Complement-mediated Cell Death Research Articles

The Bioluminescent Modified Ham Test Identifies the Classical Pathway As the Major Driver of Complement Activation in Atypical Hemolytic Uremic Syndrome and Primary Antiphospholipid Syndrome

Introduction: The role of complement is increasingly recognized in disease pathogenesis, however diagnostic assays are limited by low predictive value, technical requirements, and inability to assess relative contributions of each pathway of complement (classical, alternative, or lectin). The traditional mHam was developed as a functional assay of complement-mediated eukaryotic cell death in atypical hemolytic uremic syndrome (aHUS). However, broader use of the assay has been limited by inter-operator dependence and technical requirements. We have developed a bioluminescent mHam that is robust and easily performed (no cell washes or exogenous reagent addition) and utilizes an autonomously bioluminescent HEK293 cell line to allow for real-time assessment of changes in metabolic health due to complement activation. Given both HEK293 and endothelial cells lack complement receptor 1 (CR1), a crucial regulator of the classical pathway (CP), this assay measures complement activity of greater relevance to disease pathogenesis. Methods: The bioluminescent mHam uses LiveLight (LL) HEK293 cells (490 BioTech) engineered to stably express the bacterial luciferase ( lux) and flavin reductase genes ( luxF) to create autonomous bioluminescence. A PIGA null clone (LL PIGA- HEK293) was created through proaerolysin selection (removing GPI-anchored complement regulators CD55 and CD59). To perform the assay, 40,000 cells were harvested with trypsin/EDTA and plated as triplicates in gelatin veronal buffer (GVB ++,80 µL). Serum (20 µL) was added after specified treatments (heat inactivation or inhibitor addition). Activity was monitored by serial luminescence measurements every 5 min at 37 °C. A relative luminescence of <25% at one hour is designated as positive based upon lowest replicate of any healthy control. Results: The bioluminescent mHam, unlike the original mHam, provides continuous measurement of complement activity (example tracing Figure 1). The complement specificity of the luminescent changes were demonstrated with heat inactivation, terminal complement blockade with eculizumab, and calcium/magnesium dependence. The relative contribution of CP to changes in luminescence was confirmed with the specific CP inhibitor sutimlimab as well as use of pathway specific buffers. Healthy control (HC) samples show a relative luminescence of 53.7% (95%CI 44.0-63.4) with no difference observed in acute thrombotic thrombocytopenic purpura (TTP) (mean 56.2%, 95%CI 41.2-71.2, p=>0.9999). We next utilized samples of aHUS patients known to be in remission, off complement inhibitor therapy. A large proportion of these patients have ongoing assay positivity even in the traditional mHam (9 of 13 in one unpublished cohort). The bioluminescent mHam shows that 5 of 6 aHUS patients in remission have ongoing positivity (mean 17.56%, 95%CI -10.5-45.6, p=0.0131). An acute aHUS sample showed similar results (17.8% after plasmapheresis). Unexpectedly, the relative luminescence is rescued by addition of either C1s or C5 inhibitors, but use of alternative pathway (AP) inhibitors has no significant rescuing effect ( Figure 1-2). Of aHUS patients tested while on complement inhibition, 91% (10/11) show greater than 88% blockade ( Figure 2 aHUS on therapy) . Finally, in one cohort, the traditional mHam was positive in 52% (14/27) of primary antiphospholipid syndrome (PAPS) cases and 15% (10/66) of secondary APS (SAPS) (unpublished data). Thus far, the bioluminescent mHam shows abnormal complement activity in 92% (12/13) of PAPS and 17% (2/12) of patients in SAPS ( Figure 2). Again, complement inhibition with C1s or C5 inhibitors abrogated the effect in a dose dependent manner. Conclusions: The bioluminescent mHam test is an exquisitely sensitive real-time measure of abnormal complement activity. It is easy to perform, more closely approximates the complement regulators on renal endothelium, can be utilized for therapeutic monitoring, and has already offered novel disease insights through the use of specific pathway inhibitors. Although aHUS is traditionally categorized as a disorder of the AP, these results suggest the “trigger” mediating disease pathology is the classical pathway. Also, we show that a large proportion of primary APS patients have abnormal CP activation responsive to C1s and C5 inhibition, suggesting the disease may be responsive to complement-based therapeutics.

Temporal lobe epilepsy with GAD antibodies: neurons killed by T cells not by complement membrane attack complex.

