Publications by authors named "Ming-Lang Zhao"

17 Publications

  • Page 1 of 1

The Role of IgM Antibodies in T Cell Lymphoma Protection in a Novel Model Resembling Anaplastic Large Cell Lymphoma.

J Immunol 2021 05 21;206(10):2468-2477. Epub 2021 Apr 21.

Somatic Hypermutation Group, Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC

MRL/lpr mice typically succumb to immune complex-mediated nephritis within the first year of life. However, MRL/lpr mice that only secrete IgM Abs because of activation-induced deaminase deficiency (AIDMRL/lpr mice) experienced a dramatic increase in survival. Further crossing of these mice to those incapable of making secretory IgM (μS mice) generated mice lacking any secreted Abs but with normal B cell receptors. Both strains revealed no kidney pathology, yet Ab-deficient mice still experienced high mortality. In this article, we report Ab-deficient MRL/lpr mice progressed to high-grade T cell lymphoma that can be reversed with injection of autoreactive IgM Abs or following adoptive transfer of IgM-secreting MRL/lpr B cells. Anti-nuclear Abs, particularly anti-dsDNA IgM Abs, exhibited tumor-killing activities against a murine T cell lymphoma cell line. Passive transfers of autoreactive IgM Abs into p53-deficient mice increased survival by delaying onset of T cell lymphoma. The lymphoma originated from a double-negative aberrant T cell population seen in MRL/lpr mice and most closely resembled human anaplastic large cell lymphoma. Combined, these results strongly implicate autoreactive IgM Abs in protection against T cell lymphoma.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4049/jimmunol.2001279DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8113120PMC
May 2021

Lysines in the lyase active site of DNA polymerase β destabilize nonspecific DNA binding, facilitating searching and DNA gap recognition.

J Biol Chem 2020 08 9;295(34):12181-12187. Epub 2020 Jul 9.

Genome Integrity and Structural Biology Laboratory, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina, USA. Electronic address:

DNA polymerase (pol) β catalyzes two reactions at DNA gaps generated during base excision repair, gap-filling DNA synthesis and lyase-dependent 5´-end deoxyribose phosphate removal. The lyase domain of pol β has been proposed to function in DNA gap recognition and to facilitate DNA scanning during substrate search. However, the mechanisms and molecular interactions used by pol β for substrate search and recognition are not clear. To provide insight into this process, a comparison was made of the DNA binding affinities of WT pol β, pol λ, and pol μ, and several variants of pol β, for 1-nt-gap-containing and undamaged DNA. Surprisingly, this analysis revealed that mutation of three lysine residues in the lyase active site of pol β, 35, 68, and 72, to alanine (pol β KΔ3A) increased the binding affinity for nonspecific DNA ∼11-fold compared with that of the WT. WT pol μ, lacking homologous lysines, displayed nonspecific DNA binding behavior similar to that of pol β KΔ3A, in line with previous data demonstrating both enzymes were deficient in processive searching. In fluorescent microscopy experiments using mouse fibroblasts deficient in PARP-1, the ability of pol β KΔ3A to localize to sites of laser-induced DNA damage was strongly decreased compared with that of WT pol β. These data suggest that the three lysines in the lyase active site destabilize pol β when bound to DNA nonspecifically, promoting DNA scanning and providing binding specificity for gapped DNA.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.RA120.013547DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7443498PMC
August 2020

Mitochondrial dysfunction and DNA damage accompany enhanced levels of formaldehyde in cultured primary human fibroblasts.

Sci Rep 2020 03 27;10(1):5575. Epub 2020 Mar 27.

Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC, 27709, USA.

