Roberta Bulla

Protection against inflammation- and autoantibody-caused fetal loss by the chemokine decoy receptor D6

Fetal loss in animals and humans is frequently associated with inflammatory conditions. D6 is a promiscuous chemokine receptor with decoy function, expressed in lymphatic endothelium, that recognizes and targets to degradation most inflammatory CC chemokines. Here, we report that D6 is expressed in placenta on invading extravillous trophoblasts and on the apical side of syncytiotrophoblast cells, at the very interface between maternal blood and fetus. Exposure of D6−/− pregnant mice to LPS or antiphospholipid autoantibodies results in higher levels of inflammatory CC chemokines and increased leukocyte infiltrate in placenta, causing an increased rate of fetal loss, which is prevented by blocking inflammatory chemokines. Thus, the promiscuous decoy receptor for inflammatory CC chemokines D6 plays a nonredundant role in the protection against fetal loss caused by systemic inflammation and antiphospholipid antibodies.

Platelet-Activating Factor and Kinin-Dependent Vascular Leakage as a Novel Functional Activity of the Soluble Terminal Complement Complex

The infrequent occurrence of septic shock in patients with inherited deficiencies of the terminal complement components experiencing meningococcal disease led us to suspect that the terminal complement complex is involved in vascular leakage. To this end, the permeabilizing effect of the cytolytically inactive soluble terminal complement complex (SC5b-9) was tested in a Transwell system measuring the amount of fluorescein-labeled BSA (FITC-BSA) leaked through a monolayer of endothelial cells. The complex caused increased permeability to FITC-BSA after 15 min as opposed to the prompt response to bradykinin (BK). The effect of SC5b-9 was partially reduced by HOE-140 or CV-3988, two selective antagonists of BK B2 and platelet-activating factor receptors, respectively, and was completely neutralized by the mixture of the two antagonists. Also, DX-88, a specific inhibitor of kallikrein, partially inhibited the activity of SC5b-9. The permeabilizing factor(s) released after 30 min of incubation of endothelial cells with SC5b-9 caused a prompt leakage of albumin like BK. Intravital microscopy confirmed both the extravasation of circulating FITC-BSA across mesenteric microvessels 15 min after topical application of SC5b-9 and the complete neutralization by the mixture of HOE-140 and CV-3988. SC5b-9 induced opening of interendothelial junctions in mesenteric endothelium documented by transmission electron microscopy.

A non–complement-fixing antibody to β2 glycoprotein I as a novel therapy for antiphospholipid syndrome

A single-chain fragment variable (scFv) recognizing β2-glycoprotein 1 (β2GPI) from humans and other species was isolated from a human phage display library and engineered to contain an IgG1 hinge-CH2-CH3 domain. The scFv-Fc directed against β2GPI domain I-induced thrombosis and fetal loss, thus mimicking the effect of antibodies from patients with antiphospholipid syndrome (APS). Complement is involved in the biological effect of anti-β2GPI scFv-Fc, as demonstrated by its ability to promote in vitro and in vivo complement deposition and the failure to induce vascular thrombosis in C6-deficient rats and fetal loss in C5-depleted mice. A critical role for complement was also supported by the inability of the CH2-deleted scFv-Fc to cause vessel occlusion and pregnancy failure. This antibody prevented the pathological effects of anti-β2GPI antibodies from APS patients and displaced β2GPI-bound patient antibodies. The CH2-deleted antibody represents an innovative approach potentially useful to treat APS patients refractory to standard therapy.

An Alternative Role of C1q in Cell Migration and Tissue Remodeling: Contribution to Trophoblast Invasion and Placental Development

Fetal trophoblast cells invading the decidua in the early phase of pregnancy establish complex interaction with the maternal extracellular matrix. We discovered that C1q was widely distributed in human decidual stroma in the absence of C4 and C3 and was actively synthesized by migrating extravillous trophoblasts. The cells expressed the messages for the three chains of C1q and secreted this complement component that interacted with the proteins of the decidual extracellular matrix. Solid phase-bound C1q promoted trophoblast adhesion and migration, and cell binding to C1q resulted in activation of ERK1/2 MAPKs. Ab inhibition experiments showed that the receptors for the globular head of C1q/p33 and α4β1 integrin were both involved in this process and were colocalized on the cell surface following binding of C1q to trophoblasts. We also found that C1q−/− mice manifested increased frequency of fetal resorption, reduced fetal weight, and smaller litter sizes compared with wild-type mice. C1q deficiency was associated with impaired labyrinth development and decidual vessel remodeling. Collectively, these data suggest that C1q plays an important role in promoting trophoblast invasion of decidua and that defective local production of C1q may be involved in pregnancy disorders, such as pre-eclampsia, characterized by poor trophoblast invasion.

