Important Effector Mechanism Research Articles

Anti-SIV Cytolytic Molecules in Pigtail Macaques

Release of granzymes and perforin from the cytolytic granules of SIV-specific CD8 T cells is a critically important effector mechanism facilitating the elimination of SIV-infected cells. We sequenced granzyme A, B, and K and perforin in pigtail macaques and defined polymorphisms between humans, rhesus macaques, and pigtail macaques. The pigtail macaque sequences were similar to the corresponding rhesus sequences at the mRNA and protein level and (0.4-1.1% sequence differences) but substantially different from human sequences (3.8-8.1% sequence differences). We used this sequence information to develop multiplex PCR assays to detect these genes. We also successfully studied the release of perforin and granzyme B from deregulating SIV-specific CD8 T cells by flow cytometry. These sequences and tools enable further study of the cytolytic control of SIV in pigtail macaques.

The action of MBL-associated serine protease 1 (MASP1) on factor XIII and fibrinogen

The complement system is an important recognition and effector mechanism of the innate immune system that upon activation leads to the elimination of foreign bodies. It can be activated through three pathways of which the lectin pathway is one. The lectin pathway relies on the binding of mannan-binding lectin (MBL) or the ficolins and the subsequent activation of the MBL-associated serine proteases (MASPs), namely, MASP1, 2 and 3 which all form complexes with both MBL and the ficolins. Major substrates have only been identified for MASP2 i.e. C4 and C2. For MASP1 only a few protein substrates which are cleaved at a low rate have been identified while none are known for MASP3. Since chromogenic substrate screenings have shown that MASP1 has thrombin-like activity, we wanted to investigate the catalytic potential of MASP1 towards two major proteins involved in the clotting process, fibrinogen and factor XIII, and compare the activity directly with that of thrombin. We found that rMASP1 and thrombin cleave factor XIII A-chain and the fibrinogen β-chain at identical sites, but differ in cleavage of the fibrinogen α-chain. The thrombin turnover rate of factor XIII is approximately 650 times faster than that of rMASP1 at 37°C, pH 7.4. rMASP1 cleavage of fibrinogen leads to the release of the proinflammatory peptide fibrinopeptide B. Thus rMASP1 has similar, but not identical specificity to thrombin and its catalytic activity for factor XIII and fibrinogen cleavage is much lower than that of thrombin. Nevertheless, rMASP1 can drive the formation of cross-linked fibrinogen. Since MASP1 is activated on contact of MBL or the ficolins with microorganisms, fibrinogen and factor XIII may be involved in the elimination of invading pathogens.

An Essential Role for ras in LPS Mediated Amplification of TREM-1 Induced Neutrophil Activation.

The triggering receptor expressed on myeloid cells 1 (TREM-1) is constitutively expressed on polymorphonuclear neutrophils (PMN), monocytes and macrophage subsets and has been recently identified as an important player in the innate inflammatory response to microbial infections and sepsis. Expression of TREM-1 and activation of PMN and monocytes is regulated in concert with microbial products via the recognition of Toll-like receptor (TLR) ligands such as bacterial lipopolysaccaride (LPS). However, it is currently unclear how the amplification of the inflammatory responses by TREM-1 and TLR agonists is mediated on an intracellular level. To this end, we were interested to identify signalling events leading to the synergistic activation of PMN upon TREM-1 and TLR ligation. Using pharmacologic inhibitors and western blot analyses we demonstrate that PI3 kinase, PLC-g and p38 MAP kinase are essential for the initiation of the respirator burst as an important and immediate effector mechanism of human PMN. Furthermore, the phosphorylation of PKB/Akt and MAP kinase ERK show a biphasic pattern upon TREM-1/TLR4 coligation indicating distinct activation mechanisms for these receptors. Interestingly, the TLR4, but not TREM-1 mediated respiratory burst was dependent on the activation of ras leading downstream to an augmented calcium flow and subsequently synergistic burst activity. Taken together, we provide a new mechanism how TREM-1 and TLR interact in PMN mediating enhanced activation. These results shed a new light on our understanding how the innate inflammatory responses are regulated and might contribute to the development of future concept for the treatment of a dysregulated immune response in severe inflammatory conditions such as sepsis.