Temporal lobe epilepsy (TLE) is one of the syndromes linked to antibodies against glutamic acid decarboxylase (GAD). It has been questioned whether 'limbic encephalitis with GAD antibodies' is a meaningful diagnostic entity. The immunopathogenesis of GAD-TLE has remained enigmatic. Improvement of immunological treatability is an urgent clinical concern. We retrospectively assessed the clinical, MRI and CSF course as well as brain tissue of 15 adult patients with GAD-TLE who underwent temporal lobe surgery. Brain tissue was studied by means of immunohistochemistry, multiplex fluorescent microscopy and transcriptomic analysis for inflammatory mediators and neuronal degeneration. In 10 patients, there was a period of mediotemporal swelling and T2 signal increase; in nine cases this occurred within the first 6 years after symptom onset. This resulted in unilateral or bilateral hippocampal sclerosis; three cases developed hippocampal sclerosis within the first 2 years. All CSF studies done within the first year (n = 6) revealed intrathecal synthesis of immunoglobulin G. Temporal lobe surgeries were done after a median disease duration of 9 years (range 3 weeks to 60 years). Only two patients became seizure-free. Brain parenchyma collected during surgery in the first 6 years revealed high numbers of plasma cells but no signs of antibody-mediated tissue damage. Even more dense was the infiltration by CD8+ cytotoxic T lymphocytes (CTLs) that were seen to locally proliferate. Further, a portion of these cells revealed an antigen-specific resident memory T cell phenotype. Finally, CTLs with cytotoxic granzyme B+ granules were also seen in microglial nodules and attached to neurons, suggesting a CTL-mediated destruction of these cells. With longer disease duration, the density of all lymphocytes decreased. Whole transcriptome analysis in early/active cases (but not in late/inactive stages) revealed 'T cell immunity' and 'Regulation of immune processes' as the largest overrepresented clusters. To a lesser extent, pathways associated with B cells and neuronal degeneration also showed increased representation. Surgically treated patients with GAD-TLE go through an early active inflammatory, 'encephalitic' stage (≤6 years) with CTL-mediated, antigen-driven neuronal loss and antibody-producing plasma cells but without signs of complement-mediated cell death. Subsequently, patients enter an apparently immunologically inactive or low-active stage with ongoing seizures, probably caused by the structural damage to the temporal lobe. 'Limbic encephalitis' with GAD antibodies should be subsumed under GAD-TLE. The early tissue damage explains why immunotherapy does not usually lead to freedom from seizures.

Complement Activation Drives Progression of Pre-Eclampsia to HELLP Syndrome

Background: Pre-eclampsia is a hypertensive disorder of pregnancy that affects about 5% of all pregnancies; however, its pathogenesis remains poorly understood. About 1% of pre-eclampsia cases will progress to a severe form termed HELLP syndrome characterized by Hemolysis, Elevated Liver enzymes, and Low Platelet counts. We have previously shown that abnormal activation of complement is a driver of disease in a subset of patients with HELLP syndrome (Vaught et al. 2016 and 2018). Based on these studies, we proposed that germline variants in complement regulatory genes predispose to HELLP in the setting of pregnancy, a complement amplifying event. However, whether complement dysregulation correlates to disease severity in HELLP syndrome is unclear. In addition, the contribution of complement activation to the onset or severity of pre-eclampsia remains unknown. We hypothesized that patients with pre-eclampsia harboring complement gene variants are at increased risk for progression to HELLP syndrome and worse maternal and fetal outcomes due to dysregulation of the complement system.Methods: In this prospective cohort study, patients with the pregnancy-associated hypertensive disorders including HELLP or pre-eclampsia were enrolled after diagnostic validation with the Tennessee and ACOG classification criteria, respectively. In total, 24 women with HELLP and 42 women with pre-eclampsia were enrolled and followed through their hospital course and compared with 47 healthy normotensive pregnant women. Both maternal and fetal clinical outcomes were recorded. Delivery before estimated gestational age < 23 weeks was considered previable, and intra-uterine fetal death (IUFD) was characterized as death of the fetus in utero during the pregnancy. Targeted sequencing of 13 genes known to regulate the alternative complement pathway was performed, and non-synonymous, heterozygous germline variants with a minor allele frequency less than 0.01 were identified. A functional assay for complement activation, the modified Ham assay (mHam), was performed in a subset of cases with available serum samples. The mHam is based on the principle that a cell line lacking surface complement regulators is susceptible to complement mediated cell death; non-viable cells > 20.5% is considered a positive test.Results: The frequency of rare germline variants in the HELLP cohort was 45.8%, comparable to a well-established complement mediated disease (aHUS, Table 1). By comparison, the frequency of germline variants in the pre-eclampsia and control cohort was 28.6% and 23.4%, respectively. Of the 14 patients with sera available to perform the mHam, 57% of HELLP syndrome patients had a positive mHam as compared to 0% of the controls (p = incalculable). Among patients with HELLP syndrome, those with a complement gene variant were more likely to have a poor clinical outcome compared to those without a variant. Specifically, 3/11 patients with variants versus 1/13 patients without variants were diagnosed at a previable stage and/or experienced IUFD (p = 0.19). There was no difference in the rate of HELLP onset after 28 and 34 weeks between the two groups. Notably, when comparing the cohort of HELLP patients harboring variants to the cohort without, patients with variants had higher rates of acute renal failure, hypoxia, and ICU care, but a lower frequency of blood transfusions (Table 2).Conclusion: Patients with HELLP syndrome harbor rare germline variants in complement genes at a greater frequency than controls (p = 0.05) and a similar frequency to aHUS, a known complement mediated disease. HELLP syndrome patients also carry mutations at a higher frequency than women with pre-eclampsia (p = 0.16) who have a similar rate of variants as controls. This suggests that complement gene variants predispose to progression of pre-eclampsia to HELLP syndrome where the gene variant and pregnancy provide a first and second ‘hit’ similar to other complement disorders. Patients with rare complement-associated variants are also more likely more susceptible to severe disease manifestations. [Display omitted] DisclosuresChaturvedi: UCB: Other: Advisory board participation; Sanofi Genzyme: Other: Advisory board member; Alexion: Other: Advisory board member; Dova: Other: Advisory board member; Argenx: Other: Advisory board member.