Controlling complement resistance in cancer by using human monoclonal antibodies that neutralize complement-regulatory proteins CD55 and CD59.

Expression of the complement‐regulatory proteins (CRP) CD46, CD55 and CD59 represents a strategy used by tumor cells to evade complement‐dependent cell cytoxicity stimulated by monoclonal antibodies. We have isolated two single‐chain variable fragments (scFv) to CD55 and CD59 from a human phage‐display library and from these scFv we have produced two miniantibodies (MB), MB‐55 (against CD55) and MB‐59 (against CD59), containing the human hinge–CH2–CH3 domains of IgG1. The specificity of the two MB for the corresponding CRP was assessed by ELISA using purified CD46, CD55 and CD59. MB‐55 and MB‐59 neutralized the inhibitory activity of CD55 and CD59, respectively, restoring the complement‐mediated lysis of sheep and guinea pig erythrocytes that was otherwise inhibited by the two CRP. FACS analysis revealed binding of MB‐55 and MB‐59 to the lymphoma cell line Karpas 422. The two MB induced a two‐fold increase in the complement‐dependent killing of these cells stimulated by Rituximab, a chimeric anti‐CD20 monoclonal antibody. Transfection of HEK293T cells with vectors encoding MB‐55 or MB‐59 markedly reduced the expression of CD55 and CD59. We conclude that the human antibodies MB‐55 and MB‐59 may represent a therapeutic tool to increase the complement‐dependent killing activity of Rituximab in the treatment of non‐Hodgkins lymphoma.

In vivo distribution of β2 glycoprotein I under various pathophysiologic conditions

In vitro studies have documented β2 glycoprotein I (β2GPI) binding to endothelial cells (ECs) and trophoblast using antiphospholipid antibodies. The in vivo binding of β2GPI to these cells and the conditions that favor their interaction have not been investigated. We analyzed the in vivo distribution of cyanine 5.5-labeled β2GPI in mice and evaluated the effect of pregnancy and circulating antibodies on its tissue localization. The signal was detected in the liver by whole body scan and ex vivo analysis. The β2GPI failed to bind to the vascular endothelium and reacted only with the ECs of uterine vessels. In pregnant mice the protein was localized on ECs and trophoblast at the embryo implantation sites. Immunized mice showed a similar β2GPI biodistribution to naive mice but the immunized pregnant animals exhibited a significant increase in fetal loss associated with C3 and C9 deposition at the implantation sites. Treatment of mice with LPS after β2GPI-Cy5.5 injection promoted protein localization on gut and brain ECs associated with IgG, C1q, and C9 deposition in immunized mice. These findings indicate that β2GPI binding to EC requires priming with pro-inflammatory factors which is not needed for uterine and placental localization probably dependent on hormonal changes.

Fluvastatin treatment inhibits leucocyte adhesion and extravasation in models of complement-mediated acute inflammation

Complement activation plays a relevant role in the development of tissue damage under inflammatory conditions, and clinical and experimental observations emphasize its contribution to inflammatory vasculitides. Statins have recently been shown to reduce cardiovascular morbidity independently of plasma cholesterol lowering and in vitro studies support a direct anti‐inflammatory action of these drugs. The aim of this study was to verify the in vivo effect of fluvastatin on complement‐mediated acute peritoneal inflammation. The effect of oral treatment with fluvastatin was investigated in normo‐cholesterolaemic rats that received intraperitoneal injection of either yeast‐activated rat serum (Y‐act RS) or lipopolysaccharide to induce peritoneal inflammation monitored by the number of PMN recruited in peritoneal fluid washes. In addition, vascular adherence and extravasation of leucocytes were evaluated by direct videomicroscopy examination on mesentery postcapillary venules topically exposed to Y‐act RS. The number of PMN in the peritoneal washes of rats treated with fluvastatin was 38% lower than that of untreated animals (P < 0·05)  12  h after LPS injection, and was even lower (56%) in rats treated with Y‐act RS already 8 h after injection (P < 0·02). Firm adhesion to endothelium and extravasation of leucocytes evaluated under direct videomicroscopy observation were significantly inhibited in fluvastatin treated rats (77% and 72%, respectively; P < 0·01), 120 min after treatment with Y‐act RS. Our results demonstrate that fluvastatin inhibits in vivo complement‐dependent acute peritoneal inflammation and suggest a role for statins in preventing the inflammatory flares usually associated with complement activation in chronic diseases, such as SLE or rheumatoid arthritis.

