Innate recognition of apoptotic cells:novel apoptotic cell-associated molecular patterns revealed by crossreactivity of anti-LPS antibodies

Cells dying by apoptosis are normally cleared by phagocytes through mechanisms that can suppress inflammation and immunity. Molecules of the innate immune system, the pattern recognition receptors (PRRs), are able to interact not only with conserved structures on microbes (pathogen-associated molecular patterns, PAMPs) but also with ligands displayed by apoptotic cells. We reasoned that PRRs might therefore interact with structures on apoptotic cells-apoptotic cell-associated molecular patterns (ACAMPs)-that are analogous to PAMPs. Here we show that certain monoclonal antibodies raised against the prototypic PAMP, lipopolysaccharide (LPS), can crossreact with apoptotic cells. We demonstrate that one such antibody interacts with a constitutively expressed intracellular protein, laminin-binding protein, which translocates to the cell surface during apoptosis and can interact with cells expressing the prototypic PRR, mCD14 as well as with CD14-negative cells. Anti-LPS cross reactive epitopes on apoptotic cells colocalised with annexin V-and C1q-binding sites on vesicular regions of apoptotic cell surfaces and were released associated with apoptotic cell-derived microvesicles (MVs). These results confirm that apoptotic cells and microbes can interact with the immune system through common elements and suggest that anti-PAMP antibodies could be used strategically to characterise novel ACAMPs associated not only with apoptotic cells but also with derived MVs. © 2013 Macmillan Publishers Limited All rights reserved.

The role of the innate immune system in the clearance of apoptotic cells

Rapid clearance of dying cells is a vital feature of apoptosis throughout development, tissue homeostasis and resolution of inflammation. The phagocytic removal of apoptotic cells is mediated by both professional and amateur phagocytes, armed with a series of pattern recognition receptors that participate in host defence and apoptotic cell clearance. CD14 is one such molecule. It is involved in apoptotic cell clearance (known to be immunosuppressive and anti-inflammatory) and binding of the pathogen-associated molecular pattern, lipopolysaccharides (a pro-inflammatory event). Thus CD14 is involved in the assembly of two distinct ligand-dependent macrophage responses. This project sought to characterise the involvement of the innate immune system, particularly CD14, in the removal of apoptotic cells. The role of non-myeloid CD14 was also considered and the data suggests that the expression of CD14 by phagocytes may define their professional status as phagocytes. To assess if differential CD14 ligation causes the ligand-dependent divergence in macrophage responses, a series of CD14 point mutants were used to map the binding of apoptotic cells and lipopolysaccharides. Monoclonal antibodies, 61D3 and MEM18, known to interfere with ligand-binding and responses, were also mapped. Data suggests that residue 11 of CD14, is key for the binding of 61D3 (but not MEM18), LPS and apoptotic cells, indicating lipopolysaccharides and apoptotic cells bind to similar residues. Furthermore using an NF-kB reporter, results show lipopolysaccharides but not apoptotic cells stimulate NF-kB. Taken together these data suggests ligand-dependent CD14 responses occur via a mechanism that occurs downstream of CD14 ligation but upstream of NF-?B activation. Alternatively apoptotic cell ligation of CD14 may not result in any signalling event, possibly by exclusion of TLR-4, suggesting that engulfment receptors, (e.g. TIM-4, BAI1 and Stablin-2) are required to mediate the uptake of apoptotic cells and the associated anti-inflammatory response.

The effect of ageing on macrophage Toll-like receptor-mediated responses in the fight against pathogens

The cellular changes during ageing are incompletely understood yet immune system dysfunction is implicated in the age-related decline in health. The acquired immune system shows a functional decline in ability to respond to new pathogens whereas serum levels of cytokines are elevated with age. Despite these age-associated increases in circulating cytokines, the function of aged macrophages is decreased. Pathogen-associated molecular pattern receptors such as Toll-like receptors (TLRs) are vital in the response of macrophages to pathological stimuli. Here we review the evidence for defective TLR signalling in normal ageing. Gene transcription, protein expression and cell surface expression of members of the TLR family of receptors and co-effector molecules do not show a consistent age-dependent change across model systems. However, there is evidence for impaired downstream signalling events, including inhibition of positive and activation of negative modulators of TLR induced signalling events. In this paper we hypothesize that despite a poor inflammatory response via TLR activation, the ineffective clearance of pathogens by macrophages increases the duration of their activation and contributes to perpetuation of inflammatory responses and ageing.