Complement dysregulation is associated with severe COVID-19 illness.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) may manifest as thrombosis, stroke, renal failure, myocardial infarction, and thrombocytopenia, reminiscent of other complement- mediated diseases. Multiple clinical and preclinical studies have implicated complement in the pathogenesis of COVID-19 illness. We previously found that the SARS-CoV-2 spike protein activates the alternative pathway of complement (APC) in vitro through interfering with the function of complement factor H, a key negative regulator of APC. Here, we demonstrated that serum from 58 COVID-19 patients (32 patients with minimal oxygen requirement, 7 on high flow oxygen, 17 requiring mechanical ventilation and 2 deaths) can induce complementmediated cell death in a functional assay (the modified Ham test) and increase membrane attack complex (C5b-9) deposition on the cell surface. A positive modified Ham assay (>20% cell-killing) was present in 41.2% COVID-19 patients requiring intubation (n=7/17) and only 6.3% in COVID-19 patients requiring minimal oxygen support (n=2/32). C5 and factor D inhibition effectively mitigated the complement amplification induced by COVID-19 patient serum. Increased serum factor Bb level was associated with disease severity in COVID-19 patients, suggesting that APC dysregulation plays an important role. Moreover, SARS-CoV-2 spike proteins directly block complement factor H from binding to heparin, which may lead to complement dysregulation on the cell surface. Taken together, our data suggest that complement dysregulation contributes to the pathogenesis of COVID-19 and may be a marker of disease severity.

SMR peptide antagonizes mortalin promoted release of extracellular vesicles and affects mortalin protection from complement-dependent cytotoxicity in breast cancer cells and leukemia cells.

Background: Mortalin/GRP-75/mt-hsp70 is a mitochondrial chaperone protein, found in the cytoplasm, endoplasmic reticulum and cytoplasmic vesicles. It functions in many cellular processes such as mitochondrial biogenesis, intracellular trafficking, cell proliferation, signaling, immortalization and tumorigenesis. Thus, inhibition of mortalin is a promising avenue for cancer therapy. Previous studies in our lab have suggested that mortalin contributes to breast cancer development and progression. We showed that tumor extracellular vesicle secretion was decreased by knockdown of mortalin expression using HIV-1 Nef SMR peptides. Specifically, these peptides can block extracellular vesicle secretion and mediate cell cycle arrest in MDA-MB-231 and MCF-7 breast cancer cells. Aims: This study aims to investigate further the function and mechanism of interaction of PEG-SMR-CLU and SMR-CPP peptides with the chaperone protein mortalin and to explore the effect of SMR-derived peptides and mortalin expression on extracellular vesicle release and complement dependent cell toxicity in human breast cancer and leukemia cell lines. Results: Our results demonstrated additional effects reversing the tumorigenicity of these cells. First, the modified SMRwt peptides reduced the expression of the mesenchymal marker vimentin (VIM). Second, exposure to the SMRwt peptide inhibited mortalin and complement C9 expression in MDA-MB-231, MCF-7 breast cancer cells and K562 leukemia cells as measured by the Western blot analysis. Third, the SMRwt peptides blocked the cancer cells’ ability to release extracellular vesicles, which we observed blocked extracellular vesicle-mediated release of complement, re-establishing complements mediated cell death in those peptide-treated cells. Methods: We developed a series of peptides derived from the Secretion Modification Region (SMR) of HIV-1 Nef protein, modified by the addition of either a cell-penetrating peptide (CPP), a positively charged arginine-rich peptide derived from HIV-1 regulatory protein Tat, or a Clusterin-binding peptide (CLU), a molecular chaperone involved in protein secretion. Both CPP and CLU peptide sequences were added at the C-terminus of the Nef SMR peptide. The CLU-containing peptides were also modified with polyethylene glycol (PEG) to enhance solubility. After treatment of cells with the peptides, we used the MTT cell viability and complement-mediated cytotoxicity assays to confirm the inhibitory role of modified SMRwt peptides on the proliferation of MDA-MB-231 and MCF-7 breast cancer cells and K562 leukemia cells. Flow cytometry was used to determine complement mediated cell apoptosis and death. Western blot analysis was used to track SMR peptides impact on expression of mortalin, vimentin and complement C9 and to measure the expression of extracellular vesicle proteins. NanoSight analysis and acetylcholinesterase (AChE) assay were used for measuring extracellular vesicles particle size and concentration and acetylcholinesterase. Conclusions: Mortalin promotes cell proliferation, metastasis, angiogenesis, downregulate apoptotic signaling. Thus, mortalin is a potential therapeutic target for cancer immunotherapy. The novel SMRwt peptides antagonize the functions of mortalin, blocking tumor extracellular vesicle release and extracellular vesicle-mediated release of complement. This leads to decreases in breast cancer cell metastasis and allows standard treatment of these late stage tumor cells, thus having important clinical implications for late stage breast cancer chemotherapy. These findings support further investigation into the therapeutic value of the SMR peptide in cancer metastasis.

