Barbara A. Benson

Resistance Against the Membrane Attack Complex of Complement Induced in Porcine Endothelial Cells with a Galα(1–3)Gal Binding Lectin: Up-Regulation of CD59 Expression

Endothelial cells (EC) play central roles in vascular physiology and pathophysiology. EC activation results in proinflammatory activities with production of cytokines and expression of adhesion molecules. However, we have shown before in a model of xenotransplantation that prolonged stimulation of porcine EC with human anti-porcine IgM natural Abs can activate the cells to become resistant against cytotoxicity by the membrane attack complex of complement (MAC). Now we report the major characteristics of induction and maintenance of resistance elicited in porcine EC with Bandeiraea simplicifolia lectin that binds terminal galα(1–3)gal. Lectin-treated cells underwent little or no cytotoxicity and PGI2 release when exposed to MAC. Induction of resistance required incubation of the EC with lectin for 4 h but was not fully manifested until 16 h later. Most of the initially bound lectin remained on the cell surface for >60 h. EC-bound lectin did not inhibit binding of IgM natural Abs or activation and binding of C components, including C9, but a C-induced permeability channel of reduced size was present. Induction of resistance required protein synthesis, developed slowly, and was associated with up-regulation of expression of mRNA for the MAC inhibitor CD59 and membrane-associated CD59 protein. Resistance lasted at least 3 days, and the cells regained normal morphology and were metabolically active. This induced resistance may have a physiologic counterpart that might be amenable to pharmacologic manipulation in vascular endothelium pathophysiology.

Human IgM xenoreactive natural antibodies can induce resistance of porcine endothelial cells to complement-mediated injury

Abstract: It is thought that human IgM xenoreactive natural antibodies (nAbs) can induce activation of porcine endothelial cells independent of complement. Therefore we hypothesized that pretreatment of porcine endothelial cells with anti‐pig nAbs may affect the ability of the endothelial cells, when subsequently incubated with a source of nAbs and complement, to bind antibodies and complement components and to undergo complement‐mediated cytotoxicity. We preincubated porcine endothelial cells at 37°C for 1 hr or 40 hr with a source of nAbs. We then incubated these pretreated endothelial cells with a complement source that contained a normal complement level and a low level of IgM nAbs for 1 hr to measure bound IgM and IgG and complement components, and for 4 hr to measure cytotoxicity. We found that preincubation for as long as 40 hr did not impair the binding of IgM and IgG, implying no antibody‐induced loss of membrane antigens from the endothelial cells, or the binding of C3bi, C4d, C6, and C9 upon complement activation. In contrast, preincubation for 40 hr with a nAb source induced in the endothelial cells marked resistance to complement‐mediated killing. Resistance could be induced with purified human IgM but not with purified IgG or IgM‐depleted human serum. The ability of purified IgM to induce resistance was abrogated by removal of anti‐pig xenoreactive nAbs by absorption with pig endothelial cells, and induction of resistance required protein synthesis. We conclude that prolonged incubation of human anti‐pig nAbs with pig endothelial cells does not cause loss of endothelial cell membrane antigens or impairment in binding of nAbs or complement components; instead, it induces marked resistance to complement‐mediated cytotoxicity. These observations may be of value to develop strategies that enhance survival of a xenograft.

IL-4 and IL-13 Induce Protection of Porcine Endothelial Cells from Killing by Human Complement and from Apoptosis through Activation of a Phosphatidylinositide 3-Kinase/Akt Pathway

Vascular endothelial cells (EC) perform critical functions that require a balance of cell survival and cell death. EC death by apoptosis and EC activation and injury by the membrane attack complex of complement are important mechanisms in atherosclerosis and organ graft rejection. Although the effects of various cytokines on EC apoptosis have been studied, little is known about their effects on complement-mediated EC injury. Therefore, we studied the abilities of various cytokines to induce protection of porcine aortic EC against apoptosis and killing by human complement, a model of pig-to-human xenotransplantation. We found that porcine EC incubated with IL-4 or IL-13, but not with IL-10 or IL-11, became protected from killing by complement and apoptosis induced by TNF-α plus cycloheximide. Maximal protection required 10 ng/ml IL-4 or IL-13, developed progressively from 12 to 72 h of incubation, and lasted 48–72 h after cytokine removal. Protection from complement was not associated with reduced complement activation, C9 binding, or changes in CD59 expression. Inhibition of PI3K prevented development of protection; however, inhibition of p38 MAPK or p42/44 MAPK had no effect. IL-4 and IL-13 induced rapid phosphorylation of Akt. Although protection was inhibited by an Akt inhibitor and a dominant negative Akt mutant transduced into EC, it was induced by transduction of EC with the constitutively active Akt variant, myristylated Akt. We conclude that IL-4 and IL-13 can induce protection of porcine EC against killing by apoptosis and human complement through activation of the PI3K/Akt signaling pathway.

