C5a Regulates IL-1ß Production and Leukocyte Recruitment in a Murine Model of Monosodium Urate Crystal-Induced Peritonitis.

Gouty arthritis results from the generation of monosodium urate (MSU) crystals within joints. These MSU crystals elicit acute inflammation characterized by massive infiltration of neutrophils and monocytes that are mobilized by the pro-inflammatory cytokine IL-1ß. MSU crystals also activate the complement system, which regulates the inflammatory response; however, it is unclear whether or how MSU-mediated complement activation is linked to IL-1ß release in vivo, and the various roles that might be played by individual components of the complement cascade. Here we show that exposure to MSU crystals in vivo triggers the complement cascade, leading to the generation of the biologically active complement proteins C3a and C5a. C5a, but not C3a, potentiated IL-1ß and IL-1α release from LPS-primed MSU-exposed peritoneal macrophages and human monocytic cells in vitro; while in vivo MSU-induced C5a mediated murine neutrophil recruitment as well as IL-1ß production at the site of inflammation. These effects were significantly ameliorated by treatment of mice with a C5a receptor antagonist. Mechanistic studies revealed that C5a most likely increased NLRP3 inflammasome activation via production of reactive oxygen species (ROS), and not through increased transcription of inflammasome components. Therefore we conclude that C5a generated upon MSU-induced complement activation increases neutrophil recruitment in vivo by promoting IL-1 production via the generation of ROS, which activate the NLRP3 inflammasome. Identification of the C5a receptor as a key determinant of IL-1-mediated recruitment of inflammatory cells provides a novel potential target for therapeutic intervention to mitigate gouty arthritis.

Gamma interferon regulates contraction of the influenza virus-specific CD8 T cell response and limits the size of the memory population.

The factors that regulate the contraction of the CD8 T cell response and the magnitude of the memory cell population against localized mucosal infections such as influenza are important for generation of efficient vaccines but are currently undefined. In this study, we used a mouse model of influenza to demonstrate that the absence of gamma interferon (IFN-γ) or IFN-γ receptor 1 (IFN-γR1) leads to aberrant contraction of antigen-specific CD8 T cell responses. The increased accumulation of the effector CD8 T cell population was independent of viral load. Reduced contraction was associated with an increased fraction of CD8 T cells expressing the interleukin-7 receptor (IL-7R) at the peak of the response, resulting in enhanced numbers of memory/memory precursor cells in IFN-γ(-/-) and IFN-γR(-/-) compared to wild-type (WT) mice. Blockade of IL-7 within the lungs of IFN-γ(-/-) mice restored the contraction of influenza virus-specific CD8 T cells, indicating that IL-7R is important for survival and is not simply a consequence of the lack of IFN-γ signaling. Finally, enhanced CD8 T cell recall responses and accelerated viral clearance were observed in the IFN-γ(-/-) and IFN-γR(-/-) mice after rechallenge with a heterologous strain of influenza virus, confirming that higher frequencies of memory precursors are formed in the absence of IFN-γ signaling. In summary, we have identified IFN-γ as an important regulator of localized viral immunity that promotes the contraction of antigen-specific CD8 T cells and inhibits memory precursor formation, thereby limiting the size of the memory cell population after an influenza virus infection.

Cutting edge: the NLRP3 inflammasome links complement-mediated inflammation and IL-1ß release.

The complement system is a potent component of the innate immune response, promoting inflammation and orchestrating defense against pathogens. However, dysregulation of complement is critical to several autoimmune and inflammatory syndromes. Elevated expression of the proinflammatory cytokine IL-1ß is often linked to such diseases. In this study, we reveal the mechanistic link between complement and IL-1ß secretion using murine dendritic cells. IL-1ß secretion occurs following intracellular caspase-1 activation by inflammasomes. We show that complement elicits secretion of both IL-1ß and IL-18 in vitro and in vivo via the NLRP3 inflammasome. This effect depends on the inflammasome components NLRP3 and ASC, as well as caspase-1 activity. Interestingly, sublethal complement membrane attack complex formation, but not the anaphylatoxins C3a and C5a, activated the NLRP3 inflammasome in vivo. These findings provide insight into the molecular processes underlying complement-mediated inflammation and highlight the possibility of targeting IL-1ß to control complement-induced disease and pathological inflammation.