Expression of Complement Regulatory Protein CD55 on Tumor Cells Has an Adverse Effect on the Outcome of Follicular Lymphoma Patients Treated with Immunochemotherapy

The addition of rituximab to chemotherapy has considerably improved the outcome of follicular lymphoma (FL) patients, but the lengths of remissions are still variable. Activation of the C system has been shown to be an important effector mechanism of rituximab. The aim of this study was to evaluate whether differences in the expression of CD20 and C regulatory molecules (C-REG) in lymphoma cells correlate to the clinical course of FL. We used oligonucleotide microarray analysis to study mRNA levels of CD20, CD46 (MCP), CD55 (DAF) and CD59 (protectin) in the FL tissue of 23 patients treated with R-CHOP. The median follow-up time was 55 months. The patients were divided into long-term (progression free survival (PFS) >35 mo) and short-term (PFS <21 mo) responders, and mRNA levels were compared between the groups. High CD55 expression was observed more often in FL patients who relapsed early resulting in a shorter progression free survival (p=0.027). Median PFS time for patients with a low CD55 level was significantly longer than for those with high CD55 expression (not reached vs. 20 mo, p=0.003). The results were validated prospectively by analyzing the protein levels of CD20 and C-REGs with flow cytometry. The results of a pilot study of 12 FL patients showed that relative expression of CD20 to CD55 in CD20 positive cell population was higher in patients in remission in comparison to ones who relapsed. According to Kaplan-Meier estimates, the patients with low CD20/CD55 and CD20/C-REGave (average C regulator expression) ratios relapsed more often than the other patients. The results suggest that the expression of C regulators, especially of CD55, affects the efficacy of immunochemotherapy, and that C regulatory molecules represent an effective escape mechanism of immunochemotherapy in FL.

Molecular cloning, promoter analysis and induced expression of the complement component C9 gene in the grass carp Ctenopharyngodon idella

Complement-mediated killing of pathogens through lytic pathway is an important effector mechanism of innate immune response. C9 is the ninth member of complement components, creating the membrane attack complex (MAC). In the present study, a putative cDNA sequence encoding the 650 amino acids of C9 and its genomic organization were identified in grass carp Ctenopharyngodon idella. The deduced amino acid sequence of grass carp C9 (gcC9) showed 48% and 38.5% identity to Japanese flounder and human C9, respectively. Domain search revealed that gcC9 contains a LDL receptor domain, an EGF precursor domain, a MACPF domain and two TSP domain located in the N-terminal and C-terminal, respectively. Phylogenetic analysis demonstrated that gcC9 is clustered in a same clade with Japanese flounder, pufferfish and rainbow trout C9. The gcC9 gene consists of 11 exons with 10 introns, spacing over approximately 7 kb of genomic sequence. Analysis of gcC9 promoter region revealed the presence of a TATA box and some putative transcription factor such as C/EBP, HSF, NF-AT, CHOP-C, HNF-3B, GATA-2, IK-2, EVI-1, AP-1, CP2 and OCT-1 binding sites. The first intron region contains C/EBPb, HFH-1 and Oct-1 binding sites. RT-PCR and Western blotting analysis demonstrated that the mRNA and protein of gcC9 gene have similar expression patterns, being constitutively expressed in all organs examined of healthy fish, with the highest level in hepatopancreas. By real-time quantitative RT-PCR analysis, gcC9 transcripts were significantly up-regulated in head kidney, spleen, hepatopancreas and down-regulated in intestine from inactivated fish bacterial pathogen Flavobacterium columnare-stimulated fish, demonstrating the role of C9 in immune response.

