Macrophages are present in nearly all tissues of the human body . These cells, whose foremost function is phagocytosis, are of importance in maintaining steady-state tissue homeostasis [1-3]; macrophages participate in, among others, wound healing, the production of growth factors and removal of apoptotic bodies and cellular debris originating from tissue remodeling [1, 3]; boek the immune system p. 13). Another example of macrophage activity that is favorable to human health, is the metabolic clearance of erythrocytes (± 2 x 1011 each day) enabling the reuse of both iron and hemoglobulin  boek essential cell biology alberts et al. p 524). Additionally to the involvement in tissue homeostasis, macrophages play an indispensable role in immunity such as involvement in the primary response against invading microorganisms, coordination of the adaptive immune response and inflammation [1-2].
1.2 Differentiation of macrophages
Macrophages differentiate from circulating monocytes [1, 4]; boek the immune system p. 13). The migration of monocytes from blood to tissue can occur either in steady state or during inflammation [5-7]. Monocytes originate from a common myeloid progenitor in the bone marrow [1, 8-9]. When released from the bone marrow circulating blood monocytes can migrate into virtually all tissues of the body, in response to chemotactic signals such as cytokines, and subsequently differentiate into macrophages [4, 9]. Three different types of macrophages can be indicated after monocyte-to-macrophage differentiation: endothelium-resident macrophages, tissue-resident macrophages, and recruited macrophages [9-10].
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Kupffer cells (liver), Langerhans cells (skin), microglia (central nervous system), and osteoclasts (bone) all belong to tissue-resident macrophage populations that have been adapted to their local microenvironment [9-10]. Several studies have indicated that numerous tissue-resident macrophage populations (i.e. alveolar macrophages, splenic white-pulp and metallophilic macrophages, liver Kupffer cells, and brain microglia) are predominantly sustained trough local proliferation during steady state instead of replenishment by circulating monocytes (Reviewed in ). Conversely, in response to injury or infection, tissue-resident macrophages are locally activated and a fast recruitment of circulatory monocytic precursors into the corresponding infected or inflamed tissue is observed (Reviewed in ) . These macrophages do not only have an increased turnover rate, but also show an altered phenotype . Diverse stimuli from immunological and inflammatory processes may generate different types of recruited macrophages: antigen-non-specific responses may generate 'elicited' macrophages, whereas antigen-specific immune responses contribute to the formation of 'classically activated' and 'alternatively activated' macrophages [9-10]. According to Gordon et al., 2003, 'it is difficult to distinguish between originally resident macrophages from more recently recruited, elicited, or activated macrophages, because cells adapt to a particular microenvironment' . A detailed overview of macrophage differentiation is displayed in Figure 1.
Figure 1â”‚ Monocyte-to-macrophage differentiation . The development of monocytes from a common myeloid progenitor requires the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF), which stimulates the proliferation and maturation of these cells. Moreover, interleukin 3 (IL-3), KIT, tumor-necrosis-factor (TNF)-family proteins, TNF-receptor-related molecules, as well as PU.1 initiate the determination of macrophages. Bone-marrow monocytes are released into the peripheral blood from where they can enter virtually all tissues of the body. Monocyte migration through the endothelial layer is controlled by integrins, selectins, immunoglobulin super family molecules, and epidermal growth factor seven-transmembrane spanning (EGF-TM7)-type receptors. In tissues, monocytes may differentiate further into tissue-resident macrophages or recruited macrophages, the latter in response to inflammatory or immune stimuli. Resident-tissue macrophages may develop into different types of cells such as Langerhans cells or osteoclasts, dependent on signals from their local microenvironment (i.e. surface and secretory products of neighboring cells, and extracellular matrix). Tissue-resident macrophages that do not die in situ may migrate into draining lymph nodes after which they are filtered from the afferent lymph. Additionally, tissue-resident macrophages can enter the circulatory system again and consequently differentiate into dendritic cells (DCs).
1.3 Classically and alternatively activated macrophages
As mentioned, classically and alternatively activated macrophages are both formed in response to antigen-specific immunity. However, as a result of their extensive plasticity, macrophages may express phenotypic alterations in response to diverse environmental signals by which dissimilar (functional) populations of macrophages are formed ; different microbial stimuli and cytokines contribute to the formation of either classically or alternatively activated macrophages [9, 11-12]. Apart from cytokines such as GM-CSF and tumor-necrosis factor (TNF) and microbial products like lipopolysaccharide (LPS), IFN-É£ is considered as the predominant inducer of classically activated macrophages [12-14]. Alternatively activated macrophages can be formed in response to anti-inflammatory molecules such as IL-4, IL-13 or IL-10, immune complexes, vitamin D3, and glucocorticoid hormones [11, 15-16].
