Membrane-bound Complement Inhibitors Research Articles

Elevated expression of complement factor I in lung cancer cells associates with shorter survival–Potentially via non-canonical mechanism

While numerous membrane-bound complement inhibitors protect the body's cells from innate immunity's autoaggression, soluble inhibitors like complement factor I (FI) are rarely produced outside the liver. Previously, we reported the expression of FI in non-small cell lung cancer (NSCLC) cell lines. Now, we assessed the content of FI in cancer biopsies from lung cancer patients and associated the results with clinicopathological characteristics and clinical outcomes. Immunohistochemical staining intensity did not correlate with age, smoking status, tumor size, stage, differentiation grade, and T cell infiltrates, but was associated with progression-free survival (PFS), overall survival (OS) and disease-specific survival (DSS). Multivariate Cox analysis of low vs. high FI content revealed HR 0.55, 95 % CI 0.32-0.95, p=0.031 for PFS, HR 0.51, 95 % CI 0.25-1.02, p=0.055 for OS, and HR 0.32, 95 % CI 0.12-0.84, p=0.021 for DSS. Unfavorable prognosis might stem from the non-canonical role of FI, as the staining pattern did not correlate with C4d - the product of FI-supported degradation of active complement component C4b. To elucidate that, we engineered three human NSCLC cell lines naturally expressing FI with CRISPR/Cas9 technology, and compared the transcriptome of FI-deficient and FI-sufficient clones in each cell line. RNA sequencing revealed differentially expressed genes engaged in intracellular signaling pathways controlling proliferation, apoptosis, and responsiveness to growth factors. Moreover, in vitro colony-formation assays showed that FI-deficient cells formed smaller foci than FI-sufficient NSCLC cells, but their size increased when purified FI protein was added to the medium. We postulate that a non-canonical activity of FI influences cellular physiology and contributes to the poor prognosis of lung cancer patients.

MAP-2:CD55 chimeric construct effectively modulates complement activation.

The complement system is a complex, tightly regulated protein cascade involved in pathogen defense and the pathogenesis of several diseases. Thus, the development of complement modulators has risen as a potential treatment for complement-driven inflammatory pathologies. The enzymatically inactive MAP-2 has been reported to inhibit the lectin pathway by competing with its homologous serine protease MASP-2. The membrane-bound complement inhibitor CD55 acts on the C3/C5 convertase level. Here, we fused MAP-2 to the four N-terminal domains of CD55 generating a targeted chimeric inhibitor to modulate complement activation at two different levels of the complement cascade. Its biological properties were compared in vitro with the parent molecules. While MAP-2 and CD55 alone showed a minor inhibition of the three complement pathways when co-incubated with serum (IC50MAP-2+CD55 1-4 = 60.98, 36.10, and 97.01 nM on the classical, lectin, and alternative pathways, respectively), MAP-2:CD551-4 demonstrated a potent inhibitory activity (IC50MAP-2:CD55 1-4 = 2.94, 1.76, and 12.86 nM, respectively). This inhibitory activity was substantially enhanced when pre-complexes were formed with the lectin pathway recognition molecule mannose-binding lectin (IC50MAP-2:CD55 1-4 = 0.14 nM). MAP-2:CD551-4 was also effective at protecting sensitized sheep erythrocytes in a classical hemolytic assay (CH50 = 13.35 nM). Finally, the chimeric inhibitor reduced neutrophil activation in full blood after stimulation with Aspergillus fumigatus conidia, as well as phagocytosis of conidia by isolated activated neutrophils. Our results demonstrate that MAP-2:CD551-4 is a potent complement inhibitor reinforcing the idea that engineered fusion proteins are a promising design strategy for identifying and developing drug candidates to treat complement-mediated diseases.

