreign material encounters a highly integrated system of natural and acquired defence mechanisms, that prevent injury, infections, and invasion of host tissue ^44-46. This system of host defence includes mechanical (filtration, cough, mucociliary clearance), molecular (airway secretions), phagocytic, and antigen-specific immune mechanisms (B- and T-cell-mediated reactions) of resistance (reviewed in ⁴⁷).

Introduction

1.3.1.1 The alveolar macrophage

The alveolar macrophage (AM) is the primary resident respiratory cell responsible for maintaining the sterility of the alveolar space and thus represents the first line of defence. As a mobile cell and representative of the mononuclear phagocyte system, the AM is believed to be the central modulator, as both regulator and effector of the degree of inflammation and anti-inflammation within the alveolar space ⁴⁸. This metabolically highly active cell scavenges particulate matter, removes macromolecular debris, kills microorganisms, functions as an accessory cell in immune response, maintains and repairs the lung parenchyma and provides surveillance against neoplasms ⁴⁹. Since the pulmonary alveolar macrophages are enveloped by surfactant in vivo, and since their lysosomes contain hydrolytic enzymes necessary for surfactant degradation ⁵⁰, it is believed that alveolar macrophages are also involved in the clearance of surfactant from the alveoli. When confronted with a particularly large inoculum of microorganisms, the AM supplements its direct antimicrobial capabilities by recruiting and activating polymorphonuclear leukocytes (PMN) from the bloodstream, which may represent the second line of defence. Approximately 40% of the body’s PMN’s are marginated within the microvasculature of the lungs, facilitating the recruitment to the alveolus, or to other sides of the body ⁵¹.

1.3.1.2 Location and origin

Pulmonary macrophages constitute the most abundant non-parenchymal cell type in the lung

52. Macrophages are present in the alveoli (alveolar macrophages, AM), interstitial spaces (interstitial macrophages), intravascular spaces (intravascular macrophages), conducting airways (airway macrophages), pleura (pleural macrophages), and lymph nodes (lymph note macrophages) ^53,54.

Alveolar macrophages initially encounter materials, that reach gas exchange units and subsequently perform phagocytosis and microbial killing. Interstitial macrophages serve as precursors for physiologic renewal of alveolar macrophages and for their expansion during pulmonary inflammation ⁵³. Adhering to the pulmonary endothelial cells, intravascular macrophages remove circulating particles, but may also contribute to pathogenesis during sepsis ^48,55. Airway macrophages are responsible for the reactivity of the conductive airways in response to inhaled stimuli in patients with asthma ⁴⁷.

The ultimate source of pulmonary macrophages are monocyte progenitor cells in the bone marrow ⁵⁶, which enter the lung from the vascular bed, and adapt to the local environment by maturation into tissue macrophages. Maturation results in an increase in cell size, in the number of cytoplasmatic organelles, lysosomal enzyme activity, phagocytosis capacity, and expression of Fc, complement and cytokine receptors on surface membranes ⁵⁴. Alveolar

Introduction

macrophages vary considerable in size and morphologic features. They represent both phenotypically and functionally a heterogenous population of cells ⁵⁷. Pulmonary macrophages live for weeks to months and possess some limited replicative potential ⁵⁸. It is believed that the effector arm of the cell-mediated immune response depends on both the activation of resident macrophages as well as on the recruitment of circulating monocytes into the alveoli

58. However, the proportion of monocyte influx and local replication during the pulmonary inflammation has to be defined.

Chemotactic factors like complement component C5a, transforming growth factor beta (TGF-β), formylated peptides and monocyte-chemotactic peptide (MCP-1), stimulate the influx of monocytes into the lung (reviewed in ⁵⁰). To exert their effector functions, alveolar macrophages have to be either activated non-specifically by adherence ⁵⁹ or usually through ligand-to-receptor coupling. Among a large number of receptors expressed on alveolar macrophages ⁵⁷, immunoglobulin (Ig) and complement (C) receptors (CR1, CR3) participate in receptor-mediated phagocytosis. The former bind to the Fc portion of Ig molecules (IgG, IgA, IgE), whereas the latter are important natural opsonins for microbial organisms and particulates ⁶⁰. Macrophages further express C5a, a potent chemotactic, cell-activating and pro-inflammatory molecule, as well as various cytokine receptors for interleukin-1 (IL-1), tumor necrosis factor (TNF), interferon γ (INF-γ), growth factor receptors and cell activation factors e.g. lectins, lipoproteins and glucocorticoids (reviewed in ^50,57).

