A2A receptors in inflammation and injury: lessons learned from transgenic animals

Adenosine regulates the function of the innate and adaptive immune systems through targeting virtually every cell type that is involved in orchestrating an immune/inflammatory response. Of the four adenosine receptors (A(1), A(2A), A(2B), A(3)), A(2A) receptors have taken center stage as the primary anti-inflammatory effectors of extracellular adenosine. This broad, anti-inflammatory effect of A(2A) receptor activation is a result of the predominant expression of A(2A) receptors on monocytes/macrophages, dendritic cells, mast cells, neutrophils, endothelial cells, eosinophils, epithelial cells, as well as lymphocytes, NK cells, and NKT cells. A(2A) receptor activation inhibits early and late events occurring during an immune response, which include antigen presentation, costimulation, immune cell trafficking, immune cell proliferation, proinflammatory cytokine production, and cytotoxicity. In addition to limiting inflammation, A(2A) receptors participate in tissue remodeling and reparation. Consistent with their multifaceted, immunoregulatory action on immune cells, A(2A) receptors have been shown to impact the course of a wide spectrum of ischemic, autoimmune, infectious, and allergic diseases. Here, we review the regulatory roles of A(2A) receptors in immune/inflammatory diseases of various organs, including heart, lung, gut, liver, kidney, joints, and brain, as well as the role of A(2A) receptors in regulating multiple organ failure and sepsis.     

Secreted phospholipases A2: A proinflammatory connection between macrophages and mast cells in the human lung

Secretory phospholipases A₂ (sPLA₂) are an emerging class of mediators of inflammation. These enzymes accumulate in plasma and other biological fluids of patients with inflammatory, autoimmune and allergic diseases. sPLA₂s are secreted at low levels in the normal airways and tend to increase during inflammatory lung diseases (e.g. bronchial asthma, chronic obstructive pulmonary disease, interstitial lung fibrosis, and sarcoidosis) as the result of plasma extravasation and/or local production. Such immune resident cells as macrophages and mast cells can be a source of sPLA₂s in the lung. However, these cells are also targets for sPLA₂s that sustain the activation programs of macrophages and mast cells with mechanism related to their enzymatic activity as well as to their capacity to interact with surface molecules (e.g., heparan sulfate proteoglycans, M-type receptor, mannose receptor). Recent evidence suggests that mast cells are a better source of extracellular sPLA₂s than macrophages. On the other hand, macrophages appear to be a preferential target for sPLA₂s. Anatomical association between macrophages and mast cells in the airways suggest that sPLA₂s released by mast cells may activate in a paracrine fashion several macrophage functions relevant to the modulation of lung inflammation. Thus, sPLA₂s may play a major role in inflammatory lung diseases by acting as a proinflammatory connection between macrophages and mast cells.     

In Vitro Generation of Eosinophil Chemotactic Factor from Human and Murine Mononuclear Phagocytes

Low molecular weight eosinophil chemotactic factor (ECF), which has previously been demonstrated in mast cells, basophils, neutrophils and eosinophils, was shown to be released by several types of mononuclear phagocytes. Highly purified rat peritoneal macrophages and human monocytes produced ECF on stimulation with the calcium ionophore A23187 (Ion) and with phagocytic stimuli in a time-dependent fashion, whereas lymphocyte- or mast cell-specific stimuli were ineffective. Two murine macrophage lines and a fibroblast cell line (L cells) also generated and secreted ECF with the different stimuli. ECF from macrophages was similar to that from neutrophils in its target cell specificity (eosinophils and neutrophils) and its elution profile on Sephadex G-10 columns (300-500 dalton). ECF secretion from monocytes was not affected by mitomycin C or cycloheximide, whereas indomethacin enhanced and a phospholipase A inhibitor decreased its production. These in vitro findings suggest that, through ECF, mononuclear phagocytes may potentially regulate eosinophil and neutrophil influx to sites of inflammatory reactions.     

