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Cytotoxic T cells (CTL) induce cell death of their target cells either by the surface interaction between Fas ligand and Fas or by the release of perforin and granzymes. Both lytic pathways induce apoptosis yet it is not known whether identical or distinct apoptotic pathways are activated. The protooncogene bcl-2 is known to protect various hematopoietic cells from apoptosis induced by diverse agents. Here we show that overexpression of the Bcl-2 protein in the murine mastocytoma line P815 or in concanavalin A-activated splenocytes suppresses apoptotic cell death induced by allospecific primary cytotoxic T lymphocytes (CTL) in which only the Fas lytic pathway was functional. Bcl-2 also reduced target cell killing induced by CTL whose lytic activity was dependent on the perforin/granzyme pathway only. These data provide evidence that, in the target cells studied here, both perforin/granzyme and Fas apoptotic pathways are modulated by Bcl-2 and suggest that these two pathways converge at a step prior to Bcl-2 inhibition.

Interaction of the widely expressed Fas with its membrane-bound ligand (FasL) leads to rapid cell death via apoptosis. To avoid pathological tissue damage, the activity of FasL requires tight regulation. Here, we report that the Src homology 3 (SH3) domain of Fyn binds to the proline-rich cytoplasmic region of FasL. Binding of the SH3 domain occurs between amino acid residues 44–71 which contains several potential SH3 interaction sites. This binding is specific, as SH3 domains of Lck, Grb2 and ras-GAP bind only weakly or not at all. We suggest that FasL activity may be modulated by SH3 domains of the src-like Fyn kinase.

Fas was recently demonstrated to be the major target molecule engaged by CD4+ cytolytic T lymphocytes (CTL). We examined Fas expression on various cloned T cell subpopulations and their susceptibility to lysis by CD4+ or CD8+ CTL. A reciprocal relationship in Fas and Fas-ligand expression was observed in CD4+ T helper (Th)1- and Th2-type clones, and Fas mRNA was predominantly detected in Th2 clones, whereas Fas-ligand mRNA was principally found in Th1 clones. The two Th0 clones tested expressed both Fas and Fas-ligand, but only one exhibited cytolytic activity, whereas both were sensitive to CD4-mediated lysis. A functional consequence of the inverse Fas-Fas-ligand expression pattern was that Th2 and Th0 cells were sensitive to lysis by both Th1 CD4+ CTL and a CD8+ CTL clone in a Fas-dependent manner. These results suggest that cytolytic CD4+ Th1 cells may play an immunomodulatory role, regulating a Th2/Th0 response by Fas-mediated lysis.

Deoxyribonuclease I (DNase I) has recently been implicated in cell death by apoptosis, a process which is frequently accompanied by chromatin DNA degradation. Despite extensive studies on DNase I, its genomic organisation remained unknown. Here we report for the first time on the intron-exon structure of the DNase I gene. The coding region of mouse DNase I is composed of eight introns and eight exons, spanning 2315 base pairs. The deduced protein sequence is 91.5% identical to its rat counterpart, but does not carry the two mutations (Glu13 to D and V67 to I) responsible for the decrease in actin-binding of rat DNase I. The enzymatic activity of mouse DNase I is found in striated muscle, kidney, intestine, liver, lymphnodes, but not in the heart, spleen or pancreas.

List of Contents.- Perforin and Granzymes: Crucial Effector Molecules in Cytolytic T Lymphocyte and Natural Killer Cell-Mediated Cytotoxicity.- Fas-Based T Cell-Mediated Cytotoxicity.- Structure and Biogenesis of Lytic Granules.- The Role of CD28 Costimulation Cytotoxic in the Generation of T Lymphocytes.- Degranulating Cytotoxic Lymphocytes Inflict Multiple Damage Pathways on Target Cells.- Granzymes and Apoptosis: Targeting the Cell Cycle.- Perforin, Killer Cells and Gene Transfer Immunotherapy for Cancer.- TIA-1: Structural and Functional Studies on a New Class of Cytolytic Effector Molecule.- Calreticulin: A Granule-Protein by Default or Design?.- A New Function for an Old Enzyme: The Role of DNase I in Apoptosis.- The Ligands arid Receptors of the Lymphotoxiri System.