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Related to the Th1/Th2 nomenclature, classically activated and alternatively activated macrophages can be categorized as M1 and M2 macrophages respectively [1, 11, 13-14]. In total, three different sub-forms of M2 macrophages can be identified, each expressing a characteristic set of cytokines and cell surface markers: M2a, M2b, M2c (Figure 2).
Besides their contribution to Il-12 mediated Th1 responses, M1 macrophages are able of killing intracellular pathogens and tumor cells [11-13]. Additionally, M1 macrophages can produce numerous amounts of pro-inflammatory cytokines such as IL-12, Il-1β and TNF . M2 macrophages on the other hand, are involved in Th2-type inflammatory processes, allergy, tumor promotion, killing and encapsulation of parasites, and tissue remodeling and repair . The activation of M2 macrophages is accompanied by a reduction in pro-inflammatory cytokine production compared to M1 macrophage activation; M2 macrophages predominantly produce anti-inflammatory cytokines such as Il-10 and IL-1 receptor antagonist (Il-1ra). In addition to the dissimilar patterns of cytokine production, both types of macrophages express different phenotypically characteristics, receptors as well as effector functions (Figure 2) [11, 15, 17]. For instance, M1 macrophages are hallmarked by an IL-10low, IL-12high, Il-23high phenotype [11-13]. Conversely, a phenotype comprising IL-10high, IL-12low, and IL-23low is characteristic for M2 polarized macrophages [11, 13]). M1 macrophages express opsonic receptors (e.g. FcÉ£RIII), whereas M2 macrophages express high levels of non-opsonic receptors such as scavenger, mannose and galactose-type receptors [11, 13]. It has been shown that the arginine pathway in M2 macrophages is shifted towards the formation of ornithine which is a key component of collagen, and polyamines involved in cell growth [11, 13, 15]. Nevertheless, the arginine metabolism in M1 macrophages shows predominant levels of inducible nitric oxide synthase (iNOS) followed by the release of effector molecules such as nitric oxide (NO) and related reactive nitrogen intermediates (RNI) [9, 11, 13, 15, 18].
Figure 2â”‚ Overview of M1 and M2 classification . Macrophages can differentiate in response to endogenous or exogenous signals. Exposure to IFN-É£ in the absence or presence of LPS or TNF gives rise to M1 macrophages, while M2 differentiation is driven by a variety of stimuli. M1 macrophage activity is related to Th1 responses, killing of intracellular pathogens and tumor resistance. M2a (generated in the presence of IL-4 and IL-13) and M2b (formed in response to a combined exposure to immune complexes as well as TLR or IL-1R agonists) are involved in among others Th2 responses, allergy and tumor promotion. The different forms of M2 macrophages share a characteristic phenotype (IL-12low, IL-23low, and IL-10high). However, the production of cytokines differs and depends on the type of stimulus by which the M2 macrophage is generated. On the contrary, M1 macrophages express an IL-10low, IL-12high, Il-23high phenotype.
1.4 Macrophages and disease
It has been shown that the pro-inflammatory classically activated macrophages are of importance in host defense during acute infectious diseases among others [1, 17]. However, macrophages are not only important in health but also in disease. When M1 macrophage activation is not effectively regulated, an uncontrolled release of both pro-inflammatory cytokines and mediators can result in tissue damage ; it has been shown that an excessive or a prolonged M1 differentiation in response to acute infection may result in urinary tract infections, gastroenteritis, neonatal meningitis and sepsis among others . Moreover, M1 macrophages are considered as 'key mediators of the immune-pathology that occurs during several autoimmune diseases, including rheumatoid arthritis and inflammatory bowel disease' . Besides the beneficial effects for the host such as tissue repair and the encapsulation of parasites, M2 macrophages are also involved in many diseases. An example is their role in tumor promotion and progression [15, 19].