Secretory phospholipases A2 as multivalent mediators of inflammatory and allergic disorders

Phospholipases A(2) (PLA(2)s) are enzymes responsible for mobilization of fatty acids, including arachidonic acid (AA), from phospholipids. These enzymes are classified as high-molecular-weight cytosolic PLA(2)s (cPLA(2)s) and low-molecular-weight secretory PLA(2)s (sPLA(2)s). There is increasing evidence that large quantities of sPLA(2)s are released in the plasma of patients with systemic inflammatory and autoimmune diseases. In addition, high levels of sPLA(2)s can be detected at sites of allergic inflammation including the upper airways of patients with rhinitis and the lower airways of patients with asthma. These extracellular enzymes play an important role in inflammation by releasing AA, which can be subsequently converted to proinflammatory prostaglandins and leukotrienes. Generation of AA mediated by sPLA(2)s occurs through different mechanisms, including (1) the direct hydrolysis of outer cell membrane phospholipids, (2) internalization and transfer of sPLA(2)s to intracellular pools of phospholipids enriched in AA, and (3) activation of cPLA(2)s. In addition, sPLA(2)s induce degranulation and production of cytokines and chemokines from a variety of cells involved in inflammatory and immune responses. These effects are exerted by mechanisms that are independent of the enzymatic activity and are mediated by the interaction of sPLA(2)s with specific or promiscuous membrane receptors. Therefore, sPLA(2)s may have an important role in inflammatory and allergic reactions by activating multiple mechanisms within inflammatory and immune cells, leading to the production of eicosanoids, cytokines and chemokines.     

Extracellular DNA traps in allergic,infectious, and autoimmune diseases

Extracellular DNA traps are part of the innate immune response and are seen with many infectious, allergic, and autoimmune diseases. They can be generated by several different leukocytes, including neutrophils, eosinophils, and monocytes, as well as mast cells. Here, we review the composition of these extracellular DNA‐containing structures as well as potential mechanisms for their production and function. In general, extracellular DNA traps have been described as binding to and killing pathogens, particularly bacteria, fungi, but also parasites. On the other hand, it is possible that DNA traps contribute to immunopathology in chronic inflammatory diseases, such as bronchial asthma. In addition, it has been demonstrated that they can initiate and/or potentiate autoimmune diseases. Extracellular DNA traps represent a frequently observed phenomenon in inflammatory diseases, and they appear to participate in the cross‐talk between different immune cells. These new insights into the pathogenesis of inflammatory diseases may open new avenues for targeted therapies.     

Secretory phospholipases A2 induce cytokine release from blood and synovial fluid monocytes.

Secretory phospholipases A2 (sPLA2) are released in the blood of patients with various inflammatory diseases and exert proinflammatory activities by releasing arachidonic acid (AA), the precursor of eicosanoids. We examined the ability of four sPLA2 to activate blood and synovial fluid monocytes in vitro. Monocytes were purified from blood of healthy donors or from synovial fluid of patients with rheumatoid arthritis by negative immunoselection and by adherence to plastic dishes, respectively. The cells were incubated with group IA, IB, IIA and III sPLA2 and the release of TNF-alpha, IL-6 and IL-12 was determined by ELISA. Group IA, IB and IIA sPLA2 induced a concentration-dependent release of TNF-alpha and IL-6 from blood monocytes. These sPLA2 activated IL-12 production only in monocytes preincubated with IFN-gamma. Group IA and IIA sPLA2 also induced TNF-alpha and IL-6 release from synovial fluid monocytes. TNF-alpha and IL-6 release paralleled an increase in their mRNA expression and was independent from the capacity of sPLA2 to mobilize AA. These results indicate that sPLA2 stimulate cytokine release from blood and synovial fluid monocytes by a mechanism at least partially unrelated to their enzymatic activity. This effect may concur with the generation of AA in the proinflammatory activity of sPLA2 released during inflammatory diseases.     

Macrophage inflammatory protein-1alpha and C-C chemokine receptor-1 in allergen-induced skin late-phase reactions: relationship to macrophages, neutrophils, basophils, eosinophils and T lymphocytes.