Comparison of Cytotoxic Flow Cytometric Cross Match With Complement Dependent Lymphocytotoxicity and Flow Cytometric Cross Match in Renal Transplant Patients

Cytotoxic flow cytometric crossmatch (cFCXM), identified by detecting complement-mediated cytotoxic cell death in addition to the capability of showing the alloantibodies binding onto lymphocytes at the same time, can reduce the necessary time and workload in evaluating alloantibodies. More data from clinical samples are needed for cFCXM to be accepted by tissue typing laboratories. In this study, we compared cFCXM with complement-dependent lymphocytotoxicity and standard flow cytometric crossmatch in 41 renal pretransplant patients. A comparison of the obtained data was performed using Spearman's correlation test. We found that cFCXM showed no statistically significant differences with complement-dependent lymphocytotoxicity and flow cytometric crossmatch. We believe that cFCXM can be used in clinical laboratories in the near future following intra-laboratory validation.

An Alternative Pathway Specific Flow Cytometric Assay to Detect Complement Activation in Atypical Hemolytic Uremic Syndrome (aHUS)

Introduction aHUS is thrombotic microangiopathy associated with excess activation of alternative complement pathway (AP). Virtually all germline mutations that predispose to aHUS [e.g., factor H (fH)] occur in genes that regulate the amplification loop in the AP. The modified Ham test (mHam) measures the ability of a nucleated cell to protect itself from complement-mediated cell death (in vitro) in the absence of downstream cell-surface complement regulators, CD55 and CD59. Thus, the mHam exposes defects in serum-based complement regulators/activators that involve the amplification loop. The mHam is useful in distinguishing aHUS from thrombotic thrombocytopenic purpura (TTP); limitations are the lack of a reliable positive control and the lack of a confirmatory assay. In order to develop a positive control for the mHam, three complement activators, cobra venom factor (CVF), lipopolysaccharide (LPS) and sialidase (neuraminidase) were studied. CVF and LPS can activate and deplete serum of complement. Sialidase (Sia) makes cells more susceptible to complement mediated killing by removing sialic acid residues that are critical for binding the complement regulator, fH. Here, we show that Sia is a reliable positive control for the mHam and that cell surface C5b-9 accumulation correlates with a positive mHam.Methods/Results: The mHam assay was performed utilizing the PIGAnull TF1 cell line as previously described. CVF and LPS in dosages of 1.0 to 10ug/mL were added to 20% normal human serum (NHS) individually for 15 minutes at 37°C, and then added to cells in GVB++ buffer for 30 minutes. Sia (6.25unit/mL to 200unit/mL) was added to cells in GVB++ buffer for 15 minutes at 37°C first, and then incubated with 20% normal human serum (NHS) for 30 minutes. Complement-mediated cell killing in the mHam was evaluated using WST-1 absorbance as described previously. We found that sia treatment was more effective and reliable than LPS and CVF at inducing complement-mediated killing. A sia dose of 50U/mL or greater increased complement mediated killing by more than 3-fold in the mHam. Cell killing correlated with increased C5b-9 staining and was abrogated by the addition of an anti-C5 antibody and a factor D inhibitor. Next, we loaded PIGAnull TF1 cells with increasing amounts of C3b, added 20% NHS, and simultaneously performed the mHam and stained cells with an anti-C5b-9 monoclonal antibody for FACS staining to see if C5b-9 accumulation correlates with mHam killing. Briefly, PIGAnull TF1 cells in GVB+ buffer were incubated with 17.5ug C3, 4ug factor B and 0.1ug factor D for 20 minutes at room temperature (RT). Cells were washed and then incubated with 2ug factor B and 0.1ug factor D at RT for 3 minutes first, and treated with 0.25M EDTA in GVB0 buffer. Then 17.5ug C3 was added to cells for 15 minutes at 37°C. After three consecutive cycles of C3b loading, 20% NHS was added and complement activity was measured in the mHam and by flow cytometry. Cells loaded with one or two cycles of C3b showed minimal C5b-9 staining and minimal cell death (<15%) in the mHam; however, after three cycles there was a marked increase in C5b-9 staining (Figure 1A) that correlated with increased killing in the mHam. The addition of 2.5uM factor D inhibitor to NHS blocked C5b-9 accumulation on the PIGAnull TF1 cells. Based on these data we hypothesized that serum from aHUS patients would lead to an increase in C5b-9 staining on PIGAnull TF1 cells and also correlate with increased cell killing in the mHam. For these studies, 20% NHS from healthy controls, and aHUS was incubated with TF1PIGAnull cells. The mHam was performed as above using GVB++ buffer. For flow cytometry experiments AP pathway buffer (Mg++EGTA) was used and cells were stained with anti-C5b-9 antibody and analyzed by FACs. TF1PIGAnull cells treated with 50U/mL sialidase incubated with NHS was used as a positive control. aHUS serum from patients (with and without germline APC mutations) resulted in a marked increase in cell surface C5b-9 staining in AP buffer and correlated with increased cell death in the mHam. C5b-9 staining was blocked by addition of a factor D inhibitor (Figure 1B).Conclusion We describe an AP pathway specific flow cytometric assay that correlates with the mHam and may further aid in the diagnosis of aHUS. Sialidase treatment of TF1PIGAnull cells serves as a reliable positive control for the mHam by making cells more susceptible to AP killing. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Alternative Complement System Mediates Cell Death in Retinal Ischemia Reperfusion Injury.

