Biomedical subjects

Mark J Walport

Publications and source records attributed to Mark J Walport.

At least 19 recordsLinked to original sources

PubMed2006

A novel mechanism for complement activation at the surface of B cells following antigen binding.

Coligation of CD21 with BCR on the surface of B cells provides a costimulatory signal essential for efficient Ab responses to T-dependent Ags. To achieve this, Ag must be directly linked to C3 fragments, but how this occurs in vivo is not fully understood. Using BCR transgenic mice, we demonstrated that C3 was deposited on the surface of B cells following both high- and moderate-affinity Ag binding. This was dependent on the specific binding of IgM to the BCR-bound Ag and can occur independently of soluble immune complex formation. Based on these data, we propose a novel model in which immune complexes can form directly on the surface of the B cell following Ag binding. This model has implications for our understanding of B lymphocyte activation.

Animals↗

PubMed2006

The in vivo expression of actin/salt-resistant hyperactive DNase I inhibits the development of anti-ssDNA and anti-histone autoantibodies in a murine model of systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is characterised by the production of autoantibodies against ubiquitous antigens, especially nuclear components. Evidence makes it clear that the development of these autoantibodies is an antigen-driven process and that immune complexes involving DNA-containing antigens play a key role in the disease process. In rodents, DNase I is the major endonuclease present in saliva, urine and plasma, where it catalyses the hydrolysis of DNA, and impaired DNase function has been implicated in the pathogenesis of SLE. In this study we have evaluated the effects of transgenic over-expression of murine DNase I endonucleases in vivo in a mouse model of lupus. We generated transgenic mice having T-cells that express either wild-type DNase I (wt.DNase I) or a mutant DNase I (ash.DNase I), engineered for three new properties - resistance to inhibition by G-actin, resistance to inhibition by physiological saline and hyperactivity compared to wild type. By crossing these transgenic mice with a murine strain that develops SLE we found that, compared to control non-transgenic littermates or wt.DNase I transgenic mice, the ash.DNase I mutant provided significant protection from the development of anti-single-stranded DNA and anti-histone antibodies, but not of renal disease. In summary, this is the first study in vivo to directly test the effects of long-term increased expression of DNase I on the development of SLE. Our results are in line with previous reports on the possible clinical benefits of recombinant DNase I treatment in SLE, and extend them further to the use of engineered DNase I variants with increased activity and resistance to physiological inhibitors.

Animals↗

PubMed2005

Predominant role of IgM-dependent activation of the classical pathway in the clearance of dying cells by murine bone marrow-derived macrophages in vitro.

Soluble molecules including complement components have been shown to facilitate the clearance of dying cells by phagocytes, a process that is important in preventing tissue damage and autoimmunity. However, the extent to which complement is involved in this process and the relative contribution of each of the complement activation pathways is not fully understood. We examined the role of complement in the recognition/uptake of apoptotic thymocytes by murine bone marrow-derived macrophages (BMDM) in vitro using sera from gene-targeted mice. We found this process to be IgM- and complement-dependent, especially when the apoptotic cell-to-BMDM ratio was low, and the level of C3 deposition on apoptotic cells correlated closely with their uptake. The addition of C1q rectified the phagocytic defect seen in the presence of C1q-deficient serum in vitro but had no effect on the phagocytic defect observed with serum deficient in both IgM antibodies and C1q. Similarly, complement activation by IgM antibodies was essential for in vivo C3 deposition on apoptotic cells and their uptake by peritoneal macrophages. Hence, the efficient uptake of dying cells by BMDM requires IgM antibodies and complement.

Animals↗

PubMed2005

Research funding, partnership and strategy - a UK perspective.

In July 2004, the UK government published a 10-year science and innovation framework aimed at providing a more strategic, partnership-based approach to delivering science. With the aim of creating a UK society that is confident about the governance, regulation and use of science and technology, how can we sustain or increase the supply of money for research, how should funding agencies dispense money, and how can we optimize the partnership arrangements for the funding of research?

Forecasting↗

PubMed2004

Dissection of BXSB lupus phenotype using mice congenic for chromosome 1 demonstrates that separate intervals direct different aspects of disease.