Lymphocyte-mediated cytotoxicity has been proposed to consist of the polarized secretion of granule-stored perforin leading to target-cell lysis. Nevertheless, perforin-independent pathways were postulated to explain the cytolytic activity of apparently perforin-free lymphocytes and the DNA degradation found in dying target cells. To evaluate the role of perforin, we used gene targeting in embryonic stem cells to produce mice lacking perforin. Mice homozygous for the disrupted gene have no perforin mRNA. The mice are healthy. Activation and granzyme A secretion of perforin-free cytolytic T cells are unaltered. The killing activity of cytolytic T cells as well as natural killer (NK) cells, however, is impaired but not abolished. Approximately one-third of the killing activity remains when lysis of 3T3 fibroblast targets and the apoptotic cell death of YAC-1 NK targets are analyzed. We conclude that perforin is a crucial effector molecule in T cell- and NK cell-mediated cytolysis. However, alternative perforin-independent lytic mechanisms also exist.

Bcl-2α is a mitochondrial or perinuclear-associated oncoprotein that prolongs the life span of a variety of cell types by interfering with programmed cell death. How Bcl-2 confers cell survival is unknown, although antioxidant and antiprotease functions have been proposed. In addition, protein structures of Bcl-2 that are crucial for its survival activity are still ill-defined. Bcl-2 can occur as Bcl-2α or Bcl-2β, two alternatively spliced forms which solely differ in their carboxyl termini. The finding that Bcl-2α is active and membrane bound, but Bcl-2β is inactive and cytosolic, indicates that the carboxyl terminus contributes to the survival activity of Bcl-2. This region contains two subdomains, a domain X with unknown function and a hydrophobic stretch reported to mediate membrane assocation of Bcl-2α. Recently Bcl-2-related proteins have been identified. These include Bax that heterodimerizes with Bcl-2 and, when overxpressed, counteracts Bcl-2. Bax contains two highly conserved regions of sequence homology with Bcl-2, referred to as Bcl-2 homology 1 and 2 (BH1 and BH2) domains. Site-directed mutagenesis studies have revealed that both domains are not only novel dimerization motifs for the interaction of Bax with Bcl-2 but also crucial for the survival activity of Bcl-2. Interestingly, the C-terminal end of BH2 encompasses the Bcl-2α/β splice site, as well as part of domain X in Bcl-2α. To better define the role of domain X and the hydrophobic C-terminal stretch of Bcl-2α for its survival activity, we created various deletion and truncation mutations in these regions by site-directed mutagenesis. We show here that membrane attachment and therefore the hydrophobic stretch is not required for the survival activity of Bcl-2, but part of domain X appears to be indispensable.Key words: apoptosis, Bcl-2, mutagenesis, cell survival, functional domains.

Granzymes are a family of serine proteases that are harbored in cytoplasmic granules of activated T lymphocytes and are released upon target cell interaction. Immediate and complete neurite retraction was induced in a mouse neuronal cell line when total extracts of granule proteins were added. This activity was isolated and identified as granzyme A. This protease not only induced neurite retraction at nanomolar concentrations but also reversed the stellation of astrocytes. Both effects were critically dependent on the esterolytic activity of granzyme A. As neurite retraction is known to be induced by thrombin, possible cleavage and activation of the thrombin receptor were investigated. A synthetic peptide spanning the N-terminal thrombin receptor activation sequence was cleaved by granzyme A at the authentic thrombin cleavage site Leu-Asp-Pro-Arg-Ser. Antibodies to the thrombin receptor inhibited both thrombin and granzyme A-mediated neurite retraction. Thus, T-cell-released granzyme A induces cellular responses by activation of the thrombin receptor. As brain-infiltrating CD4+ lymphocytes are the effector cells in experimental allergic encephalomyelitis, granzyme A released in the brain may contribute to the etiology of autoimmune disorders in the nervous system.

Macrophage precursor cells, derived from mouse bone marrow culture with granulocyte-macrophage colony-stimulating factor or colony-stimulating factor 1 (CSF-1) as growth factor and interleukin-2 (IL-2) as stimulating factor, were activated by IL-2 to exert strong cytolytic activity against Yac-1 cells. In response to IL-2 stimulation these bone marrow macrophage precursor cells produced perforin as lytic molecules. The purity of the precursor cells for the study was proved as homogeneous positivity for Mac-1, NK-1.1 and negativity for Lyt 1 and 2. The cells express CSF-1 receptors on their surface, are able to proliferate and differentiate into typical macrophages when stimulated with CSF-1, and are therefore members of the macrophage lineage. Perforin transcripts were identified by Northern blot analysis of IL-2–treated macrophage precursor cells, and the presence of perforin protein in the cytoplasmic granules was demonstrated by immunhistochemical staining using a monoclonal antiperforin antibody. In addition, the biological activity of the perforin contained in the macrophage precursor’s granules could be documented as calcium-dependent lytic activity using Yac-1 and sheep red blood cells as targets. The results presented in this paper imply the existence of a bipotent precursor cell, which can mature into a typical macrophage if CSF-1 or phorbol 12-myristate 13-acetate is supplied as differentiation stimulating factor but develops into an NK/LAK cell when early activation with IL-2 is provided. J. Leukoc. Biol. 56: 117–123; 1994.