A soluble chimeric inhibitor of C3 and C5 convertases, complement activation blocker-2, prolongs graft survival in pig-to-rhesus monkey heart transplantation

Complement plays a critical role in many pathologic processes and in xenograft rejection. Therefore, effective complement inhibitors are of great interest. In pig‐to‐primate organ transplantation, hyperacute rejection results from antibody deposition and complement activation. Complement activation blocker‐2 (CAB‐2), a recombinant soluble chimeric protein derived from human decay accelerating factor (DAF) and membrane cofactor protein, inhibits C3 and C5 convertases of both classical and alternative pathways. CAB‐2 reduces complement‐mediated tissue injury of a pig heart perfused ex vivo with human blood. Therefore, we studied the efficacy of CAB‐2 when a pig heart is transplanted heterotopically into rhesus monkeys receiving no immunosuppression. Graft survival in three control monkeys was 1.26 ± 0.2 h; it was markedly prolonged in eight monkeys that received CAB‐2. Of the six monkeys that received a single dose of CAB‐2 (15 mg/kg i.v.), four had graft survivals of 21, 95, 96, and 108 h, and two died at 7 to11 h post‐transplant with a beating graft, as a result of technical complications. The two monkeys given multiple doses of CAB‐2 had graft survivals of 95 and 96 h. CAB‐2 markedly inhibited complement activation, as shown by a strong reduction in generation of C3a and SC5b‐9. At graft rejection, tissue deposition of iC3b, C4 and C9 was similar or slightly reduced from controls, and deposition of IgG, IgM, C1q and fibrin did not change. Thus, complement inhibition with CAB‐2 abrogates hyperacute rejection of pig hearts transplanted into rhesus monkeys, but does not prevent delayed/acute vascular rejection. These studies demonstrate that the beneficial effects of complement inhibition on survival of a pig heart xenograft in rhesus monkeys are similar to those in other primate species and that CAB‐2 may be useful in xenotransplantation and other complement‐mediated conditions.

Characteristics of CD59 up-regulation induced in porcine endothelial cells by alphaGal ligation and its association with protection from complement.

Abstract: Background: Activation of endothelial cells may result in proinflammatory and procoagulant changes, or in changes that protect the endothelial cells (EC) from injurious insults. Stimulation of porcine EC with human anti‐porcine antibodies, or lectins from Bandeiraea simplicifolia that bind terminal Galα(1–3)Gal (abbreviated αGal), can induce EC protection from cytotoxicity by human complement. These EC also exhibit up‐regulation of CD59 protein and mRNA expression. Porcine CD59 has been reported to protect porcine cells from human complement. Therefore we investigated the specificity requirements and other characteristics of the induced CD59 up‐regulation, as well as the role of up‐regulated CD59 in lectin‐induced protection of EC from human complement. Methods: Aortic EC were incubated in vitro with αGal‐binding lectins B. simplicifolia lectin I isolectin B4 (IB4) and B. simplicifolia lectin I (BS‐I) and CD59 expression was assessed by flow cytometry and enzyme linked immunosorbent assay (ELISA). Binding requirement was studied using disaccharides containing either αgalactosyl or βgalactosyl moieties to inhibit CD59 up‐regulation. Protection from complement killing was assessed after incubation of EC with human serum as a source of anti‐porcine antibodies and complement. The role of CD59 in lectin‐induced protection was studied in the presence of an anti‐pig CD59 antibody and after removal of CD59 using phosphatidylinositol (PI)‐specific phospholipase C (PI‐PLC). Results: We found that induction of CD59 up‐regulation required specific binding of the lectin to terminal αGal and was not induced either by soluble factors that may be released from EC by stimulation with the lectin or by TNF‐α, IFN‐γ, or IL‐1α. Unstimulated or BS‐I‐treated EC showed little or no expression of decay accelerator factor (DAF). Removal of membrane‐associated CD59 (and other proteins that are associated with the membrane through PI linkage) with PI‐PLC from EC that had been exposed to lectin restored their complement sensitivity to various degrees, depending on the extent of lectin‐induced protection. Cytotoxicity was completely restored in cells that exhibited partial protection induced with lectin at low doses or for a short period of time. However, EC that were fully resistant to complement did not regain sensitivity to complement after removal of CD59. Changes in CD59 expression did not modify the degree of C9 binding. Conclusion: Induction of CD59 expression required specific binding of the lectin to terminal αGal and was not induced by soluble factors that may be released from EC by lectin stimulation. Increased CD59 expression may contribute to this form of protection from complement; however, mechanisms other than CD59 up‐regulation appear to be essential for the development of full protection.

