Jörg Zwirner

C5a anaphylatoxin is a major regulator of activating versus inhibitory FcγRs in immune complex–induced lung disease

IgG Fc receptors (FcgammaRs, especially FcgammaRIII) and complement (in particular, C5a anaphylatoxin) are critical effectors of the acute inflammatory response to immune complexes (ICs). However, it is unknown whether and how these two key components can interact with each other in vivo. We use here a mouse model of the acute pulmonary IC hypersensitivity reaction to analyze their potential interaction. FcgammaRIII and C5aR are coexpressed on alveolar macrophages (AMs), and both FcgammaRIII and C5aR mutant mice display impaired immune responses. We find that recombinant human C5a (rhC5a) can control inverse expression of various FcgammaRs, and costimulation of ICs with rhC5a results in strong enhancement of FcgammaRIII-triggered cellular activation in vitro and in vivo. Moreover, we show here that early IC-induced bioactive C5a, and its interaction with C5aR, causes induction of activating FcgammaRIII and suppression of inhibitory FcgammaRII on AMs that appears crucial for efficient cytokine production and neutrophil recruitment in lung pathology. Therefore, C5a, which is a potent chemoattractant, has a broader critical function in regulating the inhibitory/activating FcgammaRII/III receptor pair to connect complement and FcgammaR effector pathways in immune inflammation.

A regulatory role for the C5a anaphylatoxin in type 2 immunity in asthma

Complement component 5 (C5) has been described as either promoting or protecting against airway hyperresponsiveness (AHR) in experimental allergic asthma, suggesting pleomorphic effects of C5. Here we report that local pharmacological targeting of the C5a receptor (C5aR) prior to initial allergen sensitization in murine models of inhalation tolerance or allergic asthma resulted in either induction or marked enhancement of Th2-polarized immune responses, airway inflammation, and AHR. Importantly, C5aR-deficient mice exhibited a similar, increased allergic phenotype. Pulmonary allergen exposure in C5aR-targeted mice resulted in increased sensitization and accumulation of CD4+ CD69+ T cells associated with a marked increase in pulmonary myeloid, but not plasmacytoid, DC numbers. Pulmonary DCs from C5aR-targeted mice produced large amounts of CC chemokine ligand 17 (CCL17) and CCL22 ex vivo, suggesting a negative impact of C5aR signaling on pulmonary homing of Th2 cells. In contrast, C5aR targeting in sensitized mice led to suppressed airway inflammation and AHR but was still associated with enhanced production of Th2 effector cytokines. These data suggest a dual role for C5a in allergic asthma, i.e., protection from the development of maladaptive type 2 immune responses during allergen sensitization at the DC/T cell interface but enhancement of airway inflammation and AHR in an established inflammatory environment.

Complement: A novel factor in basal and ischemia-induced neurogenesis

Through its involvement in inflammation, opsonization, and cytolysis, the complement protects against infectious agents. Although most of the complement proteins are synthesized in the central nervous system (CNS), the role of the complement system in the normal or ischemic CNS remains unclear. Here we demonstrate for the first time that neural progenitor cells and immature neurons express receptors for complement fragments C3a and C5a (C3a receptor (C3aR) and C5a receptor). Mice that are deficient in complement factor C3 (C3−/−) lack C3a and are unable to generate C5a through proteolytic cleavage of C5 by C5‐convertase. Intriguingly, basal neurogenesis is decreased both in C3−/− mice and in mice lacking C3aR or mice treated with a C3aR antagonist. The C3−/− mice had impaired ischemia‐induced neurogenesis both in the subventricular zone, the main source of neural progenitor cells in adult brain, and in the ischemic region, despite normal proliferative response and larger infarct volumes. Thus, in the adult mammalian CNS, complement activation products promote both basal and ischemia‐induced neurogenesis.

6-Sulfo LacNAc, a Novel Carbohydrate Modification of PSGL-1, Defines an Inflammatory Type of Human Dendritic Cells

The monoclonal antibody M-DC8 defines a major subset of human blood dendritic cells (DCs). Here we identify the M-DC8 structure as 6-sulfo LacNAc, a novel carbohydrate modification of the P selectin glycoprotein ligand 1 (PSGL-1). In contrast to previously described blood DCs, M-DC8+ DCs lack the cutaneous lymphocyte antigen (CLA) on PSGL-1 and fail to bind P and E selectin. Yet they express anaphylatoxin receptors (C5aR and C3aR) and the Fcgamma receptor III (CD16), which recruit cells to inflammatory sites. While sharing with DC1 the expression of myeloid markers and a potent capacity to prime T cells in vitro, M-DC8+ DCs produce far more TNF-alpha in response to the bacterial endotoxin lipopolysaccharide (LPS). Thus, 6-sulfo LacNAc-expressing DCs appear as a novel proinflammatory DC subset.