The innate immune system and the clearance of apoptotic cells

Apoptosis, programmed cell death, is used by multicellular organisms to remove cells that are in excess, damaged or diseased. Activation of the apoptosis programme generates "eat me" signals on the surface of the apoptotic cell that mediate recognition and clearance by the innate immune system. CD14, a pattern recognition receptor expressed on macrophages, is widely known for its ability to recognise the pathogen-associated molecular pattern lipopolysaccharide (LPS) and promote inflammation. However, CD14 has also been shown to mediate binding and removal of apoptotic cells in a process that is anti-inflammatory suggesting CD14 is capable of producing two distinct, ligand-dependent macrophage responses. Whilst the molecular basis for this dichotomy has yet to be defined it is clear that CD14 defines a point of interest on the macrophage surface where we may study ligand-specific responses of macrophages. Our work seeks to define the molecular mechanisms underlying the involvement of CD14 in the non-inflammatory clearance of apoptotic cells. Here we used three different differentiation strategies to generate macrophages from the monocytic cell line THP-1. The resultant macrophage models were characterised to assess the expression and function of CD14 within each model system. Whilst each macrophage model shows increased levels of surface CD14 expression, our results demonstrate significant differences in the various models’ abilities to respond to LPS and clear apoptotic cells in a CD14-dependent manner. TLR4 levels correlated positively with LPS responsiveness but not CD14-dependent apoptotic cell clearance or anti-inflammatory responses to apoptotic cells. These observations suggest CD14-dependent apoptotic cell clearance is not dependent on TLR4. Taken together our data support the notion that the CD14 ligand-dependent responses to LPS and apoptotic cells derive from changes at the macrophage surface. The nature and composition of the CD14-co-receptor complex for LPS and apoptotic cell binding and responses is the subject of further study.

CD14 and innate immune clearance of apoptotic cells

Rapid elimination of cells undergoing programmed cell death (apoptosis) is vital to maintain tissue homeostasis. The phagocytic removal of apoptotic cells (AC) is mediated by innate immune molecules, professional phagocytes and amateur phagocytes that recognise "eat me" signals on the surface of the AC. CD14, a pattern recognition receptor expressed on macrophages, is widely known for its ability to recognise the pathogen-associated molecular pattern lipopolysaccharide (LPS) and promote inflammation. CD14 also mediates the binding and removal of AC, a process that is considered to be anti-inflammatory therefore suggesting CD14 is capable of producing two distinct ligand-dependent responses. Our work seeks to define the molecular mechanisms underlying the involvement of CD14 in the non-inflammatory clearance of AC. Here we describe three different differentiation strategies used to generate macrophages from the monocytic cell line THP-1. Whilst CD14 expression was increased in each macrophage model we demonstrate significant differences in the various macrophage models' abilities to respond to LPS and clear AC. We show that CD14 expression correlates with CD14-dependent AC clearance and anti-inflammatory responses to AC. However LPS responsiveness correlates, as expected, with TLR4 but not CD14 expression. These observations suggest CD14-dependent AC clearance is not dependent on TLR4. Taken together our data support the notion that CD14 ligand-dependent responses to LPS and AC are derived from changes at the macrophage surface. The nature and composition of the CD14-co-receptor complex for LPS and AC binding and consequent responses is the subject of further study.