1.5 Macrophages in rheumatoid arthritis and osteoarthritis
Rheumatoid arthritis (RA) is an autoimmune disease characterized by joint inflammation and destruction of cartilage and bone often leading to joint disability [20-22]. Macrophages are known to play a key role during inflammation in RA [23-24]; macrophages are abundantly present in the synovial layer as well as the cartilage-pannus junction in RA . Previous research even indicated a positive correlation between the incidence of cartilage destruction and the amount of synovial macrophages being activated . These infiltrated macrophages produce considerable amounts of pro-inflammatory cytokines (e.g. TNFα, IL-10), chemokines and chemoattractants such as IL-8, and over express major histocompatibility complex class II molecules, indicating macrophage activation (reviewed in ). Macrophages are related to RA in both acute and chronic phases of disease as these cells have extensive pro-inflammatory, destructive and remodeling competences and are able of contributing to inflammation as well as cartilage destruction [23, 26]. Indeed, previous research indicated that both synovial inflammation [27-28] and cartilage destruction  were diminished when macrophages were removed from the synovium before onset of experimental arthritis. However, it is unlikely that macrophages are involved in the initiation process of the pathogenesis in RA .
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Macrophages are also of importance during osteoarthritis (OA). OA is a degenerative joint disease comprising less inflammation compared to RA. OA usually occurs after middle age [29-30]. However, this disease is not only predominant in elderly as a result of the ageing process, it is also considered as a leading cause of chronic disability in younger people since mechanical factors (e.g. history of joint trauma, obesity) are important risk factors in OA [29-30]. Table 1 displays an overview of the similarities and dissimilarities between synovial macrophages present in either RA and OA.
Table 1. Role of synovial macrophages and their mediators in RA and OA* 
Synovial macrophages play an important part in driving pathology.
Macrophages drive the production of several proinflammatory cytokines (e.g., IL-6, IL-8).
Macrophages drive the production of several MMPs.
There are fewer macrophages in OA synovium.
IL-1 production is driven by TNFα in RA but not in OA.
IL-1 is NF-KB dependent in RA but not in OA.
TNFα is more strongly NF-KB dependent in RA.
There is differential expression of FAK family kinases in RA and OA synovium.
Osteophyte formation is specific for OA.
In experimental OA, osteophyte formation is mediated by macrophages.
* RA = rheumatoid arthritis; OA = osteoarthritis; IL-6 = interleukin-6; MMPs = matrix metalloproteinases; TNFα = tumor necrosis factor α; FAK = focal adhesion kinase.
1.6 Damage associated molecular patterns; S100A8 and S100A9.
The recognition of tissue damage in response to damage associated molecular patterns (DAMPs) and reaction to invading microorganisms through pathogen associated molecular patterns (PAMPs) is essential in macrophage activation . Both PAMP and DAMP molecules are suggested to be recognized by pattern recognition-receptors (PRRs)  such as multiligand receptors for advanced glycation end products (RAGE) and toll-like receptors (TLRs) (Reviewed in ). Most DAMPS, such as heat shock proteins and HMGB1, are known to express diverse functionalities within the human body [31, 33]. On the one hand they are involved in cell homeostasis by acting as calcium-binding proteins or chaperones [31, 33-34]. However, during conditions of cell stress these DAMPS are secreted into the extracellular milieu in response to activated or damaged cells and subsequently activate both immune cells and vascular endothelial cells by acting as danger signals .
S100 proteins are considered as endogenous DAMPs after being released by phagocytes . Three Ca2+-binding proteins belonging to the group of S100 proteins -S100A8, S100A9 and S100A12- are predominant within the cytoplasma of meyelomonocytic cells [33, 35-36] . Although indications exist that phagocyte-S100 proteins are involved in host defense, these S100 proteins principally encompass pro-inflammatory functions such as the induction of pro-inflammatory cytokine responses and leukocyte recruitment [33, 37]. High concentrations of these phagocyte-specific S100 proteins can be found at local sites of inflammation (reviewed in ).