Cell-derived anaphylatoxins as key mediators of antibody-dependent type II autoimmunity in mice

Complement C5a, a potent anaphylatoxin, is a candidate target molecule for the treatment of inflammatory diseases, such as myocardial ischemia/reperfusion injury, RA, and the antiphospholipid syndrome. In contrast, up until now, no specific contribution of C5a and its receptor, C5aR, was recognized in diseases of antibody-dependent type II autoimmunity. Here we identify C5a as a novel key mediator of autoimmune hemolytic anemia (AIHA) and show that mice lacking C5aR are partially resistant to this IgG autoantibody-induced disease model. Upon administration of anti-erythrocyte antibodies, upregulation of activating Fcgamma receptors (FcgammaRs) on Kupffer cells, as observed in WT mice, was absent in C5aR-deficient mice, and FcgammaR-mediated in vivo erythrophagocytosis was impaired. Surprisingly, in mice deficient in FcgammaRI and FcgammaRIII, anti-erythrocyte antibody-induced C5 and C5a production was abolished, demonstrating the existence of a previously unidentified FcgammaR-mediated C5a-generating pathway. These results show that the development of a full-blown antibody-dependent autoimmune disease requires C5a--produced by and acting on FcgammaR--and may suggest therapeutic benefits of C5 and/or C5a/C5aR blockade in AIHA and other diseases closely related to type II autoimmune injury.

Macrophages Induce the Inflammatory Response in the Pulmonary Arthus Reaction through Gαi2 Activation That Controls C5aR and Fc Receptor Cooperation

Complement and FcγR effector pathways are central triggers of immune inflammation; however, the exact mechanisms for their cooperation with effector cells and their nature remain elusive. In this study we show that in the lung Arthus reaction, the initial contact between immune complexes and alveolar macrophages (AM) results in plasma complement-independent C5a production that causes decreased levels of inhibitory FcγRIIB, increased levels of activating FcγRIII, and highly induced FcγR-mediated TNF-α and CXCR2 ligand production. Blockade of C5aR completely reversed such changes. Strikingly, studies of pertussis toxin inhibition show the essential role of Gi-type G protein signaling in C5aR-mediated control of the regulatory FcγR system in vitro, and analysis of the various C5aR-, FcγR-, and Gi-deficient mice verifies the importance of Gαi2-associated C5aR and the FcγRIII-FcγRIIB receptor pair in lung inflammation in vivo. Moreover, adoptive transfer experiments of C5aR- and FcγRIII-positive cells into C5aR- and FcγRIII-deficient mice establish AM as responsible effector cells. AM lacking either C5aR or FcγRIII do not possess any such inducibility of immune complex disease, whereas reconstitution with FcγRIIB-negative AM results in an enhanced pathology. These data suggest that AM function as a cellular link of C5a production and C5aR activation that uses a Gαi2-dependent signal for modulating the two opposing FcγR, FcγRIIB and FcγRIII, in the initiation of the inflammatory cascade in the lung Arthus reaction.

Chemoattraction of Macrophages, T Lymphocytes, and Mast Cells Is Evolutionarily Conserved within the Human α-Defensin Family

Human defensins are natural peptide antibiotics. On the basis of the position and bonding of six conserved cysteine residues, they are divided into two families, designated α- and β-defensins. Human α-defensins are expressed predominantly in neutrophils (human neutrophil peptides (HNP) 1–4) or intestinal Paneth cells (human defensins (HD) 5 and 6). Although α-defensins have been implicated in the pathogenesis of inflammatory bowel disease, their immunomodulatory functions are poorly understood. In the present study, HNP-1, HNP-3, and HD5 were found to be potent chemotaxins for macrophages but not dendritic cells using Gαi proteins and MAPK as signal transducers. α-Defensins were also chemoattractive for the human mast cell line HMC-1 but lacked, in contrast to β-defensins, the ability to induce intracellular calcium fluxes. Furthermore, HNP-1, HNP-3, and HD5 comparably mobilized naive as well as memory T lymphocytes. Using the protein kinase C (PKC) inhibitors GF109 and Gö6976, we observed a PKC-independent functional desensitization to occur between human α-defensins, which suggests a common receptor for HNP-1, HNP-3, and HD5 on immune cells. This α-defensin receptor was subject to heterologous desensitization by the PKC activator PMA and to PKC-dependent cross-desensitization by human β-defensins. Conversely, α-defensins desensitized β-defensin-mediated migration of immune cells in a PKC-dependent manner, suggesting unique receptors for both defensin families. Taken together, our observations indicate that chemoattraction of macrophages, T lymphocytes, and mast cells represents an immunomodulatory function which is evolutionarily conserved within the human α-defensin family and tightly regulated by β-defensins.