Vascular cell responsiveness to toll-like receptor ligands in carotid atheroma

Background Atherosclerosis is potentiated by stimulation of Toll-like receptors (TLRs), which serve to detect pathogen associated molecular patterns (PAMPs). However little is known of which PAMPs may be present in atheroma, or capable of stimulating inflammatory signalling in vascular cells. Materials and Methods DNA extracted from human carotid atheroma samples was amplified and sequenced using broad-range 16S gene specific primers to establish historical exposure to bacterial PAMPs. Responsiveness of primary human arterial and venous endothelial and smooth muscle cells to PAMPs specific for each of the TLRs was assessed by measurement of interleukin-8 secretion and E-selectin expression. Results Extracts of atheromatous tissue stimulated little or no signalling in TLR-transfected HEK-293 cells. However, sequencing of bacterial DNA amplified from carotid atheroma revealed the presence of DNA from 17 different bacterial genera, suggesting historical exposure to bacterial lipopeptide, lipopolysaccharide and flagellin. All cells examined were responsive to the ligands of TLR3 and TLR4, poly inosine:cytosine and lipopolysaccharide. Arterial cells were responsive to a wider range of PAMPs than venous cells, being additionally responsive to bacterial flagellin and unmethylated cytosine-phosphate-guanosine DNA motifs, the ligands of TLR5 and TLR9, respectively. Cells were generally unresponsive towards the ligands of human TLR7 and TLR8, loxoribine and single stranded RNA. Only coronary artery endothelial cells expressed TLR2 mRNA and responded to the TLR2 ligand Pam3CSK4. Conclusions Vascular cells are responsive to a relatively diverse range of TLR ligands and may be exposed, at least transiently, to ligands of TLR2, TLR4, TLR5 and TLR9 during the development of carotid atheroma.

Protein oxidative modifications:new mass spectrometry approaches to detection in biological samples

In inflammatory diseases, release of oxidants leads to oxidative damage to proteins. The precise nature of oxidative damage to individual proteins depends on the oxidant involved. Chlorination and nitration are markers of modification by the myeloperoxidase-H2O2-Cl- system and nitric oxide-derived oxidants, respectively. Although these modifications can be detected by western blotting, currently no reliable method exists to identify the specific sites damage to individual proteins in complex mixtures such as clinical samples. We are developing novel LCMS2 and precursor ion scanning methods to address this. LC-MS2 allows separation of peptides and detection of mass changes in oxidized residues on fragmentation of the peptides. We have identified indicative fragment ions for chlorotyrosine, nitrotyrosine, hydroxytyrosine and hydroxytryptophan. A nano-LC/MS3 method involving the dissociation of immonium ions to give specific fragments for the oxidized residues has been developed to overcome the problem of false positives from ions isobaric to these immonium ions that exist in unmodified peptides. The approach has proved able to identify precise protein modifications in individual proteins and mixtures of proteins. An alternative methodology involves multiple reaction monitoring for precursors and fragment ions are specific to oxidized and chlorinated proteins, and this has been tested with human serum albumin. Our ultimate aim is to apply this methodology to the detection of oxidative post-translational modifications in clinical samples for disease diagnosis, monitoring the outcomes of therapy, and improved understanding of disease biochemistry.

Impact of senescence and disease on the structural and functional performance of the visual pathway