Abrogation of Antibody-Mediated Allograft Rejection by Regulatory CD4 T Cells with Indirect Allospecificity

Alloantibody is an important effector mechanism for allograft rejection. In this study, we tested the hypothesis that regulatory T cells with indirect allospecificity can prevent humoral rejection by using a rat transplant model in which acute rejection of MHC class I-disparate PVG.R8 heart grafts by PVG.RT1(u) recipients is mediated by alloantibody and is dependent upon help from CD4 T cells that can recognize the disparate MHC alloantigen only via the indirect pathway. Pretransplant treatment of PVG.RT1(u) recipients with anti-CD4 mAb plus donor-specific transfusion abrogated alloantibody production and prolonged PVG.R8 graft survival indefinitely. Naive syngeneic splenocytes injected into tolerant animals did not effect heart graft rejection, suggesting the presence of regulatory mechanisms. Adoptive transfer experiments into CD4 T cell-reconstituted, congenitally athymic recipients confirmed that regulation was mediated by CD4 T cells and was alloantigen-specific. CD4 T cell regulation could be broken in tolerant animals either by immunizing with an immunodominant linear allopeptide or by depleting tolerant CD4 T cells, but surprisingly this resulted in neither alloantibody generation nor graft rejection. These findings demonstrate that anti-CD4 plus donor-specific transfusion treatment results in the development of CD4 regulatory T cells that recognize alloantigens via the indirect pathway and act in an Ag-specific manner to prevent alloantibody-mediated rejection. Their development is associated with intrinsic tolerance within the alloantigen-specific B cell compartment that persists after T cell help is made available.

Complement-Induced Cell Death by Rituximab Depends on CD20 Expression Level and Acts Complementary to Antibody-Dependent Cellular Cytotoxicity

The use of the CD20-specific antibody rituximab has greatly improved the response to treatment of CD20+ follicular lymphoma. Despite the success of rituximab, resistance has been reported and prognostic markers to predict individual response are lacking. The level of CD20 expression on tumors has been related to response, but results of several studies are contradictory and no clear relationship could be established. Complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) are thought to be important effector mechanisms, but the exact mechanism of rituximab-mediated cell kill is still unknown. Importantly, no data have been reported on the combined contribution of CDC and ADCC. We have developed a system of clonally related CEM-CD20 cells by retroviral transfer of the human CD20 cDNA (n = 90). This set of cells, with the CD20 molecule as the only variable, was used to study the importance of CD20 expression level on rituximab-mediated CDC, ADCC, and the combination. We show a sigmoidal correlation of CD20 expression level and rituximab-mediated killing via CDC but not ADCC. On both high and low CD20-expressing cells, all CD20 molecules were translocated into lipid rafts after rituximab binding. Furthermore, CDC and ADCC act simultaneously and CDC-resistant cells are sensitive to ADCC and vice versa. These findings suggest that CDC depends on CD20 expression level and that both CDC and ADCC act complementary. These data give new insights into novel strategies to improve the efficacy of CD20-specific antibodies for the treatment of CD20+ tumors.

Production of Nitric Oxide During Graft Rejection Is Regulated by the Th1/Th2 Balance, the Arginase Activity, and l-arginine Metabolism

Production of nitric oxide (NO) by graft infiltrating macrophages has been proposed as an important effector mechanism of allograft rejection. Although high levels of NO are generated during allograft rejection, undetectable or only limited amounts of NO were found in rejected skin xenografts. BALB/c mice were grafted with skin transplants from syngeneic, allogeneic or xenogeneic (rat) donors. The production of NO, cytokines and arginase in the grafts was determined by spectrophotometry, enzyme-linked immunosorbent assay, or polymerase chain reaction. Effects of depletion of CD4+ cells, neutralization of interleukin (IL)-4 or application of arginase inhibitors N(omega)-hydroxy-L-arginine (L-NOHA) and L-valine on production of NO in rejected xenografts were evaluated. Rejection of rat skin xenografts, on the contrary to rejection of allografts, was associated with a local high production of Th2 cytokines IL-4 and IL-10, overexpression of arginase genes, strongly enhanced arginase activity and attenuated NO generation in the graft. The supernatants obtained after cultivation of skin xenograft (but not allograft or syngeneic graft) explants contained a high arginase activity and strongly suppressed NO production by activated macrophages. This suppression was completely inhibited by L-NOHA or was overcome by an excess of exogenous L-arginine, a substrate for NO synthesis. Cocultivation of xenograft explants that did not produce NO with arginase inhibitors L-NOHA or L-valine restored NO generation in the graft. The results suggest that upregulation of arginase activity by Th2 cytokines during xenograft rejection limits the bioavailability of L-arginine for the inducible NO synthase and thus attenuates generation of NO by the graft-infiltrating macrophages.

