J. Pugin
Introduction
Ilia Metchnikoff made the first description of macrophages and their function in innate immunity at the end of the 19thcentury, and was awarded the Nobel Prize in 1908 for this discovery. Macrophages are phagocytic cells of myeloid lineage that originate from circulating monocytes and have transmigrated into tissues. They are present in virtually all tissues of the body where they carry out essential functions in maintaining normal homeostasis but also participate in pathological condi- tions [1]. Tissue macrophages are responsible for the non-inflammatory clearance of dying cells and debris [2]. Macrophages also sense their surrounding milieu for the presence of unusual stresses and/or the presence of non-self molecules and micro-organisms, recognized as dangerous to the body [3]. The engagement and activation of macrophages lead to responses that are typical of innate immunity, such as the rapid generation of an inflammatory response, and they also play a role as efficient effectors for the clearance of microorganisms [1]. Macrophages are also important in communicating with the adaptive arm of immunity, either as macrophages or as monocyte- or macrophage-derived dendritic cells [3]. They also participate in the presentation of non-self antigens to lymphocytes and secrete mediators, boosting adaptive immune responses. Finally, macrophages play a key role in wound healing and tissue repair, and possess natural tumoricidal activity.
Origin of Tissue Macrophages
Circulating monocytes originate from myeloid bone marrow progenitors after a differentiation process under the control of cytokines and growth factors, par- ticularly the stem cell factor, macrophage colony stimulating factor (M-CSF), and interleukin (IL)-3 [1]. Two different monocyte subpopulations are found in the circulation, recognizable by their expression of certain chemokine receptors and L-selectins [1]. The first population naturally migrates into tissue where they can settle for several months up to years, and acquire tissue specificity as resi- dent peritoneal macrophages, alveolar macrophages, Kupffer cells, or microglial cells). Alternatively, under the control of cytokines and growth factors (essentially IL-4 and granulocyte-macrophage colony stimulating factor [GM-CSF]), mono- cytes can also transform into undifferentiated dendritic cells, such as Langerhans
cells in the skin [3]. These cells will eventually migrate to lymphoid organs and become ‘professional antigen-presenting cells’ after adequate stimulation. An- other phenotypically distinct monocyte sub-population can be recruited to tissues during inflammatory processes [1]. The recruitment process involves a series of precise mechanisms (specific chemokines of the CC family) and upregulation of leukocyte-endothelial adhesion molecules [4]. Such adhesion molecules are, at least in part, different under constitutive conditions compared to those involved in inflammation-induced trafficking of monocytes to tissues. The tissue microenvi- ronment is key to the differentiation of the macrophage into a tissue-specific cell.
The end-function of a hepatic Kupffer cell will be markedly different from that of a microglial cell or an alveolar macrophage, for example. The level of activation of these sentinel cells is also determined by the local balance between activator and de-activator cytokines (such as interferon [IFN]
γ
and IL-10, respectively). These mediators originate principally from epithelial cells and other cell populations of adaptive immunity in the surrounding tissue.Macrophages Express an Armada of Receptors
One of the major functions of the macrophage is to sense molecules and physical stresses in their microenvironment, in order to recognize the presence of foreign molecules [3]. Macrophages express a wide variety of receptors of innate immunity that recognize generally conserved microbial molecules. For example, they express high levels of CD14, a glycosyl-phosphatidylinositol glycoprotein, which binds several pathogen-associated molecular patterns (PAMPs), such as lipopolysac- charide (LPS), peptidoglycan, lipopeptides, mycobacterial lipoarabinomannan, and double-stranded RNA [5, 6]. It is believed that CD14 ‘concentrates’ microbial molecules at the macrophage surface allowing interactions with signaling receptors such as Toll-like receptors (TLRs). CD14 also enhances endocytosis of PAMPs and phagocytosis of osponized bacteria and yeasts [7]. The scavenger, complement, beta-glucan, mannose, and Fc receptors are among the receptors important for the clearance of microbial products. Importantly, although macrophages are capable of ingesting bacteria, this is performed more efficiently by another myeloid cell, the polymorphonuclear neutrophil, or PMN. Conversely, monocyte/macrophages are by far the best producers of inflammatory mediators in the body. Among the various monocyte/macrophage products are pro-inflammatory cytokines (tumor necrosis factor [TNF], IL-1, IL-6, macrophage migration inhibitory factor [MIF]), anti- inflammatory cytokines (IL-4, IL-10, IL-13, transforming growth factor [TGF]-
β
);chemokines (IL-8), growth factors (G-CSF), anti-microbial peptides (defensins, cathelicidins), lipid mediators (prostanoids, leukotrienes, platelet-activating fac- tor [PAF]), oxygen and nitrogen radicals, and enzymes (lipases, proteases) [1].
