C3-deficient Animals Research Articles

Decreased sialylation elicits complement-related microglia response and bipolar cell loss in the mouse retina.

Sialylation plays an important role in self-recognition, as well as keeping the complement and innate immune systems in check. It is unclear whether the reduced sialylation seen during aging and in mice heterozygous for the null mutant of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (Gne+/-), an essential enzyme for sialic acid biosynthesis, contributes to retinal inflammation and degeneration. We found a reduction of polysialic acid and trisialic acid expression in several retinal layers in Gne+/- mice at 9 months of age compared to Gne+/+ wildtype (WT) mice, which was associated with a higher microglial expression of the lysosomal marker CD68. Furthermore, the total number of rod bipolar cells was reduced in 12 months old Gne+/- mice in comparison to WT mice, demonstrating loss of these retinal interneurons. Transcriptome analysis showed up-regulation of complement, inflammation, and apoptosis-related pathways in the retinas of Gne+/- mice. Particularly, increased gene transcript levels of the complement factors C3 and C4 and the pro-inflammatory cytokine Il-1β were observed by semi-quantitative real-time polymerase chain reaction (sqRT-PCR) in 9 months old Gne+/- mice compared to WT mice. The increased expression of CD68, loss of rod bipolar cells, and increased gene transcription of complement factor C4, were all prevented after crossing Gne+/- mice with complement factor C3-deficient animals. In conclusion, our data show that retinal hyposialylation in 9 and 12 months old Gne+/- mice was associated with complement-related inflammation and lysosomal microglia response, as well as rod bipolar cells loss, which was absent after genetic deletion of complement factor C3.

Complement C4-deficient mice have a high mortality rate during PTZ-induced epileptic seizures, which correlates with cognitive problems and the deficiency in the expression of Egr1 and other immediate early genes.

Complement system plays an important role in the immune defense against pathogens; however, recent studies demonstrated an important role of complement subunits C1q, C4, and C3 in normal functions of the central nervous system (CNS) such as non-functional synapse elimination (synapse pruning), and during various neurologic pathologies. Humans have two forms of C4 protein encoded by C4A and C4B genes that share 99.5% homology, while mice have only one C4B gene that is functionally active in the complement cascade. Overexpression of the human C4A gene was shown to contribute to the development of schizophrenia by mediating extensive synapse pruning through the activation C1q-C4-C3 pathway, while C4B deficiency or low levels of C4B expression were shown to relate to the development of schizophrenia and autism spectrum disorders possibly via other mechanisms not related to synapse elimination. To investigate the potential role of C4B in neuronal functions not related to synapse pruning, we compared wildtype (WT) mice with C3- and C4B- deficient animals for their susceptibility to pentylenetetrazole (PTZ)- induced epileptic seizures. We found that C4B (but not C3)-deficient mice were highly susceptible to convulsant and subconvulsant doses of PTZ when compared to WT controls. Further gene expression analysis revealed that in contrast to WT or C3-deficient animals, C4B-deficient mice failed to upregulate expressions of multiple immediate early genes (IEGs) Egrs1-4, c-Fos, c-Jus, FosB, Npas4, and Nur77 during epileptic seizures. Moreover, C4B-deficient mice had low levels of baseline expression of Egr1 on mRNA and protein levels, which was correlated with the cognitive problems of these animals. C4-deficient animals also failed to upregulate several genes downstream of IEGs such as BDNF and pro-inflammatory cytokines IL-1β, IL-6, and TNF. Taken together, our study demonstrates a new role of C4B in the regulation of expression of IEGs and their downstream targets during CNS insults such as epileptic seizures.

Antibody-enhanced cross-presentation of self antigen breaks T cell tolerance

We have developed a model of autoimmunity to investigate autoantibody-mediated cross-presentation of self antigen. RIP-mOVA mice, expressing OVA in pancreatic beta cells, develop severe autoimmune diabetes when given OT-I cells (OVA-specific CD8(+) T cells) and anti-OVA IgG but not when given T cells alone. Anti-OVA IgG is not directly injurious to the islets but rather enhances cross-presentation of apoptotic islet antigen to the OT-I cells, leading to their differentiation into potent effector cells. Antibody-driven effector T cell activation is dependent on the presence of activating Fc receptors for IgG (FcgammaRs) and cross-priming DCs. As a consequence, diabetes incidence and severity was reduced in mice lacking activating FcgammaRs. An intact complement pathway was also required for disease development, as C3 deficiency was also partially protective. C3-deficient animals exhibited augmented T cell priming overall, indicating a proinflammatory role for complement activation after the T cell priming phase. Thus, we show that autoreactive antibody can potently enhance the activation of effector T cells in response to cross-presented self antigen, thereby contributing to T cell-mediated autoimmunity.