Pro- and anti-apoptotic activities of protozoan parasites

During infection, programmed cell death, i.e. apoptosis, is an important effector mechanism of innate and adaptive host responses to parasites. In addition, it fulfils essential functions in regulating host immunity and tissue homeostasis. Not surprisingly, however, adaptation of parasitic protozoa to their hosts also involves modulation or even exploitation of cell death in order to facilitate parasite survival in a hostile environment. During recent years, considerable progress has been made in our understanding of apoptosis during parasitic infections and there is now convincing evidence that apoptosis and its modulation by protozoan parasites has a major impact on the parasite-host interaction and on the pathogenesis of disease. This review updates our current knowledge on the diverse functions apoptosis may fulfil during infections with diverse protozoan parasites including apicomplexans, kinetoplastids and amoebae. Furthermore, we also summarize common mechanistic themes of the pro- and anti-apoptotic activities of protozoan parasites. The diverse and complex effects which parasitic protozoa exert on apoptotic cell death within the host highlight fascinating interactions of parasites and their hosts. Importantly, they also stress the importance of further investigations before the modulation of host cell apoptosis can be exploited to combat parasitic infections.

Alternative Signaling Pathways Regulating Type I Interferon-Induced Apoptosis

Type I interferons (IFNs) are pleiotropic cytokines that exert multiple effects on normal and tumor cells. These effects are supposedly mediated through the stimulation of several signal transduction pathways by type I IFNs. These include the well-studied canonical Jak-Stat pathway, largely responsible for the antiviral and growth-inhibitory activities of IFNs, as well as the phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways, whose importance in IFN-induced biologic outcomes has not been precisely established. One of the effects of type I IFNs on tumor cells is the induction of programmed cell death, apoptosis, which has been studied extensively over the last decade and has been suggested to be an important effector mechanism for IFN's antitumor effects in the treatment of cancer. The aim of this review is to summarize and discuss the recent data in the field of type I IFN-induced apoptosis, with special emphasis on the molecular mechanisms of apoptosis and on the role of alternative noncanonical signaling pathways stimulated by type I IFNs in this process.

Inhibiting complement regulators in cancer immunotherapy with bispecific mAbs

Although monoclonal antibody (mAb)-mediated immunotherapy of cancer has been proven to be feasible for clinical use, success rates until now have been disappointing. One reason for this might be the overexpression of membrane-bound complement regulatory proteins (mCRPs) by tumour cells. As complement activation is an important effector mechanism induced by therapeutic mAbs, inhibition of complement activation by tumour cells might reduce therapeutic efficacy by decreasing direct complement-mediated lysis as well as complement-dependent cellular cytotoxicity. Modulation of the function of these mCRPs might be achieved with therapeutic bispecific (bi-)mAbs that target a tumour antigen and simultaneously block a major mCRP. Clinical results will probably increase with such bi-mAbs compared with monovalent antitumour mAbs. In this review the feasibility of this approach is discussed.

Lymphocytes of dogs immunised with purified excreted-secreted antigens of Leishmania infantum co-incubated with Leishmania infected macrophages produce IFN gamma resulting in nitric oxide-mediated amastigote apoptosis

The role of nitric oxide (NO) in the anti-leishmanial activity has been confirmed both in vitro and in vivo. Recently, we demonstrated that NO-mediated apoptosis-like amastigote death pathway is an important and highly regulated mechanism used for the clearance of Leishmania within infected murine macrophages stimulated to produce NO endogenously. To further characterize these important effector mechanisms in dog, a natural host-reservoir of L. infantum/ L. chagasi, we have developed an ex vivo infection model of canine macrophages. Exposure of L. infantum-infected macrophages to autologous peripheral lymphocytes derived from dogs immunised with purified excreted-secreted antigens of L. infantum promastigotes ( LiESAp) formulated with muramyl dipeptide (MDP) as adjuvant resulted in a significant leishmanicidal effect due to interferon (IFN)-γ dependent macrophage activation. Concomitant accumulation of NO 3 −/NO 2 − in supernatants of co-cultured cells and in situ staining of parasites with terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling (TUNEL) and YOPRO-1 showed that NO-mediated apoptosis of intracellular L. infantum amastigotes is occurring in canine macrophages as previously observed in mouse models. Monitoring these parameters in dogs after immunisation and before experimental challenge can represent a useful and easy way to rapidly evaluate vaccine candidates against canine visceral leishmaniasis.