CD14, a 55-kDa phosphatidylinositiol-anchored membrane glycoprotein expressed on the surface of monocytes, macrophages, and, to a lesser extent on neutrophils ⁶¹ has been identified as an important receptor for LPS, in association with LPS-binding protein (LBP). The stimulation of CD14 triggers a concentration-dependent macrophage activation ⁶². This activation results in the release of TNF, IL-6 and IL-8 ^63,64 and has been described as both LBP-dependent and -independent ⁶³. Haugen and coworkers reported a higher expression of CD14 on monocytes (MO) than on AM, with AM of different size and maturity having different CD14 expression levels. The expression of CD14 can be modulated in response to LPS, but the LPS binding capacity of AM and MO does not correlate with their CD14 levels ⁶⁵. Indeed, several other macrophage receptors that function as LPS receptors have been described ⁶⁶.

1.3.1.3 Secretory function

Macrophages are pluripotent cells. The range of products secreted by alveolar macrophages is broad, with over 100 molecular species having been identified, including reactive oxygen species, proteases and anti-proteases, bioactive lipids, cytokines as well as growth factors (reviewed in ⁵⁰).

Introduction

Reactive oxygen radicals are generated via the “respiratory burst”. Together with several proteases and other enzymes that function intracellularly, reactive oxygen species are involved in microbial killing. Secreted proteinases (metalloproteinases, serine proteinases) affect fibrinolysis and extracellular matrix remodelling ⁵⁰. Stimulation of alveolar macrophages with IgG or IgE immune-complexes leads to the production of both cyclooxygenase-derived (thromboxane A₂ and prostaglandin E₂ and D₂) and lipoxygenase-derived (leukotriene B₄) compounds ⁶⁷. In the IgG immune complex animal model of acute lung injury ⁶⁸, bronchoalveolar lavage fluids contain large amounts of biologically active TNF and IL-1. These

“early response cytokines” function in a pro-inflammatory manner, by playing a key role in the upregulation or induction of adhesion molecules in the lung vasculature, such as ICAM-1 and E-selectin, which are critically necessary for neutrophil recruitment. In this regard, the α -chemokine family (IL-8 or its family relatives such as MIP-2, CINC) are described to have chemotactic activity primarily for neutrophils and to a lesser extent for T-cell subsets, while the β-chemokines (e.g. MIP-1a, b; MCP-1, 2 and 3) are thought to be predominantly chemotactic for monocytes and lymphocytes. In contrast, there are various anti-inflammatory cytokines such as IL-10, IL-4, IL-6 or IL-13 produced by alveolar macrophages to suppress the cytokine production and therefore regulate the intensity of the inflammatory response (reviewed in ⁶⁹).

In addition to cytokines, also lipid mediators such as leukotrienes (LT) upregulate directly the antimicrobial potential of macrophages as well as PMN’s from different species against both bacteria and fungi (reviewed in ⁷⁰). Leukotriene levels have been shown to be elevated in bronchoalveolar lavage fluid from patients with bacterial pneumonia and also in lung tissue in animal models of pneumonia ⁷⁰.

1.3.1.4 Phagocytic function

Macrophages remove materials present in the local environment through both pinocytosis and phagocytosis. In contrast to pinocytosis, phagocytosis is an energy-dependent process, characterized by adherence through receptors and engulfment, leading further to internalisation of the particulates and finally to digestion in phagolysosomal vacuoles. Efficient phagocytosis depends on opsonization. Opsonins defined in the lung include IgG1, IgG3, C3b, surfactant protein A/ D (SP-A/ D) ^71,72, mannose and lipopolysaccharide-binding proteins ⁶⁰. Furthermore, SP-A has been reported to enhance the phagocytosis of C1q-coated particles by alveolar macrophages ⁷³. However, binding of inhaled environmental particles must be accomplished without the benefit of opsonization by specific antibodies. The identities of receptors on AM’s that mediate unopsonized particle binding are not fully known, whereas the role of some scavenger receptors (SR) have been recently reported ^66,74,75.