BACKGROUND: Macrophage inflammatory protein (MIP)-1alpha binds to C-C chemokine receptor (CCR)-1 with high affinity. CCR-1 is expressed on neutrophils, eosinophils, monocytes, T lymphocytes and basophils; cells characteristic of atopic allergic inflammation. In vitro, MIP-1alpha is chemotactic for monocytes, T cells and basophils and is also a potent histamine-releasing factor for basophils and mast cells. Although increased levels of MIP-1alpha were shown in atopic allergic disorders, the kinetics of expression of these CC chemokines in vivo is largely unknown. OBJECTIVE: To investigate the kinetics of expression of MIP-1alpha and receptor CCR-1 and the relationships between the expression and infiltration of inflammatory cells in allergen-induced cutaneous late-phase reactions in atopic subjects. METHODS: Cryostat sections, obtained from skin biopsies from 10 human atopic subjects at 6, 24, 48, 72 h and 7 days after allergen challenge, were processed for immunohistochemistry and in situ hybridization using 35S-labelled riboprobes. RESULTS: The peak expression of allergen-induced mRNA for MIP-1alpha and CCR-1 was 6 h. This was maintained at 24 h, and gradually returned to base line at 7 days. At 6 h, the number of cells expressing MIP-1alpha mRNA significantly correlated with elastase+ neutrophils and BB-1+ basophils. At 24 h, the MIP-1alpha mRNA+ cells significantly correlated with CD68+ macrophages. There were significant inverse correlations between the numbers of MIP-1alpha mRNA cells and the numbers of Tryptase+ mast cells at 6 and 24 h after allergen challenge. CONCLUSION: Allergen-induced cutaneous late-phase reactions in humans were associated with increased expression of MIP-1alpha and CCR-1. This may be relevant to the infiltration of neutrophils, eosinophils, basophils and macrophages.     

Immune metabolism in allergies,does it matter?—A review of immune metabolic basics and adaptations associated with the activation of innate immune cells in allergy

Type I allergies are pathological, type 2 inflammatory immune responses against otherwise harmless environmental allergens that arise from complex interactions between different types of immune cells. Activated immune cells undergo extensive changes in phenotype and function to fulfill their effector functions. Hereby, activation, differentiation, proliferation, migration, and mounting of effector responses require metabolic reprogramming. While the metabolic changes associated with activation of dendritic cells, macrophages, and T cells are extensively studied, data about the metabolic phenotypes of the other cell types critically involved in allergic responses (epithelial cells, eosinophils, basophils, mast cells, and ILC2s) are rather limited. This review briefly covers the basics of cellular energy metabolism and its connection to immune cell function. In addition, it summarizes the current state of knowledge in terms of dendritic cell and macrophage metabolism and subsequently focuses on the metabolic changes associated with activation of epithelial cells, eosinophils, basophils, mast cells, as well as ILC2s in allergy. Interestingly, the innate key cell types in allergic inflammation were reported to change their metabolic phenotype during activation, shifting to either glycolysis (epithelial cells, M1 macrophages, DCs, eosinophils, basophils, acutely activated mast cells), oxidative phosphorylation (M2 macrophages, longer term activated mast cells), or fatty acid oxidation (ILC2s). Therefore, immune metabolism is of relevance in allergic diseases and its connection to immune cell effector function needs to be considered to better understand induction and maintenance of allergic responses. Further progress in this field will likely improve both our understanding of disease pathology and enable new treatment targets/strategies.     

Cysteinyl leukotrienes: multi-functional mediators in allergic rhinitis

Cysteinyl leukotrienes (CysLTs) are a family of inflammatory lipid mediators synthesized from arachidonic acid by a variety of cells, including mast cells, eosinophils, basophils, and macrophages. This article reviews the data for the role of CysLTs as multi-functional mediators in allergic rhinitis (AR). We review the evidence that: (1) CysLTs are released from inflammatory cells that participate in AR, (2) receptors for CysLTs are located in nasal tissue, (3) CysLTs are increased in patients with AR and are released following allergen exposure, (4) administration of CysLTs reproduces the symptoms of AR, (5) CysLTs play roles in the maturation, as well as tissue recruitment, of inflammatory cells, and (6) a complex inter-regulation between CysLTs and a variety of other inflammatory mediators exists.     