The activity of inducible nitric oxide synthase in rejected skin xenografts is selectively inhibited by a factor produced by grafted cells

Production of nitric oxide (NO) by graft infiltrating macrophages has been suggested as an important effector mechanism of allograft rejection. Expression of the gene for the inducible NO synthase (iNOS) and the production of NO in rejected graft has been demonstrated in various models of allotransplantation. However, whether NO plays a role in rejection of skin xenografts has not been documented. Explants of rejected skin allografts or xenografts (rat to mouse) were cultivated in vitro and the production of NO, interleukin (IL)-2, IL-4, IL-10 and interferon-gamma (IFN-gamma) by graft infiltrating cells was determined by the Griess reaction or ELISA. Effects of supernatants from cultures of xenograft explants on the expression of gene for iNOS, accumulation of iNOS protein and NO production were determined by RT-PCR or Western blots. Molecular mass of the factor with the suppressive activity was characterized by filtration on chromatography Sephacryl S-200 Superfine column. In addition, the effects of 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT), a selective iNOS inhibitor, on survival of skin xenografts were tested. While explants of rejected mouse skin allografts produced substantial amounts of NO, undetectable or only very low levels of NO were found in supernatants from cultured rat skin xenografts. Cocultivation of bacterial lipopolysaccharide (LPS)-stimulated mouse macrophages which produce high quantities of NO, with pieces of rejected xenografts, but not of syngeneic grafts, allografts or normal rat skin, completely inhibited production of NO. Production of IL-6 and IL-10 by LPS-stimulated macrophages was not inhibited under the same conditions. The inhibition of NO production was mediated by a factor which was produced by rejected rat xenograft and which was eluted from chromatography Sephacryl S-200 Superfine column in a fraction representing a molecular mass of 67 kDa. The factor did not inhibit the expression of the gene for iNOS, reduce the level of iNOS protein in stimulated macrophages, or function as a scavenger of NO. Rather, the factor inhibited the function of iNOS. The finding that NO does not play an important role during rejection of skin xenografts is supported by the observation that treatment of graft recipients with AMT, a specific iNOS inhibitor, did not enhance xenograft survival, while the same treatment resulted in prolongation of survival of skin allografts. The results thus demonstrate that a 67-kDa molecule produced by rejected rat skin xenografts selectively inhibits iNOS activity in graft infiltrating macrophages. We suggest that NO does not play a significant role in rejection of skin xenografts as it does in the case of allograft rejection.

Macrophage-derived interleukin-18 in experimental renal allograft rejection

Interleukin 18 (IL-18) is primarily a macrophage-derived, pro-inflammatory cytokine. As macrophages can act as effector cells in acute rejection, we examined the role of IL-18 in a rat model of acute renal allograft rejection. Life-sustaining orthotopic DA to Lewis allograft and Lewis-Lewis isograft kidney transplants were performed. In the same model, macrophage-depleted animals, achieved with liposomal-clodronate therapy, were also studied. Macrophage (ED1+) accumulation and IL-18 expression was assessed by immunohistochemistry. CD11b+ cells (macrophages) were isolated from kidney and spleen by micro beads. Real-time PCR was used to assess IL-18 and INF-gamma mRNA expression in tissue and cell isolates. Allografts, but not isografts, developed severe tubulo-interstitial damage and increased serum creatinine by day 5 (P<0.001). Immunohistochemistry revealed a greater ED1+ cell accumulation in day 5 allografts compared with isografts (P<0.001). IL-18 mRNA expression was increased 3-fold in allografts compared to isografts (P<0.001). Accordingly, IL-18 protein was increased in allografts (P<0.001), and was predominantly expressed by ED1+ macrophages. CD11b+ macrophages isolated from allografts had a 6-fold upregulation of IL-18 mRNA expression compared to isograft macrophages (P<0.001). Macrophage depletion resulted in a marked attenuation of allograft rejection, ED1+ and IL-18+ cells were significantly reduced (P<0.05) as was IL-18 mRNA expression (29.28+/-2.85 vs 62.48+/-3.05, P<0.001). INF-gamma mRNA expression (P<0.01) and iNOS (P<0.001) production were also significantly reduced in the macrophage-depleted animals. This study demonstrates that IL-18 is significantly increased during acute rejection and is principally produced by intra-graft macrophages. We hypothesize that IL-18 upregulation may be an important macrophage effector mechanism during the acute rejection process.

