Hector Molina

Complement C3 Activation Is Required for Antiphospholipid Antibody-induced Fetal Loss

The antiphospholipid syndrome (APS) is characterized by recurrent fetal loss, vascular thrombosis, and thrombocytopenia occurring in the presence of antiphospholipid (aPL) antibodies. The pathogenesis of fetal loss and tissue injury in APS is incompletely understood, but is thought to involve platelet and endothelial cell activation as well as procoagulant effects of aPL antibodies acting directly on clotting pathway components. Recent studies have shown that uncontrolled complement activation in the placenta leads to fetal death in utero. We hypothesized that aPL antibodies activate complement in the placenta, generating split products that mediate placental injury and lead to fetal loss and growth retardation. To test this hypothesis, we used a murine model of APS in which pregnant mice are injected with human IgG containing aPL antibodies. We found that inhibition of the complement cascade in vivo, using the C3 convertase inhibitor complement receptor 1–related gene/protein y (Crry)-Ig, blocks fetal loss and growth retardation. Furthermore, mice deficient in complement C3 were resistant to fetal injury induced by aPL antibodies. While antigenic epitopes recognized by aPL antibodies are important in the pathogenesis of APS, our data show that in vivo complement activation is required for aPL antibody-induced fetal loss and growth retardation.

Complement facilitates early prion pathogenesis

New-variant Creutzfeldt–Jakob disease and scrapie are typically initiated by extracerebral exposure to the causative agent, and exhibit early prion replication in lymphoid organs. In mouse scrapie, depletion of B-lymphocytes prevents neuropathogenesis after intraperitoneal inoculation, probably due to impaired lymphotoxin-dependent maturation of follicular dendritic cells (FDCs), which are a major extracerebral prion reservoir. FDCs trap immune complexes with Fc-γ receptors and C3d/C4b-opsonized antigens with CD21/CD35 complement receptors. We examined whether these mechanisms participate in peripheral prion pathogenesis. Depletion of circulating immunoglobulins or of individual Fc-γ receptors had no effect on scrapie pathogenesis if B-cell maturation was unaffected. However, mice deficient in C3, C1q, Bf/C2, combinations thereof or complement receptors were partially or fully protected against spongiform encephalopathy upon intraperitoneal exposure to limiting amounts of prions. Splenic accumulation of prion infectivity and PrPSc was delayed, indicating that activation of specific complement components is involved in the initial trapping of prions in lymphoreticular organs early after infection.

Tolerance is dependent on complement C3 fragment iC3b binding to antigen-presenting cells.

Systemic tolerance can be induced by the introduction of antigen into an immune-privileged site. Here we investigated the role of complement in the induction of tolerance after intraocular injection. We found that the development of antigen-specific tolerance is dependent on a complement activation product. The ligation of the complement C3 activation product iC3b to complement receptor type 3 (the iC3b receptor) on antigen-presenting cells resulted in the sequential production of transforming growth factor-β2 and interleukin-10, which is essential for the induction of tolerance. These observations may extend to the development of both neonatal tolerance and other forms of acquired tolerance.

Lymphotoxin‐α‐deficient and TNF receptor‐I‐deficient mice define developmental and functional characteristics of germinal centers

Summary: Mice deficient in LTa (LTα‐/‐) lack lymph nodes and Peyers patches. This action of LTa in lymph node organogenesis appears to be mediated by the membrane form of LT using a mechanism independent of TNF receptor I (TNFR‐I) or II (TNFR‐II). In contrast, normal Peyers patch development appears to require both LTa and TNFR‐I, with TNFR‐I‐/‐ mice showing hypoplastic Peyers patch structures. LTα‐/‐ mice also fail to support the normal segregation of T‐cell and B‐cell zones within the splenic white ptilp. Again, this occurs via a mechanism independent of TNFR‐I or TNFR‐II. Additionally, follicular dendritic cell (FDC) dusters or germinal centers fail to develop in the spleen of LTα‐/‐ animals. Mice defi‐cient in either TNFα or TNFR‐I also fail to develop splenic FDC dusters and germinal centers, indicating that signaling by both LTα and TNFα is required for development of these speciahzed lymphoid tissue structures. Finally, the splenic white pulp areas in LTα‐/‐ mice lack the marginal zone of monoclonal antibody MOMA‐1‐staining metallophilic macrophages, whereas TNFR‐I‐deficient mice have preserved MOMA‐1 staining. Thus, certain actions of LTa to regulate spleen white pulp architecture are medi‐ated by receptors other than TNFR‐I, most likely by the LTβR or a closely related receptor. We tested whether germinal centers are essential for mat‐uration of T‐cell‐dependent antibody responses. When LTα‐/‐ mice were immunized with low doses of NP‐ovalbumin (NP‐OVA) adsorbed to alum, there was dramatically impaired production of high afTmity anti‐NP IgG; however, after immunization with high doses of NP‐OVA adsorbed to alum, LTα‐/‐ mice mounted a high affinity NF‐specific serum IgG response similar to wild‐type mice, all in the absence of germinal centers or clus‐tered FDC. Thus, although germinal centers enhance the processes required for maturation of the humoral immune response, the mecha‐nisms responsible for affinity maitiration are not absolutely dependent on the presence of germinal centers.

The evolution of mouse and human complement C3-binding proteins: divergence of form but conservation of function.

Despite the fact that the early components of the mouse and human complement cascades are very similar, there are marked differences between the two species in the structure of C3 receptors and the molecules that control homologous lysis. Here, Michael Holers, Taroh Kinoshita and Hector Molina compare and contrast the mouse and human RCA region products and conclude that the receptor and regulatory roles are conserved despite the structural variation.