Complement Plays an Important Role in Spinal Cord Injury and Represents a Therapeutic Target for Improving Recovery following Trauma

Initiation of an inflammatory cascade following traumatic spinal cord injury (SCI) is thought to cause secondary injury and to adversely impact functional recovery, although the mechanisms involved are not well defined. We report on the dynamics of complement activation and deposition in the mouse spinal cord following traumatic injury, the role of complement in the development of SCI, and the characterization of a novel targeted complement inhibitor. Following traumatic injury, mice deficient in C3 had a significantly improved locomotor score when compared with wild-type controls, and analysis of their spinal cords revealed significantly more tissue sparing, with significantly less necrosis, demyelination, and neutrophil infiltration. Wild-type mice were also treated with CR2-Crry, a novel inhibitor of complement activation that targets to sites of C3 deposition. A single intravenous injection of CR2-Crry 1 hour after traumatic injury improved functional outcome and pathology to an extent similar to that seen in C3-deficient animals. CR2-Crry specifically targeted to the injured spinal cord in a distribution pattern corresponding to that seen for deposited C3. As immunosuppression is undesirable in patients following SCI, targeted CR2-Crry may provide appropriate bioavailability to treat SCI at a dose that does not significantly affect systemic levels of serum complement activity.

Immunodeficient Mice Are Poor Mobilizers -Novel Evidence That Demonstrates a Pivotal Role of Complement in Triggering Mobilization of HSPC.

Complement (C) is activated by immunoglobulin-dependent classical and immunoglobulin-independent alternative pathways, and we recently reported that the activation of C enhances the responsiveness of hematopoietic stem/progenitor cells (HSPC) to an SDF-1 gradient cascade by releasing C3a and desArgC3a cleavage fragments (Blood 2003; 101:3784; Blood 2004; 103: 2071). We also found that bone marrow (BM) concentration of C3a and desArgC3a increases during mobilization with G-CSF or cyclophosphamide (CY). To explain this phenomenon we envisioned that these mobilizing agents expose a neo-antigen in BM tissue by turning BM into a highly proteolytic microenvironment. As a consequence of this, the newly exposed neo-epitope becomes bound by natural IgM antibodies leading to the activation of the classical C cascade. In our studies to elucidate the role of C activation in triggering the mobilization of HSPC, we found that C is effectively activated in the BM of G-CSF- or CY- mobilized wild-type (wt) but not IgM-deficient (RAG2null) mice. More importantly we found that several immunodeficient murine strains (RAG2null, SCID and Xid) displayed severely reduced G-CSF-induced mobilization of HSPC which we believe is a result of lack of B lymphocytes and complement-activating immunoglobulins. Supporting this, we found T cell depletion in wt mice did not affect mobilization. Moreover, G-CSF-induced mobilization in RAG2null, SCID and Xid animals was restored after infusion of murine inmmunoglobulins. Furthermore, since mobilization by zymosan or sulfated glycans activates C via the alternative immunoglobulin-independent pathway, we focused on the role of C activation during zymosan-induced mobilization. As expected, zymosan-induced immunoglobulin-independent C activation and mobilization of HSPC were unaffected in immunodeficient (RAG2null, SCID and Xid) mice which have a normal serum level of C3. However, these processes were severely reduced in C3-deficient animals. Thus our data strongly support the notion that C and innate immunity is an important trigger of mobilization of HSPC. While G-CSF and cyclophosphamide activate C by a classical IgM-dependent pathway, zymosan and sulfated polyglycans activate it by employing an alternative pathway. In conclusion, mobilization of HSPC could be envisioned as part of a more global immune response that is triggered/mediated by C3 activation/cleavage.

Transplantation studies in C3-deficient animals reveal a novel role of the third complement component (C3) in engraftment of bone marrow cells.