The diagnosis and monitoring of ocular disease presents considerable clinical difficulties for two main reasons i) the substantial physiological variation of anatomical structure of the visual pathway and ii) constraints due to technical limitations of diagnostic hardware. These are further confounded by difficulties in detecting early loss or change in visual function due to the masking of disease effects, for example, due to a high degree of redundancy in terms of nerve fibre number along the visual pathway. This thesis addresses these issues across three areas of study: 1. Factors influencing retinal thickness measures and their clinical interpretation As the retina is the principal anatomical site for damage associated with visual loss, objective measures of retinal thickness and retinal nerve fibre layer thickness are key to the detection of pathology. In this thesis the ability of optical coherence tomography (OCT) to provide repeatable and reproducible measures of retinal structure at the macula and optic nerve head is investigated. In addition, the normal physiological variations in retinal thickness and retinal nerve fibre layer thickness are explored. Principal findings were: • Macular retinal thickness and optic nerve head measurements are repeatable and reproducible for normal subjects and diseased eyes • Macular and retinal nerve fibre layer thickness around the optic nerve correlate negatively with axial length, suggesting that larger eyes have thinner retinae, potentially making them more susceptible to damage or disease • Foveola retinal thickness increases with age while retinal nerve fibre layer thickness around the optic nerve head decreases with age. Such findings should be considered during examination of the eye with suspect pathology or in long-term disease monitoring 2. Impact of glucose control on retinal anatomy and function in diabetes Diabetes is a major health concern in the UK and worldwide and diabetic retinopathy is a major cause of blindness in the working population. Objective, quantitative measurements of retinal thickness. particularly at the macula provide essential information regarding disease progression and the efficacy of treatment. Functional vision loss in diabetic patients is commonly observed in clinical and experimental studies and is thought to be affected by blood glucose levels. In the first study of its kind, the short term impact of fluctuations in blood glucose levels on retinal structure and function over a 12 hour period in patients with diabetes are investigated. Principal findings were: • Acute fluctuations in blood glucose levels are greater in diabetic patients than normal subjects • The fluctuations in blood glucose levels impact contrast sensitivity scores. SWAP visual fields, intraocular pressure and diastolic pressure. This effect is similar for type 1 and type 2 diabetic patients despite the differences in their physiological status. • Long-term metabolic control in the diabetic patient is a useful predictor in the fluctuation of contrast sensitivity scores. • Large fluctuations in blood glucose levels and/or visual function and structure may be indicative of an increased risk of development or progression of retinopathy 3. Structural and functional damage of the visual pathway in glaucomatous optic neuropathy The glaucomatous eye undergoes a number of well documented pathological changes including retinal nerve fibre loss and optic nerve head damage which is correlated with loss of functional vision. In experimental glaucoma there is evidence that glaucomatous damage extends from retinal ganglion cells in the eye, along the visual pathway, to vision centres in the brain. This thesis explores the effects of glaucoma on retinal nerve fibre layer thickness, ocular anterior anatomy and cortical structure, and its correlates with visual function in humans. Principal findings were: • In the retina, glaucomatous retinal nerve fibre layer loss is less marked with increasing distance from the optic nerve head, suggesting that RNFL examination at a greater distance than traditionally employed may provide invaluable early indicators of glaucomatous damage • Neuroretinal rim area and retrobulbar optic nerve diameter are strong indicators of visual field loss • Grey matter density decreases at a rate of 3.85% per decade. There was no clear evidence of a disease effect • Cortical activation as measured by fMRI was a strong indicator of functional damage in patients with significant neuroretinal rim loss despite relatively modest visual field defects These investigations have shown that the effects of senescence are evident in both the anterior and posterior visual pathway. A variety of anatomical and functional diagnostic protocols for the investigation of damage to the visual pathway in ocular disease are required to maximise understanding of the disease processes and thereby optimising patient care.

Therapeutic potential of statins and the induction of heme oxygenase-1 in preeclampsia

Heme oxygenase (Hmox) is an endogenous system that offers protection against placental cytotoxic damage associated with preeclampsia. The Hmox1/carbon monoxide (CO) pathway inhibits soluble Flt-1 (sFlt-1) and soluble Endoglin (sEng). More importantly, statins induce Hmox1 and suppress the release of sFlt-1 and sEng; thus, statins and Hmox1 activators are potential novel therapeutic agents for treating preeclampsia. The contribution of the Hmox system to the pathogenesis of preeclampsia has been further indicated by the incidence of preeclampsia being reduced by a third in smokers, who had reduced levels of circulating sFlt-1. Interestingly, preeclamptic women exhale less CO compared with women with healthy pregnancies. Hmox1 is reduced prior to the increase in sFlt-1 as Hmox1 mRNA expression in the trophoblast is decreased in the first trimester in women who go on to develop preeclampsia. Induction of Hmox1 or exposure to CO or bilirubin has been shown to inhibit the release of sFlt-1 and sEng in animal models of preeclampsia. The functional benefit of statins and Hmox1 induction in women with preeclampsia is valid not only because they inhibit sFlt-1 release, but also because statins and Hmox1 are associated with anti-apoptotic, anti-inflammatory, and anti-oxidant properties. The StAmP trial is the first randomized control trial (RCT) evaluating the use of pravastatin to ameliorate severe preeclampsia. This proof-of-concept study will pave the way for future global RCT, the success of which will greatly contribute to achieving the United Nations Millennium Development Goals (MDG4 and MDG5) and offering an affordable and easily accessible therapy for preeclampsia. © 2014 The Authors.