Expression of cysteinyl leukotriene synthetic and signalling proteins in inflammatory cells in active seasonal allergic rhinitis

BACKGROUND: Cysteinyl leukotrienes (CysLTs) are bioactive lipids that have been shown to contribute to allergic and inflammatory diseases. Eosinophils and mast cells have the capacity to produce large amounts of CysLTs after allergic or non-allergic stimulation. Molecular identification of both the synthetic and signalling proteins in the CysLT pathway allows the investigation of expression of the CysLT enzymes and receptors in active allergic rhinitis. OBJECTIVE: We examined the expression of the proteins involved in the synthesis of CysLTs and the cysteinyl leukotriene-1 (CysLT1) and cysteinyl leukotriene-2 (CysLT2) receptors in inflammatory cells from patients with active seasonal allergic rhinitis. METHODS: Nasal lavage samples were obtained from patients during active seasonal allergic rhinitis. Specific cellular immunocytochemical techniques were used to detect the cysteinyl leukotriene synthetic proteins, namely 5-lipoxygenase (5-LO), 5-lipoxygenase-activating protein (FLAP) and leukotriene C4 synthase (LTC4S). In situ hybridization and immunocytochemical techniques were used to identify the mRNA and proteins for the CysLT1 and CysLT2 receptors. RESULTS: 5-LO, FLAP and LTC4S, and the CysLT1 and CysLT2 receptors were expressed in the majority of eosinophils and in subsets of mast cells and mononuclear cells. 5-LO, FLAP and the CysLT1 receptor, but not LTC4S or the CysLT2 receptor, were expressed in a subset of nasal neutrophils. CONCLUSIONS: Our study demonstrates the presence of CysLT pathway proteins in key allergic and inflammatory cells from the upper airway of patients with active seasonal allergic rhinitis. Our expression data highlight the potential of CysLT-modifying agents to treat both upper and lower airway symptoms in patients suffering from allergic rhinitis and asthma.     

Prolonged nasal eosinophilia in allergic patients after common cold

BACKGROUND: Viral respiratory tract infections may cause both harmless common colds and severe asthma exacerbations; the differences in disease expression probably depend on the allergic status of the patient. To determine whether altered immunologic mechanisms underlie these differences, we investigated nasal inflammation during naturally acquired common cold. METHODS: In a group of 16 patients (eight allergic), nasal brush samples were taken, and nasal symptoms were recorded during common cold, 2 weeks later (convalescence), and at baseline (>4 weeks without nasal symptoms). Nasal brush cells were stained immunohistochemically for Langerhans cells, T cells, monocytes, neutrophils, B cells, macrophages, natural killer (NK) cells, mast cells, eosinophils, eotaxin, and RANTES. RESULTS: Four rhinovirus, four coronavirus, three RSV, one Mycoplasma pneumoniae, and one influenza A/enterovirus double infection were confirmed. Increased numbers of T cells, monocytes, macrophages, NK cells, eosinophils, and RANTES- and eotaxin-positive cells, but not neutrophils, were observed during common cold in allergic and nonallergic patients, and increased numbers of mast cells in allergic patients. Compared to nonallergic patients, in allergic patients eosinophil influx persisted into convalescence. CONCLUSION: Prolonged nasal eosinophil influx was observed in allergic patients after common cold. What immunologic factors can induce prolonged eosinophil influx and whether this may increase the risk of subsequent allergen-induced hypersensitivity reactions must be studied further.     

Role of cysteinyl leukotriene receptor antagonists in the management of allergic rhinitis

The cysteinyl leukotrienes (CysLTs) are lipid mediators that have been implicated in the pathogenesis of allergic diseases. Pharmacological studies using CysLTs indicate that there exist two classes of receptors named CysLT₁ and CysLT₂. The former is sensitive to CysLT₁ receptor antagonists currently used for the treatment of asthma and allergic rhinitis. Our previous immunohistochemical and autoradiographic studies showed that anti-CysLT₁ receptor antibody adhered to eosinophils, mast cells, macrophages, neutrophils, and vascular endothelial cells in human nasal mucosa, and that a novel radioactive CysLT₁ receptor antagonist, [³H]-pranlukast, bound specifically to CysLT₁ receptors in human inferior turbinate and its binding sites were localized to vascular endothelium and interstitial cells. These data suggest that in allergic rhinitis, the major targets of CysLT₁ receptor antagonists are the vascular bed and infiltrating leucocytes such as mast cells, eosinophils, and macrophages. Clinical trials have demonstrated that CysLT₁ receptor antagonists are as effective as antihistamines, but are less effective than intranasal steroids for the treatment of allergic rhinitis. The use of CysLT₁ receptor antagonists in combination with antihistamines has generally resulted in greater efficacy than when these agents were used alone.     