β‐Defensins chemoattract macrophages and mast cells but not lymphocytes and dendritic cells: CCR6 is not involved

β‐Defensins are natural peptide antibiotics whose immunomodulatory functions are poorly understood. In the present study, macrophages were found to migrate to human β‐defensins (HBD)‐1 to ‐4 using Gαi proteins as well as MAPK ERK, p38 and JNK as signal transducers. In addition, mast cells responded to HBD‐1 to ‐4 with calcium fluxes as well as chemotaxis, which increased upon stimulation with IgE plus antigen or ionomycin. In contrast, human β‐defensins were unable to induce migration of memory lymphocytes and dendritic cells (DC). Similar to HBD, the murine β‐defensin (mBD)‐8 mobilized macrophages and lacked the ability to recruit memory T cells. These findings were unexpected as CCR6 on memory T cells and DC has been previously observed to be a receptor for human β‐defensins. In support of our findings, however, RBL‐2H3 as well as 300.19 cells stably expressing CCR6 proved to be unresponsive to HBD‐2 and ‐3. Intriguingly, our observation of a PKC‐independent homologous desensitization between HBD‐1 to ‐4 suggests a common receptor for HBD. In summary, chemoattraction of macrophages and mast cells is evolutionary conserved within the β‐defensin family despite a considerable sequence variation and distinct antimicrobial activities. However, CCR6 is not a functional receptor for β‐defensins.

C5a Initiates the Inflammatory Cascade in Immune Complex Peritonitis

Immune complex (IC)-induced inflammation is integral to the pathogenesis of several autoimmune diseases. ICs activate the complement system and interact with IgG FcγR. In this study, we demonstrate that activation of the complement system, specifically generation of C5a, initiates the neutrophilic inflammation in IC peritonitis. We show that ablation of C5a receptor signaling abrogates neutrophil recruitment in wild-type mice and prevents the enhancement of neutrophil migration seen in FcγRIIB−/− mice, suggesting that C5aR signaling is the crucial initial event upstream of FcγR signaling. We also provide evidence that C5a initiates the inflammatory cascade both directly, through C5aR-mediated effector functions on infiltrating and resident peritoneal cells, and indirectly, through shifting the balance between activating and inhibitory FcγRs on resident cells toward an inflammatory phenotype. We conclude that complement activation and C5a generation are prerequisites for IC-induced inflammation through activating FcγR, which amplifies complement-induced inflammation in autoimmunity.

Activated Human T Lymphocytes Express a Functional C3a Receptor

The C3a molecule is an anaphylatoxin of the C system with a wide spectrum of proinflammatory effects predominantly on cells of myeloid origin. In this study we investigated the expression of the high affinity receptor for C3a (C3aR) in human T lymphocytes using receptor-specific mAb. C3aR expression was detected in CD4+ and CD8+ blood- or skin-derived T cell clones (TCC) from birch pollen-sensitized patients with atopic dermatitis. No significant difference in C3aR expression in CD4+ or CD8+ TCCs could be observed. In contrast to C3a(desArg), C3a led to a transient calcium flux in TCCs expressing the C3aR, whereas C3aR-negative TCCs were unreactive. Circulating T cells from patients suffering from severe inflammatory skin diseases expressed the C3aR, whereas no expression of C3aR could be found in unstimulated T lymphocytes from patients with mild inflammatory skin diseases or from healthy individuals. Type I IFNs, which are potent stimulators of cellular immunity, were identified as up-regulators of C3aR expression in vitro in freshly isolated or cloned T lymphocytes. Moreover, C3aR+ T cells were found at the sites of injection in IFN-β-treated patients with multiple sclerosis. These data provide direct evidence for the expression of C3aR on activated human T lymphocytes; this may point to a biological function of C3a in T cell-dependent diseases.