To dissect the individual effects of the four non-MHC, autosomal loci (Bxs1 to Bxs4) that contribute to SLE susceptibility in BXSB mice, we generated congenic lines from chromosome 1 on a C57BL/10.Y(BXSB) (B10.Yaa) background for the intervals (values in megabases (Mb)) Bxs1 (46.3-89.2 Mb), Bxs1/4 (20.0-65.9 Mb), Bxs1/2 (64.4-159.0 Mb), and Bxs2/3 (105.4-189.0 Mb). Glomerulonephritis, qualitatively similar to that seen in the parental BXSB strain, developed in three of these congenic strains. Early onset, severe disease was observed in B10.Yaa.BXSB-Bxs2/3 congenic mice and caused 50% mortality by 12 mo. In B10.Yaa.BXSB-Bxs1/4 mice disease progressed more slowly, resulting in 13% mortality at 12 mo. The progression of renal disease in both of these strains was correlated with the level of anti-dsDNA Abs. B10.Yaa.BXSB-Bxs1 mice, despite their genetic similarity to B10.Yaa.BXSB-Bxs1/4 mice, developed a low-grade glomerulonephritis in the absence of anti-dsDNA Abs. Thus, Bxs4 directed an increase in titer and spectrum of autoantibodies, whereas Bxs1 promoted the development of nephritis. The Bxs2 interval was linked to the production of anti-dsDNA Abs without concomitant glomerulonephritis. In contrast, the Bxs3 interval was sufficient to generate classic lupus nephritis in a nonautoimmune-prone strain. Immune phenotype differed between controls and congenics with a significant increase in B220(+) cells in BXSB and B10.Yaa.BXSB-Bxs2/3, and an increase in CD4 to CD8 ratio in both BXSB and B10.Yaa.BXSB-Bxs1/4. Disease in the Bxs3 mice was delayed in comparison to the BXSB parental strain, emphasizing the necessity for multiple interactions in the production of the full BXSB phenotype.

Animals↗

PubMed2004

Spontaneous autoimmunity in 129 and C57BL/6 mice-implications for autoimmunity described in gene-targeted mice.

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disorder in which complex genetic factors play an important role. Several strains of gene-targeted mice have been reported to develop SLE, implicating the null genes in the causation of disease. However, hybrid strains between 129 and C57BL/6 mice, widely used in the generation of gene-targeted mice, develop spontaneous autoimmunity. Furthermore, the genetic background markedly influences the autoimmune phenotype of SLE in gene-targeted mice. This suggests an important role in the expression of autoimmunity of as-yet-uncharacterised background genes originating from these parental mouse strains. Using genome-wide linkage analysis, we identified several susceptibility loci, derived from 129 and C57BL/6 mice, mapped in the lupus-prone hybrid (129 x C57BL/6) model. By creating a C57BL/6 congenic strain carrying a 129-derived Chromosome 1 segment, we found that this 129 interval was sufficient to mediate the loss of tolerance to nuclear antigens, which had previously been attributed to a disrupted gene. These results demonstrate important epistatic modifiers of autoimmunity in 129 and C57BL/6 mouse strains, widely used in gene targeting. These background gene influences may account for some, or even all, of the autoimmune traits described in some gene-targeted models of SLE.

Animals↗

PubMed2004

Murine CD93 (C1qRp) contributes to the removal of apoptotic cells in vivo but is not required for C1q-mediated enhancement of phagocytosis.