Formaldehyde (FA) is a simple biological aldehyde that is produced inside cells by several processes such as demethylation of DNA and proteins, amino acid metabolism, lipid peroxidation and one carbon metabolism (1-C). Although accumulation of excess FA in cells is known to be cytotoxic, it is unknown if an increase in FA level might be associated with mitochondrial dysfunction. We choose to use primary human fibroblasts cells in culture (foreskin, FSK) as a physiological model to gain insight into whether an increase in the level of FA might affect cellular physiology, especially with regard to the mitochondrial compartment. FSK cells were exposed to increasing concentrations of FA, and different cellular parameters were studied. Elevation in intracellular FA level was achieved and was found to be cytotoxic by virtue of both apoptosis and necrosis and was accompanied by both G2/M arrest and reduction in the time spent in S phase. A gene expression assessment by microarray analysis revealed FA affected FSK cells by altering expression of many genes including genes involved in mitochondrial function and electron transport. We were surprised to observe increased DNA double-strand breaks (DSBs) in mitochondria after exposure to FA, as revealed by accumulation of γH2A.X and 53BP1 at mitochondrial DNA foci. This was associated with mitochondrial structural rearrangements, loss of mitochondrial membrane potential and activation of mitophagy. Collectively, these results indicate that an increase in the cellular level of FA can trigger mitochondrial DNA double-strand breaks and dysfunction.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-020-61477-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7101401PMC
March 2020

Oxidative DNA Damage Modulates DNA Methylation Pattern in Human Breast Cancer 1 (BRCA1) Gene via the Crosstalk between DNA Polymerase β and a DNA Methyltransferase.

Cells 2020 01 16;9(1). Epub 2020 Jan 16.

Biochemistry Ph.D. Program, Florida International University, Miami, FL 33199, USA.

DNA damage and base excision repair (BER) are actively involved in the modulation of DNA methylation and demethylation. However, the underlying molecular mechanisms remain unclear. In this study, we seek to understand the mechanisms by exploring the effects of oxidative DNA damage on the DNA methylation pattern of the tumor suppressor breast cancer 1 (BRCA1) gene in the human embryonic kidney (HEK) HEK293H cells. We found that oxidative DNA damage simultaneously induced DNA demethylation and generation of new methylation sites at the CpGs located at the promoter and transcribed regions of the gene ranging from -189 to +27 in human cells. We demonstrated that DNA damage-induced demethylation was mediated by nucleotide misincorporation by DNA polymerase β (pol β). Surprisingly, we found that the generation of new DNA methylation sites was mediated by coordination between pol β and the DNA methyltransferase, DNA methyltransferase 3b (DNMT3b), through the interaction between the two enzymes in the promoter and encoding regions of the BRCA1 gene. Our study provides the first evidence that oxidative DNA damage can cause dynamic changes in DNA methylation in the BRCA1 gene through the crosstalk between BER and DNA methylation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cells9010225DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7016758PMC
January 2020

Shining light on the response to repair intermediates in DNA of living cells.

DNA Repair (Amst) 2020 01 12;85:102749. Epub 2019 Nov 12.

Genome Integrity and Structural Biology Laboratory, NIEHS, National Institutes of Health, Research Triangle Park, NC 27709, USA. Electronic address:

Fluorescently-tagged repair proteins have been widely used to probe recruitment to micro-irradiation-induced nuclear DNA damage in living cells. Here, we quantify APE1 dynamics after micro-irradiation. Markers of DNA damage are characterized and UV-A laser micro-irradiation energy conditions are selected for formation of oxidatively-induced DNA base damage and single strand breaks, but without detectable double strand breaks. Increased energy of laser micro-irradiation, compared with that used previously in our work, enables study of APE1 dynamics at the lesion site. APE1 shows rapid transient kinetics, with recruitment half-time of less than 1 s and dissociation half-time of less than 15 s. In cells co-transfected with APE1 and PARP1, the recruitment half-time of PARP1 was slower than that of APE1, indicating APE1 is a rapid responder to the damage site. While recruitment of APE1 is unchanged in the presence of co-transfected PARP1, APE1 dissociation is 3-fold slower, revealing PARP1 involvement in APE1 dynamics. Further, we find that APE1 dissociation kinetics are strongly modified in the absence of DNA polymerase β (pol β). After unchanged recruitment to the damage site, dissociation of APE1 became undetectable. This indicates a necessary role for pol β in APE1 release after its recruitment to the damage site. These observations represent an advance in our understanding of in vivo dynamics of base excision repair factors APE1, PARP1 and pol β.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.dnarep.2019.102749DOI Listing
January 2020

In silico structure prediction and inhibition mechanism studies of AtHDA14 as revealed by homology modeling, docking, molecular dynamics simulation.