The term apoptosis describes the predictable structural changes associated with many forms of programmed cell death. One of the first visible events of apoptosis is the collapse of the nucleus. Nuclear degradation is manifested by digestion of chromatin into nucleosome-sized fragments or multiples of these. This digestion of DNA is enzymatic, and several attempts have been made to characterize apoptosis-specific endodeoxyribonucleases. Although there are strong candidates for such enzymes, direct evidence for their role in apoptosis is yet to be provided.

Tumor microenvironment plays an essential role in modulating and supporting tumor growth. Solid tumors are frequently infiltrated with innate immune cells as illustrated by the strong macrophages infiltration observed in breast tumors. In fact, infiltrating macrophages in breast tumors are associated with poor prognosis. IL-1beta (IL-1 b) is a proinflammatory cytokine produced mostly by macrophages and dendritic cells which detection in advance breast tumor stage is also correlated with bad prognosis. Furthermore, mouse studies using the orthotopic implantation of the mammary tumor cell line 4T1 have suggested a role for IL-1b in tumor growth and vascularization. IL-1b is a peculiar cytokine as its production is tightly controlled. First, proinflammatory signals induce the production of the proIL-1 b. To be active, the cytokine has to be cleaved by the caspase-1 within a complex named the inflammasome. Finally the cleaved cytokine is secreted outside the cells. By and large, the inflammasome is composed of a scaffold receptor, for instance NLRP3, an adaptor, ASC, and caspase-1. The NLRP3 inflammasome is mainly expressed by macrophages and dendritic cells and gets activated upon detection of pattern-associated molecular pattern but also danger-associated molecular pattern such as extracellular ATP or uric acid. As the inflammasome controls the biological activity of IL-1b, we speculated that the inflammasome should play an important role in the breast tumor microenvironment. To test our hypothsesis, we used the mouse mammary model of breast cancer using orthotopic implantation of 4T1 cells within the mammary gland of either wild-type or inflammasome deficient mice. Our results show that tumor growth is delayed in caspase-1 deficient mice suggesting that the presence of the inflammasome in the tumor microenvironment supports tumor growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A14.

Nature 469, 221–225 (2011) On page 2 of the print and PDF version of this Letter, there are two citations to Fig. 2f. The first citation to Fig. 2e, f, which refers to active IL-1β in the culture supernatant after knockdown of beclin 1 and ATG5, should instead be to Fig. 2d, e; the second citation to Fig.

The interleukin-1 (IL-1) family of cytokines has been implicated in the pathogenesis of atherosclerosis in previous studies. The NLRP3 inflammasome has recently emerged as a pivotal regulator of IL-1β maturation and secretion by macrophages. Little is currently known about a possible role for the NLRP3 inflammasome in atherosclerosis progression in vivo. We generated ApoE-/- Nlrp3-/-, ApoE-/- Asc-/- and ApoE-/- caspase-1-/- double-deficient mice, fed them a high-fat diet for 11 weeks and subsequently assessed atherosclerosis progression and plaque phenotype. No differences in atherosclerosis progression, infiltration of plaques by macrophages, nor plaque stability and phenotype across the genotypes studied were found. Our results demonstrate that the NLRP3 inflammasome is not critically implicated in atherosclerosis progression in the ApoE mouse model.

Clusterin (gene symbol: CLI) is a post-translationally nicked, two-chain plasma and tissue glycoprotein of 80 kDa. It forms high-density lipoprotein complexes with apolipoprotein A-I in plasma, functions as an inhibitor of the cytolytic reaction of the terminal complement proteins C5 to C9, and is secreted by Sertoli cells in large amounts into the seminal fluid. By isolating and characterizing three partially overlapping cosmid clones, the authors have established the complete physical map of the clusterin gene which spans about 20 kb. The subchromosomal position of the clusterin gene (CLI) and the order of CLI and the lipoprotein lipase (LPL) gene were determined by fluorescence in situ hybridization. They show that CLI, previously assigned to chromosome 8, is located on 8p21 proximal to the LPL locus. Based on this localization they consider clusterin as a novel candidate gene determining susceptibility to atherosclerosis. 23 refs., 2 figs.