Human CD93 (known as C1qRp) has been shown to be a phagocytic receptor involved in the in vitro C1q-dependent enhancement of phagocytosis. However, binding of CD93 to C1q and its function remain controversial. In this study, we have generated CD93-deficient mice (CD93(-/-)) to investigate its biological role(s). The CD93(-/-) mice were viable and showed no gross abnormalities in their development. Thioglycolate-elicited peritoneal macrophages deficient in CD93 showed a similar enhancement in complement- and FcgammaR-dependent uptake of RBC to the wild-type macrophages when plated on C1q-coated surfaces suggesting that the lack of this receptor had no effect on these C1q-mediated events. There was no impairment in either complement- or FcgammaR-dependent phagocytic assays in vivo. By contrast, the CD93(-/-) mice had a significant phagocytic defect in the clearance of apoptotic cells in vivo (human Jurkat T cells and murine thymocytes: p=0.0006 and p=0.0079, respectively) compared with strain-matched controls. However, in vitro, the CD93(-/-) macrophages showed similar engulfment of apoptotic cells to wild-type macrophages. Furthermore, no supporting evidence for a role of CD93 as an adhesion molecule was found using intravital microscopy or analyzing peritoneal cell recruitment in response to three different inflammatory stimuli (thioglycolate, zymosan A, and IL-1beta). Thus, our findings indicate that murine CD93 is expressed on the peritoneal macrophage, especially on thioglycolate-elicited cells, but does not appear to play a key role in C1q-mediated enhancement of phagocytosis or in the intercellular adhesion events tested. However, our results suggest that it may contribute to the in vivo clearance of dying cells.

Adjuvants, Immunologic↗

PubMed2004

Restoration of C1q levels by bone marrow transplantation attenuates autoimmune disease associated with C1q deficiency in mice.

C1q deficiency in both humans and mice is strongly associated with autoimmunity. We have previously shown that bone marrow transplantation (BMT) restored C1q levels in C1q-deficient (C1qa(-/-)) mice. Here, we studied the effect of BMT on autoimmunity in C1qa(-/-) mice. Following irradiation, young C1qa(-/-) or wild-type MRL/Mp mice received bone marrow cells (BMC) from strain-matched wild-type or C1qa(-/-) animals. C1q levels increased rapidly when C1qa(-/-) mice received BMC from wild-type mice. Conversely, they decreased slowly in wild-type mice transplanted with C1qa(-/-) BMC. C1qa(-/-) animals transplanted with C1qa(-/-) BMC demonstrated accelerated disease when compared with wild-type mice given wild-type BMC. In contrast, a significant delay in the development of autoantibodies and glomerulonephritis was observed in C1qa(-/-) mice reconstituted with wild-type BMC, and the impaired clearance of apoptotic cells, previously described in C1qa(-/-) mice, was rectified. Moreover, the autoimmune disease was accelerated in wild-type mice given C1qa(-/-) BMC compared to animals transplanted with wild-type cells. These results provide supporting evidence that BMT may be a therapeutic option in the treatment of autoimmunity associated with human C1q deficiency.

Animals↗

PubMed2004

CD59a deficiency exacerbates ischemia-reperfusion injury in mice.

The terminal complement components C5a and the membrane attack complex are involved in the pathogenesis of ischemia-reperfusion injury in many organs. CD59 is the major regulator of membrane attack complex formation. Mice deficient in the Cd59a gene (mCd59a-/-) were used to investigate the role of CD59 in renal ischemia-reperfusion injury. Unilateral ischemia-reperfusion injury was induced by clamping the left renal pedicle for 30 minutes under general anesthetic. Mice were studied at 72 hours and 2 weeks after ischemia-reperfusion injury. mCd59a-/- mice developed significantly greater tubular injury (P = 0.01), tubulointerstitial apoptosis (P = 0.02), and neutrophil influx (P = 0.04) than controls at 72 hours after ischemia-reperfusion. Two weeks after ischemia-reperfusion, mCd59a-/- mice exhibited more severe tubular damage predominantly in a corticomedullary distribution than controls (P = 0.02). Quantification of interstitial leukocytes revealed significantly greater numbers of infiltrating lymphocytes (but not macrophages) in mCd59a-/- mice than controls (P = 0.04) at 2 weeks. At both time points, significantly more C9 (as a marker of membrane attack complex) deposition occurred in a peritubular distribution in mCd59a-/- mice than controls. In conclusion, these results demonstrate that the lack of CD59a, by allowing unregulated membrane attack complex deposition, exacerbates both the tubular injury and the interstitial leukocyte infiltrate after ischemia-reperfusion injury in mice.

Animals↗

PubMed2004

Monocytosis and accelerated activation of lymphocytes in C1q-deficient autoimmune-prone mice.