HLA class I, NKG2D, and natural cytotoxicity receptors regulate multiple myeloma cell recognition by natural killer cells

AbstractThe role of natural killer (NK) cells in multiple myeloma is not fully understood. Here, NK susceptibility of myeloma cells derived from distinct disease stages was evaluated in relation to major histocompatibility complex (MHC) class I, MHC class I chain-related protein A (MICA), MHC class I chain-related protein B (MICB), and UL16 binding protein (ULBP) expression. MHC class I molecules were hardly detectable on bone marrow cells of early-stage myeloma, while late-stage pleural effusion-derived cell lines showed a strong MHC class I expression. Conversely, a high MICA level was found on bone marrow myeloma cells, while it was low or not measurable on pleural effusion myeloma cells. The reciprocal surface expression of these molecules on bone marrow- and pleural effusion-derived cell was confirmed at mRNA levels. While bone marrow-derived myeloma cells were readily recognized by NK cells, pleural effusion-derived lines were resistant. NK protection of pleural effusion cells was MHC class I dependent. Receptor blocking experiments demonstrated that natural cytotoxicity receptor (NCR) and NK receptor member D of the lectinlike receptor family (NKG2D) were the key NK activating receptors for bone marrow-derived myeloma cell recognition. In ex vivo experiments patient's autologous fresh NK cells recognized bone marrow-derived myeloma cells. Our data support the hypothesis that NK cell cytotoxicity could sculpture myeloma and represents an important immune effector mechanism in controlling its intramedullary stages. (Blood. 2005;105:251-258)

Regulation of arterial blood pressure

In each cardiac cycle arterial blood pressure fluctuates between diastolic and systolic pressure. However, the body behaves from day to day as if it regulated the mean arterial blood pressure, which is the average between diastolic and systolic pressures. Such regulation is achieved by interdependent adjustments of only 3 parameters: Heart rate (HR), ventricular stroke volume (SV) and total peripheral vascular resistance (TPVR). These are related as follows: HR - SV = Cardiac Output (CO); CO - TPVR = Mean Arterial Blood Pressure. The regulatory system includes stretch-sensitive sensors, central nervous integrators/evaluators and neuro-humoral effector mechanisms. Central nervous integration and evaluation of incoming signals occurs mostly in the pons/medulla regions of the midbrain. The most important effector mechanisms are the parasympathetic and sympathetic divisions of the autonomic nervous system, the renin-angiotensin system and vasopressin. Short-term regulation of arterial blood pressure is dominated by the baroreceptor mechanism, whereby pressure is sensed by both cardio-pulmonary nerve endings and stretch-sensitive cells in renal afferent arterioles. Long-term regulation involves mainly the regulation of extracellular fluid volume by pressure natriuresis mechanisms residing in the kidney and by widespread actions of angiotensin 2. Studies in hypertensives have suggested that the long-term-controlled variable is not arterial blood pressure, but the balance between intake and output of fluid and electrolytes. If the kidney requires a higher perfusion pressure to achieve that balance then daily blood pressure regulation occurs around an appropriately higher setpoint.

Protection of Renal Epithelial Cells against Oxidative Injury by Endoplasmic Reticulum Stress Preconditioning Is Mediated by ERK1/2 Activation