Porcine endothelial cells and iliac arteries transduced with AdenoIL-4 are intrinsically protected, through Akt activation, against immediate injury caused by human complement.

Vascular endothelial cells (ECs) can be injured in a variety of pathologic processes that involve activated complement. We reported previously that porcine ECs incubated with exogenous IL-4 or IL-13 are protected from cytotoxicity by human complement and also from apoptosis by TNF-α. The resistance to complement consists of an intrinsic mechanism that is lost a few days after cytokine removal. In our current study, we investigated whether transfer of the IL-4 gene into porcine ECs in vitro and into porcine vascular tissues in vivo would induce efficient and durable protection from human complement. We found that ECs transduced with adenoIL-4 or adenoIL-13 exhibited continuous production of the cytokine and prolonged protection from complement-mediated killing. IL-4 also protected ECs from activation: ECs incubated with IL-4 did not develop cell retraction and intercellular gaps upon stimulation with sublytic complement. The endothelium and subendothelium of pig iliac arteries that were transduced with the IL-4 gene were effectively protected from complement-dependent immediate injury after perfusion with human blood. However, after similar perfusion, the endothelium was immediately lost from arteries that were transduced with a control adenovirus. The protection was not due to up-regulation of the complement regulators decay accelerating factor, membrane cofactor protein, and CD59, or to reduced complement activation, but required the participation of Akt. Although our studies model protection in pig-to-primate xenotransplantation, our findings of IL-4 induction of Akt-mediated protection may be more broadly applicable to EC injury as manifested in ischemia-reperfusion, allotransplantation, and various vascular diseases.

Cannabinoid receptor-specific mechanisms to alleviate pain in sickle cell anemia via inhibition of mast cell activation and neurogenic inflammation.

Sickle cell anemia is a manifestation of a single point mutation in hemoglobin, but inflammation and pain are the insignia of this disease which can start in infancy and continue throughout life. Earlier studies showed that mast cell activation contributes to neurogenic inflammation and pain in sickle mice. Morphine is the common analgesic treatment but also remains a major challenge due to its side effects and ability to activate mast cells. We, therefore, examined cannabinoid receptor-specific mechanisms to mitigate mast cell activation, neurogenic inflammation and hyperalgesia, using HbSS-BERK sickle and cannabinoid receptor-2-deleted sickle mice. We show that cannabinoids mitigate mast cell activation, inflammation and neurogenic inflammation in sickle mice via both cannabinoid receptors 1 and 2. Thus, cannabinoids influence systemic and neural mechanisms, ameliorating the disease pathobiology and hyperalgesia in sickle mice. This study provides ‘proof of principle’ for the potential of cannabinoid/cannabinoid receptor-based therapeutics to treat several manifestations of sickle cell anemia.

Spinal glial activation and oxidative stress are alleviated by treatment with curcumin or coenzyme Q in sickle mice.

Sickle cell anemia (SCA) is accompanied by unpredictable episodes of recurrent acute pain during vaso-occlusive crises (VOC), superimposed on chronic pain.1 Pain in SCA can start in infancy and may continue throughout life, leading to sustained activation of the nociceptive mechanisms resulting in poor therapeutic outcomes. Pain is an outcome of nociceptive processing in the central nervous system (CNS), triggered by peripheral nervous system response to exogenous and endogenous stimuli. Activation of transient receptor potential vanilloid 1 (TRPV1) channels on C-fibers, neurogenic inflammation, mast cell activation, systemic inflammation, and oxidative stress in the periphery have been demonstrated in SCA.2,3 However, the extent and mechanisms of CNS involvement remain unknown in SCA. The activation of inflammatory and neuronal cells in the CNS plays a pivotal role in nociception.4 We recently observed that spinal nociceptive neurons are sensitized in sickle mice, suggestive of central sensitization.5 Bidirectional signaling occurs between neurons and immunocompetent cells present in the CNS, including microglia, astrocytes and oligodendrocytes.4 Activated microglia release reactive oxidative species (ROS), inflammatory cytokines, neurotrophic factors, and prostaglandins that excite nociceptive neurons and contribute to the persistence of chronic pain.4 It is therefore likely that the activation of central nociceptive mechanisms contributes to chronic pain in SCA. Remarkable decreases in inflammation, thiobarbituric acid reactive substances (TBARS, an indicator of oxidative stress), and VOC have been observed in SCA patients receiving coenzyme Q10 (CoQ10).6 Additionally, curcumin reduced markers of oxidative stress in thalassemia patients and also ameliorates pain hypersensitivity in rats with monoarthritis by decreasing spinal neuroinflammation.7,8 Since excess free iron due to hemolysis contributes to oxidative stress and inflammation in SCA, we examined glial activation, inflammation and oxidative stress in the spinal cords of sickle mice and tested the possibility of a synergistic effect of CoQ10 and/or curcumin to ameliorate spinal oxidative stress, glial activation and hyperalgesia. To examine our hypotheses, we used female transgenic HbSS-BERK sickle mice with murine α and β globin knockouts and expressing human sickle, or normal haemoglobin A (designated sickle or control mice, henceforth, respectively). We bred and characterized these mice by pheno- and genotyping (see Online Supplementary Appendix for details).3,9 These sickle mice have severe hematologic disease, organ damage and tonic hyperalgesia similar to that observed in human SCA.9–11 Sickle mice received either vehicle (olive oil), curcumin (15 mg/kg), CoQ10 (45 mg/kg), or both CoQ10 and curcumin (cotreatment) daily for 4 weeks by gavage. Female mice were used because BERK female mice show more hyperalgesia as compared to males.11 Pain behaviors were evaluated during the proestrous/estrous cycle before treatment and weekly. The mice were tested for mechanical hyperalgesia using paw withdrawal frequency (PWF) in response to von Frey filaments, paw withdrawal latency (PWL) in response to a heat stimulus using a Hargreave’s apparatus; and sensitivity to cold was determined by PWL and PWF per 2 min on a cold plate (see detailed procedures in the Online Supplementary Appendix).11 Spinal cords were harvested after 4 weeks of treatment. Sections were examined by laser scanning confocal microscopy (LSCM) for Iba1, a microglial marker (Wako, Richmond, VA, USA), glial fibrillary acidic protein (GFAP), an astrocyte marker (Abcam, Cambridge, MA, USA), neuropeptide substance P (SP, Abcam) and detection of ROS with dihydroethidium (DHE, Life Technologies, Grand Island, NY, USA).