IRF4 transcription factor-dependent CD11b+ dendritic cells in human and mouse control mucosal IL-17 cytokine responses.

Mouse and human dendritic cells (DCs) are composed of functionally specialized subsets, but precise interspecies correlation is currently incomplete. Here, we showed that murine lung and gut lamina propria CD11b+ DC populations were comprised of two subsets: FLT3- and IRF4-dependent CD24(+)CD64(-) DCs and contaminating CSF-1R-dependent CD24(-)CD64(+) macrophages. Functionally, loss of CD24(+)CD11b(+) DCs abrogated CD4+ T cell-mediated interleukin-17 (IL-17) production in steady state and after Aspergillus fumigatus challenge. Human CD1c+ DCs, the equivalent of murine CD24(+)CD11b(+) DCs, also expressed IRF4, secreted IL-23, and promoted T helper 17 cell responses. Our data revealed heterogeneity in the mouse CD11b+ DC compartment and identifed mucosal tissues IRF4-expressing DCs specialized in instructing IL-17 responses in both mouse and human. The demonstration of mouse and human DC subsets specialized in driving IL-17 responses highlights the conservation of key immune functions across species and will facilitate the translation of mouse in vivo findings to advance DC-based clinical therapies.

Complement mediated signaling on pulmonary CD103(+) dendritic cells is critical for their migratory function in response to influenza infection.

Trafficking of lung dendritic cells (DCs) to the draining lymph node (dLN) is a crucial step for the initiation of T cell responses upon pathogen challenge. However, little is known about the factors that regulate lung DC migration to the dLN. In this study, using a model of influenza infection, we demonstrate that complement component C3 is critically required for efficient emigration of DCs from the lung to the dLN. C3 deficiency affect lung DC-mediated viral antigen transport to the dLN, resulting in severely compromised priming of virus-specific T cell responses. Consequently, C3-deficient mice lack effector T cell response in the lungs that affected viral clearance and survival. We further show that direct signaling by C3a and C5a through C3aR and C5aR respectively expressed on lung DCs is required for their efficient trafficking. However, among lung DCs, only CD103(+) DCs make a significant contribution to lung C5a levels and exclusively produce high levels of C3 and C5 during influenza infection. Collectively, our findings show that complement has a profound impact on immune regulation by controlling tissue DC trafficking and highlights a potential utility for complement as an adjuvant in novel vaccine strategies.

Complement component c1q mediates mitochondria-driven oxidative stress in neonatal hypoxic-ischemic brain injury.

Hypoxic-ischemic (HI) brain injury in infants is a leading cause of lifelong disability. We report a novel pathway mediating oxidative brain injury after hypoxia-ischemia in which C1q plays a central role. Neonatal mice incapable of classical or terminal complement activation because of C1q or C6 deficiency or pharmacologically inhibited assembly of membrane attack complex were subjected to hypoxia-ischemia. Only C1q(-/-) mice exhibited neuroprotection coupled with attenuated oxidative brain injury. This was associated with reduced production of reactive oxygen species (ROS) in C1q(-/-) brain mitochondria and preserved activity of the respiratory chain. Compared with C1q(+/+) neurons, cortical C1q(-/-) neurons exhibited resistance to oxygen-glucose deprivation. However, postischemic exposure to exogenous C1q increased both mitochondrial ROS production and mortality of C1q(-/-) neurons. This C1q toxicity was abolished by coexposure to antioxidant Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid). Thus, the C1q component of complement, accelerating mitochondrial ROS emission, exacerbates oxidative injury in the developing HI brain. The terminal complement complex is activated in the HI neonatal brain but appeared to be nonpathogenic. These findings have important implications for design of the proper therapeutic interventions against HI neonatal brain injury by highlighting a pathogenic priority of C1q-mediated mitochondrial oxidative stress over the C1q deposition-triggered terminal complement activation.

Interplay between REST and nucleolin transcription factors: a key mechanism in the overexpression of genes upon increased phosphorylation.