In the first phase of infection by the Human Immunodeficiency Virus type 1 (HIV-1), persistent generalised lymphadenopathy can be the only feature of the disease, with or without associated clinical symptoms. The main histologic abnormality observed in the swollen lymph node is hyperplasia of follicles. The network comprising of follicular dendritic cells (FDC) appears to be damaged. Immunohistochemistry shows signs of fragmentations and indentations. HIV-1 components have been suggested to play a role in this process, as proteins, 1,2 virions3 and mRNA2,4 of the virus are concentrated in the germinal centers. We5 and others6 have provided evidence that FDC can be infected by HIV-1. Some authors have proposed a role for the immune response towards the virus. This was supported by the demonstration of cytotoxic cells, documented by the presence of mRNA encoding serine esterase Granzyme B, which occurs in granules of cytotoxic cells.7

Complement protein C9 assembles with C5, C6, C7, C8 on the surface of target cells to form the lytic membrane attack complex (MAC). During MAC assembly and insertion into the target membrane, the hydrophilic, globular C9 partially unfolds to expose a hydrophobic lipid interaction domain. Several copies of amphiphilic C9 subsequently polymerize to form the characteristic ring-like MAC. Using a combined photoaffinity label and computer modeling approach, two amphipathic helices in a segment encompassing the amino acids 293-334 have been predicted to interact with membrane lipids. To elucidate the mechanism of C9 lipid binding and insertion, site-directed mutagenesis was used to change the amphipathic character of the helices. While some conservative amino acid replacements such as Thr307 by a Leu were tolerated and yielded fully active C9 when expressed in COS cells, successive changes of Leu305 into Val, Ala, and Glu on the hydrophobic site of the first helix gave rise to only partly or not secreted C9. All non-conservative amino acid replacements introduced on either side of the helices resulted in non-secreted C9 that was subsequently degraded intracellularly, indicating the importance of the correct folding of the presumptive transmembrane domain during biosynthesis. A natural secretion-incompetent mutant was found in which Val293, located in the proposed lipid-binding region, was lacking. Taken together, these findings suggest that the high incidence of homozygous C9 deficiencies may be due to a blockage in intracellular transport and secretion due to point mutations in this 'hot spot' region of the molecule.

The participation of cellular serine proteinases in many physiological processes has been widely documented. These enzymes are most active at neutral, i.e., physiological pH. A number of studies have suggested that they may be involved in cellular chemotaxis, protein turnover in tissues, endocytosis and exocytosis, peptide hormone processing, cellular invasiveness or tumorigenesis, and cell proliferation (for reviews, see Mullins and Rohrlich, 1983; Neurath, 1984). These suggestions were based on the interference of proteinase inhibitors with cellular function or proliferation and the detection of cell-associated esterolytic, amidolytic, or proteolytic activities, notably in hemopoietic cells of the immune defense system, including lymphocytes, natural killer (NK) cells, mast cells, and phagocytes. This review will focus on a subfamily of serine proteases expressed in granules of lymphocytes and NK cells.

Background & Aims Clostridium difficile –associated disease (CDAD) is the leading cause of nosocomial diarrhea in the United States. C difficile toxins TcdA and TcdB breach the intestinal barrier and trigger mucosal inflammation and intestinal damage. The inflammasome is an intracellular danger sensor of the innate immune system. In the present study, we hypothesize that TcdA and TcdB trigger inflammasome-dependent interleukin (IL)-1β production, which contributes to the pathogenesis of CDAD. Methods Macrophages exposed to TcdA and TcdB were assessed for IL-1β production, an indication of inflammasome activation. Macrophages deficient in components of the inflammasome were also assessed. Truncated/mutated forms of TcdB were assessed for their ability to activate the inflammasome. The role of inflammasome signaling in vivo was assessed in ASC-deficient and IL-1 receptor antagonist–treated mice. Results TcdA and TcdB triggered inflammasome activation and IL-1β secretion in macrophages and human mucosal biopsy specimens. Deletion of Nlrp3 decreased, whereas deletion of ASC completely abolished, toxin-induced IL-1β release. TcdB-induced IL-1β release required recognition of the full-length toxin but not its enzymatic function. In vivo, deletion of ASC significantly reduced toxin-induced inflammation and damage, an effect that was mimicked by pretreatment with the IL-1 receptor antagonist anakinra. Conclusions TcdA and TcdB trigger IL-1β release by activating an ASC-containing inflammasome, a response that contributes to toxin-induced inflammation and damage in vivo. Pretreating mice with the IL-1 receptor antagonist anakinra afforded the same level of protection that was observed in ASC −/− mice. These data suggest that targeting inflammasome or IL-1β signaling may represent new therapeutic targets in the treatment of CDAD.

