In response to complement anaphylatoxin peptides C3a and C5a, human vascular endothelial cells migrate and mediate the activation of B-cells and polarization of T-cells.

The vascular endothelium has been discovered in the past several years to be important in shaping the cellular immune response. During the immune response the vascular endothelium is constantly perturbed by biologically potent molecules, including the complement activation peptides, C3a and C5a. Despite the importance of C3a and C5a in inflammation and immunity, their role in modulating lymphocyte function via activation of vascular endothelial cells is unknown. Accordingly, we investigated the regulated expression of the C3a and C5a receptors (complement anaphylatoxin C3a receptor [C3aR] and complement anaphylatoxin C5a receptor 1 [C5aR1]) on human umbilical vascular endothelial cells (HUVECs) and examined how C3a or C5a activation of HUVECs affects the activation and polarization of lymphatic cells. Our findings demonstrated that C3a and C5a increase C3aR and C5aR1 expression by HUVECs as well as directing their cellular transmigration and spreading through transwell filters. Moreover, C3a- or C5a-stimulated endothelial cells: (1) caused activation of B-lymphoblasts with significant increase in Fas Ligand (CD95L) (FasL), CD69, and IL-R1 expression, and (2) skewed T-lymphoblast cells toward a Th1 subtype, (CD4+ /CCR5+ ) that correlated with significant increase of IFN-γ. Collectively, these data indicate that C3a and C5a signaling is important in the activation and polarization of lymphocytes as they traffic through the vascular endothelium during the immune response.

Complement Receptor 1 (CR1/CD35)-expressing retinal pigment epithelial cells as a potential therapy for age-related macular degeneration.

The purpose of this study was to identify a membrane-bound complement inhibitor that could be overexpressed on retinal pigment epithelial cells (RPE) providing a potential therapy for age-related macular degeneration (AMD). This type of therapy may allow replacement of damaged RPE with cells that are able to limit complement activation in the retina. Complement Receptor 1 (CR1) is a membrane-bound complement inhibitor commonly found on erythrocytes and immune cells. In this study, QPCR and flow cytometry data demonstrated that CR1 is not well-expressed by RPE, indicating that its overexpression may provide extra protection from complement activation. To screen CR1 for this ability, a stable CR1-expressing ARPE19 line was created using a combination of antibiotic selection and FACS. Cell-based assays were used to demonstrate that addition of CR1 inhibited deposition of complement proteins C3b and C6 on the transfected line. In the end, this study identifies CR1 as a complement inhibitor that may be overexpressed on stem cell-derived RPE to create a potential "enhanced" cell therapy for AMD. A combination cell/complement therapy may create transplantable RPE better suited to avoid complement-mediated lysis and limit chronic inflammation in the retina.

The Second Receptor for C5a, C5aR2, Is Detrimental to Mice during Systemic Infection with Listeria monocytogenes.

Infection with Listeria monocytogenes is acquired through ingestion of contaminated foods and may lead to systemic infection and possible death, with an overall 20% mortality rate. Our previous work using C5aR1-/- mice and C3aR-/- mice demonstrated that C5aR1 and C3aR both play powerful anti-inflammatory and prosurvival roles during systemic infection with L. monocytogenes In our current study, we have examined the role of the third anaphylatoxin receptor, C5aR2, in the host immune response to systemic L. monocytogenes infection. C5aR2-/- mice had significantly lower bacterial burdens in the spleens and livers on both day 1 and 3 postinfection compared with C5aR2+/+ mice. The decreased bacterial burdens in the C5aR2-/- mice correlated with less liver damage and with improved survival of CD4+ and CD8+ T cells in the spleen on day 3 postinfection compared with C5aR2+/+ mice. C5aR2-/- mice also produced significantly less G-CSF, IL-6, and MCP-1 in the serum, spleen, and liver on day 1 postinfection compared with C5aR2+/+ mice. C5aR2-/- and C5aR2+/+ mice produced similar amounts of IFN-γ in their spleens on day 1 postinfection. Purified naive splenocytes from C5aR2-/- mice produced significantly more IFN-γ and IL-12p70 during in vitro infection with L. monocytogenes compared with splenocytes from C5aR2+/+ mice in an NF-κB-dependent manner. Induction of IL-12 and IFN-γ early during infection with L. monocytogenes is protective to the host, and we believe this innate increased ability to produce more IL-12 and IFN-γ provided early protection to the C5aR2-/- mice.

