Regulators of macrophage activation

Macrophages are ubiquitous phagocytes that can constitute up to 15% of the cellular content of tissues. These heterogeneous cells of the innate immune system perform important functions during health and disease. Equipped with receptors for the T helper cell cytokines INF-γ and IL-4, macrophages undergo specific activation programs during Th1 or Th2 immune responses. These activation profiles, termed classical (M1) or alternative (M2) activation respectively, are further tuned by the presence and recognition of microbial-associated molecular patterns, other cytokines, lipids, and even adhesion to the substratum. The activation of macrophages also relies on the maturation background of the cells, elicitation of complicated intracellular signalling cascades, and the crosstalk between the different signalling elements. Of interest, not all genes participating in the activation-related signalling cascades are equally important for the elicitation of functional profiles and a regulator gene hierarchy is emerging for the different types of activation. In this issue of the European Journal of Immunology, two papers add to our understanding of how cellular kinases and phosphatases, related to the PI3K pathway, regulate M1 or M2 activation programmes in macrophages.     

Natural killer cell receptor signaling

Natural killer (NK) cell immune responses are regulated by a balance of activating and inhibitory signals transmitted by cell surface receptors. Immunoreceptor tyrosine-based inhibition motifs in the cytoplasmic domains of inhibitory NK receptors recruit tyrosine or lipid phosphatases, which modulate the activation signals transmitted by receptors linked to the Syk and ZAP70 tyrosine kinases and phosphatidylinositol-3 kinases. In addition, recent studies of gene-deficient animals, in particular Syk and ZAP70 double-deficient mice, suggest that NK cells possess a robust and potentially redundant receptor system to ensure their development and function.     

Emerging role of microRNAs in regulating macrophage activation and polarization in immune response and inflammation

Diversity and plasticity are hallmarks of macrophages. Classically activated macrophages are considered to promote T helper type 1 responses and have strong microbicidal, pro‐inflammatory activity, whereas alternatively activated macrophages are supposed to be associated with promotion of tissue remodelling and responses to anti‐inflammatory reactions. Transformation of different macrophage phenotypes is reflected in their different, sometimes even opposite, roles in various diseases or inflammatory conditions. MicroRNAs (miRNAs) have emerged as critical regulators of macrophage polarization (MP). Several miRNAs are induced by Toll‐like receptors signalling in macrophages and target the 3′‐untranslated regions of mRNAs encoding key molecules involved in MP. Therefore, identification of miRNAs related to the dynamic changes of MP and understanding their functions in regulating this process are important for discussing the molecular basis of disease progression and developing novel miRNA‐targeted therapeutic strategies. Here, we review the current knowledge of the role of miRNAs in MP with relevance to immune response and inflammation.     

Differential dependence of the ingestion of necrotic cells and TNF-alpha / IL-1beta production by murine macrophages on lipid rafts

Monocytes and macrophages may encounter both pro-inflammatory and anti-inflammatory signals during their lifetime, in the form of micro-organisms or their products or as cytokines. In addition, macrophages are also exposed to apoptotic and necrotic cells. Apoptosis or 'programmed cell death' is thought to be the physiological end of developing or maturing cells, whereas necrosis is regarded as 'accidental death' or injury-associated cell death. Apoptotic cells are cleared from tissues by phagocytic cells without eliciting an inflammatory response, while necrotic cells elicit inflammation. Several cell membrane molecules from apoptotic and necrotic, as well as from phagocytic cells, have been shown to participate in the process of endocytosis of dying and potentially harmful cells. Apart from an array of cell surface receptors, it is also known that lipid rafts are key components of cell–cell communication and signalling. By using the interaction of BALB/ c mice thymus-derived apoptotic or necrotic cells with murine macrophages of the J774 cell line as a model system, we provide evidence that endocytosis of apoptotic but not of necrotic cells is inhibited by methyl-β-cyclodextrin, a cholesterol sequestering agent, able to disrupt lipid rafts. However, necrotic but not apoptotic cells co-localize with lipid rafts within macrophages. Interestingly, necrotic cell-induced secretion of TNF-α and IL-1β was also inhibited by methyl-β-cyclodextrin, thus suggesting a role for lipid rafts in the signalling of this particular inflammatory response. Taken together, our results argue in favour of differential macrophage recognition of apoptotic and necrotic cells at the level of lipid rafts, and endocytosis versus signalling for TNF-α and IL-1β synthesis.     

