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CpG oligodeoxynucleotide stimulates production of anti-neutrophil cytoplasmic antibodies in ANCA associated vasculitis.

Hurtado PR, Jeffs L, Nitschke J, Patel M, Sarvestani G, Cassidy J, Hissaria P, Gillis D, Peh CA - BMC Immunol. (2008)

Bottom Line: The stimulation of PBMC with CpG oligonucleutides resulted in the production of similar amounts of IgG in both ANCA+ patients and normal controls.Of note, CpG also induced the production of the relevant autoantibodies in patients with other types of autoimmune diseases.This phenomenon may also apply to other antibody mediated autoimmune diseases.

View Article: PubMed Central - HTML - PubMed

Affiliation: Renal Unit, Royal Adelaide Hospital, North Terrace, Adelaide, 5000, Australia. plinio.hurtado@imvs.sa.gov.au

ABSTRACT

Background: Wegener's Granulomatosis and Microscopic Polyangiitis are life-threatening systemic necrotizing vasculitides of unknown aetiology. The appearance of circulating antibodies to neutrophil cytoplasmic antigens (ANCA) is strongly associated with the development of the disease. A link between infection and disease has long been suspected, and the appearance of ANCA antibodies has been reported following bacterial and viral infections. The depletion of circulating B cells with monoclonal antibody therapy can induce remission, and this observation suggests a pathogenic role for B cells in this disease. As bacterial DNA is known to induce B cell proliferation and antibody production via TLR-9 stimulation, we have explored the possibility that unmethylated CpG oligodeoxynucleotide, as found in bacterial and viral DNA, may play a role in stimulating circulating autoreactive B cells to produce ANCA in patients with vasculitis.

Results: We have confirmed that unmethylated CpG oligonucleotide is a potent stimulator of antibody production by PBMC in vitro. The stimulation of PBMC with CpG oligonucleutides resulted in the production of similar amounts of IgG in both ANCA+ patients and normal controls. In spite of this, PR3 ANCA+ patients synthesised significantly higher amount of IgG ANCA than normal controls. In MPO ANCA+ patients, there was a tendency for patients to produce higher amount of ANCA than controls, however, the difference did not reach significance. Furthermore, we were able to detect circulating MPO-reactive B cells by ELISpot assay from the peripheral blood of 2 MPO+ ANCA vasculitis patients. Together, this indicates that circulating anti-neutrophil autoreactive B cells are present in ANCA+ vasculitis patients, and they are capable of producing antibodies in response to CpG stimulation. Of note, CpG also induced the production of the relevant autoantibodies in patients with other types of autoimmune diseases.

Conclusion: Circulating ANCA autoreactive B cells are present in patients with ANCA+ vasculitis. The production of ANCA from these cells in response to unmethylated CpG stimulation lead us to propose that stimulation of these cells by immunostimulatory DNA sequences such as CpG oligodeoxynucleotide during infection may provide a link between infection and ANCA associated vasculitis. This phenomenon may also apply to other antibody mediated autoimmune diseases.

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CpG-B induces ANCA production from vasculitis patients in vitro. PBMCs isolated from patients with active ANCA associated vasculitis, 5 PR3+ and 5 MPO+ ANCA patients, were cultured with CpG-B and IL-2. Each patient assay was paired with a healthy control. After 12 days of culture, supernatants were harvested. IgG concentration and supernatant reactivity to either PR3 or MPO was measured by ELISA. The amount of IgG detected in the supernatants was of 5.5 ± 2.2 μg mL-1 in the patients compared to 4.1 ± 1.2 μg mL-1 in the control group (A). Figure B shows the reactivity of the supernatants from PR3+ ANCA patients towards PR3 antigen. The difference against control individuals was highly significant (P = 0.0082). Figure C shows the reactivity of the supernatants from MPO+ ANCA patients towards MPO antigen. The difference was not significant (P = 0.072) although their supernatants showed a clear tendency towards higher reactivity compared to controls. There was no correlation between patients' serum ANCA titre at the time of the assay and their in vitro production of ANCA in response to CpG-B as shown in D (r2 = 0.172).
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Figure 1: CpG-B induces ANCA production from vasculitis patients in vitro. PBMCs isolated from patients with active ANCA associated vasculitis, 5 PR3+ and 5 MPO+ ANCA patients, were cultured with CpG-B and IL-2. Each patient assay was paired with a healthy control. After 12 days of culture, supernatants were harvested. IgG concentration and supernatant reactivity to either PR3 or MPO was measured by ELISA. The amount of IgG detected in the supernatants was of 5.5 ± 2.2 μg mL-1 in the patients compared to 4.1 ± 1.2 μg mL-1 in the control group (A). Figure B shows the reactivity of the supernatants from PR3+ ANCA patients towards PR3 antigen. The difference against control individuals was highly significant (P = 0.0082). Figure C shows the reactivity of the supernatants from MPO+ ANCA patients towards MPO antigen. The difference was not significant (P = 0.072) although their supernatants showed a clear tendency towards higher reactivity compared to controls. There was no correlation between patients' serum ANCA titre at the time of the assay and their in vitro production of ANCA in response to CpG-B as shown in D (r2 = 0.172).