Introduction

1.3.1.5 Microbicidal function

The production of reactive oxygen species (ROS) upon receptor-mediated stimulation of phagocytosis was first recognized in phagocytes such as macrophages, a process determining their microbicidal activity and referred to as respiratory burst. This process results from the assembly and activation of the nicotinamide dinucleotide phosphate (NADPH) oxidase, a multicomponent enzyme that catalyses the one-electron reduction of molecular oxygen to superoxide ⁷⁶, which is subsequently converted to other oxygen-derived species, by either spontaneous or enzyme-mediated reduction. ROS generated in an inflammatory milieu act in an autocrine and paracrine manner, in order to rapidly amplify the effector potential of Fcγ R on quiescent phagocytes, by means of altering signal transduction (reviewed by Pricop and Salmon, ⁷⁷). Controlled upregulation of oxygen generation is important, since dysregulation or overproduction results in acute tissue injury ^78,79. With regard to this, it has been reported that during the respiratory burst of phagocytic cells in vitro, the superoxide anion production per cell shows an inverse correlation with the cell density, a phenomenon described as autoregulation ⁸⁰. Furthermore, it has been shown that the decrease in individual cell response is due to a significant increase in the amount of basal responses of the macrophages, thus, concomitantly, the number of reactive cells remains unchanged, irrespective of the cell density of the population ⁷⁸.

In vitro, the respiratory burst can be triggered e.g. by certain soluble (PMA) or particulate opsonized zymosan (OZ) agents. Both agents produce an intense oxidative burst, although mediated by different signal transduction pathways: PMA is soluble and associated to cell membrane perturbations that is dependent on protein kinase C (PKC) activation, whereas particulate OZ reflects processes related to PKC-independent phagocytic mechanisms ⁸¹. Moreover both stimuli utilize more than one activation pathway to stimulate NADPH-oxidase

82, revealing that different stimuli produce the same reaction in terms of respiratory burst autoregulation at the single cell level. Together with the LPS-induced oxidative burst (CD14 receptor-dependent) these pathways depend on ERK 1/ 2 activation. Activated ERK kinases also control the production of TNF in both LPS and PMA membrane activation of human alveolar macrophages. Whereas LPS activates NFκB nuclear translocation, PMA does not, but rather activates alveolar macrophages through the ERK 1/ 2 MAP kinase pathway. It has been shown that IgA, a predominant Ig isotype of respiratory secretions, regulates LPS-and PMA-induced oxidative burst and TNFthrough both dependent and independent modulation of ERK pathways, revealing a role of IgA in both lung protection and inflammation ⁸³. The LPS-related oxidative burst, TNF and IL-10 release of human monocytes can also be modulated by IL-9, through an upregulation of TGF-β ⁸⁴. In contrast, ADP stimulates the respiratory burst,

Introduction

The physiological generation of ROS production has now been clearly implicated in the activation of signalling pathways, resulting in a broad array of physiological responses ranging from cell proliferation to gene expression and apoptosis ^79,86. In this regard, and as reviewed by Forman and Torres, ⁷⁹, it has been suggested that: 1. hydrogen peroxide and superoxide act as second messengers; 2. anti-oxidant enzymes are implicated in the “turn-off” phase of signal transduction; 3. the primary physiological role of the respiratory burst in macrophages may be redox signalling, rather than microbicidal activity.

In contrast to neutrophils, oxygen-dependent killing by macrophages is myeloperoxidase–

independent because they lose this enzyme during maturation ⁴⁷.

In macrophages from rodents, a second oxidant-generating pathway features inducible nitric oxide synthase (iNOS), that is inducible by TNF, IL-1, and interferon-γ ⁶⁹.

1.3.1.6 Immune function

Macrophages contribute importantly to the induction, expression and regulation of immune responses ⁴⁷. Alveolar macrophages possess the capacity to serve as antigen presenting cells (APC’s) and secrete co-stimulatory cytokines, both required for T-cell activation. In contrast to resting macrophages, alveolar macrophages activated by chronic pulmonary inflammation or by T-lymphocyte-derived cytokines are more effective in antigen presentation ⁴⁸. Furthermore, pulmonary macrophages are important effector cells for T-cell mediated immunity and regulate immune responses. Thus IFN-γ, secreted by activated T-lymphocytes potently activates resting macrophages and upregulates these host defence functions ⁸⁷. The balance between macrophage-derived enhancing and -suppressive signals determines the degree with which antigen presentation and induction of immune responses occur. Therefore, not only T-cells, but also dendritic cells (DCs), another antigen-presenting cell, are maintained in a down-modulated state in the airways, tightly controlled by high numbers of nearby macrophages ⁴⁷. Immuno-modulatory factors involved in this process include among others: AM-derived nitric oxide (NO) ⁸⁸, prostaglandin E₂ (PGE₂) ^89,90, TGFβ⁹¹ and IL-10 ⁹².

Introduction