Comput Biol Chem 2018 Aug 6;75:120-130. Epub 2018 May 6.

Key Laboratory of Bio-Resources and Eco-Environment of the Ministry of Education, College of Life Sciences, Sichuan University, Chengdu 610064, PR China. Electronic address:

Histone deacetylases (HDACs) play a significant role in the epigenetic mechanism by catalyzing deacetylation of lysine on histone in both animals and plants. HDACs involved in growth, development and response to stresses in plants. Arabidopsis thaliana histone deacetylase 14 (AtHDA14) is found to localize in the mitochondria and chloroplasts, and it involved in photosynthesis and melatonin biosynthesis. However, its mechanism of action was still unknowns so far. Therefore, in this study, we constructed AtHDA14 protein model using homology modeling method, validated using PROCHECK and presented using Ramachandran plots. We also performed virtual screening of AtHDA14 by docking with small molecule drugs and predicted their ADMET properties to select representative inhibitors. MD simulation for representative AtHDA14-ligand complexes was carried out to further research and reveal their stability and inhibition mechanism. Meanwhile, MM/PBSA method was utilized to obtain more valuable information about the residues energy contribution. Moreover, compared with four candidate inhibitors, we also found that compound 645533 and 6918837 might be a more potent AtHDA14 inhibitor than TSA (444732) and SAHA (5311). Therefore, compound 6445533 and 6918837 was anticipated to be a promising drug candidate for inhibition of AtHDA14.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.compbiolchem.2018.05.006DOI Listing
August 2018

XRCC1 phosphorylation affects aprataxin recruitment and DNA deadenylation activity.

DNA Repair (Amst) 2018 04 15;64:26-33. Epub 2018 Feb 15.

Genome Integrity and Structural Biology Laboratory, NIEHS, National Institutes of Health, Research Triangle Park, NC 27709, USA. Electronic address:

Aprataxin (APTX) is a DNA-adenylate hydrolase that removes 5'-AMP blocking groups from abortive ligation repair intermediates. XRCC1, a multi-domain protein without catalytic activity, interacts with a number of known repair proteins including APTX, modulating and coordinating the various steps of DNA repair. CK2-phosphorylation of XRCC1 is thought to be crucial for its interaction with the FHA domain of APTX. In light of conflicting reports, the importance of XRCC1 phosphorylation and APTX function is not clear. In this study, a phosphorylation mutant of XRCC1 designed to eliminate APTX binding was stably expressed in Xrcc1 cells. Analysis of APTX-GFP accumulation at micro-irradiation damage confirmed that phosphorylated XRCC1 is required for APTX recruitment. APTX-mediated DNA deadenylation activity (i.e., 5'-AMP removal) was measured in extracts of cells expressing wild-type XRCC1 or the XRCC1 phosphorylation mutant, and compared with activity in APTX-deficient and APTX-complemented human cells. APTX activity was lower in extracts from Xrcc1 and XRCC1 phosphorylation mutant cells compared to the robust activity in extract from wild-type XRCC1 expressing cells. Taken together, results verify that interaction with phosphorylated XRCC1 is a requirement for significant APTX recruitment to cellular DNA damage and enzymatic activity in cell extracts.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.dnarep.2018.02.004DOI Listing
April 2018

DNA polymerase β: A missing link of the base excision repair machinery in mammalian mitochondria.

DNA Repair (Amst) 2017 12 28;60:77-88. Epub 2017 Oct 28.