Mice deficient in complement C3 (C3(-/-)) are hematologically normal under steady-state conditions, and yet displayed a significant delay in hematopoietic recovery from either irradiation or transplantation of wild-type (WT) hematopoietic stem/progenitor cells (HSPC). Transplantation of histocompatible WT Sca-1(+) cells into C3(-/-) mice resulted in a (i) decrease in day 12 CFU-S, (ii) 5-7-day delay in platelet and leukocyte recovery, and (iii) reduced number of BM CFU-GM progenitors at day 16 after transplantation. Nevertheless, HSPC from C3(-/-) mice engrafted normally into irradiated WT mice, suggesting that there was a defect in the hematopoietic environment of C3(-/-) mice. Since C3(-/-) mice cannot activate/cleave C3, the C3 fragments C3a, C3a(des-Arg), and iC3b were examined for a role in HSPC engraftment. Liquid-phase C3a and C3a(des-Arg) increased CXCR4 incorporation into membrane lipid rafts (thus potentiating HSPC responses to SDF-1 gradients), whereas iC3b was deposited onto irradiated BM cells and functioned to tether CR3(CD11b/CD18)(+)HSPC to damaged stroma. The activity of C3a(des-Arg) suggested that C3aR(+)HSPC also expressed the C5L2 (receptor for C3a and C3a(des-Arg)) and this was confirmed. In conclusion, a novel mechanism for HSC engraftment was identified, which involves complement activation and specific C3 fragments that promote conditioning for transplantation and enhance HSPC engraftment.

Detection of two variants of complement component C3 in C3-deficient guinea pigs distinguished by the absence and presence of a thiolester

The complement system is an essential part of the innate defense, and C3 is an integral part of this powerful system. In previously identified complement C3 deficient guinea pigs only approx. 5% of the normal serum C3 level is detectable. No differences were found between in vitro C3 protein synthesis and C3 mRNA levels of cells from C3-deficient and wild-type animals and the amino acid sequences of both C3 proteins are identical as deduced from cDNA sequencing. Previously, the principal inability to form a C3 thiolester was discussed as a possible reason for this C3-deficiency. Here we report the isolation of two functionally different C3 species from the C3-deficient animals. Only one of these C3 proteins exhibits normal hemolytic activity and contains a thiolester group. The second C3 species is exclusively present in C3-deficient animals and lacks a thiolester, explaining its failure to express hemolytic activity. The presence of a second C3 species lacking a thiolester structure only in C3-deficient animals indicates that the stability of the thiolester may play a role in C3 deficiency. However further analysis of the in vitro stability of the thiolesters of C3 from normal and C3-deficient guinea pigs revealed no differences. A decreased in vivo thiolester stability might lead to the presence of C3 with and without a thiolester or alternatively the expression of two isoforms of C3 in these animals. Considering the central role of C3 in host defense, the mechanisms of C3 thiolester formation require further analysis.

Herpes simplex virus type 1 glycoprotein gC mediates immune evasion in vivo.

Many microorganisms encode proteins that interact with molecules involved in host immunity; however, few of these molecules have been proven to promote immune evasion in vivo. Herpes simplex virus type 1 (HSV-1) glycoprotein C (gC) binds complement component C3 and inhibits complement-mediated virus neutralization and lysis of infected cells in vitro. To investigate the importance of the interaction between gC and C3 in vivo, we studied the virulence of a gC-null strain in complement-intact and C3-deficient animals. Using a vaginal infection model in complement-intact guinea pigs, we showed that gC-null virus grows to lower titers and produces less severe vaginitis than wild-type or gC rescued virus, indicating a role for gC in virulence. To determine the importance of complement, studies were performed with C3-deficient guinea pigs; the results demonstrated significant increases in vaginal titers of gC-null virus, while wild-type and gC rescued viruses showed nonsignificant changes in titers. Similar findings were observed for mice where gC null virus produced significantly less disease than gC rescued virus at the skin inoculation site. Proof that C3 is important was provided by studies of C3 knockout mice, where disease scores of gC-null virus were significantly higher than in complement-intact mice. The results indicate that gC-null virus is approximately 100-fold (2 log10) less virulent that wild-type virus in animals and that gC-C3 interactions are involved in pathogenesis.