Ischemia reperfusion (IR) injury induces retinal cell death and contributes to visual impairment. Previous studies suggest that the complement cascade plays a key role in IR injury in several systemic diseases. However, the role of the complement pathway in the ischemic retina has not been investigated. The aim of this study is to determine if the alternative complement cascade plays a role in retinal IR injury, and identify which components of the pathway mediate retinal degeneration in response to IR injury. To accomplish this, we utilized the mouse model of retinal IR injury, wherein the intraocular pressure (IOP) is elevated for 45 min, collapsing the retinal blood vessels and inducing retinal ischemia, followed by IOP normalization and subsequent reperfusion. We found that mRNA expression of complement inhibitors complement receptor 1-related gene/protein-y (Crry), Cd55 and Cd59a was down-regulated after IR. Moreover, genetic deletion of complement component 3 (C3−/−) and complement factor b (Fb−/−) decreased IR-induced retinal apoptosis. Because vascular dysfunction is central to IR injury, we also assessed the role of complement in a model of shear stress. In human retinal endothelial cells (HRECs), shear stress up-regulated complement inhibitors Cd46, Cd55, and Cd59, and suppressed complement-mediated cell death, indicating that a lack of vascular flow, commonly observed in IR injury, allows for complement mediated attack of the retinal vasculature. These results suggested that in retinal IR injury, the alternative complement system is activated by suppression of complement inhibitors, leading to vascular dysfunction and neuronal cell death.

Single-Domain Antibodies and the Promise of Modular Targeting in Cancer Imaging and Treatment.

Monoclonal antibodies and their fragments have significantly changed the outcome of cancer in the clinic, effectively inhibiting tumor cell proliferation, triggering antibody-dependent immune effector cell activation and complement mediated cell death. Along with a continued expansion in number, diversity, and complexity of validated tumor targets there is an increasing focus on engineering recombinant antibody fragments for lead development. Single-domain antibodies (sdAbs), in particular those engineered from the variable heavy-chain fragment (VHH gene) found in Camelidae heavy-chain antibodies (or IgG2 and IgG3), are the smallest fragments that retain the full antigen-binding capacity of the antibody with advantageous properties as drugs. For similar reasons, growing attention is being paid to the yet smaller variable heavy chain new antigen receptor (VNAR) fragments found in Squalidae. sdAbs have been selected, mostly from immune VHH libraries, to inhibit or modulate enzyme activity, bind soluble factors, internalize cell membrane receptors, or block cytoplasmic targets. This succinct review is a compilation of recent data documenting the application of engineered, recombinant sdAb in the clinic as epitope recognition “modules” to build monomeric, dimeric and multimeric ligands that target, tag and stall solid tumor growth in vivo. Size, affinity, specificity, and the development profile of sdAbs drugs are seemingly consistent with desirable clinical efficacy and safety requirements. But the hepatotoxicity of the tetrameric anti-DR5-VHH drug in patients with pre-existing anti-drug antibodies halted the phase I clinical trial and called for a thorough pre-screening of the immune and poly-specific reactivities of the sdAb leads.