Decidual endothelial cells express surface-bound C1q as a molecular bridge between endovascular trophoblast and decidual endothelium

This study was prompted by the observation that decidual endothelial cells (DECs), unlike endothelial cells (ECs) of blood vessels in normal skin, kidney glomeruli and brain, express surface-bound C1q in physiologic pregnancy. This finding was unexpected, because deposits of C1q are usually observed in pathologic conditions and are associated with complement activation. In the case of DECs, we failed to detect immunoglobulins and C4 co-localized with C1q on the cell surface. Surprisingly, DECs expressed mRNA for the three chains of C1q and secreted detectable level of this component in serum-free medium. The ability to synthesize C1q is acquired by DECs during pregnancy and is not shared by ECs obtained from endometrium and from other sources. Cell-associated C1q has a molecular weight similar to that of secreted C1q and is released from DECs following treatment with heparinase or incubation at low pH. This suggests that C1q binds to DECs and it is not constitutively expressed on the cell surface. C1q is localized at contact sites between endovascular trophoblast and DECs and acts as an intercellular molecular bridge because adhesion of endovascular trophoblast to DECs was inhibited by antibodies to C1q and to a receptor recognizing its globular portion expressed on trophoblast.

Mutant p53 Reprograms TNF Signaling in Cancer Cells through Interaction with the Tumor Suppressor DAB2IP

Inflammation is a significant factor in cancer development, and a molecular understanding of the parameters dictating the impact of inflammation on cancers could significantly improve treatment. The tumor suppressor p53 is frequently mutated in cancer, and p53 missense mutants (mutp53) can acquire oncogenic properties. We report that cancer cells with mutp53 respond to inflammatory cytokines increasing their invasive behavior. Notably, this action is coupled to expression of chemokines that can expose the tumor to host immunity, potentially affecting response to therapy. Mechanistically, mutp53 fuels NF-κB activation while it dampens activation of ASK1/JNK by TNFα, and this action depends on mutp53 binding and inhibiting the tumor suppressor DAB2IP in the cytoplasm. Interfering with such interaction reduced aggressiveness of cancer cells in xenografts. This interaction is an unexplored mechanism by which mutant p53 can influence tumor evolution, with implications for our understanding of the complex role of inflammation in cancer.

C1q as a unique player in angiogenesis with therapeutic implication in wound healing

Significance C1q is a well-known initiator of the complement classical pathway and interacts with several immune and nonimmune cells inducing complement activation-independent functions. Endothelial cells represent one of the potential targets of C1q that binds to cell surface-expressed receptors and stimulates inflammation. Here we report a unique and hitherto unrecognized function of C1q to promote angiogenesis acting through the globular heads. The angiogenic activity of C1q was supported by its ability to induce new vessel formation in in vitro and in vivo models of wound healing. These findings have important implications for the treatment of clinical diseases associated with impaired angiogenesis such as chronic skin ulcers in diabetic patients. We have previously shown that C1q is expressed on endothelial cells (ECs) of newly formed decidual tissue. Here we demonstrate that C1q is deposited in wound-healing skin in the absence of C4 and C3 and that C1q mRNA is locally expressed as revealed by real-time PCR and in situ hybridization. C1q was found to induce permeability of the EC monolayer, to stimulate EC proliferation and migration, and to promote tube formation and sprouting of new vessels in a rat aortic ring assay. Using a murine model of wound healing we observed that vessel formation was defective in C1qa−/− mice and was restored to normal after local application of C1q. The mean vessel density of wound-healing tissue and the healed wound area were significantly increased in C1q-treated rats. On the basis of these results we suggest that C1q may represent a valuable therapeutic agent that can be used to treat chronic ulcers or other pathological conditions in which angiogenesis is impaired, such as myocardial ischemia.