Contribution of survivin to the immune system,allergies and autoimmune diseases

In addition to malignancies, survivin (a member of the apoptosis inhibitor family) has been implicated in the pathogenesis of inflammatory disorders, including autoimmune and allergic diseases. Survivin is constantly expressed in the proliferating hematopoietic progenitor cells, and it is re-expressed in the mature cells of the innate and adaptive immunity, upon activation. Survivin enhances the expression of co-stimulatory molecules and MHC class II molecules in dendritic cells, and promotes the lifespan of macrophages, neutrophils, and eosinophils, while suppressing natural killer (NK) cell activity. Survivin has been implicated in T cell maturation, T cell expansion, effector CD4⁺ T cell differentiation, maintenance of memory CD4⁺ T and CD8⁺ T cells, as well as antibody production. Upregulated expression of survivin was indicated in the T cells as well as various samples collected from allergic patients. Survivin can contribute to the pathogenesis of allergic diseases via the promotion of the Th2 polarization, promoting IL-4 expression, compromising activation-induced cell death (AICD) in Th2 cells, and preventing apoptosis of eosinophils, as well as, amplification of eosinophilia. Moreover, survivin can interfere with clonal deletion of autoreactive T and B cells, as well as suppress Treg cell development and activity supporting the development of autoimmune diseases. This review discusses the role of survivin in immunity, allergy and autoimmunity as well as provides evidence that survivin may be considered as a novel therapeutic target for the treatment of allergic and autoimmune diseases.     

Measurement of NK cell and macrophage activation

FcgammaRIII(CD16), one of the low-affinity IgG Fc receptors exists in two forms. FcgammaRIIIa is expressed on NK cells, a subset of T lymphocytes, a subpopulation of monocytes and macrophages, and shows a cell type specific glycosylation pattern. FcgammaRIIIb is expressed exclusively on neutrophils in two allotypes, NA1/2, and it can be induced on eosinophils. Both FcyRIIIs are released from the cell surface. FcgammaRIIIa is released by the action of a metalloprotease upon the in vitro activation of NK cells and macrophages. FcgammaRIIIb is released upon activation and during the apoptosis of neutrophils by proteolytic activity. So, the amount of each type of soluble FcgammaRIII means the activation of each type of cell. We measured three types of soluble FcgammaRIII in plasma and also urine with newly developed anti-FcgammaRIIIa and anti-FcgammaRIIIa(Mphi) monoclonal antibodies. We found that sFcgammaRIIIa may be a novel marker of inflammatory activity in rheumatoid arthritis. sFcgammaRIIIa(M phi) may serve as predictive marker for atherosclerosis. Further, urinary sFcgammaRIIIa and sFcgammaRIIIa(Mphi) may be novel markers for the assessment of disease activity in nephritis.     

Regulatory roles of mast cells in immune responses

Mast cells are important immune cells for host defense through activation of innate immunity (via toll-like receptors or complement receptors) and acquired immunity (via FcεRI). Conversely, mast cells also act as effector cells that exacerbate development of allergic or autoimmune disorders. Yet, several lines of evidence show that mast cells act as regulatory cells to suppress certain inflammatory diseases. Here, we review the mechanisms by which mast cells suppress diseases.     