Derivation and characterization of the human embryonic stem cell line CR-4: Differentiation to human retinal pigment epithelial cells.

The CR-4 human embryonic stem cell line was derived from the inner cell mass of a developing blastocyst. This cell line has been adapted to grow in feeder-free conditions and is especially well-suited for differentiation to retinal pigment epithelium. The line demonstrates a normal human 46,XX female karyotype. Pluripotency was assessed through qRT-PCR for expression of NANOG, OCT-4, and SOX-2. A teratoma assay was performed and results were positive for all three germ layers. Testing for Mycoplasma was negative.

The Complement Anaphylatoxins C5a and C3a Suppress IFN-ß Production in Response to Listeria monocytogenes by Inhibition of the Cyclic Dinucleotide-Activated Cytosolic Surveillance Pathway.

Listeria monocytogenes is an intracellular Gram-positive bacterium that induces expression of type I IFNs (IFN-α/IFN-ß) during infection. These cytokines are detrimental to the host during infection by priming leukocytes to undergo L. monocytogenes-mediated apoptosis. Our previous studies showed that C5aR1-/- and C3aR-/- mice are highly susceptible to L. monocytogenes infection as a result of increased IFN-ß-mediated apoptosis of major leukocyte cell populations, including CD4+ and CD8+ T cells. However, the mechanisms by which C3a and C5a modulate IFN-ß expression during L. monocytogenes infection were not examined in these initial investigations. Accordingly, we report in this article that C5a and C3a suppress IFN-ß production in response to L. monocytogenes via cyclic di-AMP (c-di-AMP), a secondary messenger molecule of L. monocytogenes, in J774A.1 macrophage-like cells and in bone marrow-derived dendritic cells (BMDCs). Moreover, C5a and C3a suppress IFN-ß production by acting through their respective receptors, because no inhibition was seen in C5aR1-/- or C3aR-/- BMDCs, respectively. C5a and C3a suppress IFN-ß production in a manner that is dependent on Bruton's tyrosine kinase, p38 MAPK, and TANK-binding kinase 1 (TBK1), as demonstrated by the individual use of Bruton's tyrosine kinase, p38 MAPK, and TBK1 inhibitors. Pretreatment of cells with C5a and C3a reduced the expression of the IFN-ß signaling molecules DDX41, STING, phosphorylated TBK1, and phosphorylated p38 MAPK in wild-type BMDCs following treatment with c-di-AMP. Collectively, these data demonstrate that C3a and C5a, via direct signaling through their specific receptors, suppress IFN-ß expression by modulation of a distinct innate cytosolic surveillance pathway involving DDX41, STING, and other downstream molecular targets of L. monocytogenes-generated c-di-AMP.

Innate and adaptive immunologic functions of complement in the host response to Listeria monocytogenes infection.

Listeria monocytogenes is a leading cause of foodborne-illness associated mortality that has attracted considerable attention in recent years due to several significant outbreaks. It has also served as a model organism for the study of intracellular pathogens. For these reasons the host response to L. monocytogenes has long been the subject of investigation. A potent innate and adaptive immune response is required for containment and clearance of L. monocytogenes. However, some elements of this response, such as type 1 interferons, can be detrimental to the host. Recent studies have revealed novel functions for the complement system, an ancient arm of innate immunity, in this process. Here we review the role of complement in the host response to L. monocytogenes.

The C5a anaphylatoxin receptor (C5aR1) protects against Listeria monocytogenes infection by inhibiting type 1 IFN expression.

