Extracellular Mitochondrial DNA and Oxidatively Damaged DNA
Extracellular Mitochondrial DNA and Oxidatively Damaged DNA
We investigated whether plasma and synovial fluid (SF) samples from patients with rheumatoid arthritis (RA) contained extracellular mitochondrial DNA (mtDNA) or the oxidatively damaged DNA adduct 8-hydroxy-2'-deoxyguanosine (8-oxodG). Moreover, we correlated the laboratory findings of the patients with RA with their levels of mtDNA and 8-oxodG. SF and plasma samples from 54 patients with RA, SF from 30 non-arthritic control subjects, and plasma from 22 healthy volunteers were collected. The samples were subjected to polymerase chain reaction (PCR) using mitochondrial genomic primers, and the products were analyzed by SDS-polyacrylamide-gel electrophoresis. The intensities of the PCR-amplified bands were quantified and normalized to a reference sample. Furthermore, the SF samples were assayed by enzyme-linked immunosorbent assay for 8-oxodG. Extracellular PCR-amplifiable mtDNA was detected in the SF of 38 of 54 (70%) patients with RA, but not in any of the SF controls. PCR-amplifiable mtDNA was detected in the plasma of 30 of 54 (56%) of patients with RA and in 6 of 22 (27%) of the healthy volunteers. The levels of mtDNA in the plasma and SF samples of patients with RA were significantly higher (P < 0.0001) than in the respective control samples. The presence of both mtDNA and 8-oxodG in SF was significantly correlated with the presence of rheumatoid factor in the patients with RA. Extracellular mtDNA and oxidized DNA were detected in the SF of the great majority of patients with RA, but were absent or present at low levels in the control SF. These findings indicate that endogenous nucleic acid compounds might participate in joint inflammation by activating immune cells in the joints to produce proinflammatory cytokines.
The mitochondria of human cells contain supercoiled double-stranded circular genomes that comprise 16.5 kilobases of mitochondrial DNA (mtDNA). mtDNA is similar to bacterial DNA and distinct from eukaryotic nuclear DNA (nDNA) in that it is not protected by histones and contains CpG motifs in which the cytosine is unmethylated. Bacterial DNA that contains CpG motifs has been shown to have powerful immunostimulatory effects , and we have shown that bacterial DNA containing unmethylated CpG motifs induces arthritis . Reactive oxygen species (ROS), such as hydroxyl radicals, superoxide radicals, and hydrogen peroxide, are generated from aerobic metabolism , mitochondrial oxidative phosphorylation , and from other sources, such as neutrophils and macrophages . mtDNA is more susceptible to ROS-induced damage than nDNA because the mitochondrion lacks an effective system for repairing oxidative lesions. Unrepaired damage to mtDNA leading to mutations and energetic dysfunction has been linked to several debilitating diseases, such as diabetes mellitus, neoplasias, and Parkinson's and Alzheimer's diseases.
When injected intra-articularly in mice, purified mtDNA induced arthritis whereas nDNA from the same cells did not (LV Collins, S Hajizadeh, E Holme, I-M Jonsson, A Tarkowski, unpublished work). By the same token, oxidatively damaged DNA, irrespective of its origin, has the capacity to induce arthritis (LV Collins, S Hajizadeh, E Holme, I-M Jonsson, A Tarkowski, unpublished work). The release of damaged nuclear and mtDNA from cells during tissue damage might therefore have an initiating or potentiating role in inflammation. We examined whether immunostimulatory mtDNA was involved in human arthritis by assaying the levels of extracellular mtDNA in plasma and synovial fluid (SF) samples from patients with rheumatoid arthritis (RA) and from control subjects using amplification by polymerase chain reaction (PCR) with mitochondrial genomic primers. We also investigated whether the levels of 8-hydroxy-2'-deoxyguanosine (8-oxodG), which is a marker for oxidative DNA damage, differed in the joints of patients with RA and control subjects. Our results indicate highly significant differences in the SF levels of both 8-oxodG and mtDNA between patients with RA and healthy subjects, which suggests that these two DNA species might participate in joint inflammation.