Genome-Wide Association Study Identifies Variants Associated with Clinical Outcomes in Pediatric Patients with Newly Diagnosed Acute Myeloid Leukemia

Genetic aberrations in leukemic blasts are associated with clinical outcomes in patients with AML. However, the role of inherited genetic variants with regards to variation in AML treatment outcome remains obscure. To that end, we conducted a genome wide association study (GWAS) on 400 newly diagnosed pediatric AML patients to identify single nucleotide polymorphism (SNPs) that may be associated with minimal residual disease (MRD1) after induction 1 chemotherapy, event-free survival (EFS) and overall survival (OS). In this study, we utilized whole-genome genotyping with Illumina's Omni2.5Exome-8 BeadChip array to investigate SNPs for association with clinical outcome in 2 pediatric AML cohorts: the multi-site AML02 [NCT00136084, n=167] and AML08 [NCT00703820, n=233] trials. Post GWAS quality control, 1,365,439 unphased SNP genotypes were selected for imputation using TOPMed Imputation Server. Following imputation and additional quality control filtering, 6,741,200 SNPs evaluated for association with clinical outcomes (MRD1, EFS and OS). To improve statistical rigor, two GWAS approaches were utilized: i) A one-stage GWAS with both clinical trial cohorts combined (n=400) and ii) A two-stage GWAS where we utilized a two-step iterative resampling procedure (PMID: 26377241) by splitting the cohort at a 70:30 ratio into discovery and replication cohorts, balanced by clinical trial, treatment arm and initial risk group at diagnosis (100 randomizations performed). For each of the 100 iterations of the two-stage GWAS, we used the discovery cohort to perform a genome-wide analysis, followed by a replication screen. SNPs replicated in at least 20 of 100 rounds of discovery-replication were prioritized. A comparison of the two approaches showed all significant SNPs in the two-stage GWAS were also captured in the one-stage GWAS. For EFS, a Bonferroni-adjusted genome-wide significant association was observed for a SNP in CSMD1 and this SNP was replicated in 83 out of 100 discovery-replication rounds for two-stage GWAS. For CSMD1 SNP, presence of variant A allele has significantly reduced EFS (HR=3.29, 95% CI (2.177-4.975), P=1.61x10-8, Figure 1A). CSMD1 is a membrane-bound complement inhibitor that has been shown to act as a putative tumor suppressor gene in other malignancies (PMIDs:27764775, 35623420). Another significant marker worth mentioning was an intronic SNP in CDKN2B-AS1 that associated with poor outcome [EFS: HR=2.37, 95% CI (1.700-3.317), P=4.40x10-7] and [OS: HR=2.87, 95% CI (1.954-4.225), P=8.06x10-8, Figure 1B]. This marker is in high linkage disequilibrium with 26 other SNPs within the CDKN2B and CDKN2B-AS1 cluster. The product of this gene is an RNA molecule that interacts with polycomb repressive complex-1 (PRC1) and -2 (PRC2) involved in silencing other genes within this cluster through epigenetic mechanisms (PMID: 20716771). CDKN2B-AS1 has been reported to play a role in tumorigenesis and chemoresistance in pediatric T-cell acute lymphoblastic leukemia and with the risk of chronic lymphocytic leukemia (PMIDs: 32976214/23770605). Though limited by numbers, this is one of the largest GWAS conducted in pediatric AML to identify SNPs associated with clinical outcomes. Our top results show that genetic variation in several genes of biological relevance may impact MRD1, EFS and OS. Future studies are aimed at looking at SNP/gene and association with gene expression collected from leukemic cells at time of diagnosis to establish AML-specific eQTLs and functional validation of the most promising candidates. Additionally, future analyses will include other subgroups such as cytogenetic risk group and validation of these findings in larger cohort of patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

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.

Complement therapeutics meets nanomedicine: overcoming human complement activation and leukocyte uptake of nanomedicines with soluble domains of CD55

Complement activation plays an important role in pharmacokinetic and performance of intravenously administered nanomedicines. Significant efforts have been directed toward engineering of nanosurfaces with low complement activation, but due to promiscuity of complement factors and redundancy of pathways, it is still a major challenge. Cell membrane-anchored Decay Accelerating Factor (DAF, a.k.a. CD55) is an efficient membrane bound complement regulator that inhibits both classical and alternative C3 convertases by accelerating their spontaneous decay. Here we tested the effect of various short consensus repeats (SCRs, “sushi” domains) of human CD55 on nanoparticle-mediated complement activation in human sera and plasma. Structural modeling suggested that SCR-2, SCR-3 and SCR-4 are critical for binding to the alternative pathway C3bBb convertase, whereas SCR-1 is dispensable. Various domains were expressed in E.coli and purified by an affinity column. SCRs were added to lepirudin plasma or sera from different healthy subjects, to monitor nanoparticle-mediated complement activation as well as C3 opsonization. Using superparamagnetic iron oxide nanoworms (SPIO NWs), we found that SCR-2-3-4 was the most effective inhibitor (IC50 ~0.24 μM for C3 opsonization in sera), followed by SCR-1-2-3-4 (IC50 ~0.6 μM), whereas shorter domains (SCR-3, SCR-2-3, SCR-3-4) were ineffective. SCR-2-3-4 also inhibited C5a generation (IC50 ~0.16 μM in sera). In addition to SPIO NWs, SCR-2-3-4 effectively inhibited C3 opsonisation and C5a production by clinically approved nanoparticles (Feraheme, LipoDox and Onivyde). SCR-2-3-4 inhibited both lectin and alternative pathway activation by nanoparticles. When added to lepirudin-anticoagulated blood from healthy donors, it significantly reduced the uptake of SPIO NWs by neutrophils and monocytes. These results suggest that soluble domains of membrane-bound complement inhibitors are potential candidates for preventing nanomedicine-mediated complement activation in human subjects.