Complement-Dependent Cytotoxicity Assay.

A useful feature of therapeutic antibodies is the ability to kill the cells to which they bind. Antibodies are capable of mediating cell killing in a variety of ways. Apoptosis, complement-mediated mechanisms, and antibody-dependent cellular cytotoxicity are all effects that can be assayed to characterize lead antibody candidates. Extensive, multidose characterizations of a series of candidates can be performed in a short amount of time using assays developed for high-throughput flow cytometry systems. Antibodies that contain the Fc portion of the human IgG1 can activate complement-mediated cell death. One way in which they do this is via direct complement killing of tumor cells by the membrane attack complex, a process usually called complement-dependent cytotoxicity.

ST8Sia6 deficiency is insufficient to induce complement-mediated attack on recent thymic emigrants

Abstract T cells require additional developmental steps after positive selection before they are functionally competent and can enter the long-lived peripheral T cell pool. During maturation, T cells gain the ability to produce cytokines upon stimulation, and the ability to protect themselves from TNF and complement-mediated cell death. In three different mouse models with defects in T cell maturation (CD4-cre NKAP conditional knockout mice, CD4-cre HDAC3 conditional knockout mice and CD4-cre RUNX1 conditional knockout mice), recent thymic emigrant (RTE) numbers are greatly reduced due to complement-mediated attack initiated by the binding of IgM and activation of the classical pathway. These models have deficiencies in the addition of sialic acid modifications, which is normally increased during the later stages of T cell development. As stripping cell surface sialic acids by neuraminidase on peripheral T cells leads to binding of natural IgM and complement activation, it was hypothesized complement-mediated attack is due to a defect in sialylation. Transcriptionally, the sialic acid transferase ST8Sia6 is upregulated concurrently with T cell maturation after positive selection. To understand the role of ST8Sia6 in T cell maturation, we generated ST8Sia6 knockout mice. These mice are healthy and fertile. RTEs and mature T cells are present in the periphery in normal numbers, and do not show evidence of complement deposition. Therefore, loss of ST8Sia6 alone is insufficient to induce complement-mediated attack on RTEs and other co-expressed sialyltransferases may also be required to protect from complement deposition.

Cytotoxic Antibody Detection by Means of Flow-Cytometric Cross-Match

BackgroundComplement-dependent lymphocytotoxicity (CDC-XM) and flow-cytometric (FCXM) cross-match are analyzed individually for each donor and recipient pair, because these techniques have fundamental differences for the evaluation of histocompatibility. Lately, cytotoxic flow-cytometric cross-match (cFCXM) has been developed as an alternative to both CDC-XM and FCXM techniques. We evaluated the limits of cFCXM with the use of different positive serum dilutions. MethodsCDC-XM, FCXM, and cFCXM tests were performed with the use of commercially available negative and positive serum samples and lymphocytes from healthy donors. ResultsComplement-dependent cell death was successfully detected with the use of cFCXM. Complement-dependent cell death ratios in cFCXM were similar those in CDC-XM. With cFCXM, not only complement-dependent cell death but also IgG binding could be detected within a single assay. At higher concentrations of the positive serum, IgG–fluorescein isothiocyanate (FITC) mean fluorescent intensity (MFI) values detected with the use of cFCXM were less than those of conventional FCXM. Correspondingly, for dead cells, MFI values of IgG-FITC were less than those of live cells in higher positive serum concentrations in the cFCXM assay. Moreover, our results demonstrated that in cFCXM analysis, the decreasing ratio of dead cells at increasing positive serum dilutions was not in parallel with the same decrease in IgG-FITC MFI values. ConclusionsThe cFCXM technique detects complement-mediated cytotoxic cell death with the additional ability to show IgG binding in the same tube and therefore may reduce the necessary bench time and workload.

Inhibition of the alternative complement pathway preserves photoreceptors after retinal injury.

Degeneration of photoreceptors is a primary cause of vision loss worldwide, making the underlying mechanisms surrounding photoreceptor cell death critical to developing new treatment strategies. Retinal detachment, characterized by the separation of photoreceptors from the underlying retinal pigment epithelium, is a sight-threatening event that can happen in a number of retinal diseases. The detached photoreceptors undergo apoptosis and programmed necrosis. Given that photoreceptors are nondividing cells, their loss leads to irreversible visual impairment even after successful retinal reattachment surgery. To better understand the underlying disease mechanisms, we analyzed innate immune system regulators in the vitreous of human patients with retinal detachment and correlated the results with findings in a mouse model of retinal detachment. We identified the alternative complement pathway as promoting early photoreceptor cell death during retinal detachment. Photoreceptors down-regulate membrane-bound inhibitors of complement, allowing for selective targeting by the alternative complement pathway. When photoreceptors in the detached retina were removed from the primary source of oxygen and nutrients (choroidal vascular bed), the retina became hypoxic, leading to an up-regulation of complement factor B, a key mediator of the alternative pathway. Inhibition of the alternative complement pathway in knockout mice or through pharmacological means ameliorated photoreceptor cell death during retinal detachment. Our current study begins to outline the mechanism by which the alternative complement pathway facilitates photoreceptor cell death in the damaged retina.