Non-malignant cells can be transformed via the activation of kinases that control degradation of neural-restrictive silencer factor (REST). Here, we identify a mechanism that contributes to the activation of genes, expression of which is controlled by responsive elements containing overlapping binding sites for REST and nucleolin. We demonstrate that both phosphorylated and non-phosphorylated nucleolin-bound DNA; however, only phosphorylated nucleolin successfully competed with either full-length REST or a REST-derived DNA-binding peptide, REST68, for binding to the overlapping binding sites. We show that this interplay between the two transcription factors regulates the activation of cell survival and immunomodulatory genes in tumors and non-malignant cells with activated protein kinase C, which is accompanied with alterations in cell proliferation and apoptosis. We propose a model for the regulation of these genes, which brings a new insight into the molecular mechanisms that control cellular transformation driven by activation of protein kinases.

CD59 blockade enhances antigen-specific CD4+ T cell responses in humans: a new target for cancer immunotherapy?

CD59, a broadly expressed GPI-anchored molecule, regulates formation of the membrane attack complex of the complement cascade. We previously demonstrated that mouse CD59 also down-modulates CD4(+) T cell activity in vivo. In this study, we explored the role of CD59 on human CD4(+) T cells. Our data demonstrate that CD59 is up-regulated on activated CD4(+) T cells and serves to down-modulate their activity in response to polyclonal and Ag-specific stimulation. The therapeutic potential of this finding was explored using T cells isolated from colorectal cancer patients. The findings were striking and indicated that blockade of CD59 significantly enhanced the CD4(+) T cell response to two different tumor Ags. These data highlight the potential for manipulating CD59 expression on T cells for boosting weak immune responses, such as those found in individuals with cancer.

Defective B cell ontogeny and humoral immune response in mice prematurely expressing human complement receptor 2 (CR2, CD21) is similar to that seen in aging wild type mice.

Mice prematurely expressing human CR2 (hCR2) in the B cell lineage have a defective B cell ontogeny and humoral immune response. We have previously determined altered tyrosine phosphorylation patterns within hCR2 transgenic mice, suggesting that irreversible changes in B cell signaling pathways had occurred, which could explain the B cell unresponsiveness associated with hCR2 transgene expression. In support of that assertion, we found that increasing antigen dose or addition of adjuvant had a minimal impact on the ability of B cells to respond to antigen. However, analysis of aged hCR2(high) mice (1 year plus) revealed that both B cell numbers, B cell sub-population distribution including expansion of a newly described B regulatory cell subset, and immune responses were comparable with age-matched hCR2 negative mice. Finally, we established that B cell unresponsiveness to antigen in aging wild type mice (1 year plus) was equivalent to that noted in 3-month-old hCR2(high) mice. This data provides evidence that 3-month-old hCR2(high) mice have a humoral immune system resembling aged mice and suggests that further examination of the precise molecular and cellular parallels between aged wild type mice and 3-month-old hCR2(high) mice could provide an important insight into the mechanisms which lead to B cell unresponsiveness in the aging immune system.

Identification of the high affinity binding site in the Streptococcus intermedius toxin intermedilysin for its membrane receptor, the human complement regulator CD59.

The unique species specificity of the bacterial cytolysin intermedilysin is explained by its requirement for the human complement regulator CD59 as the primary receptor. Binding studies using individual domains of intermedilysin mapped the CD59-binding site to domain 4 and swap mutants between human and rabbit (non-intermedilysin-binding) CD59 implicated a short sequence (residues 42-59) in human CD59 in binding intermedilysin. We set out to map more closely the CD59 binding site in intermedilysin. We first looked for regions of homology between domain 4 in intermedilysin and the terminal complement components that bind CD59, C8 and C9. A nine amino acid sequence immediately adjacent the undecapeptide segment in intermedilysin domain 4 matched (5 of 9 identical, 3 of 9 conserved) a sequence in C9. A peptide containing this sequence caused dose-dependent inhibition of intermedilysin-mediated lysis of human erythrocytes and rendered erythrocytes more susceptible to complement lysis. Surface plasmon resonance analysis of intermedilysin binding to immobilized CD59 revealed saturable fast-on, fast-off binding and a calculated affinity of 4.9 nM. Substitution of three residues from the putative binding site caused a 5-fold reduction in lytic potency of intermedilysin and reduced affinity for immobilized CD59 by 2.5-fold. The demonstration that a peptide modeled on the CD59-binding site inhibits intermedilysin-mediated haemolysis leads us to suggest that such peptides might be useful in treating infections caused by intermedilysin-producing bacteria.

Crry deficiency in complement sufficient mice: C3 consumption occurs without associated renal injury.