Evidence for the opposing roles of different gamma delta T cell subsets in macrophage homeostasis

To ensure invading pathogens are eliminated with minimal damage to host tissues it is essential that macrophage activation be tightly regulated. Previously we demonstrated that a subset of gammadelta T cells (Vgamma1(+)) contributes to resolving pathogen-induced immune responses by killing activated macrophages. However, the exaggerated macrophage response seen in infected Vgamma1(+) T cell-deficient mice suggests that gammadelta T cells play a broader role in macrophage homeostasis and other subsets might promote macrophage activation. Using a macrophage:gammadelta T cell co-culture system we have shown that gammadelta T cells increase the activity of macrophages activated in vivo by Listeria monocytogenes infection. In a dose-dependent manner, gammadelta T cells up-regulated production of cytokines (TNF-alpha, IL-6, IL-10) and chemokines (MIP-1alpha, MIP-1beta) by Listeria-elicited macrophages. The ability to increase macrophage cytokine production was prominent among Vgamma4(+) gammadelta T cells. Reciprocally, Vgamma4(+) gammadelta T cells were activated by Listeria-elicited macrophages, resulting in production of the anti-inflammatory cytokine, IL-10. gammadelta T cell adoptive transfer experiments showed that Vgamma4(+) T cells protected TCRdelta(-/-) mice against Listeria-induced liver injury and necrosis. These findings identify distinct and non-overlapping roles for gammadelta T cell subsets in regulating macrophage function during pathogen-induced immune responses.     

MT1-MMP regulates the PI3Kδ·Mi-2/NuRD-dependent control of macrophage immune function

Macrophages play critical roles in events ranging from host defense to obesity and cancer, where they infiltrate affected tissues and orchestrate immune responses in tandem with the remodeling of the extracellular matrix (ECM). Despite the dual roles played by macrophages in inflammation, the functions of macrophage-derived proteinases are typically relegated to tissue-invasive or -degradative events. Here we report that the membrane-tethered matrix metalloenzyme MT1-MMP not only serves as an ECM-directed proteinase, but unexpectedly controls inflammatory gene responses wherein MT1-MMP(-/-) macrophages mount exaggerated chemokine and cytokine responses to immune stimuli both in vitro and in vivo. MT1-MMP modulates inflammatory responses in a protease-independent fashion in tandem with its trafficking to the nuclear compartment, where it triggers the expression and activation of a phosphoinositide 3-kinase δ (PI3Kδ)/Akt/GSK3β signaling cascade. In turn, MT1-MMP-dependent PI3Kδ activation regulates the immunoregulatory Mi-2/NuRD nucleosome remodeling complex that is responsible for controlling macrophage immune response. These findings identify a novel role for nuclear MT1-MMP as a previously unsuspected transactivator of signaling networks central to macrophage immune responses.     

Receptor signaling in immune cell development and function

Immune cell development and function must be tightly regulated through cell surface receptors to ensure proper responses to pathogen and tolerance to self. In T cells, the signal from the T-cell receptor is essential for T-cell maturation, homeostasis, and activation. In mast cells, the high-affinity receptor for IgE transduces signal that promotes mast cell survival and induces mast cell activation. In dendritic cells and macrophages, the toll-like receptors recognize microbial pathogens and play critical roles for both innate and adaptive immunity against pathogens. Our research explores how signaling from these receptors is transduced and regulated to better understand these immune cells. Our recent studies have revealed diacylglycerol kinases and TSC1/2-mTOR as critical signaling molecules/regulators in T cells, mast cells, dendritic cells, and macrophages.     

CD45, CD148, and Lyp/Pep: critical phosphatases regulating Src family kinase signaling networks in immune cells

Summary:  Reciprocal regulation of tyrosine phosphorylation by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) is central to normal immune cell function. Disruption of the equilibrium between PTK and PTP activity can result in immunodeficiency, autoimmunity, or malignancy. Src family kinases (SFKs) play a central role in both immune cell function and disease due to their proximal position in numerous signal transduction cascades including those emanating from integrin, T and B-cell antigen receptors, Fc, growth factor, and cytokine receptors. Given that tight regulation of SFKs activity is critical for appropriate responses to stimulation of these various signaling pathways, it is perhaps not surprising that multiple PTPs are involved in their regulation. Here, we focus on the role of three phosphatases, CD45, CD148, and LYP/PEP, which are critical regulators of SFKs in hematopoietic cells. We review our current understanding of their structures, expression, functions in different hematopoietic cell subsets, regulation, and putative roles in disease. Finally, we discuss remaining questions that must be addressed if we are to have a clearer understanding of the coordinated regulation of tyrosine phosphorylation and signaling networks in hematopoietic cells and how they could potentially be manipulated therapeutically in disease.     