Membrane-bound complement inhibitors mediate binding of periodontal pathogens but prevent invasion of gingival epithelial cells

Membrane-bound complement inhibitors mediate binding of periodontal pathogens but prevent invasion of gingival epithelial cells

Novel potential inhibitors of complement system and their roles in complement regulation and beyond

The complement system resembles a double-edged sword since its activation can either benefit or harm the host. Thus, regulation of this system is of utmost importance and performed by several circulating and membrane-bound complement inhibitors. The pool of well-established regulators has recently been enriched with proteins that either share structural homology to known complement inhibitors such as Sushi domain-containing (SUSD) protein family and Human CUB and Sushi multiple domains (CSMD) families or extracellular matrix (ECM) macromolecules that interact with and modulate complement activity. In this review, we summarize the current knowledge about newly discovered complement inhibitors and discuss their implications in complement regulation, as well as in processes beyond complement regulation such cancer development. Understanding the behavior of these proteins will introduce new mechanisms of complement regulation and may provide new avenues in the development of novel therapies.

Potential Mechanisms Underlying TGF-β-mediated Complement Activation in Lung Fibrosis.

While our previous studies suggest that limiting bleomycin-induced complement activation suppresses TGF-β signaling, the specific hierarchical interactions between TGF-β and complement in lung fibrosis are unclear. Herein, we investigated the mechanisms underlying TGF-β-induced complement activation in the pathogenesis of lung fibrosis. C57-BL6 mice were given intratracheal instillations of adenoviral vectors overexpressing TGF-β (Ad-TGFβ) or the firefly gene-luciferase (Ad-Luc; control). Two weeks later, mice with fibrotic lungs were instilled RNAi specific to receptors for C3a or C5a-C3ar or C5ar, and sacrificed at day 28. Histopathological analyses revealed that genetic silencing of C3ar or C5ar arrested the progression of TGF-β-induced lung fibrosis, collagen deposition and content (hydroxyproline, col1a1/2); and significantly suppressed local complement activation. With genetic silencing of either C3ar or C5ar, in Ad-TGFβ-injured lungs: we detected the recovery of Smad7 (TGF-β inhibitor) and diminished local release of DAF (membrane-bound complement inhibitor); in vitro: TGF-β-mediated loss of DAF was prevented. Conversely, blockade of the TGF-β receptor prevented C3a-mediated loss of DAF in both normal primary human alveolar and small airway epithelial cells. Of the 52 miRNAs analyzed as part of the Affymetrix array, normal primary human SAECs exposed to C3a, C5a or TGF-β caused discrete and overlapping miRNA regulation related to epithelial proliferation or apoptosis (miR-891A, miR-4442, miR-548, miR-4633), cellular contractility (miR-1197) and lung fibrosis (miR-21, miR-200C, miR-31HG, miR-503). Our studies present potential mechanisms by which TGF-β activates complement and promotes lung fibrosis.

Complement inhibitor CSMD1 acts as tumor suppressor in human breast cancer.