Complement-mediated immune mechanisms in allergy

Allergic conditions are associated with canonical and noncanonical activation of the complement system leading to the release of several bioactive mediators with inflammatory and immunoregulatory properties that regulate the immune response in response to allergens during the sensitization and/or the effector phase of allergic diseases. Further, immune sensors of complement and regulator proteins of the cascade impact on the development of allergies. These bioactive mediators comprise the small and large cleavage fragments of C3 and C5. Here, we provide an update on the multiple roles of immune sensors, regulators, and bioactive mediators of complement in allergic airway diseases, food allergies, and anaphylaxis. A particular emphasis is on the anaphylatoxins C3a and C5a and their receptors, which are expressed on many of the effector cells in allergy such as mast cells, eosinophils, basophils, macrophages, and neutrophils. Also, we will discuss the multiple pathways, by which the anaphylatoxins initiate and control the development of maladaptive type 2 immunity including their impact on innate lymphoid cell recruitment and activation. Finally, we briefly comment on the potential to therapeutically target the complement system in different allergic conditions.     

Protective roles for myeloid cells in neuroinflammation

Myeloid cells represent the major cellular component of innate immune responses. Myeloid cells include monocytes and macrophages, granulocytes (neutrophils, basophils and eosinophils) and dendritic cells (DC). The role of myeloid cells has been broadly described both in physiological and in pathological conditions. All tissues or organs are equipped with resident myeloid cells, such as parenchymal microglia in the brain, which contribute to maintaining homeostasis. Moreover, in case of infection or tissue damage, other myeloid cells such as monocytes or granulocytes (especially neutrophils) can be recruited from the circulation, at first to promote inflammation and later to participate in repair and regeneration. This review aims to address the regulatory roles of myeloid cells in inflammatory diseases of the central nervous system (CNS), with a particular focus on recent work showing induction of suppressive function via stimulation of innate signalling in multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE).     

DAP10- and DAP12-associated receptors in innate immunity

Summary:  The DAP10 and DAP12 signaling subunits are highly conserved in evolution and associate with a large family of receptors in hematopoietic cells, including dendritic cells, plasmacytoid dendritic cells, neutrophils, basophils, eosinophils, mast cells, monocytes, macrophages, natural killer cells, and some B and T cells. Some receptors are able to associate with either DAP10 or DAP12, which contribute unique intracellular signaling functions. Studies of humans and mice deficient in these signaling subunits have provided surprising insights into the physiological functions of DAP10 and DAP12, demonstrating that they can either activate or inhibit immune responses. DAP10- and DAP12-associated receptors have been shown to recognize both host-encoded ligands and ligands encoded by microbial pathogens, indicating that they play an important role in innate immune responses.     

Nerve growth factor: an important molecule in allergic inflammation and tissue remodelling

BACKGROUND: Several studies provide evidence that nerve growth factor (NGF) has an expanding role in neuroimmune interactions. METHODS: We review our data on circulation levels of NGF in allergic diseases as well as on the relationships between this neurotrophin and primary and secondary effector cells of allergic inflammation. RESULTS: In vernal keratoconjunctivitis, a close relationship exists between the increased NGF plasma values and the number of mast cells infiltrating the conjunctiva. NGF serum values are also increased in other allergic diseases and asthma, and are related to the severity of the inflammatory process and disease. Human CD4+ T cell clones (preferentially of activated Th2 type) produce and release NGF, and express high-affinity NGF receptors. NGF is preformed in and can act on human peripheral blood eosinophils to preferentially release inflammatory mediators. Immunoreactivity for high affinity NGF receptors is present both in basal epithelial cells and in the inflamed stroma of the allergic conjunctiva. Topical administration of NGF results in a complete healing of neurotophic corneal ulcers in man, thus suggesting a profound effect of NGF on human fibroblasts and extracellular matrix. CONCLUSION: Data presented suggest that NGF is an important molecule in allergic inflammation and tissue remodelling occurring in allergic diseases.     

The uptake by blood-borne phagocytes of monosodium urate is dependent on heat-labile serum factor(s) and divalent cations

Accumulation in tissues of post-apoptotic cells is a feature frequently observed in patients with systemic lupus erythematosus and in murine models of systemic autoimmune diseases. One of the endogenous danger molecules released by secondarily necrotic cells is monosodium urate (MSU), which is already established to be the causative agent of gout. Here, we show that MSU is taken up by eosinophils, neutrophils and monocytes in a process involving (a) heat-labile serum factor(s) and divalent cations. The uptake induces the release of the pro-inflammatory cytokines IL-1β/IL-18/TNFα and IL-6/IL-8 by monocytes and PMN, respectively.