Sodium sensing in the brain.

Sodium (Na) homeostasis is crucial for life, and the Na+ level ([Na+]) of body fluids is strictly maintained at a range of 135–145 mM. However, the existence of a [Na+] sensor in the brain has long been controversial until Nax was identified as the molecular entity of the sensor. This review provides an overview of the [Na+]-sensing mechanism in the brain for the regulation of salt intake by summarizing a series of our studies on Nax. Nax is a Na channel expressed in the circumventricular organs (CVOs) in the brain. Among the CVOs, the subfornical organ (SFO) is the principal site for the control of salt intake behavior, where Nax populates the cellular processes of astrocytes and ependymal cells enveloping neurons. A local expression of endothelin-3 in the SFO modulates the [Na+] sensitivity for Nax activation, and thereby Nax is likely to be activated in the physiological [Na+] range. Nax stably interacts with Na+/K+-ATPase whereby Na+ influx via Nax is coupled with activation of Na+/K+-ATPase associated with the consumption of ATP. The consequent activation of anaerobic glucose metabolism of Nax-positive glial cells upregulates the cellular release of lactate, and this lactate functions as a gliotransmitter to activate GABAergic neurons in the SFO. The GABAergic neurons presumably regulate hypothetic neurons involved in the control of salt intake behavior. Recently, a patient with essential hypernatremia caused by autoimmunity to Nax was found. In this case, the hypernatremia was considered to be induced by the complement-mediated cell death in the CVOs, where Nax specifically populates.

Construction and characterization of recombinant human C9 or C7 linked to single Chain Fv directed to CD25

Complement-dependent cytotoxicity (CDC) is a potent promoter of tumor clearance during monoclonal antibody therapy. Complement activation on antibody-bearing tumor cells results in formation of the membrane attack complex (MAC), which activates cell death. The complement activation cascade that bridges between antibody binding and MAC formation is regulated by complement inhibitors that are over-expressed on tumor cells. In order to bypass those complement regulators, we have designed an immunoconjugate composed of a humanized single chain Fv of an anti-Tac (CD25) monoclonal antibody fused at its C terminus either to complement protein C9 (scFv-C9) or to complement C7 (scFv-C7) and tagged with six histidines at the C terminal end. Recombinant scFv-C9 and scFv-C7 were expressed in 293T cells and purified. Both are shown to efficiently bind to CD25-positive tumor cells. In addition, scFv-C9, but not scFv-C7, increases MAC deposition on the cells and enhances complement-mediated cell death of target CD25-positive cells. Thus, scFv-C9 fusion protein is potentially a novel reagent for application in cancer immunotherapy.

Complement activation mediates cetuximab inhibition of non-small cell lung cancer tumor growth in vivo

BackgroundCetuximab, an antibody targeting the epidermal growth factor receptor (EGFR), increases survival in patients with advanced EGFR-positive non-small cell lung cancer when administrated in combination with chemotherapy. In this study, we investigated the role of complement activation in the antitumor mechanism of this therapeutic drug.ResultsEGFR-expressing lung cancer cell lines were able to bind cetuximab and initiate complement activation by the classical pathway, irrespective of the mutational status of EGFR. This activation led to deposition of complement components and increase in complement-mediated cell death. The influence of complement activation on the activity of cetuximab in vivo was evaluated in xenografts of A549 lung cancer cells on nude mice. A549 cells express wild-type EGFR and have a KRAS mutation. Cetuximab activity against A549 xenografts was highly dependent on complement activation, since complement depletion completely abrogated the antitumor efficacy of cetuximab. Moreover, cetuximab activity was significantly higher on A549 cells in which a complement inhibitor, factor H, was genetically downregulated.ConclusionsWe demonstrate for the first time that the in vivo antitumor activity of cetuximab can be associated with a complement-mediated immune response. These results may have important implications for the development of new cetuximab-based therapeutic strategies and for the identification of markers that predict clinical response.