Genome Integrity and Structural Biology Laboratory, National Institutes of Health, NIEHS, 111 T.W. Alexander Drive, P.O. Box 12233, Research Triangle Park, NC 27709, USA. Electronic address:

Mitochondrial genome integrity is fundamental to mammalian cell viability. Since mitochondrial DNA is constantly under attack from oxygen radicals released during ATP production, DNA repair is vital in removing oxidatively generated lesions in mitochondrial DNA, but the presence of a strong base excision repair system has not been demonstrated. Here, we addressed the presence of such a system in mammalian mitochondria involving the primary base lesion repair enzyme DNA polymerase (pol) β. Pol β was localized to mammalian mitochondria by electron microscopic-immunogold staining, immunofluorescence co-localization and biochemical experiments. Extracts from purified mitochondria exhibited base excision repair activity that was dependent on pol β. Mitochondria from pol β-deficient mouse fibroblasts had compromised DNA repair and showed elevated levels of superoxide radicals after hydrogen peroxide treatment. Mitochondria in pol β-deficient fibroblasts displayed altered morphology by electron microscopy. These results indicate that mammalian mitochondria contain an efficient base lesion repair system mediated in part by pol β and thus pol β plays a role in preserving mitochondrial genome stability.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.dnarep.2017.10.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5919216PMC
December 2017

XRCC1-mediated repair of strand breaks independent of PNKP binding.

DNA Repair (Amst) 2017 12 19;60:52-63. Epub 2017 Oct 19.

Genomic Integrity and Structural Biology Laboratory, NIEHS, National Institutes of Health, Research Triangle Park, NC 27709, USA. Electronic address:

Repair of DNA-protein crosslinks and oxidatively damaged DNA base lesions generates intermediates with nicks or gaps with abnormal and blocked 3'-phosphate and 5'-OH ends that prevent the activity of DNA polymerases and ligases. End cleaning in mammalian cells by Tdp1 and PNKP produces the conventional 3'-OH and 5'-phosphate DNA ends suitable for completion of repair. This repair function of PNKP is facilitated by its binding to the scaffold protein XRCC1, and phosphorylation of XRCC1 by CK2 at several consensus sites enables PNKP binding and recruitment to DNA damage. To evaluate this documented repair process, a phosphorylation mutant of XRCC1, designed to eliminate PNKP binding, was stably expressed in Xrcc1 mouse fibroblast cells. Analysis of PNKP-GFP accumulation at micro-irradiation induced damage confirmed that the XRCC1 phosphorylation mutant failed to support efficient PNKP recruitment, whereas there was rapid recruitment in cells expressing wild-type XRCC1. Recruitment of additional fluorescently-tagged repair factors PARP-1-YFP, GFF-XRCC1, PNKP-GFP and Tdp1-GFP to micro-irradiation induced damage was assessed in wild-type XRCC1-expressing cells. PARP-1-YFP recruitment was best fit to two exponentials, whereas kinetics for the other proteins were fit to a single exponential. The similar half-times of recruitment suggest that XRCC1 may be recruited with other proteins possibly as a pre-formed complex. Xrcc1 cells are hypersensitive to the DNA-protein cross-link inducing agent camptothecin (CPT) and the DNA oxidative agent HO due in part to compromised PNKP-mediated repair. However, cells expressing the PNKP interaction mutant of XRCC1 demonstrated marked reversal of CPT hypersensitivity. This reversal represents XRCC1-dependent repair in the absence of the phosphorylation-dependent PNKP recruitment and suggests either an XRCC1-independent mechanism of PNKP recruitment or a functional back-up pathway for cleaning of blocked DNA ends.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.dnarep.2017.10.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5696015PMC
December 2017

Role of the oxidized form of XRCC1 in protection against extreme oxidative stress.

Free Radic Biol Med 2017 06 4;107:292-300. Epub 2017 Feb 4.