Molecular Analysis of the Third Component of Canine Complement (C3) and Identification of the Mutation Responsible for Hereditary Canine C3 Deficiency

Genetically determined deficiency of the third component of complement (C3) in the dog is characterized by a predisposition to recurrent bacterial infections and to type 1 membranoproliferative glomerulonephritis. The current studies were undertaken to characterize the cDNA for wild-type canine C3 and identify the molecular basis for hereditary canine C3 deficiency. Amplification, cloning, and sequence analysis indicated that canine C3 is highly conserved in comparison with human, mouse, and guinea pig C3. Southern blot analysis failed to show any gross deletions or rearrangements of DNA from C3-deficient animals. Northern blot analysis indicated that the livers of these animals contain markedly reduced quantities of a normal length C3 mRNA. The full-length 5.1-kb canine C3 cDNA was amplified in overlapping PCR fragments. Sequence analysis of these fragments has shown a deletion of a cytosine at position 2136 (codon 712), leading to a frameshift that generates a stop codon 11 amino acids downstream. The deletion has been confirmed in genomic DNA, and its inheritance has been demonstrated by allele-specific oligonucleotide hybridization.

C3-Independent Glomerulonephritis in Guinea Pigs: Dependence upon Primary Humoral Response

We used C3-deficient (C3D) guinea pigs to evaluate the role of C3 in an active model of experimental nephritis. Normal strain 2 (C3N, n = 13) and C3D (n = 6) guinea pigs were immunized with cationized bovine γ-globulin (CBGG). Fourteen days later (Day 0), daily intravenous injections of CBGG were given for 3 to 7 days and the animals were sacrificed on Day 10 or 21. Immunofluorescence (IF) microscopy of renal tissue revealed two patterns of glomerular IgG deposition: granular loop (11/13 C3N, 3/6 C3D), and predominantly mesangial (2/13 C3N, 3/6 C3D). Codeposited C3 was seen in all C3N and in no C3D animals. Electron microscopy showed subepithelial deposits in all. A significant correlation (P < 0.005) was seen between an animal's IF pattern and its level of serum antibodies to CBGG; those with lower antibody levels exhibited the mesangial pattern. C3D animals had lower mean antibody levels than C3N (P < 0.01), but both IF patterns were represented. Urine protein concentration, which was increased relative to controls, did not differ between C3N and C3D groups, but was significantly greater in those with loop IF. Serum albumin was significantly reduced in animals with loop IF. C3N animals showed a significant reduction in mean serum C3. In this model, immune deposit location and degree of proteinuria are independent of C3 deposition and dependent upon the level of antibody response to CBGG. Induction of antibody to CBGG is impaired in the absence of C3.

The third component of complement protects against Escherichia coli endotoxin-induced shock and multiple organ failure.

We investigated whether the third component of complement (C3) is involved in the pathophysiology of endotoxic shock, and if it is involved, whether it plays a protective role or whether it mediates shock and multiple organ failure. In a prospective, controlled investigation, six Brittany spaniels that were homozygous for a genetically determined deficiency of C3 (C3 deficient, < 0.003% of normal serum C3 levels) and six heterozygous littermates (controls, approximately 50% of mean normal serum C3 level) were given 2 mg/kg of reconstituted Escherichia coli 026:B6 acetone powder as a source of endotoxin, intravenously. All animals were given similar fluid and prophylactic antibiotic therapy, and had serial hemodynamic variables obtained. After E. coli endotoxin infusion, C3-deficient animals had higher peak levels of endotoxin and less of a rise in temperature than controls (P < 0.05). During the first 4 h after E. coli endotoxin infusion, C3-deficient animals had significantly greater decreases in mean central venous pressure and mean pulmonary artery pressure than controls (P < 0.02). During the first 48 h after E. coli endotoxin infusion, C3-deficient animals had significantly greater decreases in mean arterial pH, left ventricular ejection fraction, and mean pulmonary capillary wedge pressure, and greater increases in mean arterial lactate, arterial-alveolar O2 gradient, and transaminases (aspartate aminotransferase and alanine aminotransferase) than controls, (all P < 0.05). After E. coli endotoxin infusion, C3-deficient animals compared to controls had significantly less of a decrease in mean C5 levels (P < 0.01), but similar (P = NS) increases in circulating tumor necrosis factor levels, bronchoalveolar lavage neutrophils, and protein, and similar (P = NS) decreases in blood leukocytes and platelets. Two of six C3-deficient animals and two of six controls died. In summary, after intravenous infusion of E. coli endotoxin, canines with C3 deficiency have decreased endotoxin clearance and worse E. coli endotoxin-induced shock and organ damage. Thus, the third component of the complement system plays a beneficial role in the host defense against E. coli endotoxic shock.