Mentions: PBMCs were isolated from 10 patients with biopsy-proven ANCA+ vasculitis, each paired to a normal control individual, and cultured in the presence of CpG-B and IL-2. Patient clinical details are shown in Table 1. Notably, all but one of these patients were not receiving immunosuppressive medications at the time of assay. Cell culture supernatants were harvested after 12 days of culture, and concentration of IgG measured by ELISA as well as their reactivity against PR3 or MPO (Fig. 1). The supernatants from PR3+ and MPO+ ANCA patients were tested for reactivity against PR3 or MPO antigen respectively. We found that CpG B at 3.2 mg μg mL-1 to be a strong stimulant of IgG production in vitro. The amount of IgG detected in the supernatants were similar in both patient and control groups, with a concentration of 5.5 ± 2.2 and 4.1 ± 1.2 μg mL-1 respectively (Fig. 1A). The reactivity against PR3 and MPO antigens in the patients' supernatants were consistently greater than those observed in normal controls. In PR3+ ANCA patients, this difference achieved significant levels (P = 0.0082) (Fig. 1B). In MPO+ patients the difference was not significant (P = 0.072) although their supernatants showed a clear tendency towards higher reactivity (Fig. 1C). There was no correlation between serum ANCA titre and the amount of ANCA produced in vitro (r2 = 0.172) (Fig. 1D). In addition to CpG-B, PBMCs from 5 different pairs were also stimulated with LPS, pokeweed mitogen (PWM) or inactivated staphylococcus aureus (Fig. 2). The production of IgG under these conditions was significantly lower compared to CpG-B and we were not able to detect ANCA in these conditions. The inactivated staphylococcus aureus used in these experiments had been formalin fixed. In this form, they are unlikely to provide freely available unmethylated CpG, even though they should contain unmethylated CpG within.


CpG oligodeoxynucleotide stimulates production of anti-neutrophil cytoplasmic antibodies in ANCA associated vasculitis.

Hurtado PR, Jeffs L, Nitschke J, Patel M, Sarvestani G, Cassidy J, Hissaria P, Gillis D, Peh CA - BMC Immunol. (2008)

CpG-B induces ANCA production from vasculitis patients in vitro. PBMCs isolated from patients with active ANCA associated vasculitis, 5 PR3+ and 5 MPO+ ANCA patients, were cultured with CpG-B and IL-2. Each patient assay was paired with a healthy control. After 12 days of culture, supernatants were harvested. IgG concentration and supernatant reactivity to either PR3 or MPO was measured by ELISA. The amount of IgG detected in the supernatants was of 5.5 ± 2.2 μg mL-1 in the patients compared to 4.1 ± 1.2 μg mL-1 in the control group (A). Figure B shows the reactivity of the supernatants from PR3+ ANCA patients towards PR3 antigen. The difference against control individuals was highly significant (P = 0.0082). Figure C shows the reactivity of the supernatants from MPO+ ANCA patients towards MPO antigen. The difference was not significant (P = 0.072) although their supernatants showed a clear tendency towards higher reactivity compared to controls. There was no correlation between patients' serum ANCA titre at the time of the assay and their in vitro production of ANCA in response to CpG-B as shown in D (r2 = 0.172).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 1: CpG-B induces ANCA production from vasculitis patients in vitro. PBMCs isolated from patients with active ANCA associated vasculitis, 5 PR3+ and 5 MPO+ ANCA patients, were cultured with CpG-B and IL-2. Each patient assay was paired with a healthy control. After 12 days of culture, supernatants were harvested. IgG concentration and supernatant reactivity to either PR3 or MPO was measured by ELISA. The amount of IgG detected in the supernatants was of 5.5 ± 2.2 μg mL-1 in the patients compared to 4.1 ± 1.2 μg mL-1 in the control group (A). Figure B shows the reactivity of the supernatants from PR3+ ANCA patients towards PR3 antigen. The difference against control individuals was highly significant (P = 0.0082). Figure C shows the reactivity of the supernatants from MPO+ ANCA patients towards MPO antigen. The difference was not significant (P = 0.072) although their supernatants showed a clear tendency towards higher reactivity compared to controls. There was no correlation between patients' serum ANCA titre at the time of the assay and their in vitro production of ANCA in response to CpG-B as shown in D (r2 = 0.172).
Mentions: PBMCs were isolated from 10 patients with biopsy-proven ANCA+ vasculitis, each paired to a normal control individual, and cultured in the presence of CpG-B and IL-2. Patient clinical details are shown in Table 1. Notably, all but one of these patients were not receiving immunosuppressive medications at the time of assay. Cell culture supernatants were harvested after 12 days of culture, and concentration of IgG measured by ELISA as well as their reactivity against PR3 or MPO (Fig. 1). The supernatants from PR3+ and MPO+ ANCA patients were tested for reactivity against PR3 or MPO antigen respectively. We found that CpG B at 3.2 mg μg mL-1 to be a strong stimulant of IgG production in vitro. The amount of IgG detected in the supernatants were similar in both patient and control groups, with a concentration of 5.5 ± 2.2 and 4.1 ± 1.2 μg mL-1 respectively (Fig. 1A). The reactivity against PR3 and MPO antigens in the patients' supernatants were consistently greater than those observed in normal controls. In PR3+ ANCA patients, this difference achieved significant levels (P = 0.0082) (Fig. 1B). In MPO+ patients the difference was not significant (P = 0.072) although their supernatants showed a clear tendency towards higher reactivity (Fig. 1C). There was no correlation between serum ANCA titre and the amount of ANCA produced in vitro (r2 = 0.172) (Fig. 1D). In addition to CpG-B, PBMCs from 5 different pairs were also stimulated with LPS, pokeweed mitogen (PWM) or inactivated staphylococcus aureus (Fig. 2). The production of IgG under these conditions was significantly lower compared to CpG-B and we were not able to detect ANCA in these conditions. The inactivated staphylococcus aureus used in these experiments had been formalin fixed. In this form, they are unlikely to provide freely available unmethylated CpG, even though they should contain unmethylated CpG within.