Genomic Integrity and Structural Biology Laboratory, NIEHS, National Institutes of Health, Research Triangle Park, NC 27709, USA. Electronic address:

The multi-domain protein XRCC1 is without catalytic activity, but can interact with a number of known repair proteins. The interaction between the N-terminal domain (NTD) of XRCC1 and DNA polymerase β (pol β) is critical for recruitment of pol β to sites of DNA damage and repair. Crystallographic and NMR approaches have identified oxidized and reduced forms of the XRCC1 NTD, and the corresponding forms of XRCC1 have been identified in cultured mouse fibroblast cells. Both forms of NTD interact with pol β, but the interaction is much stronger with the oxidized form. The potential for formation of the C12-C20 oxidized conformation can be removed by alanine substitution at C12 (C12A) leading to stabilized reduced XRCC1 with a lower pol β binding affinity. Here, we compare cells expressing C12A XRCC1 (XRE8) with those expressing wild-type XRCC1 (XC5). Reduced C12A XRCC1 is detected at sites of micro-irradiation DNA damage, but provides slower recruitment of pol β. Expression of reduced XRCC1 does not affect sensitivity to MMS or HO In contrast, further oxidative stress imposed by glutathione depletion results in increased sensitization of reduced XRCC1-expressing cells to HO compared with wild-type XRCC1-expressing cells. There is no indication of enhanced HO-generated free radicals or DNA strand breaks in XRE8 cells. However, elevated cellular PAR is found following HO exposure, suggesting BER deficiency of HO-induced damage in the C12A expressing cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.freeradbiomed.2017.02.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5457714PMC
June 2017

DNA polymerase β contains a functional nuclear localization signal at its N-terminus.

Nucleic Acids Res 2017 02;45(4):1958-1970

National Institute of Environmental Health Sciences, Genome Integrity and Structural Biology Laboratory, National Institutes of Health, Research Triangle Park, NC 27709, USA.

DNA polymerase β (pol β) requires nuclear localization to fulfil its DNA repair function. Although its small size has been interpreted to imply the absence of a need for active nuclear import, sequence and structural analysis suggests that a monopartite nuclear localization signal (NLS) may reside in the N-terminal lyase domain. Binding of this domain to Importin α1 (Impα1) was confirmed by gel filtration and NMR studies. Affinity was quantified by fluorescence polarization analysis of a fluorescein-tagged peptide corresponding to pol β residues 2-13. These studies indicate high affinity binding, characterized by a low micromolar Kd, that is selective for the murine Importin α1 (mImpα1) minor site, with the Kd strengthening to ∼140 nM for the full lyase domain (residues 2-87). A further reduction in Kd obtains in binding studies with human Importin α5 (hImpα5), which in some cases has been demonstrated to bind small domains connected to the NLS. The role of this NLS was confirmed by fluorescent imaging of wild-type and NLS-mutated pol β(R4S,K5S) in mouse embryonic fibroblasts lacking endogenous pol β. Together these data demonstrate that pol β contains a specific NLS sequence in the N-terminal lyase domain that promotes transport of the protein independent of its interaction partners. Active nuclear uptake allows development of a nuclear/cytosolic concentration gradient against a background of passive diffusion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkw1257DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5389473PMC
February 2017

Altered pattern of immunoglobulin hypermutation in mice deficient in Slip-GC protein.

J Biol Chem 2012 Sep 25;287(38):31856-65. Epub 2012 Jul 25.

Somatic Hypermutation Group, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.