Measurement of apoptotic cell clearance in vitro

The phagocytic clearance of apoptotic cells is a highly efficient and nonphlogistic process in vivo. Research in this area has been limited, at least in part, by technical difficulties associated with the techniques used in the detailed study of apoptotic cell clearance mechanisms. This chapter provides details of methods that may be used to study apoptotic cell clearance in vitro. Such methods have been used successfully to identify phagocyte-associated or apoptotic cell-associated molecular players in the recognition process.

Regional variability in visual field sensitivity during hypercapnia

PURPOSE: Previous investigations have demonstrated a relative vascular autoregulatory inefficiency of the inferior compared to the superior retina in healthy subjects breathing increased CO2. The purpose of this study was to determine whether the superior and inferior visual field sensitivities of healthy eyes are similarly affected during mild hypercapnia. DESIGN: Experimental study. METHODS: Visual field analysis (Humphrey Field Analyser; SITA standard 24-2 program) was carried out on one randomly selected eye of 22 subjects (mean age, 27.7 ± 5 years) during normal room air breathing and isoxic hypercapnia. The Student paired t-tests were used to compare the visual field indices mean deviation (MD) and pattern standard deviation (PSD) for each breathing condition. A secondary, sectoral analysis of mean pointwise sensitivity was performed for each condition. In each case a P value of <.01 was considered statistically significant (Bonferroni corrected). RESULTS: Visual field MD was -0.23 ± 0.95dB during room air breathing and -0.49 ± 1.04dB during hypercapnia (P = .034). Sectoral pointwise mean sensitivity deteriorated by 0.46dB (P = .006) in the upper visual hemifield during hypercapnia, whereas no significant difference was observed for the lower hemifield (P = .331). CONCLUSIONS: The upper visual hemifield exhibited a significantly greater degree of deterioration in pointwise visual field mean sensitivity compared to the lower hemifield during hypercapnic conditions. This suggests that the upper visual hemifield and hence inferior retina is more susceptible to insult during hypercapnia than the superior retina in healthy individuals. A regional susceptibility of inferior retinal function to altered vascular or metabolic effects may account for the earlier and more frequent inferior nerve fibre damage associated with glaucomatous optic neuropathy. © 2003 by Elsevier Science Inc. All rights reserved.

The role of protein oxidation in ageing & disease

Ageing is a natural phenomenon of the human lifecycle, yet it is still not understood what causes the deterioration of the human body near the end of the lifespan. One popular theory is the Free Radical Theory of Ageing, which proposes that oxidative damage to biomolecules causes ageing of tissues. The ageing population is affected by many chronic diseases. This study focused on sarcopenia (muscle loss in ageing) and obesity as two models for comparison of oxidative damage in muscle proteins in mice. The aim of the study was to develop advanced mass spectrometry methods to detect specific oxidative modifications to mouse muscle proteins, including oxidation, nitration, chlorination, and carbonyl group formation, but western blotting was also used to provide complementary information on the oxidative state of proteins from aged and obese muscle. Mass spectrometry proved to be a powerful tool, enabling identification of the types of modifications present, the sites at which they were present and percentage of the peptide populations that were modified. Targeted and semi-targeted mass spectrometry methods were optimised for the identification and quantitation of the oxidised residues in muscle proteins. The development of the quantitative methods enabled comparisons of mass spectrometry instruments. Both the Time of Flight and QTRAP systems showed advantages of using the different mass analysers to quantify oxidative modifications. Several oxidised residues were characterised and quantified in both the obese and sarcopenic models, and higher levels of oxidation were found compared to their control counterparts. Residues found to be oxidised were oxidation of proline, tyrosine and tryptophan, dioxidation of methionine, allysine and nitration of tyrosine. However quantification was performed on methionine dioxidation and cysteine trioxidation containing residues in SERCA. The combination of measuring residue susceptibility and functional studies could contribute to understanding the overall role of oxidation in ageing and obesity.