C1q deficiency has been shown to accelerate spontaneous autoimmunity in mice. We studied the time course of activation of monocytes and lymphocytes in autoimmune and non-autoimmune mice in the presence or absence of C1q as a disease accelerator. Autoimmune MRL\Mp.C1qa-\- and non-autoimmune C57BL\6.C1qa-\- mice were analysed at various time points between 6 and 33 weeks of age and compared to strain- and age-matched C1q-sufficient controls. Splenic and peritoneal leucocytes were analysed by flow cytometry and plasma levels of immunoglobulin M (IgM), total IgG, IgG subclasses and IgM autoantibodies were measured. Both C1q-deficient strains had significantly more splenic monocytes than their controls at all time points analysed. In addition, MRL\Mp.C1qa-\- but not C57BL/6.C1qa-\- mice developed splenic hypercellularity starting at about 12-17 weeks old, had signs of accelerated CD4+ T-cell activation and showed a marked increase in splenic plasma cells and total serum IgM levels from about 22 weeks of age. The accelerated CD4+ T-cell activation was not due to a direct inhibitory effect of C1q on T cells. These data show that C1q deficiency causes splenic monocytosis together with accelerated T-cell activation in an autoimmune-prone mouse strain.

Animals↗

PubMed2004

The role of complement in the development of systemic lupus erythematosus.

Complement has both beneficial and deleterious roles in the pathogenesis of systemic lupus erythematosus (SLE). On the one hand, patients with SLE present with decreased complement levels and with complement deposition in inflamed tissues, suggestive of a harmful role of complement in the effector phase of disease. On the other hand, homozygous deficiency of any of the classical pathway proteins is strongly associated with the development of SLE. There are two main hypotheses to explain these observations. The first invokes an important role for complement in the physiological waste-disposal mechanisms of dying cells and immune complexes. The second hypothesis is based around the role of complement in determining the activation thresholds of B and T lymphocytes, with the proposal that complement deficiency causes incomplete maintenance of peripheral tolerance. These two hypotheses are not mutually exclusive. In addition, there is evidence for a contribution from other genetic factors in determining the phenotype of disease in the absence of complement.

Animals↗

PubMed2003

Polymorphism at the C-reactive protein locus influences gene expression and predisposes to systemic lupus erythematosus.

Relative deficiency of pentraxin proteins is implicated in the pathogenesis of systemic lupus erythematosus. The C-reactive protein (CRP) response is defective in patients with acute flares of disease, and mice with targeted deletions of the serum amyloid P component gene (Sap) develop a lupus-like illness. In humans, the genes for CRP (CRP) and SAP (APCS) map to 1q23.2 within an interval linked with SLE. We have investigated the candidate genes CRP and APCS in two cohorts totalling 586 UK simplex SLE families. The inheritance of an intronic dinucleotide repeat and seven single nucleotide polymorphisms in the CRP and APCS genes was examined by application of family-based tests of association and linkage. Basal levels of CRP were influenced independently by two polymorphisms at the CRP locus, CRP 2 and CRP 4. Furthermore, the latter polymorphism was linked/associated with SLE and antinuclear autoantibody production. Thus, the polymorphism associated with reduced basal CRP was also associated with the development of SLE. These data support the hypothesis that defective disposal of potentially immunogenic material is a contributory factor in lupus pathogenesis. The identification of polymorphisms that determine basal CRP levels has implications in ischaemic heart disease, where CRP level is an important predictor of risk.

C-Reactive Protein↗

PubMed2003

Lupus-prone mice have an abnormal response to thioglycolate and an impaired clearance of apoptotic cells.

Deficiency of complement in humans and mice is associated with the development of lupus and with abnormal repair of inflammatory and immune complex-mediated tissue injury. Here we ask whether similar defects in the resolution of inflammation are found in mice prone to spontaneous lupus. We compared the response to an i.p. injection of thioglycolate between two lupus-prone strains (MRL/Mp and NZB/W) and two non lupus-prone strains of mice (C57BL/6 and BALB/c). In all four strains the influx of polymorphonuclear neutrophils (PMN) was similar. However, by 96 h clearance of PMN in the control strains was complete, whereas in the autoimmune-prone strains PMN were still detectable. The number of mononuclear cells recruited was markedly reduced in the lupus-prone strains compared with the controls, and their phenotype was different. The lupus-prone strains had significantly fewer elicited macrophages that were CD11b-high and Ly6C-negative. In lupus-prone mice at 24 h there was a significantly increased number of apoptotic PMN free in the peritoneum, accompanied by a reduced percentage of macrophages containing apoptotic bodies, suggesting a defect in their uptake. An impaired ability of resident peritoneal macrophages from lupus-prone mice to engulf apoptotic cells was demonstrated by in vivo and in vitro cell clearance assays. These observations indicate that lupus-prone strains have an abnormal inflammatory response to thioglycolate and an intrinsic impairment in apoptotic cell uptake. These findings have implications for the initiation of autoimmunity, as lupus autoantigens are expressed on dying cells, and impaired disposal of these could enhance the development of autoimmunity.