Human CUB and Sushi multiple domains 1 (CSMD1) is a membrane-bound complement inhibitor suggested to act as a putative tumor suppressor gene, since allelic loss of this region encompassing 8p23 including CSMD1 characterizes various malignancies. Here, we assessed the role of CSMD1 as a tumor suppressor gene in the development of breast cancer in vitro and in vivo. We found that human breast tumor tissues expressed CSMD1 at lower levels compared to that in normal mammary tissues. The decreased expression of CSMD1 was linked to a shorter overall survival of breast cancer patients. We also revealed that expression of CSMD1 in human breast cancer cells BT-20 and MDA-MB-231 significantly inhibited their malignant phenotypes, including migration, adhesion and invasion. Conversely, stable silencing of CSMD1 expression in T47D cells enhanced cancer cell migratory, adherent and clonogenic abilities. Moreover, expression of CSMD1 in the highly invasive MDA-MB-231 cells diminished their signaling potential as well as their stem cell-like properties as assessed by measurement of aldehyde dehydrogenase activity. In a xenograft model, expression of CSMD1 blocked the ability of cancer cells to metastasize to secondary sites in vivo, likely via inhibiting local invasion but not the extravasation into distant tissues. Taken together, these findings demonstrate the role of CSMD1 as a tumor suppressor gene in breast cancer.

Effect of IL-6 and IL-8 on the expression of the complement activation inhibitors MAC-inhibitory protein and decay-accelerating factor in ovarian cancer A2780 cells.

The aim of the present study was to evaluate the role of interleukin (IL)-6 and IL-8 on the expression of the membrane-bound complement inhibitors membrane attack complex-inhibitory protein (CD59) and decay-accelerating factor (CD55), in the human ovarian carcinoma A2780 cell line, which is a non-producing IL-6 cell line that does exhibit IL-6 responsiveness, due to the presence of IL-6 receptors. Extracellular levels of complement system inhibitors were evaluated by western blotting and reverse transcription-quantitative polymerase chain reaction. Cellular localization of CD55 and CD59 in the ovarian cancer cells was assessed by immunofluorescence. The detection of a soluble form of CD55 and CD59 released by the A2780 cells following stimulation with IL-6 and IL-8 was detected by enzyme-linked immunosorbent assay. The present data revealed that A2780 cells express CD55 and CD59 at the mRNA and protein level, but do not secrete these proteins to the culture medium. Results of western blotting demonstrated that the protein level of CD59 was regulated by IL-6 and IL-8 in a dose-dependent manner. Immunofluorescence analysis revealed that the ovarian cancer A2780 cell line expresses the membrane bound form of CD55 protein. The present results indicate that CD55 and CD59 may affect the efficiency of complement-mediated immunotherapies.

Local expression of complement factor I in breast cancer cells correlates with poor survival and recurrence.

Tumor cells often evade killing by the complement system by overexpressing membrane-bound complement inhibitors. However, production of soluble complement inhibitors in cells other than hepatocytes was rarely reported. We screened several breast cancer cell lines for expression of soluble complement inhibitor, complement factor I (FI). We also analyzed local production of FI in tissue microarrays with tumors from 130 breast cancer patients by in situ hybridization and immunohistochemistry. We found expression of FI in breast adenocarcinoma cell line MDA-MB-468 and confirmed its functional activity. Expression of FI at mRNA and protein levels was also confirmed in tumor cells and tumor stroma, both in fibroblasts and infiltrating immune cells. Multivariate Cox regression analyses revealed that high expression of FI protein in tumor cells was correlated with significantly shorter cancer-specific survival (HR 2.8; 95 % CI 1.0-7.5; p = 0.048) and recurrence-free survival (HR 3.4; 95 % CI 1.5-7.4; p = 0.002). High FI expression was positively correlated with tumor size (p < 0.001), and Nottingham histological grade (p = 0.015) and associated with estrogen and progesterone receptor status (p = 0.03 and p = 0.009, respectively). Our data show that FI is expressed in breast cancer and is associated with unfavorable clinical outcome.

The immunohistochemical analysis of membrane-bound CD55, CD59 and fluid-phase FH and FH-like complement inhibitors in cancers of ovary and corpus uteri origin.