We investigated whether plasma and synovial fluid (SF) samples from patients with rheumatoid arthritis (RA) contained extracellular mitochondrial DNA (mtDNA) or the oxidatively damaged DNA adduct 8-hydroxy-2'-deoxyguanosine (8-oxodG). Moreover, we correlated the laboratory findings of the patients with RA with their levels of mtDNA and 8-oxodG. SF and plasma samples from 54 patients with RA, SF from 30 non-arthritic control subjects, and plasma from 22 healthy volunteers were collected. The samples were subjected to polymerase chain reaction (PCR) using mitochondrial genomic primers, and the products were analyzed by SDS-polyacrylamide-gel electrophoresis. The intensities of the PCR-amplified bands were quantified and normalized to a reference sample. Furthermore, the SF samples were assayed by enzyme-linked immunosorbent assay for 8-oxodG. Extracellular PCR-amplifiable mtDNA was detected in the SF of 38 of 54 (70%) patients with RA, but not in any of the SF controls. PCR-amplifiable mtDNA was detected in the plasma of 30 of 54 (56%) of patients with RA and in 6 of 22 (27%) of the healthy volunteers. The levels of mtDNA in the plasma and SF samples of patients with RA were significantly higher (P < 0.0001) than in the respective control samples. The presence of both mtDNA and 8-oxodG in SF was significantly correlated with the presence of rheumatoid factor in the patients with RA. Extracellular mtDNA and oxidized DNA were detected in the SF of the great majority of patients with RA, but were absent or present at low levels in the control SF. These findings indicate that endogenous nucleic acid compounds might participate in joint inflammation by activating immune cells in the joints to produce proinflammatory cytokines.
The mitochondria of human cells contain supercoiled double-stranded circular genomes that comprise 16.5 kilobases of mitochondrial DNA (mtDNA). mtDNA is similar to bacterial DNA and distinct from eukaryotic nuclear DNA (nDNA) in that it is not protected by histones and contains CpG motifs in which the cytosine is unmethylated. Bacterial DNA that contains CpG motifs has been shown to have powerful immunostimulatory effects , and we have shown that bacterial DNA containing unmethylated CpG motifs induces arthritis . Reactive oxygen species (ROS), such as hydroxyl radicals, superoxide radicals, and hydrogen peroxide, are generated from aerobic metabolism , mitochondrial oxidative phosphorylation , and from other sources, such as neutrophils and macrophages . mtDNA is more susceptible to ROS-induced damage than nDNA because the mitochondrion lacks an effective system for repairing oxidative lesions. Unrepaired damage to mtDNA leading to mutations and energetic dysfunction has been linked to several debilitating diseases, such as diabetes mellitus, neoplasias, and Parkinson's and Alzheimer's diseases.
When injected intra-articularly in mice, purified mtDNA induced arthritis whereas nDNA from the same cells did not (LV Collins, S Hajizadeh, E Holme, I-M Jonsson, A Tarkowski, unpublished work). By the same token, oxidatively damaged DNA, irrespective of its origin, has the capacity to induce arthritis (LV Collins, S Hajizadeh, E Holme, I-M Jonsson, A Tarkowski, unpublished work). The release of damaged nuclear and mtDNA from cells during tissue damage might therefore have an initiating or potentiating role in inflammation. We examined whether immunostimulatory mtDNA was involved in human arthritis by assaying the levels of extracellular mtDNA in plasma and synovial fluid (SF) samples from patients with rheumatoid arthritis (RA) and from control subjects using amplification by polymerase chain reaction (PCR) with mitochondrial genomic primers. We also investigated whether the levels of 8-hydroxy-2'-deoxyguanosine (8-oxodG), which is a marker for oxidative DNA damage, differed in the joints of patients with RA and control subjects. Our results indicate highly significant differences in the SF levels of both 8-oxodG and mtDNA between patients with RA and healthy subjects, which suggests that these two DNA species might participate in joint inflammation.
