The acquisition/exercise of the various effector functions of macrophages requires the specific activation of signaling pathways in the cells. TLR ligands, cytokines, immunocomplexes, hypoxia and CD4-positive T lymphocytes, provisional extracellular matrix (ECM) and collagen remodeling and tissue structure are important stimuli that lead to the activation of macrophages [50], e.g., via Jak/STAT, NF-κB, and redox signaling.

Cytokines represent a large group of regulatory proteins that act primarily on the immune cell differentiation, activation, and function. This also applies to macrophages. Exposure to certain cytokines results in an adequate response of macrophages to various challenges such as infection, tissue damage, tumor, or alloantigen. This is achieved by a subtle balancing of production, release, or activation of a large set of individual cytokines whose effects are defined by complex synergistic or antagonistic interactions. Besides their actions on macrophages and other immune cells, cytokines potently affect other cells and tissues.

Members of the heterologous group of cytokines bind to different types of specific receptors, among them receptor kinases (e.g., TGF-β1 receptor) and tyrosine kinase-linked receptors that lack intrinsic kinase activity. The latter group of receptors is also referred to as cytokine receptors (e.g., IL-1β and TNFα receptor).

Ligand binding to these receptors by a wide variety of cytokines or growth factors generally provokes di- or trimerization of monomeric subunits, which in turn induces rapid activation of kinases associated with the receptor, typically members of the JAK (Janus kinase) family of tyrosine kinases. Phosphorylation of the receptor itself enables recruitment and binding via SH2 (src homology 2) domains of members of the STAT (signal transducers and activators of transcription) family of transcription factors to the receptor. This allows JAKs to phosphorylate STATs, which subsequently form dimers that are capable of entering the nucleus and specifically initiate transcription of target genes, which include cytokines, SOCS1–3, and transcription factors such as GATA3, C-Maf, c-myc, NFAT1/NFAT2 [30, 33].

The NF-κB family of transcription factors plays a crucial role in inflammatory and apoptotic responses. Family members include NF-κB1 p50, NF-κB2 p52, RELA (also called p65), RELB, and c-REL1 which as homodimers or heterodimers mediate transcription of NF-κB target genes [66]. They are retained in the cytoplasm by interaction with the inhibitory molecule IκB. Two NF-κB activation cascades can be discriminated. In the classical (canonical) activation pathway, in response to various signals IκB becomes phosphorylated by serine protein kinases, mainly IKK2 but also IKK1. IκB is then degraded in the proteasome, facilitating the translocation into the nucleus of p50/p65 NFκB. This heterodimer is the most abundant complex, often referred to as being “NF-κB”. It initiates and enhances the expression of target genes including iNOS, the chemoattractant cytokine, IL-8, the inflammatory TNFα, IL-1β, and IL-6, and adhesion molecules (ICAM1) and chemokines (IP-10, MCP-1, MIP-1; Fig. 5, for details see [45]). In the alternative activation cascade, IKK1 mediates processing of p100 to p52, the latter forming heterodimers with relB which upon transfer into the nucleus initiate transcription of NF-κB target genes. C-Rel is of special importance for, e.g., the transcriptional induction of IL-12 p40 in macrophages [38].

Signal transduction pathways of Toll-like receptors. The binding of the ligand (LPS/LBP/CD14; MD-2) to TLR leads to the association of adapter molecules such as MyD88, Toll-interacting Protein (TOLLIP), the protein kinase IRAK, and TRAF6 (TNF-receptor associated factor 6). TRAF6 activates IκB kinases 1 and 2 (IKK-1/2) via the kinase TAK1 (TGF-β activated kinase). These kinases phosphorylate IκB, which leads to the degradation of the inhibitory protein and releases NF-κB as a dimer. NF-κB migrates into the cell nucleus and induces a transcriptional activation of proinflammatory and immunomodulatory genes ([76] with kind permission)

Some stimuli preferentially activate the alternative pathway; others, including lymphotoxin-β (LTβ) and LPS, activate both cascades [10, 11]. It is supposed that the two NF-κB cascades are activated in a sequential manner: the early response mediated by p65 containing dimers, the latter then continues a response with relB-containing complexes to sustain the NF-κB activation [54].

Oxidative stress, viral proteins, growth factors (angiotensin II), mitogens, the inflammatory cytokines IL-1β and TNFα via IL1 /IL-18 receptors or TNF receptors, respectively, NOD-like receptors, and TLR ligands activate IKKβ [31]. Activation of all these receptors also feeds into other signaling pathways that interfere, directly or indirectly, with the NF-κB signaling pathway [14]. Upon activation by LPS, TLR4 through Myd88 activates NF-κB, but in addition activates mitogen-activated protein kinase (MAPK), IRF5 pathways, and via endosomal TLR4 signaling, IRF3 and the production of the strong antiviral interferons become induced [14]. Autocrine effects of any cytokines released from activated macrophages further add to the complexity of resulting responses.

In humans, at least ten different TLRs exist which differ in their specificity towards distinct and characteristic pathogenic structures, like LPS, double-stranded RNA or unmethylated CpG DNA, so-called pathogen-associated molecular patterns. For this reason, TLRs are also called PRRs (pattern recognition receptors). The first TLR to be identified is TLR4, which is activated by LPS (gram-negative bacteria). LPS is bound to the macrophage surface via LBP and is bound by TLR4 in this complex (Fig. 4).

Accumulating evidence supports a signaling role for ROS that are generated by macrophages during oxidative burst.

In the last few years the view has hardened that ROS—at least when present in certain concentrations—can be viewed as a second messenger. In full agreement with this function, ROS show certain characteristics such as stimulus-dependent transient induction (e.g., via the activation of NADPH oxidase), specific function (interfering specifically with signaling components like, e.g., kinases), and the rapid termination of the signal by antioxidant enzymes/antioxidants (catalase, superoxide dismutase, glutathione peroxidases) [18]. The modulation of the intracellular concentration of calcium [Ca2+]i is a general mechanism involved here because many intracellular signaling pathways actually depend on it.

ROS have been shown to induce the activation of NF-κB, although the underlying mechanisms still remain to be elucidated fully [32]. Reversible S-glutathiolation has been established as one mechanism underlying the regulation of, e.g., the transcription factor activator protein 1 (AP1) [19].

It is well established that ROS produced by macrophages (\(}_^\) and the species subsequently formed therefrom) contributes to the oxidation of low-density lipoproteins (LDL). Oxidized LDL is rapidly taken up by macrophages leading to their transformation into “foam cells”, which are associated with an increased risk for atherosclerosis and complications that are related to this. Notably, oxidized LDL itself has been shown to interfere with many signaling pathways in macrophages [41].