Altered renal tubular expression of the complement inhibitor Crry permits complement activation after ischemia/reperfusion

Ischemia/reperfusion (I/R) of several organs results in complement activation, but the kidney is unique in that activation after I/R occurs only via the alternative pathway. We hypothesized that selective activation of this pathway after renal I/R could occur either because of a loss of complement inhibition or from increased local synthesis of complement factors. We examined the relationship between renal complement activation after I/R and the levels and localization of intrinsic membrane complement inhibitors. We found that loss of polarity of complement receptor 1-related protein y (Crry) in the tubular epithelium preceded activation of the alternative pathway along the basolateral aspect of the tubular cells. Heterozygous gene-targeted mice that expressed lower amounts of Crry were more sensitive to ischemic injury. Furthermore, inhibition of Crry expressed by proximal tubular epithelial cells in vitro resulted in alternative pathway-mediated injury to the cells. Thus, altered expression of a complement inhibitor within the tubular epithelium appears to be a critical factor permitting activation of the alternative pathway of complement after I/R. Increased C3 mRNA and decreased factor H mRNA were also detected in the outer medulla after I/R, suggesting that altered synthesis of these factors might further contribute to complement activation in this location.

Complement Component 3 Is Required for Optimal Expansion of CD8 T Cells During a Systemic Viral Infection

In addition to its established role in innate immune mechanisms, complement component C3 is also of critical importance in B cell activation and T cell-dependent Ab responses. In this study, we have examined the requirement for C3 in the generation of primary CD8 T cell responses to an acute systemic viral infection. We compared Ag-specific CD8 T cell responses to lymphocytic choriomeningitis virus (LCMV) between wild-type (+/+) and C3-deficient (C3−/−) mice on both 129/B6 and B6 backgrounds. These studies revealed that C3 activity is required for optimal expansion of LCMV-specific effector CD8 T cells in an epitope-dependent fashion, which is influenced by the genetic background of the mice. Studies in complement receptor 1/2 (CR1/CR2)-deficient mice showed that regulation of LCMV-specific CD8 T cell responses by C3 is not dependent upon CR1/CR2. These findings may have implications in vaccine development, therapy of autoimmune diseases, and prevention of graft rejection.

Impaired Affinity Maturation in Cr2−/− Mice Is Rescued by Adjuvants Without Improvement in Germinal Center Development

Cr2−/− mice have an impairment in humoral immunity, as shown by the decrease in the Ab titers against T cell-dependent Ags and abnormalities in germinal center formation. Germinal centers are present, but they are decreased in size and number, indicating problems in their development. In this study, we investigated whether this abnormality in germinal center development is associated with problems in the establishment of optimal affinity maturation and the generation of memory B cells, processes closely related to the germinal center reaction. We immunized the Cr2−/− animals with different Ags with or without adjuvants. We showed that, when immunized without adjuvants, complement receptors are absolutely required for optimal affinity maturation. Although limited affinity maturation is elicited in the Cr2−/− Ab response, it is decreased as compared with normal animals. Memory B cell generation is also impaired. In the presence of adjuvants, germinal center development in the Cr2−/− mice is still abnormal, as demonstrated by their decreased size and number. Surprisingly, adjuvants establish optimal affinity maturation and partially restore the amount of Ab produced during the primary response and memory B cell generation. However, adjuvants cannot improve the ability of follicular dendritic cells to retain Ags in the form of immune complexes. These observations indicate that immunization with inflammatory Ags offset some of the immunological abnormalities found in the Cr2−/− mice and show that optimal affinity maturation in the Cr2−/− mice can be achieved in the absence of normal germinal centers.

Negligible Role of Antibodies and C5 in Pregnancy Loss Associated Exclusively with C3-Dependent Mechanisms through Complement Alternative Pathway

Factors involved in pregnancy failure due to abnormal fetomaternal tolerance are poorly understood. Here we describe distinct defects in placenta formation and subsequent pregnancy loss solely dependent on the activation of the complement alternative pathway and the effector mechanisms provided by the maternal C3. Surprisingly, this effect is independent of other complement activation pathways and of the effector mechanisms provided by other complement components. These findings provide significant insight into the role of the innate immune system in human pregnancy failure, a frequent clinical outcome.

A Role for the Cr2 Gene in Modifying Autoantibody Production in Systemic Lupus Erythematosus

Systemic lupus erythematosus is an autoimmune disease characterized by autoantibody production against nuclear Ags. Recent studies suggest that the Cr2 gene, which encodes for complement receptor (CR)1 and CR2, is important in disease susceptibility. Because the precise disease phenotype related to this gene, in isolation or in relation to other genetic loci, is not known, we analyzed C57BL/6 mice with a targeted mutation in Cr2 (C57BL/6.Cr2−/−) with or without a concomitant mutation in Fas (C57BL/6.lpr Cr2−/−). The Cr2null mutation in a C57BL/6.lpr background markedly increases the serum concentrations of IgG1 and IgG2b and the levels of antinuclear and anti-dsDNA Abs as compared with C57BL/6.lpr controls. There is also a trend for higher concentrations of IgG2a and IgG3. In contrast, isolated deficiencies in either these CRs or Fas have a limited effect in the production of anti-dsDNA Abs. Moreover, the Cr2null mutation does not affect other disease manifestations. These findings demonstrate that abnormalities in CR1 and CR2 may be linked to the production of autoantibodies by modifying the effect of other systemic lupus erythematosus susceptibility genes. Phenotypic expression of other disease manifestations need additional Cr2-independent genetic factors.