Listeria monocytogenes is a major cause of mortality resulting from food poisoning in the United States. In mice, C5 has been genetically linked to host resistance to listeriosis. Despite this genetic association, it remains poorly understood how C5 and its activation products, C5a and C5b, confer host protection to this Gram-positive intracellular bacterium. In this article, we show in a systemic infection model that the major receptor for C5a, C5aR1, is required for a normal robust host immune response against L. monocytogenes. In comparison with wild-type mice, C5aR1(-/-) mice had reduced survival and increased bacterial burden in their livers and spleens. Infected C5aR1(-/-) mice exhibited a dramatic reduction in all major subsets of splenocytes, which was associated with elevated caspase-3 activity and increased TUNEL staining. Because type 1 IFN has been reported to impede the host response to L. monocytogenes through the promotion of splenocyte death, we examined the effect of C5aR1 on type 1 IFN expression in vivo. Indeed, serum levels of IFN-α and IFN-ß were significantly elevated in L. monocytogenes-infected C5aR1(-/-) mice. Similarly, the expression of TRAIL, a type 1 IFN target gene and a proapoptotic factor, was elevated in NK cells isolated from infected C5aR1(-/-) mice. Treatment of C5aR1(-/-) mice with a type 1 IFNR blocking Ab resulted in near-complete rescue of L. monocytogenes-induced mortality. Thus, these findings reveal a critical role for C5aR1 in host defense against L. monocytogenes through the suppression of type 1 IFN expression.

The receptor for the complement C3a anaphylatoxin (C3aR) provides host protection against Listeria monocytogenes-induced apoptosis.

Listeria monocytogenes is a Gram-positive intracellular bacterium that is acquired through tainted food and may lead to systemic infection and possible death. Despite the importance of the innate immune system in fighting L. monocytogenes infection, little is known about the role of complement and its activation products, including the potent C3a anaphylatoxin. In a model of systemic L. monocytogenes infection, we show that mice lacking the receptor for C3a (C3aR(-/-)) are significantly more sensitive to infection compared with wild-type mice, as demonstrated by decreased survival, increased bacterial burden, and increased damage to their livers and spleens. The inability of the C3aR(-/-) mice to clear the bacterial infection was not caused by defective macrophages or by a reduction in cytokines/chemokines known to be critical in the host response to L. monocytogenes, including IFN-γ and TNF-α. Instead, TUNEL staining, together with Fas, active caspase-3, and Bcl-2 expression data, indicates that the increased susceptibility of C3aR(-/-) mice to L. monocytogenes infection was largely caused by increased L. monocytogenes-induced apoptosis of myeloid and lymphoid cells in the spleen that are required for ultimate clearance of L. monocytogenes, including neutrophils, macrophages, dendritic cells, and T cells. These findings reveal an unexpected function of C3a/C3aR signaling during the host immune response that suppresses Fas expression and caspase-3 activity while increasing Bcl-2 expression, thereby providing protection to both myeloid and lymphoid cells against L. monocytogenes-induced apoptosis.

Carboxypeptidase N-deficient mice present with polymorphic disease phenotypes on induction of experimental autoimmune encephalomyelitis.

Carboxypeptidase N (CPN) is a member of the carboxypeptidase family of enzymes that cleave carboxy-terminal lysine and arginine residues from a large number of biologically active peptides and proteins. These enzymes are best known for their roles in modulating the activity of kinins, complement anaphylatoxins and coagulation proteins. Although CPN makes important contributions to acute inflammatory events, little is known about its role in autoimmune disease. In this study we used CPN(-/-) mice in experimental autoimmune encephalomyelitis (EAE), the animal model for multiple sclerosis. Unexpectedly, we observed several EAE disease phenotypes in CPN(-/-) mice compared to wild type mice. The majority of CPN(-/-) mice died within five to seven days after disease induction, before displaying clinical signs of disease. The remaining mice presented with either mild EAE or did not develop EAE. In addition, CPN(-/-) mice injected with complete or incomplete Freund's adjuvant died within the same time frame and in similar numbers as those induced for EAE. Overall, the course of EAE in CPN(-/-) mice was significantly delayed and attenuated compared to wild type mice. Spinal cord histopathology in CPN(-/-) mice revealed meningeal, but not parenchymal leukocyte infiltration, and minimal demyelination. Our results indicate that CPN plays an important role in EAE development and progression and suggests that multiple CPN ligands contribute to the disease phenotypes we observed.