Perforin (PRF1) is a cytolytic, channel-forming protein of cytolytic T cells, natural killer cells, and granulated metrial gland cells and plays a crucial role in the killer cell-mediated elimination of virally infected host cells tumor cells, and allotransplants. Two-thirds of the perforin sequence is homologous to the lytic, channel-forming complement proteins C6, C7, C8α, C8β, and C9. Using cosmid DNA containing the PRF1 gene as a probe for fluorescence in situ hybridization, we have reevaluated its chromosomal location. Previously assigned to chromosome 17q11–q21, it has now been mapped to 10q22. The human PRF1 locus lies within a conserved synteny segment present on mouse chromosome 10, consistent with the previous chromosomal assignment of mouse perforin. The perforin locus is not linked to any of the genes of the terminal complement system.

The autosomal defect in Fas antigen leads CD4-CD8-T-cells to accumulate in lymph nodes and spleen of MRL - lpr lpr mice. MRL - lpr lpr mice present increased levels of DNase I as compared to the control strain MRL-+/+. This DNase I, which most probably originates from the accumulated CD4-CD8-T-cells, cleaves nuclear DNA with a strong preference for internucleosomal sites yielding, in the presence of both Ca2+ and Mg2+, a pattern of fragments typical for apoptosis. Furthermore, we show that this “apoptosis-ladder” can be obtained with purified DNase I in presence of normal serum.

SUMMARY The membrane attack complex (MAC) of complement is activated by immune and non-immune mechanisms in the kidney. MAC has been found associated with glomerular immune deposits, but also to cell remnants, particularly along tubules and in vessel walls. Clusterin and S-protein (vitronectin) bind to MAC, rendering it cytolytically inactive. Both have been found associated with MAC in renal tissue. Here we analysed the deposition of clusterin and S-protein in 118 renal biopsies relative to the localization of the MAC using MoAbs. Statistical analysis was performed comparing no or little versus evident or strong staining by immuofluorescence (IF). In glomeruli, out of the 92 biopsies where both MAC and immunoglobulins were evaluated, deposits of MAC were found in the presence (32 out of 41) but also in the absence of immunoglobulins (20/51). Clusterin and S-protein deposits were seen, respectively, in 25 out of 61 and 36 out of 61 biopsies containing glomerular MAC and almost never in its absence (one out of 50 for both). The association of the two inhibitors with MAC was observed mainly in glomeruli containing immunoglobulin deposits (respectively, 21 out of 32 and 25 out of 32), but not when immunoglobulins were absent (three out of 20 and seven out of 20) (coefficient of concordance, K= 0·47 and 0·43). The localization of MAC along tubules and in vessels was easily identified in most biopsies (93 out of 118) and was accompaied by S-protein in most cases (tubules, 86 out of 93; vessels, 82 out of 93) (K= 0·58 and 0·57 respectively) but not by clusterin (28 out of 93 and 24 out of 93). These results suggest that clusterin does not co-localize with MAC whenever there is formation and fixation of the MAC. It seems that clusterin has a particular affinity for MAC which is associated with immunoglobulin. This observation should help to distinguish between the different forms of MAC, and might indicate that MAC associated with immunoglobulin is essentially in its cytolytically inactive form.

BACKGROUND: Characteristic symptoms of malaria include recurrent fever attacks and neurodegeneration, signs that are also found in patients with a hyperactive Nalp3 inflammasome. Plasmodium species produce a crystal called hemozoin that is generated by detoxification of heme after hemoglobin degradation in infected red blood cells. Thus, we hypothesized that hemozoin could activate the Nalp3 inflammasome, due to its particulate nature reminiscent of other inflammasome-activating agents. METHODOLOGY/PRINCIPAL FINDINGS: We found that hemozoin acts as a proinflammatory danger signal that activates the Nalp3 inflammasome, causing the release of IL-1beta. Similar to other Nalp3-activating particles, hemozoin activity is blocked by inhibiting phagocytosis, K(+) efflux and NADPH oxidase. In vivo, intraperitoneal injection of hemozoin results in acute peritonitis, which is impaired in Nalp3-, caspase-1- and IL-1R-deficient mice. Likewise, the pathogenesis of cerebral malaria is dampened in Nalp3-deficient mice infected with Plasmodium berghei sporozoites, while parasitemia remains unchanged. SIGNIFICANCE/CONCLUSIONS: The potent pro-inflammatory effect of hemozoin through inflammasome activation may possibly be implicated in plasmodium-associated pathologies such as cerebral malaria.