One of the potential therapeutic methods of cancer treatment is the immunotherapy with monoclonal antibodies. This kind of therapy, although devoid of serious side effects, has often insufficient efficacy. The presence of complement inhibitors on the cancer cells, which are able to inactivate complement-mediated immune response represents one of the main reasons for the inefficiency of such therapy. In our studies we investigated the expression of main membrane–bound and fluid-phase complement regulators: CD55, CD59 and factor H/factor H-like in tumour samples of ovarian and corpus uteri cancer. Tissue samples were collected from 50 patients and stained immunohistochemically, with the use of peroxidase-based immunodetection system. Immunohistochemical analysis revealed that complement inhibitors are present in examined tumors although their presence is heterogenous. The most prevalent is the presence of factor H/H-like, localized mostly in tumor stroma and within vascular structures. Membrane bound complement inhibitors are less prominently expressed by cancer cells. CD55 was detected in low percentage of cells, predominantly within cancer tubules. CD59 immunoreactivity was more prevalent in cancer cells, and was localized particularly at the margin of cancer cell tubules. Our results demonstrate that the most prominent complement inhibitor in cancer of ovary and corpus uteri origin is factor H/factor H-like. Blocking or downregulation of this inhibitor should be taken into consideration with regards to improving the efficiency of immunotherapy with monoclonal antibodies.

Killing of CLL and NHL cells by rituximab and ofatumumab under limited availability of complement

Rituximab and ofatumumab are anti-CD20 antibodies applicable to treatment for non-Hodgkin's lymphoma and chronic lymphocytic leukemia (CLL). Effectiveness of both immunotherapeutics may depend on exhaustible complement system. To model the efficacy of complement usage by ofatumumab and rituximab under limited complement availability, we compared complement-dependent cytotoxicity exerted by these antibodies at low (5 and 10%) and physiological (50%) serum concentration in twelve CD20-positive cell lines and six freshly isolated CLL cells. Simultaneously, we assessed the expression of CD20 and membrane-bound complement inhibitors. Ratios of CD20 to CD59 and/or CD55 distinguished highly sensitive cells lysed equally efficient by both antibodies from the moderately sensitive cells, which were killed more efficiently by ofatumumab.

Investigations of co-localization of albumin, fibrinogen and some complement components on the vascular surfaces

Albumin and fibrinogen, two independent plasma proteins are able to produce a relatively stable complex which is present on the surface of multiple cells. Existence of such structure was reported in the 1960s and 1970s by Brzosko and Nowoslawski, Groniowski, and confirmed in vitro by Lipinski in 1995. It was believed to provide protection against immunocompetent cells. In 2002, we showed the presence of a structural lining composed of albumin and fibrinogen on the surface of placental villi. Immunohistochemistry and immunoflurescence confirmed presence of this complex on the endothelial surface. We also found complement proteins such as C1q, C4d, C9, and double immunofluorescence confirmed the presence of this complex and complement components in the aortic vasa vasorum. Both membrane-bound (CD46, CD55, CD59) and soluble (H factor) complement inhibitors were present. Studies indicate that this lining is composed of plasma proteins and complement components and since it is present on the endothelial surface, it can play a role of a natural immune barrier protecting against pathological processes in the blood vessels.

Comparison of the expression of complement regulatory proteins CD46, CD55 and CD59 in primary colon cancer and synchronous/metachronous liver metastases

ENWEndNote BIBJabRef, Mendeley RISPapers, Reference Manager, RefWorks, Zotero AMA Wilczek E, Wasiutynski A, Wilczynski G, Sladowski D, Gornicka B. Comparison of the expression of complement regulatory proteins CD46, CD55 and CD59 in primary colon cancer and synchronous/metachronous liver metastases. Central European Journal of Immunology. 2013;38(4):543-548. doi:10.5114/ceji.2013.39773. APA Wilczek, E., Wasiutynski, A., Wilczynski, G., Sladowski, D., & Gornicka, B. (2013). Comparison of the expression of complement regulatory proteins CD46, CD55 and CD59 in primary colon cancer and synchronous/metachronous liver metastases. Central European Journal of Immunology, 38(4), 543-548. https://doi.org/10.5114/ceji.2013.39773 Chicago Wilczek, Ewa, Aleksander Wasiutynski, Grzegorz M. Wilczynski, Dariusz Sladowski, and Barbara Gornicka. 2013. "Comparison of the expression of complement regulatory proteins CD46, CD55 and CD59 in primary colon cancer and synchronous/metachronous liver metastases". Central European Journal of Immunology 38 (4): 543-548. doi:10.5114/ceji.2013.39773. Harvard Wilczek, E., Wasiutynski, A., Wilczynski, G., Sladowski, D., and Gornicka, B. (2013). Comparison of the expression of complement regulatory proteins CD46, CD55 and CD59 in primary colon cancer and synchronous/metachronous liver metastases. Central European Journal of Immunology, 38(4), pp.543-548. https://doi.org/10.5114/ceji.2013.39773 MLA Wilczek, Ewa et al. "Comparison of the expression of complement regulatory proteins CD46, CD55 and CD59 in primary colon cancer and synchronous/metachronous liver metastases." Central European Journal of Immunology, vol. 38, no. 4, 2013, pp. 543-548. doi:10.5114/ceji.2013.39773. Vancouver Wilczek E, Wasiutynski A, Wilczynski G, Sladowski D, Gornicka B. Comparison of the expression of complement regulatory proteins CD46, CD55 and CD59 in primary colon cancer and synchronous/metachronous liver metastases. Central European Journal of Immunology. 2013;38(4):543-548. doi:10.5114/ceji.2013.39773.