Influence of Polyclonal Antithymocyte Globulins on the Expression of Adhesion Molecules of Isolated Human Umbilical Vein Endothelial Cells

Objectives Polyclonal antithymocyte globulins (ATGs) are immunosuppressive agents applied for the treatment and prevention of organ rejection after transplantation. ATGs induce complement-mediated cell death in T lymphocytes and decrease leukocyte adhesion. However, little is known about the effects of ATGs on endothelial cells (EC). Our aim was to study the influence of ATGs upon the expression of adhesion molecules on human umbilical vein endothelial cells (HUVECs) after stimulation with tumor necrosis factor (TNF)-α. Material and Methods HUVECs obtained from umbilical cords were incubated with ATGs before and after 6-hour stimulation with TNF-α. The group incubated without ATG served as the controls. Another group was not stimulated with TNF-α. By flow cytometry, we analyzed the expression of several adhesion molecules: intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM), platelet EC adhesion molecule (PECAM), and CD62E. Statistical analysis used analysis of variance. Results After TNF-α stimulation, the EC surface expression of ICAM-1 and CD62E was reduced, although not significantly, in treated as compared with untreated cells. The expression of ICAM-1 and CD62E was similar in the unstimulated groups. The expression of VCAM, PECAM, CD55, and CD58 was not modified by ATG treatment. Conclusion Our results demonstrated that ATGs insignificantly reduced the expression of adhesion molecules in HUVECs. The effect of ATGs on stimulated HUVECs remains unclear, probably due to the lack of effector cells.

Autoimmunity to the Sodium-Level Sensor in the Brain Causes Essential Hypernatremia

Na(x) is the sodium-level sensor of body fluids in the brain involved in sodium homeostasis. Na(x)-knockout mice do not stop ingesting salt even when dehydrated. Here we report a case with clinical features of essential hypernatremia without demonstrable hypothalamic structural lesions, who was diagnosed as a paraneoplastic neurologic disorder. The patient had autoantibodies directed against Na(x), along with a ganglioneuroma composed of Schwann-like cells robustly expressing Na(x). The removal of the tumor did not reduce the autoantibody levels or relieve the symptoms. Intravenous injection of the immunoglobulin fraction of the patient's serum into mice induced abnormalities in water/salt intake and diuresis, which led to hypernatremia. In the brains of these mice, cell death was observed along with focal deposits of complement C3 and inflammatory infiltrates in circumventricular organs where Na(x) is specifically expressed. Our findings thus provide new insights into the pathogenesis of hypernatremia relevant to the sodium-level-sensing mechanism in humans.

Involvement of the c-jun N-terminal kinases JNK1 and JNK2 in complement-mediated cell death

Cell death and survival signals activated by the complement membrane attack complex C5b-9 play important roles in complement-associated diseases and in antibody-based cancer therapy. Here, we investigated the involvement of the JNK mitogen-activated protein kinase in C5b-9-induced cell lysis. Necrotic-type cell death regulation by JNK1 and JNK2 was selectively studied in mouse fibroblasts and human K562, HeLa and 293T cells. C5b-9 induced higher JNK activation than C5b-8. Pretreatment with a JNK inhibitor reduced cell sensitivity to complement-mediated lysis. KO cells deficient in either JNK1 or JNK2 were less sensitive to lysis than WT cells. This correlated with lower C3 and C5b-9 deposition on KO cells. Furthermore, silencing of JNK1 or JNK2 expression by RNA interference decreased cell lysis by complement. Reconstitution of JNK2 into JNK2−/− cells and over expression of JNK2 in WT cells increased C3 and C5b-9 deposition as well as cell sensitivity to complement-mediated lysis. Pretreatment of cells with the phosphotyrosine phosphatase inhibitor phenylarsine oxide increased JNK activation and JNK-dependent complement-mediated necrotic death of WT and JNK2−/− KO cells but not of JNK1−/− KO cells. The JNK inhibitor and PAO had no effect on complement-mediated lysis in cells lacking Bid, suggesting involvement of Bid in the JNK lytic pathway. Our results demonstrate that complement C5b-9 induce a JNK/Bid-dependent and JNK-independent necrotic cell death. Both JNK1 and JNK2 have cytotoxic potential, however JNK2 is the primary signal transducer.

Antithymocyte Globulins Bind to Human Umbilical Vein Endothelial Cells

Polyclonal antithymocyte globulins (ATGs) are immunosuppressive agents used for the treatment of organ rejection after transplantation. ATGs induce complement-mediated cell death of T lymphocytes and may decrease leukocyte adhesion. However, little is known about their effects on endothelial cells (ECs). Our aim was to study whether they bind to human umbilical vein endothelial cells (HUVECs). HUVECs obtained from umbilical cords were cultured and incubated with ATGs. A group incubated without ATG served as controls. All groups were coincubated with an anti-rabbit-IgG. Binding of ATGs to HUVECs was investigated by means of flow cytometry. Statistical analysis was performed using one-way analysis of variance (ANOVA). ATGs were bound to HUVECs with or without prior stimulation by tumor necrosis factor-alpha (TNF-alpha). Binding of ATGs to HUVECs was >99% in all treated groups. The mean intensity of fluorescence was constant in the ATG-treated groups. Our results demonstrated that ATGs bind to HUVECs before and after stimulation with TNF-alpha. Binding of ATGs to HUVECs suggests an independent endothelial mechanism of ATG action.