Neurotrophins modulate monocyte chemotaxis without affecting macrophage function

Neurotrophins nerve growth factor (NGF), brain-derived growth factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4) and their high-affinity tyrosine protein kinase receptor (Trk) family, TrkA, TrkB, TrkC, and low-affinity p75(NTR) receptor, are key molecules implicated in the development of the central nervous system. Increasing evidence suggests that they also have physiological and pathological roles outside the nervous system. In this study we examined the expression of neurotrophins and their receptors in human activated macrophages and to what extent neurotrophins themselves modulate macrophage activation, in a model of primary adult monocyte-derived macrophage. Our data indicate that macrophages express neurotrophin and neurotrophin receptor genes differentially, and respond to cell stimulation by specific inductions. Neurotrophins did not modify the antigen-presenting capacities of macrophages or their production of proinflammatory cytokines, but somehow skewed their activation phenotype. In contrast, NGF clearly increased CXCR-4 expression in macrophage and their chemotactic response to low CXCL-12 concentration. The differential effect of specific macrophage stimuli on neurotrophin expression, in particular NGF and NT-3, and the specific enhancement of CXCR-4 expression suggest that neurotrophins might participate in tissue-healing mechanisms that should be investigated further in vivo.     

Alternatively activated macrophages express the IL-27 receptor alpha chain WSX-1

The interleukin (IL)-27 receptor-alpha WSX-1 is one component of the heterodimeric IL-27 receptor that is expressed on various cell types including macrophages. We previously demonstrated that IL-27 induces STAT-3 and is able to inhibit the production of pro-inflammatory cytokines in activated macrophages suggesting a novel feed-back mechanism by which IL-27 can modulate excessive inflammation. Because IL-4 receptor-alpha (IL-4Ralpha)-induced alternatively activated macrophages have also been described to attenuate pathological inflammatory immune responses, we analyzed the contribution of IL-27 in alternative macrophage activation. In the present study, like IL-10 and IL-4, IL-27 was found to suppress IL-12/23p40 production in activated bone marrow-derived macrophages. Whereas IL-10 induced the upregulation of the IL-4Ralpha on macrophages, receptor expression was not triggered by IL-27. In contrast to IL-4, IL-27 did not induce alternative macrophage activation but IL-4 strongly upregulated the expression of WSX-1 on macrophages and alternative macrophage activation enhanced IL-27-mediated signalling. We therefore conclude from our study that IL-10, IL-4 and IL-27 collaborate in modulating macrophage activation by successive upregulation of the IL-4Ralpha and WSX-1 on alternatively activated macrophages.     

Differential regulation of the mitogen-activated protein kinases by pathogenic and nonpathogenic mycobacteria

Mycobacteria are the etiologic agents of numerous diseases which account for significant morbidity and mortality in humans and other animal species. Many mycobacteria are intramacrophage pathogens and therefore the macrophage response to infection, which includes synthesis of cytokines such as tumor necrosis factor alpha (TNF-alpha) and production of nitric oxide, has important consequences for host immunity. However, very little is known about the macrophage cell signaling pathways initiated upon infection or how pathogenic mycobacteria may modulate the macrophage responses. Using primary murine bone marrow macrophages, we established that p38 and extracellular signal-regulated kinases 1 and 2 of the mitogen-activated protein kinase (MAPK) pathways are activated upon infection with different species of mycobacteria. However, we observed decreased MAPK activity over time in macrophages infected with pathogenic Mycobacterium avium strains relative to infections with nonpathogenic mycobacteria. Furthermore, macrophages infected with M. avium produced lower levels of TNF-alpha, interleukin 1beta, and inducible nitric oxide synthase 2 than macrophages infected with nonpathogenic species. Inhibitor studies indicate that the MAPKs are required for the Mycobacterium-mediated induction of these effector proteins. Our data indicate that MAPKs are activated in macrophages upon invasion by mycobacteria and that this activation is diminished in macrophages infected with pathogenic strains of M. avium, resulting in decreased production of important immune effector proteins. The decreased MAPK activation associated with M. avium infections suggests a novel point of immune intervention by this mycobacterial species.     