The expression of membranous complement inhibitors CD46, CD55 and CD59 in the primary and metastatic colon cancer cell lines derived from the same patient

ENWEndNote BIBJabRef, Mendeley RISPapers, Reference Manager, RefWorks, Zotero AMA Wilczek E, Wasiutynski A, Sladowski D, Wilczynski G, Gornicka B. The expression of membranous complement inhibitors CD46, CD55 and CD59 in the primary and metastatic colon cancer cell lines derived from the same patient. Central European Journal of Immunology. 2013;38(4):549-555. doi:10.5114/ceji.2013.39774. APA Wilczek, E., Wasiutynski, A., Sladowski, D., Wilczynski, G., & Gornicka, B. (2013). The expression of membranous complement inhibitors CD46, CD55 and CD59 in the primary and metastatic colon cancer cell lines derived from the same patient. Central European Journal of Immunology, 38(4), 549-555. https://doi.org/10.5114/ceji.2013.39774 Chicago Wilczek, Ewa, Aleksander Wasiutynski, Dariusz Sladowski, Grzegorz M. Wilczynski, and Barbara Gornicka. 2013. "The expression of membranous complement inhibitors CD46, CD55 and CD59 in the primary and metastatic colon cancer cell lines derived from the same patient". Central European Journal of Immunology 38 (4): 549-555. doi:10.5114/ceji.2013.39774. Harvard Wilczek, E., Wasiutynski, A., Sladowski, D., Wilczynski, G., and Gornicka, B. (2013). The expression of membranous complement inhibitors CD46, CD55 and CD59 in the primary and metastatic colon cancer cell lines derived from the same patient. Central European Journal of Immunology, 38(4), pp.549-555. https://doi.org/10.5114/ceji.2013.39774 MLA Wilczek, Ewa et al. "The expression of membranous complement inhibitors CD46, CD55 and CD59 in the primary and metastatic colon cancer cell lines derived from the same patient." Central European Journal of Immunology, vol. 38, no. 4, 2013, pp. 549-555. doi:10.5114/ceji.2013.39774. Vancouver Wilczek E, Wasiutynski A, Sladowski D, Wilczynski G, Gornicka B. The expression of membranous complement inhibitors CD46, CD55 and CD59 in the primary and metastatic colon cancer cell lines derived from the same patient. Central European Journal of Immunology. 2013;38(4):549-555. doi:10.5114/ceji.2013.39774.

Both Freshly Prepared and Frozen-Stored Amniotic Membrane Cells Express the Complement Inhibitor CD59

Amniotic membrane proved to be very effective tool in the treatment of a number of ocular surface diseases. The amniotic membrane, however, has to be stored before its transplantation onto the ocular surface followed by mandatory serologic control in order to exclude the transmission of certain viruses. Therefore it is most important to study if cryopreservation of the membrane affects cell surface expression of the molecules. We measured cell surface expression of CD59, a membrane-bound complement inhibitor on the cells of freshly prepared and cryopreserved amniotic membrane. Cells of amniotic membrane were separated mechanically. Epithelial and mesenchymal cells were identified by the intracellular expression of nanog and the cell surface ICAM1 positivity, respectively. Multicolor flow cytometric immunophenotyping was used for determination of the CD59 expression. CellQuest-Pro software program (Becton Dickinson) was used both for measurements and analysis. CD59-positive cells could be detected in all investigated samples and in all investigated cell types, although the expression level of CD59 differed. CD59 was expressed both on freshly prepared and frozen-stored samples. Higher level of CD59 was detected on ICAM1+ mesenchymal cells than on nanog+ epithelial cells. Our findings indicate that amniotic membranes maintain their complement inhibiting capacity after cryopreservation.