Animals↗

PubMed2003

Both Fcgamma receptor I and Fcgamma receptor III mediate disease in accelerated nephrotoxic nephritis.

Recognition of immune complexes in glomeruli by activator Fcgamma receptors (FcgammaRI and FcgammaRIII) is an important step in the development of glomerulonephritis. The low-affinity receptor (FcgammaRIII) has previously been shown to be important in passive heterologous immune complex glomerulonephritis. However, most forms of human glomerulonephritis involve an active immune response, and the relative importance of FcgammaRI (high-affinity receptor) and FcgammaRIII in an active model of glomerulonephritis is not known. We have now studied accelerated nephrotoxic nephritis in FcgammaRIII-/- mice and FcgammaRI/III double-deficient mice, and compared them with matched wild-type controls and FcRgamma chain-deficient (FcRgamma-/-) mice. Mice were immunized against sheep IgG and injected with sheep anti-mouse glomerular basement membrane antibody 5 days later. Both FcgammaRI/III double-deficient mice and FcRgamma-/- mice were strongly protected from renal injury. In contrast, FcgammaRIII-/- mice developed substantial nephritis, although there was a dose-dependent partial protection from glomerular crescents and thrombosis. Despite this histological protection from injury, the macrophage infiltrate was not reduced, implying a dissociation of macrophage accumulation from activation in the absence of activatory FcgammaRIII. Therefore, both FcgammaRI and FcgammaRIII play a role in this active model of glomerulonephritis, because both had to be deficient to protect markedly from disease.

Animals↗

PubMed2003

CD59a deficiency exacerbates accelerated nephrotoxic nephritis in mice.

CD59 is a complement regulatory protein that inhibits the terminal part of the complement system, the membrane attack complex (MAC), a mediator of renal injury. Mice deficient in the Cd59a gene (mCd59a-/-) were used to investigate the role of CD59 in experimentally induced accelerated nephrotoxic nephritis, a model of immune complex-mediated glomerulonephritis. After accelerated nephrotoxic nephritis was induced by administration of sheep nephrotoxic globulin, mCd59a-/- mice and strain-matched controls on two genetic backgrounds, 129/Sv x C57BL/6 and 129/Sv, were examined. For both, mCd59a-/- mice developed significantly greater glomerular cellularity than wild-type (WT) mice at day 5 after administration. At day 10 post-administration, mCd59a-/- mice exhibited more glomerular thrombosis than WT mice (thrombosis score, 1.8 [range, 1.4 to 4.0] versus 0.8 [range, 0.2 to 1.5] quadrants thrombosed per glomerulus, respectively; P = 0.0006). In the majority of experiments, mCd59a-/- mice also had significantly more proteinuria than controls; however, there was no difference in serum creatinine or albumin. Quantitative immunofluorescence of kidney sections revealed significantly more C9 (as a marker of MAC) deposition within glomeruli of mCd59a-/- mice than WT controls (P < 0.001). There was no difference in deposition of C3 and sheep IgG between the two experimental groups. The lack of CD59a, by allowing unregulated MAC deposition, exacerbates the renal injury in this model of immune complex-mediated glomerulonephritis.

Animals↗

PubMed2002

The classical pathway is the dominant complement pathway required for innate immunity to Streptococcus pneumoniae infection in mice.