INTRODUCTION: MAdCAM is predominantly expressed on high endothelial venules of intestinal lymphoid tissue and expression is increased at sites of gastrointestinal inflammation. MAdCAM binds a4b7 integrin on lymphocytes facilitating their migration to the inflamed intestinal tract. PF-00547,659, a fully human anti-MAdCAM IgG2 antibody blocksMAdCAM/ a4b7 dependent lymphocyte recruitment to the gut and is therefore anticipated to reduce gut inflammation and mucosal damage. AIM: To evaluate efficacy, safety and pharmacokinetics of PF-00547,659 in patients with active UC (Mayo score > 6). METHODS: In this doubleblind placebo-controlled study a total of 80 patients were included, of whom 30 in 6 singledose (SD) cohorts (doses from 0.03 to 10 mg/kg IV and 3 mg/kg SC or placebo) and 50 in 5 multiple-dose (MD) cohorts (doses from 0.1 to 3.0 mg/kg IV and 0.3 to 1 mg/kg SC or placebo every 4-weeks). Efficacy was measured at week 4 in SD cohorts and at week 4 and week 12 in MD cohorts. Safety information was collected during the double-blind treatment and the follow-up period. Patients were followed-up until the drug plasma levels were below the detection level. RESULTS: Safety: PF-547,659 was well tolerated with an adverse event (AE)profile similar to placebo. There were no safety concerns from laboratory tests, BP and ECG measurement. No anti-drug antibodies were detected. Efficacy: Results from an interim analysis performed after all patients completed Week 12 in the MD cohorts are presented. CONCLUSION: PF-00547,659 was well tolerated with AE profile similar to placebo. Within the limitations of a small sample size, the compound performed better than placebo in clinical as well as endoscopic endpoints and biomarker levels. No dose-response was observed.

Immune defence systems (complement, cytolytic lymphocytes) make use of transmembrane pores assembled from up to 20 soluble monomers in a highly regulated process to induce cell death. Inhibitors of pore formation have been found which protect blood, endothelial and epithelial cells from the destructive effect of complement lesions. Recently, a pore-forming protein showing immunological crossreactivity to complement C9 has been found in the protozoan parasite Trypanosoma cruzi, thereby extending this protein family and generalizing its means of generating non-selective membrane permeability.

TNF–related apoptosis–inducing ligand has selective antitumor activity in mice without causing toxic side effects and may have potential as a cancer therapeutic ( 157–163 ).

Introduction Lung injury leads to pulmonary inflammation and fibrosis through myeloid differentiation primary response gene 88 (MyD88) and interleukin-1 receptor 1 (IL-1R1) signalling pathway (J Clin Invest, 2007, 117: 3786-3793). However, the molecular mechanisms by which lung injury triggers IL-1β production, inflammation and fibrosis remain poorly understood. Based on the fact that cell/tissue injury and necrosis result in production of uric acid, we hypothesised that uric acid crystals formed at the injury site might represent a key danger signal activating the inflammasome to release IL-1β thereby causing inflammatory lung pathologies. Methods Mice deficient for MyD88, IL-1R1, NALP3, ASC or Casp-1 on C57Bl/6 genetic background were used. Uric acid concentration was determined in bronchoalveolar lavages and lung homogenates. Bleomycin sulfate (10mg/kg) in saline, uric acid or allopurinol crystals (5-50mg/kg) or saline alone were given through the airways by nasal instillation. The number of cells, chemokines, cytokines and TIMP-1 in the bronchoalveolar space and MPO activity in the lung were evaluated. MMP-2 and MMP-9 levels were determined by gelatin zymography. Results Here we show that lung injury depends on the NALP3 inflammasome which is triggered by uric acid locally produced in the lung upon bleomycin-induced DNA damage and degradation. Reduction of uric acid levels using the inhibitor of uric acid synthesis allopurinol or uricase lead to a decrease in bleomycin-induced IL- 1β production, lung inflammation, repair and fibrosis. Further, local administration of exogenous uric acid crystals recapitulates lung inflammation and repair which depend on the NALP3 inflammasome, MyD88 and IL-1R1 pathways, and TLR2 and TLR4 for optimal inflammation, but are independent of the IL-18 receptor. Conclusions Uric acid released from injured cells constitutes a major endogenous danger signal which activates the NALP3 inflammasome leading to IL-1β production. Reducing uric acid tissue levels represents a novel therapeutic approach to control IL-1β production and chronic inflammatory lung pathology.