Negative regulation of pulmonary Th17 responses by C3a anaphylatoxin during allergic inflammation in mice.

Activation of complement is one of the earliest immune responses to exogenous threats, resulting in various cleavage products including anaphylatoxin C3a. In addition to its contribution to host defense, C3a has been shown to mediate Th2 responses in animal models of asthma. However, the role of C3a on pulmonary Th17 responses during allergic inflammation remains unclear. Here, we show that mice deficient in C3a receptor (C3aR) exhibited (i) higher percentages of endogenous IL-17-producing CD4(+) T cells in the lungs, (ii) higher amounts of IL-17 in the bronchoalveolar lavage fluid, and (iii) more neutrophils in the lungs than wild-type mice when challenged with intranasal allergens. Moreover, adoptive transfer experiments showed that the frequencies of antigen-specific IL-17-producing CD4(+) T cells were significantly higher in the lungs and bronchial lymph nodes of C3aR-deficient recipients than those of wild-types recipients. Bone-marrow reconstitution study indicated that C3aR-deficiency on hematopoietic cells was required for the increased Th17 responses. Furthermore, C3aR-deficient mice exhibited increased percentages of Foxp3(+) regulatory T cells; however, depletion of these cells minimally affected the induction of antigen-specific Th17 cell population in the lungs. Neutralization of IL-17 significantly reduced the number of neutrophils in bronchoalveolar lavage fluid of C3aR-deficient mice. Our findings demonstrate that C3a signals negatively regulate antigen-specific Th17 responses during allergic lung inflammation and the size of Foxp3(+) regulatory T cell population in the periphery.

Role of C3a receptors, C5a receptors, and complement protein C6 deficiency in collagen antibody-induced arthritis in mice.

The complement system, especially the alternative pathway, plays essential roles in the induction of injury in collagen Ab-induced arthritis (CAIA) in mice. The goal of the current study was to directly compare the roles of receptors for C3a and C5a, as well as the membrane attack complex, as effector mechanisms in the pathogenesis of CAIA. Clinical disease activity in C3aR(-/-), C5aR(-/-), and C6-deficient (C6-def) mice was decreased by 52, 94, and 65%, respectively, as compared with wild-type mice. Decreases in histopathologic injury as well as in IgG and C3 deposition paralleled the clinical disease activity. A decrease in the percentage of synovial neutrophils was observed in C3aR(-/-), C5aR(-/-), and C6-def mice, and a decrease in macrophages was observed in C3aR(-/-) and C5aR(-/-), but not in C6-def, mice. Synovial mRNA obtained by laser capture microdissection exhibited a decrease in TNF-α in C5aR(-/-) mice and in IL-1ß in both C5aR(-/-) and C6-def mice, whereas C3aR(-/-) mice demonstrated no change in either cytokine. Our findings show that absent C3aR-, C5aR-, or membrane attack complex-initiated effector mechanisms each decrease susceptibility to CAIA, with clinical effects most pronounced in C5aR-deficient mice. Although the absence of C3aR, C5aR, or C6 led to differential deficiencies in effector mechanisms, decreased proximal joint IgG and C3 deposition was common to all three genotypes in comparison with wild-type mice. These data suggest the existence of positive-feedback amplification pathways downstream of all three effectors that promote additional IgG deposition and C3 activation in the joint.

Targeted disruption of the gene encoding the murine small subunit of carboxypeptidase N (CPN1) causes susceptibility to C5a anaphylatoxin-mediated shock.