The rodent-specific complement regulator complement receptor 1-related gene/protein-y (Crry) is critical for complement homeostasis. Gene deletion is 100% embryonically lethal; Crry-deficient (Crry(-/-)) mice were rescued by back-crossing onto C3 deficiency, confirming that embryo loss was complement mediated. In order to rescue viable Crry(-/-) mice without deleting C3, we have tested inhibition of C5 during gestation. Crry(+/-) females were given neutralizing anti-C5 mAb immediately prior to mating with Crry(+/-) males and C5 inhibition maintained through pregnancy. A single, healthy Crry(-/-) female was obtained and mating with Crry(+/-) males yielded healthy litters containing equal numbers of Crry(+/-) and Crry(-/-) pups. Inter-crossing Crry(-/-) mice yielded healthy litters of expected size. Although the mice were not anemic, exposure of Crry(-/-) erythrocytes to normal mouse serum caused C3 deposition and lysis, while transfusion into normal or C6(-/-) mice resulted in rapid clearance. Complement activity and C3 levels in Crry(-/-) mice were markedly reduced. Comparison with factor H deficient (CfH(-/-)) mice revealed similar levels of residual C3; however, unlike the CfH(-/-) mice, Crry(-/-) mice showed no evidence of renal injury, demonstrating distinct roles for these regulators in protecting the kidney.

Modulation of CD59 expression by restrictive silencer factor-derived peptides in cancer immunotherapy for neuroblastoma.

Tumor cells escape clearance by complement by abundantly expressing CD59 and other membrane complement regulators. Existing strategies for blocking/knocking down these regulators can contribute to tumor immunoclearance in vitro; however, there are numerous difficulties restricting their use in vivo. Here, we report a new strategy for suppression of CD59 expression in neuroblastoma using peptides that target regulators of CD59 expression. We identified the neural-restrictive silencer factor (REST) as a target for modulation of CD59 expression in neuroblastoma. We next designed plasmids that encoded peptides comprising different DNA-binding domains of REST and transfected them into neuroblastoma cell lines. These peptides suppressed CD59 expression, sensitizing neuroblastoma to complement-mediated killing triggered by anti-GD2 therapeutic monoclonal antibody. These CD59-modulating peptides might be effective therapeutic adjuvants to therapeutic monoclonal antibodies used for treatment of neuroblastoma and other cancer types sharing the same mechanism for regulation of CD59 expression.

The mouse complement regulator CD59b is significantly expressed only in testis and plays roles in sperm acrosome activation and motility.

In mouse, genes encoding complement regulators CD55 and CD59 have been duplicated. The first described form of CD59, CD59a, is broadly distributed in mouse tissues, while the later identified CD59b was originally described as testis specific. Subsequent studies have been contradictory, some reporting widespread and abundant expression of CD59b. Resolution of the distribution patterns of the CD59 isoforms is important for interpretation of disease studies utilising CD59 knockout mice. Here we have performed a comprehensive distribution study of the CD59 isoforms at the mRNA and protein levels. These data confirm that expression of CD59b is essentially restricted to adult testis; trace expression in other tissues is a consequence of contamination with blood cells, shown previously to express CD59b at low level. In testis, onset of expression of CD59b coincided with puberty and was predominant on the spermatozoal acrosome. Ligation of CD59b, but not CD59a, markedly reduced spermatozoal motility, suggesting a specific role in reproductive function.

CD59a deficient mice display reduced B cell activity and antibody production in response to T-dependent antigens.

CD59a is the primary regulator of membrane attack complex in mice. Recently, we have shown that CD59a-deficient (Cd59a-/-) mice exhibit enhanced CD4+ T cell responses. Here, we explored the effects of CD59a on B cell function and antibody production. Contrary to our expectations, Cd59a-/- mice showed a decreased humoral immune response to a T cell dependent antigen, sheep red blood cells. We found that the decreased humoral immune response was associated with a reduction in plasma cell number in vivo and reduced ability to respond to stimuli during in vitro culture experiments. Using MLR studies in which purified wild type or Cd59a-/- CD4+ T cells were mixed with purified B cells from each source, we found that the reduced B cell activation was largely due to the absence of CD59a on CD4+ T cells. Furthermore, a CD59a fusion protein bound specifically to mouse B cells, and enhanced B cell proliferation in a MLR, demonstrating that B cells express an as yet unidentified ligand for CD59a that aids in B cell activation.