Upon interaction with the membrane-bound C5b 8 complex, the ninth component of complement (C9) unfolds and inserts into the membrane of cells on which surface complement has been activated. Consequently C9 oligomerization occurs and transmembrane channels of varying sizes are formed. The domain of the unfolded protein interacting with the cell membrane has so far not been identified since, unlike many integral membrane proteins, the C9 sequence does not contain a continuous stretch of hydrophobic amino acids. We studied the interaction of C9 with the lipid bilayer using the membrane-restricted photoaffinity label 3-(trifluoromethyl)-3-( m [ 125 I]iodophenyl)diazirine ( 125 I-TID). C9 was assembled on liposomes and after photoactivation, several labeled and non-labeled peptides, obtained by chemical and enzymatic cleavage or the 125 I-TID-labeled C9. were analyzed. The segment from 176 to 345 was identified as the region containing the membrane-interacting structure. By means of secondary structure predictions, we identified two amphipathic alpha-helices (292–308 and 313–333) separated by a turn (309–312). Based on these results, we constructed a molecular model for the membrane-spanning region of C9. By analogy, we also constructed a model for this domain in perform/cytolysin, a pore-forming protein found in the cytoplasmic granules of cytotoxic T-lymphocytes.

Pulsed tunable dye laser (577 nm) (PTDL) therapy induces hemoglobin coagulation and tissue necrosis, which is mainly limited to blood vessels. To define whether this treatment activates complement in normal skin and senile hemangioma, we analyzed complement deposition in blood vessels by immunofluorescence. C3 fragments, C8, and C9 were detected with specific polyclonal antibodies. The membrane attack complex of complement (MAC) was demonstrated with a monoclonal antibody which reacts only with a neoantigen of MAC. Amplification of C3 deposition by the alternative pathway was determined on cryostat sections by indirect immunofluorescence with use of C4 deficient guinea pig (GP) serum. Normal skin and hemangiomas from three individuals were studied. In PTLD-irradiated normal skin, the main findings were as follows: 1) C3 fragments, C8, C9, and MAC were deposited in vessel walls; 2) these deposits were not due to denaturation of the proteins since they became apparent only 7 min after irradiation, contrary to immediate deposition of transferrin at the sites of erythrocyte coagulates; 3) the C3 deposits were shown to amplify complement activation by the alternative pathway, a reaction which was specific since tissue necrosis itself did not lead to such amplification; 4) these reactions preceded the local accumulation of polymorphonuclear leucocytes. Tissue necrosis was more pronounced in the hemangiomas. The larger angiomatous vessels in the center of the necrosis did not fix complement significantly. By contrast, complement deposition in the vessels situated at the periphery was similar to that observed in normal skin with one exception: C8, C9, and MAC were detected in some blood vessels immediately after laser treatment, a finding consistent with assembly of the MAC occurring directly without the formation of a C5 convertase. These results indicate that complement is activated in PTDL-induced vascular necrosis, and might be responsible for the ensuing inflammatory response.

Fas-mediated programmed cell death contributes to the pathology of thyroiditis and hepatitis (pages 409−413).

Monoclonal antibodies obtained after immunization of mice with human C5 were screened for activity against native C5 and SC5b-9. Clone 568 reacted with the C5 beta-chain in western blotting and with purified native C5 in double antibody enzyme immunoassays. The reactivity of clone 568 against serum decreased markedly when serum was activated with zymosan. The residual activity was found to be against free C5, whereas the antibody did not react at all with the SC5b-9 complex. Thus, the antibody 568 reacts with an activation-dependent C5 epitope, which is concealed when C5 is incorporated into the terminal complement complex.

Complement component C9 contains two distinct cysteine-rich domains exhibiting high sequence resemblance to a domain present in the low density lipoprotein (LDL) receptor and epidermal growth factor precursor, respectively. Antibodies were raised against a peptide corresponding to the most conserved region of the LDL receptor/C9-homology segment. The antibodies were shown by immunoblotting to bind specifically to C9 but also to crossreact with C8 alpha, the alpha subunit of complement component C8. Moreover, a monoclonal antibody to a neoantigen present in polymerized C9 bound to an epitope exposed on C8 within the C5b-8 complex but buried in monomeric C8, suggesting that C8 and C9 undergo similar conformational changes during membrane-attack-complex assembly. Isolated C8 alpha-gamma exhibited the propensity to polymerize in the presence of Zn2+ and urea, as already demonstrated for C9. These data indicate that C8 alpha is closely related, both structurally and functionally, to C9.