Role of Macrophages as Sentinel Cells
Although specific roles can be attributed to subclasses of macrophages depending on their location in various organs, a common feature of the macrophage is its sentinel role. They sense various noxious stimuli in their environment and elicit responses depending on the nature of the stimulus. In addition to microbial prod- ucts, it has been shown that macrophages react to stimuli typical of tissue injury or stress, such as acidosis, extracellular ATP, tissue hypoxia, cell stretching, substance P, high mobility group box protein (HMGB)-1, uric acid, and proteolytic enzymes, including thrombin (Fig. 1). Compared with bacterial products, the magnitude of the macrophage activation is usually not as great with these latter stimuli. This introduces the important concept that these stimuli are sensed as ‘danger signals’
by macrophages. They may be considered as warning signs of tissue and cellular injury or dysfunction, and indicate that a foreign intruder, such as bacteria, might be dangerous [8, 9]. The host should then be mounting an inflammatory reaction and an immune response. Synergistic responses between danger signals and bac- terial molecules have been demonstrated both in animal and in in vitro studies.
These effects could be the result of a synergism between transcription factors at the level of the promoter region of macrophage pro-inflammatory genes. It has also been demonstrated recently that danger signals induce the assembly of a cy- toplasmic inflammasome, recruiting and activating caspase-1 [10–12]. This results in a massive increase in the production of the very potent local pro-inflammatory molecule, IL-1
β
.The tissue macrophage is also believed to play a significant role in the patho- genesis of ventilator-induced lung injury (VILI), and possibly in the subsequent remote organ dysfunction [13, 14]. The only perceptible effect of positive pressure mechanical ventilation, when applied to normal lungs with ‘reasonable’ volumes, is the recruitment of alveolar macrophages, dependent on the lung production of monocyte chemoattractant protein (MCP)-1 [15]. In these experiments, alveolar macrophages are primed by the mechanical stimulus to increase their cytoplasmic concentration of pro-inflammatory cytokine mRNAs, but do not secrete the pro- teins. It is only with a second hit, such as the presence of bacteria, for example, that they will respond with a rapid and massive local pro-inflammatory response, and a possible systemic spillover of mediators [16]. In many animal models, bacterial sepsis becomes lethal only when associated with burns, trauma, cerebral hemor- rhage, aggressive mechanical ventilation, or hypovolemic shock. This leads to the concept of synergistic effects of noxious stimuli, and the necessity for two hits on the macrophage to observe a full-blown, clinically relevant inflammatory response plus end-organ dysfunction.
Fig. 1. Macrophages sense and are activated by molecules from the microbial world and by ‘dan- ger’ signals from tissue injury. TLR: Toll-like receptor; NOD: nucleotide-binding oligomerization domain proteins; NALP: NACHT-, LRR- and pyrin domain (PYD)-containing proteins; PAR:
protease-activated receptor; ORE: oxygen-responding element; RAGE: receptor for advanced glycation endproducts; NK-1: neurokinin-1; FAK: focal adhesion kinase; MAPK: mitogen acti- vated protein kinase; NF-kB: nuclear factor-kappa B; P2X7R: purinergic P2X7 receptor; HSP: heat shock protein; HMGB-1: high-mobility group box-1 protein
Macrophages Play a Central Role in the Pathogenesis of Sepsis
Because of their involvement in innate immunity, it is not a surprise that macrophages play a critical role in bacterial sepsis and in subsequent organ dys- function. It is believed that excessive macrophage-induced inflammation is respon- sible for the loss of some organ functions, such as gas exchange in the lung [17]. Ac- tivated lung macrophages produce large amounts of pro-inflammatory cytokines, such as TNF and IL-1. In turn, these cytokines will stimulate pneumocytes and capillary endothelial cells to generate a strong chemokine gradient and recruit PMNs to the interstitium and the airspace [18]. In addition to its sentinel role, the macrophage plays a crucial role as an amplifier of the inflammatory and immune response. This inflammatory response is necessary for rapid and efficient clear- ance of a bacterial infection, but when such a response is excessive, it may become detrimental to lung integrity and function. Interestingly, neutrophilic infiltration in the lung (but also in some cases in the pleural space and the peritoneum) is not observed in other organs during severe sepsis and septic shock. Inflammatory cells are, for example, absent in the liver, the kidney, the central nervous system (CNS), or the gut despite evident dysfunction of these organs during bacterial sepsis [19].