Interleukin-4 Induces Up-regulation of Endothelial Cell Claudin-5 through Activation of FoxO1 ROLE IN PROTECTION FROM COMPLEMENT-MEDIATED INJURY

Background: IL-4 protects vascular endothelial cells (ECs) from complement-mediated junctional injury and cytotoxicity. Results: IL-4 induces up-regulated expression of junctional claudin-5 through STAT6 and FoxO1. Claudin-5 up-regulation contributes to, but is not sufficient, for protection. Conclusion: IL-4-induced claudin-5 is an important component in protection of ECs from complement. Significance: Regulation of claudin-5 expression may play a role controlling EC inflammatory injury. Injury to endothelial cells (ECs) often results in cell retraction and gap formation. When caused by antigen aggregation or complement, this injury can be prevented by pretreatment of the ECs with IL-4, suggesting that IL-4 modifies the intercellular junction. Therefore, we investigated the effects of IL-4 on expression of intercellular junction proteins and whether such effects are required for IL-4-induced resistance of ECs against complement-mediated injury. We found that IL-4 induces up-regulation of the junction protein claudin-5 in porcine ECs through activation of Jak/STAT6 and phosphorylation and translocation of FoxO1 from the nucleus to the cytoplasm. Increased claudin-5 expression resulted in increased transmembrane electrical resistance of the endothelial monolayer and participated in IL-4-induced protection of the ECs from complement injury. Down-regulation of FoxO1 using siRNA by itself caused up-regulation of claudin-5 expression and partial protection from cytotoxicity. This protection was enhanced by stimulation with IL-4. We previously reported that increased phospholipid synthesis and mitochondrial protection were required for IL-4-induced resistance of ECs against complement injury and now we demonstrate a contribution of claudin-5 expression in IL-4-induced protection.

IL-4 induces protection of vascular endothelial cells against killing by complement and melittin through lipid biosynthesis

We have shown previously that cytokines IL‐4 and IL‐13 induce protection in porcine vascular endothelial cells (EC) against killing by the membrane attack complex (MAC) of human complement. This protection is intrinsic, not due to changes in complement regulatory proteins, and requires activation of Akt and sterol receptor element binding protein‐1 (SREBP‐1), which regulates fatty acid and phospholipid synthesis. Here we report that, compared to EC incubated in medium, IL‐4‐treated EC had a profound reduction in complement‐mediated ATP loss and in killing assessed by vital dye uptake, but only a slight reduction in permeability disruption measured by calcein release. While controls exposed to complement lost mitochondrial membrane potential and subsequently died, protected EC maintained mitochondrial morphology and membrane potential, and remained alive. SREBP‐1 and fatty acid synthase activation were required for protection and fatty acid and phospholipid synthesis, including cardiolipin, were increased after IL‐4 stimulation, without increase in cholesterol content or cell proliferation. IL‐4 also induced protection of EC from killing by the channel forming protein melittin, similar to protection observed for the MAC. We conclude that IL‐4 induced activation of Akt/SREBP‐1/lipid biosynthesis in EC, resulting in protection against MAC and melittin, in association with mitochondrial protection.