CD59, a complement regulatory protein, controls choroidal neovascularization in a mouse model of wet-type age-related macular degeneration.

We have shown that membrane attack complex (MAC) formation via the activation of the alternative pathway plays a central role in the laser-induced choroidal neovascularization (CNV). This study was undertaken to understand the role of a complement regulatory protein, CD59, which controls MAC assembly and function, in this model. CNV was induced by laser photocoagulation in C57BL/6 and Cd59a(-/-) mice using an argon laser. Animals from each group were sacrificed on day 1, 3, 5, and 7 postlaser. Retinal pigment epithelium-choroid-scleral tissue was examined to determine the incidence and size of CNV complex, and semiquantitative RT-PCR and Western blot analysis for CD59a was studied. Recombinant soluble mouse CD59a-IgG2a fusion (rsCD59a-Fc) protein was injected via i.p. or intravitreal routes 24 h before laser. Our results demonstrated that CD59a (both mRNA and protein) was down-regulated during laser-induced CNV. Cd59a(-/-) mice developed CNV complex early in the disease process. Increased MAC deposition was also observed in these Cd59a(-/-) mice. Administration of rsCD59a-Fc inhibited the development of CNV complex in the mouse model by blocking MAC formation and also inhibited expression of angiogenic growth factors. These data provide strong evidence that CD59a plays a crucial role in regulating complement activation and MAC formation essential for the release of growth factors that drive the development of laser-induced CNV in mice. Thus, our results suggest that the inhibition of complement by soluble CD59 may provide a novel therapeutic alternative to current treatment.

Alternative roles for CD59.

CD59 was first identified as a regulator of the terminal pathway of complement, which acts by binding to the C8/C9 components of the assembling membrane attack complex (MAC), to inhibit formation of the lytic pore. Structurally, CD59 is a small, highly glycosylated, GPI-linked protein, with a wide expression profile. Functionally, the role of CD59 in complement regulation is well-defined but studies have also shown clear evidence for signalling properties, which are linked to its glycophosphatidyl inositol (GPI) anchor and its location within lipid rafts. Cross-linking of CD59 using specific monoclonal antibodies drives both calcium release and activation of lipid-raft associated signalling molecules such as tyrosine kinases. These observations clearly show that CD59 exhibits roles independent of its function as a complement inhibitor. In this review, we examine the progression of research in this area and explore the alternative functions of CD59 that have recently been defined.

p53 regulates cellular resistance to complement lysis through enhanced expression of CD59.

It has been recently hypothesized that the CD59 gene has two putative p53-responsive elements that may be involved in defense of host cells from damage by the complement system in inflammation. Here we have examined the roles of these putative p53-binding sequences within the CD59 gene in regulation of CD59 expression. We have shown that both of these potential responsive elements bind p53 in vitro. Knocking down expression of p53 using small interfering RNA led to a 6-fold decrease in CD59 protein expression in HeLa cells. We have previously observed a decrease of CD59 in camptothecin-induced apoptotic IMR32 cells, whereas expression was increased in the surviving fraction compared with untreated cells. Here, we have shown that these changes are associated with altered expression levels and acetylation status of p53. We have also shown that acetylation status of p53 regulates CD59 expression on cells exposed to inflammatory cytokines to model inflammation. Our data suggest that p53 and in vivo positive/negative regulators of p53 could be used to modulate susceptibility of tumor cells to complement lysis in chemotherapy.

Cutting edge: murine CD59a modulates antiviral CD4+ T cell activity in a complement-independent manner.

CD59 blocks formation of the membrane attack complex of complement by inhibiting binding of C9 to the C5b-8 complex. To investigate a role for CD59 in promoting T cell responses, we compared T cell activation in CD59a-deficient (Cd59a-/-) and wild-type (WT) mice after in vitro stimulation and after infection with rVV. Virus-specific CD4+ T cell responses were significantly enhanced in Cd59a-/- mice compared with WT mice. Similarly, Cd59a-/- T cells responded more vigorously to in vitro stimulation with CD3-specific Abs compared with WT mice. This effect of CD59a on T cell proliferation was found to be complement-independent. Collectively, these results demonstrate that CD59a down-modulates CD4+ T cell activity in vitro and in vivo, thereby revealing another link between complement regulators and T cell activation.