Regulation of ATP-gated P2X receptors by phosphoinositides

Phosphoinositides are minor membrane lipids involved in many cellular signaling processes. The addition or removal of phosphates on phosphoinositides depends on the interplay of specific lipid kinases and phosphatases. P2X receptors are cation channels that are gated by extracellular ATP. They play multiple and important physiological roles in vertebrates, ranging from pain sensation and control of smooth muscle contraction to cytokine release and immune cell death. In this article, we review recent studies that have identified phosphoinositides as critical modulators of P2X receptor function.     

What goes up must come down: A tripartite Dok‐3/Grb2/SHIP1 inhibitory module limits BCR signaling

Properly regulated immunity requires precise integration of activating and inhibitory signals. As for other lymphocytes, B cells express an antigen‐specific activating receptor, the B‐cell antigen receptor (BCR), and inhibitory receptors (e.g. FcγRIIb) that exercise checkpoint control on B‐cell activation. Moreover, following BCR engagement, CD19 recruits proteins that amplify BCR signaling, while CD22 initiates a negative feedback loop by recruiting proteins that inhibit BCR signaling. Initial BCR signaling is mediated by protein tyrosine kinases and lipid kinases; inhibitory receptors directly antagonize the actions of these enzymes by recruiting protein tyrosine phosphatases and lipid phosphatases and positioning them close to actively signaling BCRs. Previously it was thought that inhibitory receptors such as FcγRIIb and CD22 were essential for bringing these phosphatases near the BCR. In this issue of the European Journal of Immunology, Manno et al. show that a tripartite inhibitory module consisting of the adaptor proteins Dok‐3 and Grb2 and the lipid phosphatase SHIP1 binds directly to activated BCRs and limits the Ca²⁺ mobilization that is required for B lymphocyte activation. This reveals that the BCR can be both an activating and inhibitory receptor, one that activates signaling enzymes while initiating a negative feedback loop that prevents excessive signaling.     

Control of dual-specificity phosphatase-1 expression in activated macrophages by IL-10

Ligation of Toll-like receptors (TLR) on macrophages induces cytokines and mediators important for the control of pathogens. Macrophage activation has to be tightly controlled to prevent hyper-inflammation. Accordingly, the hallmarks of TLR-triggered signaling, nuclear translocation of NF-kappaB and phosphorylation of mitogen-activated protein kinases (MAPK), are transient events. We have mined microarray datasets for changes in the expression of phosphatases in resting and TLR-activated macrophages. Several members of the dual-specificity phosphatases (DUSP) were induced upon triggering TLR4 with LPS. Up-regulation of DUSP1 mRNA was transient after stimulation with LPS alone, but addition of the immunosuppressive cytokine IL-10 resulted in robust, continued DUSP1 expression. IL-10 also synergized with the anti-inflammatory glucocorticoid dexamethasone in the induction of DUSP1 mRNA expression in activated macrophages, as well as in the inhibition of IL-6 and IL-12 production. Increased expression of DUSP1 in IL-10-treated activated macrophages was correlated with a faster down-regulation of p38 MAPK activation. Thus, these data suggest an operational link between IL-10 and inhibition of p38 MAPK via sustained expression of DUSP1.     

A novel phenotype for an activated macrophage: the type 2 activated macrophage

Activated macrophages were used as antigen presenting cells (APCs) to determine the extent to which these APCs could influence an adaptive immune response. We show that activated macrophages induced a strong polarized Th1-like T cell response that was predominated by IFN-gamma. However, when antigen was targeted to Fcgamma receptors on these macrophages, their phenotype changed, and they now induced a T cell response that was predominated by IL-4. The initial biasing by activated macrophages toward a Th1-like response was a result of activation of the innate immune response, as macrophages from MyD88(-/-) mice failed to produce Th1-inducing cytokines. The reversal of the Th1 biasing was a result of FcgammaR ligation, as macrophages lacking the FcR common gamma chain failed to reverse this biasing. To show that this biasing could occur in vivo, mice were injected with activated macrophages or activated macrophages whose FcgammaR had been ligated with an irrelevant immune complex. Mice injected with FcgammaR-ligated macrophages made more antibody than those receiving conventionally activated macrophages, and the antibody was predominantly of the IgG1 isotype. These studies demonstrate that FcgammaR ligation on activated macrophages can change the phenotype of these APCs to cells that preferentially drive a Th2-like response. We have termed these cells type 2 activated macrophages.