We investigated the role of the endoplasmic reticulum (ER) stress response in intracellular Ca2+ regulation, MAPK activation, and cytoprotection in LLC-PK1 renal epithelial cells in an attempt to identify the mechanisms of protection afforded by ER stress. Cells preconditioned with trans-4,5-dihydroxy-1,2-dithiane, tunicamycin, thapsigargin, or A23187 expressed ER stress proteins and were resistant to subsequent H2O2-induced cell injury. In addition, ER stress preconditioning prevented the increase in intracellular Ca2+ concentration that normally follows H2O2 exposure. Stable transfection of cells with antisense RNA targeted against GRP78 (pkASgrp78 cells) prevented GRP78 induction, disabled the ER stress response, sensitized cells to H2O2-induced injury, and prevented the development of tolerance to H2O2 that normally occurs with preconditioning. ERK and JNK were transiently (30-60 min) phosphorylated in response to H2O2. ER stress-preconditioned cells had more ERK and less JNK phosphorylation than control cells in response to H2O2 exposure. Preincubation with a specific inhibitor of JNK activation or adenoviral infection with a construct that encodes constitutively active MEK1, the upstream activator of ERKs, also protected cells against H2O2 toxicity. In contrast, the pkASgrp78 cells had less ERK and more JNK phosphorylation upon H2O2 exposure. Expression of constitutively active ERK also conferred protection on native as well as pkAS-grp78 cells. These results indicate that GRP78 plays an important role in the ER stress response and cytoprotection. ER stress preconditioning attenuates H2O2-induced cell injury in LLC-PK1 cells by preventing an increase in intracellular Ca2+ concentration, potentiating ERK activation, and decreasing JNK activation. Thus, the ER stress response modulates the balance between ERK and JNK signaling pathways to prevent cell death after oxidative injury. Furthermore, ERK activation is an important downstream effector mechanism for cellular protection by ER stress.

Activation of complement mediates antiphospholipid antibody-induced pregnancy loss.

Although it is clear that the specific antigenic reactivity of antiphospholipid (aPL) antibodies is critical to their effect, the pathogenic mechanisms that result in injury in vivo are incompletely understood. We hyphothesized that aPL antibodies targeted to the placenta activate complement locally, generating split products that mediate placental injury and lead to foetal loss and growth retardation. To test this hypothesis, we used a murine model of APS in which pregnant mice are injected with human IgG containing aPL antibodies. Mice treated with inhibitors of complement activation and mice deficient in complement components were protected from aPL antibody-induced foetal damage. Although the cause of tissue injury in this disease is probably multifactoral, we have shown that complement activation is an absolute requirement for foetal loss and growth restriction and, therefore, thatthis pathway acts upstream of other important effector mechanisms. Identification of complement activation as a mechanism that is necessary for aPL-induced tissue damage and definition ofthe complement components necessary to trigger such injury is likely to lead to a better understanding of the pathogenesis of vascular and tissue injury in SLE and to new and improved treatments.

The role of complement in the antiphospholipid syndrome.

The anti-phospholipid syndrome (APS) is characterized by recurrent fetal loss, vascular thrombosis and thrombocytopenia occurring in the presence of antiphospholipid (aPL) antibodies. We hypothesized that aPL antibodies activate complement in the placenta, generating split products that mediate placental injury and lead to fetal loss and growth restriction, and that complement activation by aPL antibodies in other vascular areas causes inflammation and thrombophilia. Here we review studies in a murine model of APS in which human aPL antibodies are passively transferred into pregnant mice. Blockade of complement activation using a C3 convertase inhibitor or genetic deletion of C3 protected mice from pregnancy complications induced by aPL antibodies. These findings demonstrate that complement activation is a central mechanism in aPL antibody-induced pregnancy loss and fetal growth restriction. Although the cause of tissue injury in APS is likely to prove multifactorial, we have shown that complement activation is an absolute requirement for the most serious phenotypic outcomes, pregnancy complications and thrombosis, and, therefore, that this pathway acts upstream of other important effector mechanisms.

The role of iron in infections with intracellular bacteria

The requirement for iron as a critical component for cellular processes has long been appreciated. During infection with intracellular bacteria, iron is required by both the host cell and the pathogen that inhabits the host cell. Macrophages require iron as a cofactor for the execution of important antimicrobial effector mechanisms, including the NADPH dependent oxidative burst and the production of nitrogen radicals catalysed by the inducible nitric oxide synthase. On the other side of the equation, intracellular bacteria such as Salmonella typhimurium and Mycobacterium tuberculosis have an obligate requirement for iron to support their growth and survival inside cells. This brief report summarises the background to our work on iron modulation in infections with these two organisms and highlights key observations on how modulation of host iron status disturbs the equilibrium between host and pathogen and can determine the outcome of infection.