Interaction of human CR1, CR2, and h with expressed C3 and chimeric C3 molecules

The complement system is an essential part of the innate defense, and C3 is an integral part of this powerful system. In previously identified complement C3 deficient guinea pigs only approx. 5% of the normal serum C3 level is detectable. No differences were found between in vitro C3 protein synthesis and C3 mRNA levels of cells from C3-deficient and wild-type animals and the amino acid sequences of both C3 proteins are identical as deduced from cDNA sequencing. Previously, the principal inability to form a C3 thiolester was discussed as a possible reason for this C3-deficiency. Here we report the isolation of two functionally different C3 species from the C3-deficient animals. Only one of these C3 proteins exhibits normal hemolytic activity and contains a thiolester group. The second C3 species is exclusively present in C3-deficient animals and lacks a thiolester, explaining its failure to express hemolytic activity. The presence of a second C3 species lacking a thiolester structure only in C3-deficient animals indicates that the stability of the thiolester may play a role in C3 deficiency. However further analysis of the in vitro stability of the thiolesters of C3 from normal and C3-deficient guinea pigs revealed no differences. A decreased in vivo thiolester stability might lead to the presence of C3 with and without a thiolester or alternatively the expression of two isoforms of C3 in these animals. Considering the central role of C3 in host defense, the mechanisms of C3 thiolester formation require further analysis.

Phototoxic Erythema Following PUVA Treatment: Independence of Complement

The effect of PUVA treatment on normal human serum (NHS), on isolated PMN, or on C3-deficient guinea pigs and congenic (C3-competent) control animals was tested. At a concentration of 0.1 or 1 mM/l 8-MOP and UVA doses of 5-30 J/cm2, PUVA failed to induce any detectable C3-cleavage in NHS. Furthermore, when the complement (C) activation in NHS had been induced before or after PUVA treatment by various methods. PUVA did not modulate the extent of C3-cleavage. PUVA did not affect the viability of isolated PMN, nor did it induce a release of LDH or elastase. No differences between C3-deficient and C-competent guinea pig skin exposed to PUVA were observed in erythema or histologic responses. Immunohistologic examination of specimens from normal guinea pigs revealed C3b and C3d deposits on necrotic keratinocytes, findings restricted to the PUVA-treated areas. Necrosis of keratinocytes was present in skin specimens of C3-deficient animals from PUVA-treated sites to a similar extent. However, deposits of C3-related antigens were completely absent there. From these observations, we suggest that the induction of phototoxic erythema following PUVA treatment is independent of complement.

An inherited deficiency of the third component of complement, C3, in guinea pigs.

Hereditary deficiency of the third component of complement, C3, is found very seldom in the human. C3 deficiency is associated with severe bacterial infections revealing the central role of C3 in complement activation via the classical or alternative pathway. We describe a new hereditary C3 deficiency in strain 2 guinea pigs. Serum from these animals had a markedly reduced lytic activity in a standard assay for complement-dependent, antibody-mediated cytotoxicity. In functional assays of individual components, the hemolytic activity of the components C4, C2, C5 and of factors B, D and H was in the normal range. The functional C3 titer, and similarly C3 antigenic activity in the serum of these C3-deficient animals (C3D) was on average only 5.7% of normal activity. Typing the animals with alloantisera or monoclonal antibodies to guinea pig Ia-antigens revealed that the C3D animals had the major histocompatibility complex-haplotype of inbred strain 2 guinea pigs (B.1, Ia.2,4). The C3 defect is not linked to the major histocompatibility complex and, in addition, is not linked to a C3a receptor deficiency. Macrophages and hepatocytes of the C3D animals have an unimpaired capacity for synthesis and secretion of C3 as measured by enzyme-linked immunosorbent assay. There was no indication for hypercatabolism of normal C3 by the animals as shown by plasma clearance of 125I-radiolabeled C3. Thrombocytes of the C3D animals responded normally to stimulation with purified C3a in an ATP-release assay without an indication for a desensitization in vivo. Possibly the fault resides in an enhanced susceptibility of their own C3 to proteolysis. However, C3 partially purified from the plasma of the C3D animals or secreted by hepatocytes exhibited no obvious structural differences to purified normal C3 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis or in immunoblotting. The C3D serum had a reduced bactericidal activity compared to normal or to C4-deficient serum. Nevertheless, the animals are apparently healthy without an indication for increased frequency of bacterial infections. These guinea pigs provide an unique model for analysis of the biological functions of C3 in vivo and in vitro without the need for artificial C3-depletion procedures with all their known and unknown side-effects.