We recently identified a novel germinal center GTPase, SLIP-GC, that localizes to replication factories in B cells and that, when reduced, induces DNA breaks in lymphoma B cell lines in an activation-induced deaminase (AID)-dependent manner. Herein, we generated mice deficient in SLIP-GC and examined the impact of SLIP-GC deficiency in immunoglobulin hypermutation and class switch recombination, both AID-dependent mechanisms. SLIP-GC-deficient mice experienced a substantial increase in mutations at G:C base pairs at the region downstream of JH4 in the immunoglobulin heavy chain locus. This change was reflected in the overall mutation frequency, and it was associated with an increase in transitions from G:C base pairs, a hallmark of AID-mediated deamination during replication. In addition, G:C transitions at non-immunoglobulin loci also increased in these mice. Given the intracellular localization of SLIP-GC to sites of replicating DNA, these results suggest that SLIP-GC protects replicating DNA from AID-mediated deamination of cytosines in both strands.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M112.340661DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3442519PMC
September 2012

Activation-induced deaminase contributes to the antibody-independent role of B cells in the development of autoimmunity.

Autoimmunity 2012 Sep 25;45(6):440-8. Epub 2012 Jun 25.

Somatic Hypermutation Group, Laboratory of Molecular Genetics, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.

B cells contribute to autoimmunity both as secretors of pathogenic antibodies and through the activation of autoreactive T cells. B cells and antibodies acquire higher affinity to self-antigen through a process known as immunoglobulin hypermutation or SHM. The contribution of SHM to pathogenic antibody development in lupus has been established in various autoimmune mouse models and by examining antibodies from patients. However, its role in the antibody-independent contribution of B cells to autoimmunity has not been examined. Herein, we generate lupus-prone MRL/lpr mice with a limited IgM-only B cell repertoire, no secreted antibodies and no SHM. This enabled us to isolate the role of somatic hypermutation in B cell-mediated autoimmunity. We found that SHM-deficiency correlated with a reduction in autoreactive B cells, a decrease in T cell activation and a decrease in kidney lymphocytic infiltration. These data establish AID as an important contributor to the antibody-independent role of B cells in autoimmunity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3109/08916934.2012.682668DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3496248PMC
September 2012

Altered Ig hypermutation pattern and frequency in complementary mouse models of DNA polymerase ζ activity.

J Immunol 2012 Jun 30;188(11):5528-37. Epub 2012 Apr 30.

Somatic Hypermutation Group, Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, NC 27709, USA.

To test the hypothesis that DNA polymerase ζ participates in Ig hypermutation, we generated two mouse models of Pol ζ function: a B cell-specific conditional knockout and a knock-in strain with a Pol ζ mutagenesis-enhancing mutation. Pol ζ-deficient B cells had a reduction in mutation frequency at Ig loci in the spleen and in Peyer's patches, whereas knock-in mice with a mutagenic Pol ζ displayed a marked increase in mutation frequency in Peyer's patches, revealing a pattern that was similar to mutations in yeast strains with a homologous mutation in the gene encoding the catalytic subunit of Pol ζ. Combined, these data are best explained by a direct role for DNA polymerase ζ in Ig hypermutation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4049/jimmunol.1102629DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3358437PMC
June 2012

Activation-induced deaminase-deficient MRL/lpr mice secrete high levels of protective antibodies against lupus nephritis.

Arthritis Rheum 2011 Apr;63(4):1086-96

National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA.

Objective: We previously generated MRL/lpr mice deficient in activation-induced deaminase (AID) that lack isotype switching and immunoglobulin hypermutation. These mice have high levels of unmutated (germline) autoreactive IgM, yet they experienced an increase in survival and an improvement in lupus nephritis that exceeded that of MRL/lpr mice lacking IgG. The purpose of the present study was to test the hypothesis that high levels of germline autoreactive IgM in these mice confer protection against lupus nephritis.

Methods: Autoreactive IgM antibodies of various specificities, including antibodies against double-stranded DNA (dsDNA), from AID-deficient MRL/lpr mice were given to asymptomatic MRL/lpr mice, and the levels of cytokines, proteinuria, immune complex deposition in the kidneys, and glomerulonephritis were examined. Novel AID-deficient MRL/lpr mice that lack any antibodies were generated for comparison to AID-deficient MRL/lpr mice that secrete only IgM.