Aspirin and alterations in DNA repair proteins in the SW480 colorectal cancer cell line

Regular aspirin intake is associated with a reduction in the incidence of colorectal cancer. Aspirin has been shown to be cytotoxic to colorectal cancer cells in vitro. The molecular basis for this cytotoxicity is controversial, with a number of competing hypotheses in circulation. One suggestion is that the protective effect is related to the induction of expression of the DNA mismatch repair (MMR) proteins hMLH1, hMSH2, hMSH6 and hPMS2 in DNA MMR proficient cells. We report that treatment of the DNA MMR competent/p53 mutant colorectal cancer cell line SW480 with 1 mM aspirin for 48 h caused changes in mRNA expression of several key genes involved in DNA damage signalling pathways, including a significant down-regulation in transcription of the genes ATR, BRCA1 and MAPK12. Increases in the transcription of XRCC3 and GADD45alpha genes are also reported. Regulation of these genes could potentially have profound effects on colorectal cancer cells and may play a role in the observed chemo-protective effect of aspirin in vivo. Although a correlation was not seen between transcript and protein levels of ATR, BRCA1 and GADD45alpha, an increase in XRCC3 encoded protein expression upon aspirin treatment in SW480 cells was observed by immunoblotting, immunofluorescence and immunohistochemical analysis. This is the first report of XRCC3 gene transcription and encoded protein expression being susceptible to exposure to the non-steroidal anti-inflammatory drug, aspirin. Furthermore, this study indicates that alterations in gene transcription seen in microarray studies must be verified at the protein level.

8-Hydroxydeoxyguanosine. A marker of oxidative DNA damage in systemic lupus erythematosus

8-Hydroxydeoxyguanosine (80HDG) is a specific marker of oxidative damage to DNA. We have observed that patients with SLE (systemic lupus erythematosus), have undetectable levels of urinary 80HDG by HPLC. Further analysis by GC-MS confirmed that levels of 80HDG in SLE urine were 10(3)-fold lower than in an age- and sex-matched control group. Experiments utilising cultures of SLE and normal lymphocytes exposed to H2O2 confirmed the impaired ability of SLE lymphocytes to repair 80HDG. We subsequently observed in SLE patients that 80HDG had accumulated in low molecular weight DNA associated with circulating immune complexes. We suggest that oxygen radicals may induce pathology in SLE by maintaining the presence of an antigenic form of DNA in the circulation.

Hallmarks of protein oxidative damage in neurodegenerative diseases:Focus on Alzheimer's disease

The pathogenesis of several neurodegenerative diseases, including Alzheimer's disease, has been linked to a condition of oxidative and nitrosative stress, arising from the imbalance between increased reactive oxygen species (ROS) and reactive nitrogen species (RNS) production and antioxidant defences or efficiency of repair or removal systems. The effects of free radicals are expressed by the accumulation of oxidative damage to biomolecules: nucleic acids, lipids and proteins. In this review we focused our attention on the large body of evidence of oxidative damage to protein in Alzheimer's disease brain and peripheral cells as well as in their role in signalling pathways. The progress in the understanding of the molecular alterations underlying Alzheimer's disease will be useful in developing successful preventive and therapeutic strategies, since available drugs can only temporarily stabilize the disease, but are not able to block the neurodegenerative process. © 2007 Springer-Verlag.