Carboxypeptidase N (CPN) is a plasma zinc metalloprotease, which consists of two enzymatically active small subunits (CPN1) and two large subunits (CPN2) that protect the protein from degradation. Historically, CPN has been implicated as a major regulator of inflammation by its enzymatic cleavage of functionally important arginine and lysine amino acids from potent phlogistic molecules, such as the complement anaphylatoxins C3a and C5a. Because of no known complete CPN deficiencies, the biological impact of CPN in vivo has been difficult to evaluate. Here, we report the generation of a mouse with complete CPN deficiency by targeted disruption of the CPN1 gene. CPN1(-/-) mice were hypersensitive to lethal anaphylactic shock due to acute complement activation by cobra venom factor. This hypersensitivity was completely resolved in CPN1(-/-)/C5aR(-/-) but not in CPN1(-/-)/C3aR(-/-) mice. Moreover, CPN1(-/-) mice given C5a i.v., but not C3a, experienced 100% mortality. This C5a-induced mortality was reduced to 20% when CPN1(-/-) mice were treated with an antihistamine before C5a challenge. These studies describe for the first time a complete deficiency of CPN and demonstrate 1) that CPN plays a requisite role in regulating the lethal effects of anaphylatoxin-mediated shock, 2) that these lethal effects are mediated predominantly by C5a-induced histamine release, and 3) that C3a does not contribute significantly to shock following acute complement activation.

Disruption of the C5a receptor gene increases resistance to acute Gram-negative bacteremia and endotoxic shock: opposing roles of C3a and C5a.

The host response to intravascular, Gram-negative bacteria includes profound immunologic, hematologic and physiologic changes. Numerous host defense mechanisms are activated by Gram-negative bacteria, including the complement system. Activation of the complement system leads to cleavage of C5 with subsequent generation of the C5a anaphylatoxin peptide. C5a mediates potent, proinflammatory activities by binding to the C5a receptor (C5aR, CD88). In this study, we report the targeted disruption of the murine C5aR gene (C5aR-/- mice) and define the role of the C5aR in a model of Gram-negative bacteremia. Following an intravenous infusion of heat-killed Escherichia coli, the C5aR-/- mice were completely protected from the mortality suffered by their wild-type littermates (P<0.001). The C5aR-/- mice were also significantly (P=0.008) more resistant to mortality following an intravenous infusion of purified E. coli endotoxin compared to the wild-type littermates. In addition, the C5aR-/- mice were resistant to the thrombocytopenia and hemoconcentration observed in wild-type animals. Lethality in the wild-type mice was reversed by pre-treatment with either the histamine antagonist diphenhydramine or triprolidine. The wild-type littermates were also rescued following pre-treatment with the basophil and mast cell-stabilizing agent - cromolyn sodium. Collectively, these data demonstrate that not only is the absence of the C5aR protective in E. coli bacteremia, but that C5aR-dependent histamine release plays a major role in shock induced by Gram-negative septicemia. Moreover, they provide additional in vivo evidence that C3a and C5a have divergent biological functions in Gram-negative bacteremia and shock.

Ablation of the complement C3a anaphylatoxin receptor causes enhanced killing of Pseudomonas aeruginosa in a mouse model of pneumonia.

The C3a anaphylatoxin is a 77-amino acid peptide that is generated by enzymatic cleavage of C3 during activation of the complement system. C3a mediates numerous biological functions on binding its receptor (C3aR), which is present on both myeloid and nonmyeloid cells. To investigate the biological impact of C3a-mediated effects during acute pneumonia caused by Pseudomonas aeruginosa, we subjected C3aR-deficient mice and matched wild-type (WT) mice to P. aeruginosa pulmonary infection. C3aR-deficient mice exhibited increased killing of P. aeruginosa in the lungs, less dissemination of bacteria into the bloodstream, and a decreased inflammatory response to P. aeruginosa pulmonary infection compared with WT mice. To examine whether the absence of C3aR would impact the humoral immune response to P. aeruginosa, we immunized WT and C3aR-deficient mice via intraperitoneal injection with live P. aeruginosa. Both groups of mice developed similar levels of antibody specific to P. aeruginosa. Immunized C3aR-deficient and WT mice were subjected to P. aeruginosa pulmonary infection, and C3aR-deficient mice again displayed increased killing of P. aeruginosa in the lungs, less dissemination of bacteria into the bloodstream, and a decreased inflammatory response in the lungs. Collectively, these data demonstrate that independently of antibody production, absence of C3aR causes enhanced killing of P. aeruginosa despite a diminished inflammatory response in a mouse model of pneumonia.