Pure dimers, trimers, tetramers and pentamers of rabbit non-immune IgG (immunoglobulin G) or antibody IgG were prepared by polymerization in the presence of the bifunctional cross-linking reagent dithiobis (succinimidylpropionate). Oligomerization was performed either in the presence of polysaccharide antigen and specific monomeric antibody (method A) or by random cross-linking of non-immune rabbit IgG in the absence of antigen (method B). By repeated gel-filtration chromatography, samples prepared by both methods exhibited a single band in analytical sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The electrophoretic mobilities of samples prepared by method A were slightly greater than those for the corresponding samples prepared by method B. This might suggest a role played by antigen in the orientation of IgG molecules within the clusters, which may be more compact than those formed by random cross-linking. The average numbers of cross-linker molecules per oligomer varied between 3 and 6 for clusters made by method A and between 1 and 3 for clusters made by method B. Ultracentrifugal analyses of the oligomers yielded sedimentation coefficients (S20,w) of 9.6S for the dimer, 11.2S for the trimer, 13.6S for the tetramer and 16.1S for the pentamer. Comparison of the observed sedimentation coefficients with those predicted by various hydrodynamic models suggested these oligomers possessed open and linear structures. Reduction of the cross-linking molecules converted oligomers into monomeric species of IgG. C.d. spectra of some oligomers studied in the range 200-250 nm were essentially the same as that of monomeric IgG molecules, thus strongly suggesting no major conformation changes in IgG molecules within clusters. These oligomers were found to be stable for up to 2 months when stored at −70 degrees C.

Membrane attack by complement involves the self-assembly on membranes of five hydrophilic proteins (C5b, C6, C7, C8 and C9) to an amphiphilic tubular complex comprising approximately 20 subunits. The hydrophilic-amphiphilic transition of the precursor proteins is achieved by restricted unfolding and exposure of previously hidden hydrophobic domains. Restricted unfolding, in turn, is driven by high-affinity protein-protein interactions resulting in the formation of amphilic complexes. Circular polymerization of C9 to a tubular complex (poly C9) constitutes the molecular mechanism for transmembrane channel assembly and formation of ultrastructural membrane lesions.

Cytolysis mediated by complement or cytolytic lymphocytes results in the formation of morphology similar lesions in the target membrane1–6. These lesions, formed by the polymerization of C9 or perforin respectively, contribute the major killing action by causing osmotic lysis of the target cell. Following the suggestion of Mayer7 that the mechanisms of humoral and cell-mediated cytotoxicity might be related, studies into the morphology of the membrane lesions formed8–13, and the proteins responsible for causing the lesions9,14,15, have shown several similarities. While the lesion caused by natural and T-killer cells is a little larger than that caused by complement, its overall shape is similar and in both cases the cylindrical pore is formed by polymerization of a monomeric subunit, C9 (relative molecular mass, Mr = 71,000) for complement9, and perforin (Mr = 66,000) for cell-mediated cytotoxicity14,15. C9 has an absolute requirement for a receptor in the target membrane formed by the earlier membrane attack complex components, C5b, C6, C7 and C8 (ref. 8). For perforin, polymerization in a target membrane requires no receptor, specificity being derived from the specific recognition between killer and target cell. Both proteins can be made to polymerize in vitro by the addition of divalent cations (Zn2+ for C9 (ref. 16) and Ca2+ for perforin10,12,13) and the resultant complexes closely resemble their physiological counterparts. Antibodies raised against lymphocyte-killed targets have also been shown to cross-react with complement proteins17,18,33, but the antigenically related proteins were not determined in these studies. We show here using purified proteins that perforin, C9 and complexes involving C7 and C8 share a common antigenic determinant which is probably involved in polymerization.

Anti-C8 alpha-gamma specific antibodies were used to isolate cDNA clones from a human liver expression library. Antibodies affinity-purified on the expressed hybrid protein of one clone bound exclusively to the gamma-chain of reduced C8 alpha-gamma. This clone, as well as a second full length cDNA clone obtained by hybridization screening, were sequenced and the complete primary structure for C8 gamma was established. Cyanogen bromide cleavage of C8 alpha-gamma released a 12 kDa carboxy-terminal C8 gamma fragment under both reducing and nonreducing conditions which was identified by fragment-specific, affinity-purified antibodies. Our data clearly show that C8 gamma has one internal disulfide bridge between cys-76 and cys-168 within the carboxy-terminal 12 kDa fragment, whereas the remaining cysteine residue 40 forms the disulfide bridge with C8 alpha. The overall sequence homology to plasma protein HC (23% amino acid identities) and the conservation of one internal cysteine bond and one free, surface-located cysteine residue suggests a highly conserved three-dimensional structure of C8 gamma and protein HC and also a possible functional relationship between these proteins.