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The role of genetic variation near interferon-kappa in systemic lupus erythematosus.

Harley IT, Niewold TB, Stormont RM, Kaufman KM, Glenn SB, Franek BS, Kelly JA, Kilpatrick JR, Hutchings D, Divers J, Bruner GR, Edberg JC, McGwin G, Petri MA, Ramsey-Goldman R, Reveille JD, Vilá-Pérez LM, Merrill JT, Gilkeson GS, Vyse TJ, Alarcón-Riquelme ME, Cho SK, Jacob CO, Alarcón GS, Moser KL, Gaffney PM, Kimberly RP, Bae SC, Langefeld CD, Harley JB, Guthridge JM, James JA - J. Biomed. Biotechnol. (2010)

Bottom Line: Suggestive associations with skin phenotypes in EA and AA females were found, and these were also sex-specific.IFNK SNPs were associated with increased serum type I IFN in EA and AA SLE patients.The serum IFN association suggests that IFNK variants could influence type I IFN producing plasmacytoid dendritic cells in affected skin.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Immunology and Graduate Program in Immunobiology, Cincinnati Children's Hospital Research Foundation, OH 45267, USA.

ABSTRACT
Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by increased type I interferons (IFNs) and multiorgan inflammation frequently targeting the skin. IFN-kappa is a type I IFN expressed in skin. A pooled genome-wide scan implicated the IFNK locus in SLE susceptibility. We studied IFNK single nucleotide polymorphisms (SNPs) in 3982 SLE cases and 4275 controls, composed of European (EA), African-American (AA), and Asian ancestry. rs12553951C was associated with SLE in EA males (odds ratio = 1.93, P = 2.5 x 10(-4)), but not females. Suggestive associations with skin phenotypes in EA and AA females were found, and these were also sex-specific. IFNK SNPs were associated with increased serum type I IFN in EA and AA SLE patients. Our data suggest a sex-dependent association between IFNK SNPs and SLE and skin phenotypes. The serum IFN association suggests that IFNK variants could influence type I IFN producing plasmacytoid dendritic cells in affected skin.

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Related in: MedlinePlus

Stimulation of WISH reporter cells with IFN-α, IFN-κ, and SLE sera with and without anti-IFN-κ antibodies. WISH cells were assayed for type I IFN-induced gene expression as described in the methods, using the stimuli indicated on the graph. Anti-IFN-κ antibody was used at a concentration of 10 μg/mL, and anti-IFN-κ treated sera were preincubated with the antibody for 30 minutes before the sera was applied to the WISH cells. Y-axis shows the IFN-induced gene expression score calculated as described in the Methods.
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fig2: Stimulation of WISH reporter cells with IFN-α, IFN-κ, and SLE sera with and without anti-IFN-κ antibodies. WISH cells were assayed for type I IFN-induced gene expression as described in the methods, using the stimuli indicated on the graph. Anti-IFN-κ antibody was used at a concentration of 10 μg/mL, and anti-IFN-κ treated sera were preincubated with the antibody for 30 minutes before the sera was applied to the WISH cells. Y-axis shows the IFN-induced gene expression score calculated as described in the Methods.

Mentions: Serum type I IFN activity data were available for 230 EA and 158 AA SLE patients in this study. The reporter cell assay for type I IFN has been described in detail elsewhere in [9, 26]. Reporter cells were used to measure the ability of patient sera to cause IFN-induced gene expression. The reporter cells (WISH cells, ATCC number CCL-25) were cultured with 50% patient sera for 6 hours and then lysed. mRNA was purified from cell lysates, and cDNA was made from total cellular mRNA. cDNA was then quantified using real-time PCR using an Applied Biosystems 7900HT PCR machine with the SYBR Green fluorophore system. Forward and reverse primers for the genes MX1, PKR, and IFIT1, which are known to be highly and specifically induced by IFN-α, were used in the reaction [9]. GAPDH was amplified in the same samples to control for background gene expression. The amount of PCR product of the IFN-induced gene was normalized to the amount of product for the housekeeping gene GAPDH in the same sample. The relative expression of each of the three tested IFN-induced genes was calculated as a fold increase compared to its expression in WISH cells cultured with media alone. Results from the IFN assay were standardized to a healthy multiancestral reference population as previously described, and a serum IFN activity score was calculated based upon the mean and SD of the reference population [9]. This assay could theoretically measure all type I IFNs present in the sample being studied. In SLE sera, type I IFN activity is generally completely blocked by the addition of anti-IFN-α antibodies [9, 26]. Thus, the activity observed in the assay is frequently referred to as serum IFN-α activity. With regard to potential cross-reactivity with the type III IFNs, preincubation of SLE sera with anti-IFN λ1 antibodies did not diminish the capacity of those sera to cause IFN-induced gene expression in the reporter assay [26]. Given that IFN-κ is a type I IFN, we clarified the issue of whether our reporter assay was detecting circulating IFN-κ in lupus sera. Similar to published reports [17], we find that recombinant IFN-κ is capable of inducing some type I IFN-induced gene expression in our reporter assay although this is much less than that induced by the same amount of IFN-α (Figure 2). When lupus sera with high type I IFN activity are preincubated with anti-IFN-κ antibody, there is no decrease in the observed type I IFN activity, supporting the idea that IFN-κ does not contribute to circulating type I IFN activity in SLE sera (Figure 2).