S100A8 and S100A9 proteins are highly secreted into inflamed tissues as a noncovalently heterodimeric complex (S100A8/S100A9) by activated macrophages [33, 37]. S100A8 is considered as the active component within this heterodimeric complex [38-39]. On the contrary, S100A9 is suggested to play a modulating role by protecting S100A8 from early degradation [38-39]. Secretion of S100A8/S100A9 particularly occurs in response to an interaction of phagocytes with pre-activated endothelial cells (e.g. TNF-stimulated endothelial cells) which elevates intracellular calcium (Ca2+) concentrations [33, 37, 40]. Moreover, parallel activation of protein kinase C (PKC), induced by chemokines or bacterial products, is essential for S100A8/S100A9 secretion (figure …) [33, 37, 40]. S100A8 and S100A9 proteins are able of binding to the endothelial layer after they are released from phagocytic cells . Subsequently, these proteins can induce an increase in vascular permeability as well as an increased expression of pro-inflammatory chemokines (e.g. Il-8) and adhesion molecules (e.g. VCAM-1 and ICAM-1) . Consequently, a thrombogenic and inflammatory response may be instigated by these pro-inflammatory chemokines and adhesion molecules . Conversely, S100A8 and S100A9 secretion is inhibited when phagocytes come into contact with resting endothelial cells [37, 41]. It is therefore that S100A8 and S100A9 are predominantly released at sites of inflammatory tissues .
Figure 3â”‚Secretion of S100A8/S100A9 . The heterodimeric complex S100A8/S100A9 is released in response to an interaction of activated phagocytes with TNF-stimulated endothelial cells. Secretion of S100A8/S100A9 depends on two individual signals: the activation of protein kinase C (PKC), in parallel with an elevation of intracellular calcium concentrations. After being excreted into the extracellular milieu S100A8 and S100A9 can express autocrine effect on phagocytic cells. This effect can be accomplished by an up-regulation of adhesion receptor CD11b/CD18 resulting in a tight adhesion of the phagocyte to the endothelium.
1.7 Rheumatoid arthritis, osteoarthritis and S100 proteins.
S100A8 and S100A9 proteins are presumed to be concerned in the pathogenesis of several synovial inflammation and autoimmune diseases including RA . S100A8 and S100A9 proteins are comprehensively relased by activated phagocytic cells present in the synovial lining layer (Perera); Several studies reported high amounts of S100A8 and S100A9 present in the synovial fluid from RA joints [35, 41-43]. ïƒ zie Sunahori, 2006 'confirmed'.. In fact, serum levels of S100A8/S100A9 correlate with the state of disease activity within arthritis [42-43] Opzoeken….! Concentrations were found to be more profoundly present in the synovial fluid compared to the peripheral blood. Zie Sunahori Van Lent et al., showed that S100A8/S100A9 can regulate joint inflammation during antigen-induced arthritis . Additionally, it is suggested that S100A8/S100A9 is involved in the direct regulation of cartilage destruction . Moreover, it was demonstrated that
…. . Recently it is shown that these proteins are also of importance during osteoarthritis (OA). Stimulation of chondrocytes coming from OA patients with S100A8 and S100A9 seem to provoke an increase in cartilage damage. Moreover, in S100A9 knockout mice a decrease in the level of cartilage damage during OA is observed.
Heteromeric MRP8/MRP14 complexes have been shown to represent their biologically active forms.
Expression of the calcium-binding proteins MRP8 and MRP14 in monocytes is regulated by a calcium-induced suppressor mechanism. J Roth, M Goebeler, V Wrocklage, C van den Bos, and C Sorg
It is of interest to study the role of S100A8 and S100A9 in M1-M2 macrophage biology. Therefore, it was examined if M1 differentiated macrophages produce more S100A8 and S100A9 than M2 in vitro, and if S100A8 and S100A9 stimulation promotes the differentiation towards M1 or M2 macrophages. Based on literature, it was hypothesized that…. Role of S100 proteins in chondrocytes…
S100A8 and S100A9 proteins are important during rheumatoid arthritis (RA). These pro-inflammatory proteins are able of inducing the immune system and can cause inflammation and cartilage damage. Recently it is shown that these proteins are also of importance during osteoarthritis (OA). OA is a syndrome comprising less inflammation compared to RA. Stimulation of chondrocytes coming from OA patients with S100A8 and S100A9 seem to provoke an increase in cartilage damage. Moreover, in S100A9 knockout mice a decrease in the level of cartilage damage during OA is observed.
Macrophages and monocytes are important producers of both S100A8 and S100A9 and are probably related to the activation of chondrocytes and the breakdown of cartilage during OA. It is hypothesized that macrophages and monocytes of OA patients have increased production levels of S100A8 and S100A9 compared to healthy controls. Classical pro-inflammatory macrophages (M1) possibly produce more S100 protein than anti-inflammatory M2 macrophages.
S100A8 is complex because of intrecellular and extracellular functions… zie Sunahori, 2010.