The alternative activation pathway and complement component C3 are critical for a protective immune response against Pseudomonas aeruginosa in a murine model of pneumonia.

Pseudomonas aeruginosa is a leading cause of hospital-acquired pneumonia, and approximately 80% of patients with cystic fibrosis are infected with this bacterium. To investigate the overall role of complement and the complement activation pathways in the host defense against P. aeruginosa pulmonary infection, we challenged C3-, C4-, and factor B-deficient mice with P. aeruginosa via intranasal inoculation. In these studies, C3(-/-) mice had a higher mortality rate than C3(+/+) mice. Factor B(-/-) mice, but not C4(-/-) mice, infected with P. aeruginosa had a mortality rate similar to that of C3(-/-) mice, indicating that in this model the alternative pathway of complement activation is required for the host defense against Pseudomonas infection. C3(-/-) mice had 6- to 7-fold more bacteria in the lungs and 48-fold more bacteria in the blood than did C3(+/+) mice at 24 h postinfection. In vitro, phagocytic cells from C3(+/+) or C3(-/-) mice exhibited a decreased ability to bind and/or ingest P. aeruginosa in the presence of C3-deficient serum compared to phagocytic cells in the presence of serum with sufficient C3. C3(-/-) mice displayed a significant increase in neutrophils in the lungs and had higher levels of interleukin-1beta (IL-1beta), IL-6, IL-10, KC, and MIP-2 in the lungs at 24 h postinfection than did C3(+/+) mice. Collectively, these results indicate that complement activation by the alternative pathway is critical for the survival of mice infected with P. aeruginosa and that the protection provided by complement is at least in part due to C3-mediated opsonization and phagocytosis of P. aeruginosa.

Carboxypeptidase N: a pleiotropic regulator of inflammation.

Carboxypeptidase N (CPN) is a plasma zinc metalloprotease, which consists of two enzymatically active small subunits (CPN1) and two large subunits (CPN2) that protect the protein from degradation. CPN cleaves carboxy-terminal arginines and lysines from peptides found in the bloodstream such as complement anaphylatoxins, kinins, and creatine kinase MM (CK-MM). By removing only one amino acid, CPN has the ability to change peptide activity and receptor binding. CPN is a member of a larger family of carboxypeptidases, many of which also cleave arginine and lysine. Because of the highly conserved active sites and the possible redundant functions of carboxypeptidases, it has been difficult to elucidate the role of CPN in disease processes. The future use of gene ablation technology may be the most appropriate way to understand the function of CPN in vivo.

Decreased infectivity despite unaltered C3 binding by a DeltahbhA mutant of Mycobacterium tuberculosis.

HbhA of Mycobacterium tuberculosis is a multifunctional binding protein, binding to both sulfated sugars such as heparin and to human complement component C3. HbhA may therefore interact with host molecules and/or host cells during M. tuberculosis infection and play a role in the pathogenesis of this bacterium. The purpose of this study was to use allelic exchange to create an M. tuberculosis strain deficient in expression of HbhA to determine whether this protein's C3-binding activity plays a role in the pathogenesis of M. tuberculosis. An in-frame, 576-bp unmarked deletion in the hbhA gene was created using sacB as a counterselectable marker. Southern blotting and PCR analyses confirmed deletion of hbhA in the DeltahbhA mutant. The DeltahbhA mutant strain grew at a rate similar to that of the parent in broth culture and in J774.A1 murine macrophage-like cells but was deficient in growth compared to the parent strain in the lungs, liver, and spleen of infected mice. In addition, the DeltahbhA mutation did not reduce binding of M. tuberculosis to human C3 or to J774.A1 cells in the presence or absence of serum, suggesting that in the absence of HbhA, other molecules serve as C3-binding molecules on the M. tuberculosis surface. Taken together, these data indicate that HbhA is important in the infectivity of M. tuberculosis, but its ability to bind C3 is not required for mycobacterial adherence to macrophage-like cells. Using the DeltahbhA mutant strain, a second M. tuberculosis C3-binding protein similar in size to HbhA was identified as HupB, but the role of HupB as a C3-binding protein in intact organisms remains to be determined.