The complement system is an important component of the innate immune response to bacterial pathogens, including Streptococcus pneumoniae. The classical complement pathway is activated by antibody-antigen complexes on the bacterial surface and has been considered predominately to be an effector of the adaptive immune response, whereas the alternative and mannose-binding lectin pathways are activated directly by bacterial cell surface components and are considered effectors of the innate immune response. Recently, a role has been suggested for the classical pathway during innate immunity that is activated by natural IgM or components of the acute-phase response bound to bacterial pathogens. However, the functional importance of the classical pathway for innate immunity to S. pneumoniae and other bacterial pathogens, and its relative contribution compared with the alternative and mannose-binding lectin pathways has not been defined. By using strains of mice with genetic deficiencies of complement components and secretory IgM we have investigated the role of each complement pathway and natural IgM for innate immunity to S. pneumoniae. Our results show that the proportion of a population of S. pneumoniae bound by C3 depends mainly on the classical pathway, whereas the intensity of C3 binding depends on the alternative pathway. Furthermore, the classical pathway, partially targeted by the binding of natural IgM to bacteria, is the dominant pathway for activation of the complement system during innate immunity to S. pneumoniae, loss of which results in rapidly progressing septicemia and impaired macrophage activation. These data demonstrate the vital role of the classical pathway for innate immunity to a bacterial pathogen.

Animals↗

PubMed2002

Role of surfactant proteins A, D, and C1q in the clearance of apoptotic cells in vivo and in vitro: calreticulin and CD91 as a common collectin receptor complex.

Removal of cells dying by apoptosis is essential to normal development, maintenance of tissue homeostasis, and resolution of inflammation. Surfactant protein A (SP-A) and surfactant protein D (SP-D) are high abundance pulmonary collectins recently implicated in apoptotic cell clearance in vitro. Other collectins, such as mannose-binding lectin and the collectin-like C1q, have been shown to bind to apoptotic cells and drive ingestion through interaction with calreticulin and CD91 on the phagocyte in vitro. However, only C1q has been shown to enhance apoptotic cell uptake in vivo. We sought to determine the relative importance of SP-A, SP-D, and C1q in pulmonary clearance of apoptotic cells using knockout and overexpressing mice, and to determine the role of calreticulin and CD91 in this process. SP-A, SP-D, and C1q all enhanced apoptotic cell ingestion by resident murine and human alveolar macrophages in vitro. However, only SP-D altered apoptotic cell clearance from the naive murine lung, suggesting that SP-D plays a particularly important role in vivo. Similar to C1q and mannose-binding lectin, SP-A and SP-D bound to apoptotic cells in a localized, patchy pattern and drove apoptotic cell ingestion by phagocytes through a mechanism dependent on calreticulin and CD91. These results suggest that the entire collectin family of innate immune proteins (including C1q) works through a common receptor complex to enhance removal of apoptotic cells, and that collectins are integral, organ-specific components of the clearance machinery.

Animals↗

PubMed2002

Uncontrolled C3 activation causes membranoproliferative glomerulonephritis in mice deficient in complement factor H.

The alternative pathway of complement is activated continuously in vivo through the C3 'tick-over' pathway. This pathway is triggered by the hydrolysis of C3, resulting in the formation of C3 convertase. This, in turn, generates C3b, which mediates many of the biological functions of complement. Factor H, the main regulator of this activation, prevents formation and promotes dissociation of the C3 convertase enzyme, and, together with factor I, mediates the proteolytic inactivation of C3b. Factor H deficiency, described in 29 individuals from 12 families and in pigs, allows unhindered activation of fluid-phase C3 and severe depletion of plasma C3 (ref. 11). Membranoproliferative glomerulonephritis (MPGN) occurs in factor H-deficient humans and pigs. Although MPGN has been reported in other conditions in which uncontrolled activation of C3 occurs, the role of C3 dysregulation in the pathogenesis of MPGN is not understood. Here we show that mice deficient in factor H (Cfh(-/-) mice) develop MPGN spontaneously and are hypersensitive to developing renal injury caused by immune complexes. Introducing a second mutation in the gene encoding complement factor B, which prevents C3 turnover in vivo, obviates the phenotype of Cfh(-/-) mice. Thus, uncontrolled C3 activation in vivo is essential for the development of MPGN associated with deficiency of factor H.

Animals↗