Transient Pretreatment With Glucocorticoid Ablates Innate Toxicity of Systemically Delivered Adenoviral Vectors Without Reducing Efficacy

More than 300 human clinical trials utilize recombinant adenoviruses (rAds) as a gene transfer vector, confirming that rAds continue to be of high clinical interest. A primary weakness of rAds is their known propensity to trigger an innate, proinflammatory immune response rapidly after high-dose, systemic administration. In this study, we investigated what affects that pre-emptive treatment with anti-inflammatory glucocorticoids might have upon Ad vector-triggered inflammatory immune responses. We found that a simple pretreatment regimen with Dexamethasone (DEX) can significantly reduce most Ad-induced innate immune responses. DEX prevented rAd induction of systemic cytokine/chemokine releases in a dose-dependent fashion, with higher dosages preventing rAd induction of acute thrombocytopenia, endothelial cell activation, proinflammatory gene induction, and leukocyte infiltration into transduced organs. Transient glucocorticoid pretreatment also significantly reduced rAd-induced adaptive immune responses, including a decreased induction of Ad-neutralizing antibodies (NAbs). Importantly, use of DEX did not reduce the efficacy of rAd-mediated gene transduction nor rAd-derived transgene expression. Our results demonstrate that a simple, pre-emptive and transient glucocorticoid pretreatment is a viable approach to reduce rAd-associated acute toxicities that currently limit the use of Ad vectors in systemic clinical applications.

Hypoxia increases susceptibility of non-small cell lung cancer cells to complement attack.

The complement system can be specifically targeted to tumor cells due to molecular changes on their surfaces that are recognized by complement directly or via naturally occurring antibodies. However, tumor cells often overexpress membrane-bound complement inhibitors protecting them from complement attack. We have previously shown that non-small cell lung cancer (NSCLC) cells, additionally to membrane-bound inhibitors, produce substantial amounts of soluble regulators such as factor I (FI) and factor H (FH). Since low oxygen concentration is associated with rapidly growing solid tumors, we studied how NSCLC cells protect themselves from complement attack under hypoxic conditions. Unexpectedly, mRNA levels and secretion of both FI and FH were significantly decreased already after 24 h exposure to hypoxia while cell viability measured by XTT assay and annexin V/7-AAD staining was affected only marginally. Furthermore, we observed decrease of mRNA level and loss of membrane-bound complement inhibitor CD46 and increased deposition of early (C3b) and terminal (C9) complement components on hypoxic NSCLC cells. All three complement pathways (classical, lectin and alternative) were employed to deposit C3b on cell surface. Taken together, our results imply that under hypoxic conditions NSCLC give up some of their available defense mechanisms and become more prone to complement attack.

Functional activity of the membrane-associated complement inhibitor CD59 in a pig-to-human in vitro model for hyperacute xenograft rejection.

Hyperacute rejection triggered by activation of the recipient's complement system represents the major barrier to successful xenotransplantation. Transfer of human membrane-associated complement regulators to donor organs has been suggested as one strategy to interfere with complement-mediated hyperacute xenograft rejection. Pigs are discussed as potential organ donors. We therefore investigated a putative protective function of the membrane-bound complement inhibitor CD59 in a pig-to-human in vitro model of hyperacute xenograft rejection. Aortic porcine endothelial cells were transfected with human CD59 cDNA. Expression of human CD59 was demonstrated by cytofluorimetric and RNA analysis. Removal of CD59 from the cell surface by phosphatidylinositol-specific phospholipase C (PI-PLC) demonstrated its production as a glycosyl phosphatidylinositol (GPI)-anchored protein. Functional activity of the transfected CD59 was tested by a lactate dehydrogenase (LDH) release assay for complement-mediated lysis. Porcine endothelial cells expressing human CD59 were significantly protected from lysis by human serum complement compared with CD59- cells. The protective effect was abolished by preincubating the cells with anti-CD59 antibodies or PI-PLC. We calculated by Scatchard analysis that the established CD59+ cell line expressed a CD59 level comparable to that of human endothelial cells. Our results recommend the production of pigs transgenic for CD59.