The role of genetic variation near interferon-kappa in systemic lupus erythematosus.

Harley IT, Niewold TB, Stormont RM, Kaufman KM, Glenn SB, Franek BS, Kelly JA, Kilpatrick JR, Hutchings D, Divers J, Bruner GR, Edberg JC, McGwin G, Petri MA, Ramsey-Goldman R, Reveille JD, Vilá-Pérez LM, Merrill JT, Gilkeson GS, Vyse TJ, Alarcón-Riquelme ME, Cho SK, Jacob CO, Alarcón GS, Moser KL, Gaffney PM, Kimberly RP, Bae SC, Langefeld CD, Harley JB, Guthridge JM, James JA - J. Biomed. Biotechnol. (2010)

Stimulation of WISH reporter cells with IFN-α, IFN-κ, and SLE sera with and without anti-IFN-κ antibodies. WISH cells were assayed for type I IFN-induced gene expression as described in the methods, using the stimuli indicated on the graph. Anti-IFN-κ antibody was used at a concentration of 10 μg/mL, and anti-IFN-κ treated sera were preincubated with the antibody for 30 minutes before the sera was applied to the WISH cells. Y-axis shows the IFN-induced gene expression score calculated as described in the Methods.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2914299&req=5

fig2: Stimulation of WISH reporter cells with IFN-α, IFN-κ, and SLE sera with and without anti-IFN-κ antibodies. WISH cells were assayed for type I IFN-induced gene expression as described in the methods, using the stimuli indicated on the graph. Anti-IFN-κ antibody was used at a concentration of 10 μg/mL, and anti-IFN-κ treated sera were preincubated with the antibody for 30 minutes before the sera was applied to the WISH cells. Y-axis shows the IFN-induced gene expression score calculated as described in the Methods.
Mentions: Serum type I IFN activity data were available for 230 EA and 158 AA SLE patients in this study. The reporter cell assay for type I IFN has been described in detail elsewhere in [9, 26]. Reporter cells were used to measure the ability of patient sera to cause IFN-induced gene expression. The reporter cells (WISH cells, ATCC number CCL-25) were cultured with 50% patient sera for 6 hours and then lysed. mRNA was purified from cell lysates, and cDNA was made from total cellular mRNA. cDNA was then quantified using real-time PCR using an Applied Biosystems 7900HT PCR machine with the SYBR Green fluorophore system. Forward and reverse primers for the genes MX1, PKR, and IFIT1, which are known to be highly and specifically induced by IFN-α, were used in the reaction [9]. GAPDH was amplified in the same samples to control for background gene expression. The amount of PCR product of the IFN-induced gene was normalized to the amount of product for the housekeeping gene GAPDH in the same sample. The relative expression of each of the three tested IFN-induced genes was calculated as a fold increase compared to its expression in WISH cells cultured with media alone. Results from the IFN assay were standardized to a healthy multiancestral reference population as previously described, and a serum IFN activity score was calculated based upon the mean and SD of the reference population [9]. This assay could theoretically measure all type I IFNs present in the sample being studied. In SLE sera, type I IFN activity is generally completely blocked by the addition of anti-IFN-α antibodies [9, 26]. Thus, the activity observed in the assay is frequently referred to as serum IFN-α activity. With regard to potential cross-reactivity with the type III IFNs, preincubation of SLE sera with anti-IFN λ1 antibodies did not diminish the capacity of those sera to cause IFN-induced gene expression in the reporter assay [26]. Given that IFN-κ is a type I IFN, we clarified the issue of whether our reporter assay was detecting circulating IFN-κ in lupus sera. Similar to published reports [17], we find that recombinant IFN-κ is capable of inducing some type I IFN-induced gene expression in our reporter assay although this is much less than that induced by the same amount of IFN-α (Figure 2). When lupus sera with high type I IFN activity are preincubated with anti-IFN-κ antibody, there is no decrease in the observed type I IFN activity, supporting the idea that IFN-κ does not contribute to circulating type I IFN activity in SLE sera (Figure 2).

Bottom Line: Suggestive associations with skin phenotypes in EA and AA females were found, and these were also sex-specific.IFNK SNPs were associated with increased serum type I IFN in EA and AA SLE patients.The serum IFN association suggests that IFNK variants could influence type I IFN producing plasmacytoid dendritic cells in affected skin.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Immunology and Graduate Program in Immunobiology, Cincinnati Children's Hospital Research Foundation, OH 45267, USA.

ABSTRACT
Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by increased type I interferons (IFNs) and multiorgan inflammation frequently targeting the skin. IFN-kappa is a type I IFN expressed in skin. A pooled genome-wide scan implicated the IFNK locus in SLE susceptibility. We studied IFNK single nucleotide polymorphisms (SNPs) in 3982 SLE cases and 4275 controls, composed of European (EA), African-American (AA), and Asian ancestry. rs12553951C was associated with SLE in EA males (odds ratio = 1.93, P = 2.5 x 10(-4)), but not females. Suggestive associations with skin phenotypes in EA and AA females were found, and these were also sex-specific. IFNK SNPs were associated with increased serum type I IFN in EA and AA SLE patients. Our data suggest a sex-dependent association between IFNK SNPs and SLE and skin phenotypes. The serum IFN association suggests that IFNK variants could influence type I IFN producing plasmacytoid dendritic cells in affected skin.

Show MeSH
Related in: MedlinePlus