Gut translocation of bacteria is not accompanied by massive hepatic (neutrophilic) inflammation, but drives an acute phase response, that is essentially dependent
on macrophage IL-6 production [20]. In addition, Kupffer cells play an essential role in clearing bacteria and bacterial products from the portal circulation without generating a massive hepatic inflammatory reaction. This shows that the function of tissue macrophages depends on the organ involved, and that the Kupffer cell reaction to bacteria, for example, is markedly different from what is observed with alveolar macrophages.
Monocytes in the vascular compartment are also activated during bacte- rial sepsis. Interestingly, circulating monocytes are ‘reprogrammed’ and produce more anti-inflammatory mediators (IL-10, IL-1 receptor antagonist [IL-1ra], IL- 4) than pro-inflammatory cytokines [21]. When stimulated ex vivo, monocytes from critically ill patients, and particularly from septic patients, have defective pro-inflammatory responses. The net inflammatory activity in septic plasma is in fact anti-inflammatory [17, 22, 23]. This systemic anti-inflammatory response may prevent excessive, non-specific, and deleterious systemic endothelial and leuko- cyte activation where it is unwanted, i.e., in the vascular compartment [22]. It may, therefore, help modulate effector leukocytes and focus inflammation at the infected site [22]. In addition, decreased surface expression of major histocom- patibility complex (MHC) class II antigens, such as human leukocyte antigen (HLA)-DR, on the surface of circulating monocytes from patients with sepsis has been reported by several groups. This is mainly due to an IL-10-dependent se- questration of MHC class II molecules intracellularly [24], and is associated with poor outcome in patients with septic shock [25]. It remains unclear as to whether this phenotype persists in tissue macrophages after monocyte migration, and whether it is associated with impaired antigen presentation in monocyte-derived macrophages and dendritic cells.
Activation of coagulation and decreased fibrinolysis are important pathways in the pathogenesis of sepsis and related organ dysfunction [26, 27]. This has re- cently been highlighted by the PROWESS trial showing that a recombinant form of activated protein C improved survival in patients with severe sepsis and septic shock [28]. During sepsis, coagulation is activated both in the vascular compart- ment (disseminated intravascular coagulation [DIC]) and in (some) organs, such as the alveolar compartment of the lung. The ‘aberrant’ expression of tissue fac- tor is key to the initiation of DIC and in-organ coagulation [29]. Tissue factor is upregulated in monocytes and macrophages – but also in endothelial cells – af- ter exposition to bacteria, bacterial products, and pro- inflammatory cytokines, and is responsible for the observed increased in “procoagulant activity” [29, 30].
Local and systemic inhibition of the fibrinolytic pathway, mainly dependent on the plasminogen-activation inhibitor (PAI)-1 protein may also participate in in- creased procoagulant activity during sepsis. PAI-1 is produced by activated mono- cyte/macrophages, among many other cell types [31]. Finally, monocytic cells express protease-activated receptors (PARs), which are receptors for serine pro- teases, such as thrombin. These receptors, at the interface of inflammation and coagulation pathways, can modulate the inflammatory response of monocytes and tissue macrophages [32–34].
It has recently been suggested that vagus nerve stimulation attenuates macro- phage activation [35–37], as part of the newly discovered cholinergic anti- inflammatory pathway [38]. The acetylcholine
α
7-nicotinic receptor seems to be essential to modulate macrophage activation during sepsis [39].Finally, although macrophages are involved in tissue remodeling and repair in various illnesses, this has been poorly studied in sepsis and related-organ dysfunction. Conceptually, macrophages are likely to play an important role in the resolution phase of sepsis and organ failure.
Macrophage Products and Receptors: Therapeutic Targets?
Even though therapeutic strategies based on the systemic blockade of monocyte/ma- crophage-derived pro-inflammatory mediators have failed in the past, considerable interest still exists in developing modulators of macrophage function as potential therapies in sepsis and related organ dysfunction. TNF and IL-1 blockade in the lung has not been completely explored in sepsis-associated acute lung injury (ALI) or acute respiratory distress syndrome (ARDS), for example, despite ample demon- stration that an intense pro-inflammatory reaction takes place in the lungs during sepsis. Blockade of late mediators associated with mortality in pre-clinical mod- els of sepsis, such as HMGB-1, is also a valuable hypothesis to be tested [40, 41].
There may still be room in early septic shock for interventions that are directed to receptors recognizing bacterial products, but not interfering with bacterial clear- ance, such as TLRs [42]. Alternatively, therapies aimed at boosting the depressed immune functions of septic monocyte/macrophages, such as IFN
γ
and GM-CSF, have also been recently proposed and tested in small numbers of patients [43, 44].Finally, therapeutic studies based on modulation of the macrophage
α
7 nicotinic receptor or triggering receptor expressed on myeloid cells (TREM)-1 are also underway [45].References
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