Bottom Line: The stimulation of PBMC with CpG oligonucleutides resulted in the production of similar amounts of IgG in both ANCA+ patients and normal controls.Of note, CpG also induced the production of the relevant autoantibodies in patients with other types of autoimmune diseases.This phenomenon may also apply to other antibody mediated autoimmune diseases.

View Article: PubMed Central - HTML - PubMed

Affiliation: Renal Unit, Royal Adelaide Hospital, North Terrace, Adelaide, 5000, Australia. plinio.hurtado@imvs.sa.gov.au

ABSTRACT

Background: Wegener's Granulomatosis and Microscopic Polyangiitis are life-threatening systemic necrotizing vasculitides of unknown aetiology. The appearance of circulating antibodies to neutrophil cytoplasmic antigens (ANCA) is strongly associated with the development of the disease. A link between infection and disease has long been suspected, and the appearance of ANCA antibodies has been reported following bacterial and viral infections. The depletion of circulating B cells with monoclonal antibody therapy can induce remission, and this observation suggests a pathogenic role for B cells in this disease. As bacterial DNA is known to induce B cell proliferation and antibody production via TLR-9 stimulation, we have explored the possibility that unmethylated CpG oligodeoxynucleotide, as found in bacterial and viral DNA, may play a role in stimulating circulating autoreactive B cells to produce ANCA in patients with vasculitis.

Results: We have confirmed that unmethylated CpG oligonucleotide is a potent stimulator of antibody production by PBMC in vitro. The stimulation of PBMC with CpG oligonucleutides resulted in the production of similar amounts of IgG in both ANCA+ patients and normal controls. In spite of this, PR3 ANCA+ patients synthesised significantly higher amount of IgG ANCA than normal controls. In MPO ANCA+ patients, there was a tendency for patients to produce higher amount of ANCA than controls, however, the difference did not reach significance. Furthermore, we were able to detect circulating MPO-reactive B cells by ELISpot assay from the peripheral blood of 2 MPO+ ANCA vasculitis patients. Together, this indicates that circulating anti-neutrophil autoreactive B cells are present in ANCA+ vasculitis patients, and they are capable of producing antibodies in response to CpG stimulation. Of note, CpG also induced the production of the relevant autoantibodies in patients with other types of autoimmune diseases.

Conclusion: Circulating ANCA autoreactive B cells are present in patients with ANCA+ vasculitis. The production of ANCA from these cells in response to unmethylated CpG stimulation lead us to propose that stimulation of these cells by immunostimulatory DNA sequences such as CpG oligodeoxynucleotide during infection may provide a link between infection and ANCA associated vasculitis. This phenomenon may also apply to other antibody mediated autoimmune diseases.

Show MeSH
Related in: MedlinePlus