Results: Treatment with IgM anti-dsDNA resulted in a dramatic improvement in lupus nephritis. Other autoreactive IgM antibodies, such as antiphospholipid and anti-Sm, did not alter the pathologic changes. Secretion of proinflammatory cytokines by macrophages and the levels of inflammatory cells and apoptotic debris in the kidneys were lower in mice receiving IgM anti-dsDNA. Protective IgM derived from AID-deficient MRL/lpr mice displayed a distinct B cell repertoire, with a bias toward members of the V(H) 7183 family.

Conclusion: IgM anti-dsDNA protected MRL/lpr mice from lupus nephritis, likely by stopping the inflammatory cascade leading to kidney damage. A distinct repertoire of V(H) usage in IgM anti-dsDNA hybridomas from AID-deficient mice suggests that there is enrichment of a dedicated B cell population that secretes unmutated protective IgM in these mice.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/art.30230DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3079514PMC
April 2011

Activation-induced deaminase heterozygous MRL/lpr mice are delayed in the production of high-affinity pathogenic antibodies and in the development of lupus nephritis.

Immunology 2009 Jan 20;126(1):102-13. Epub 2008 Jun 20.

Laboratory of Molecular Genetics, D3-01 National Institute of Environmental Health Sciences/National Institutes of Health, Research Triangle Park, NC 27709, USA.

We previously reported that activation-induced deaminase (AID) heterozygous MRL/lpr mice have substantially lower levels of serum anti-dsDNA autoantibodies than AID wild-type littermates. Given the known functions of AID, here we examined whether this decrease in pathogenic autoantibodies in the heterozygotes was the result of a defect in class switch recombination, somatic hypermutation, or both. We report significant impairment of switch recombination to most isotypes except immunoglobulin G3 (IgG3) in vitro. However, serum levels of IgG were similar to AID wild-type levels even in very young mice. Mutation accumulation in the B cells from Peyer's patches also revealed reduced somatic hypermutation in the heterozygotes. Unlike the switch defect, the hypermutation defect probably resulted in an in vivo effect because the serum IgG antibodies from the heterozygotes were of strikingly lower affinity to dsDNA than serum IgG antibodies from wild-type littermates. This suggests that the somatic hypermutation defect resulted in impaired affinity maturation of autoantibodies in these mice and explains the low levels of specific anti-dsDNA antibodies in the heterozygotes. This correlated with a delay in the development of kidney damage. These results imply that AID levels impact the class switch recombination and somatic hypermutation mechanisms and directly implicate affinity maturation of autoantibodies in autoimmunity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1365-2567.2008.02882.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2632700PMC
January 2009

Macrophages, but not T and B lymphocytes, are critical for subepidermal blister formation in experimental bullous pemphigoid: macrophage-mediated neutrophil infiltration depends on mast cell activation.

J Immunol 2002 Oct;169(7):3987-92

Department of Dermatology, University of North Carolina, Chapel Hill, NC 27599, USA.

Bullous pemphigoid (BP) is a subepidermal blistering disease associated with autoantibodies against two hemidesmosomal proteins, BP180 and BP230. Numerous inflammatory cells infiltrate the upper dermis in BP. We have previously shown by passive transfer studies that Abs to the ectodomain of murine BP180 are capable of triggering blisters in mice that closely mimic human BP. Experimental BP depends on complement activation and neutrophil infiltration. In the present study, we investigated the relative contribution of neutrophils, mast cells (MCs), macrophages (Mphi), and lymphocytes and their functional relationship in the immunopathogenesis of this disease model by using mice deficient in these cells. Wild-type, T cell-deficient, and T and B cell-deficient mice injected intradermally with pathogenic anti-murine BP180 IgG exhibited extensive subepidermal blisters. In contrast, mice deficient in neutrophils, MCs, and Mphi were resistant to experimental BP. MCs play a major role in neutrophil recruitment into the dermis. Furthermore, Mphi-mediated neutrophil infiltration depends on MC activation/degranulation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4049/jimmunol.169.7.3987DOI Listing
October 2002
-->