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The granulocyte colony stimulating factor pathway regulates autoantibody production in a murine induced model of systemic lupus erythematosus.

Lantow M, Sivakumar R, Zeumer L, Wasserfall C, Zheng YY, Atkinson MA, Morel L - Arthritis Res. Ther. (2013)

Bottom Line: G-CSF binding by B6.Sle2c2 leukocytes was reduced as compared to B6, which was associated with a reduced expansion in response to in vivo G-CSF treatment.G-CSF in vivo treatment also failed to mobilize bone-marrow B6.Sle2c2 neutrophils as it did for B6 neutrophils.This result was corroborated by the increased anti-dsDNA IgG production in G-CSF-treated B6.TC mice, which also carry the Sle2c2 locus.

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ABSTRACT

Introduction: An NZB-derived genetic locus (Sle2c2) that suppresses autoantibody production in a mouse model of induced systemic lupus erythematosus contains a polymorphism in the gene encoding the G-CSF receptor. This study was designed to test the hypothesis that the Sle2c2 suppression is associated with an impaired G-CSF receptor function that can be overcome by exogenous G-CSF.

Methods: Leukocytes from B6.Sle2c2 and B6 congenic mice, which carry a different allele of the G-CSF receptor, were compared for their responses to G-CSF. Autoantibody production was induced with the chronic graft-versus-host-disease (cGVHD) model by adoptive transfer of B6.bm12 splenocytes. Different treatment regimens varying the amount and frequency of G-CSF (Neulasta®) or carrier control were tested on cGVHD outcomes. Autoantibody production, immune cell activation, and reactive oxygen species (ROS) production were compared between the two strains with the various treatments. In addition, the effect of G-CSF treatment was examined on the production autoantibodies in the B6.Sle1.Sle2.Sle3 (B6.TC) spontaneous model of lupus.

Results: B6.Sle2c2 and B6 leukocytes responded differently to G-CSF. G-CSF binding by B6.Sle2c2 leukocytes was reduced as compared to B6, which was associated with a reduced expansion in response to in vivo G-CSF treatment. G-CSF in vivo treatment also failed to mobilize bone-marrow B6.Sle2c2 neutrophils as it did for B6 neutrophils. In contrast, the expression of G-CSF responsive genes indicated a higher G-CSF receptor signaling in B6.Sle2c2 cells. G-CSF treatment restored the ability of B6.Sle2c2 mice to produce autoantibodies in a dose-dependent manner upon cGVHD induction, which correlated with restored CD4+ T cells activation, as well as dendritic cell and granulocyte expansion. Steady-state ROS production was higher in B6.Sle2c2 than in B6 mice. cGVHD induction resulted in a larger increase in ROS production in B6 than in B6.Sle2c2 mice, and this difference was eliminated with G-CSF treatment. Finally, a low dose G-CSF treatment accelerated the production of anti-dsDNA IgG in young B6.TC mice.

Conclusion: The different in vivo and in vitro responses of B6.Sle2c2 leukocytes are consistent with the mutation in the G-CSFR having functional consequences. The elimination of Sle2c2 suppression of autoantibody production by exogenous G-CSF indicates that Sle2c2 corresponds to a loss of function of G-CSF receptor. This result was corroborated by the increased anti-dsDNA IgG production in G-CSF-treated B6.TC mice, which also carry the Sle2c2 locus. Overall, these results suggest that the G-CSF pathway regulates the production of autoantibodies in murine models of lupus.

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Exogenous human granulocyte-colony stimulation factor (huG-CSF) restores autoantibody (autoAb) production in B6.Sle2c2 mice after induction of chronic graft vs host disease (cGVHD). (A) huG-CSF-treatment induced anti-dsDNA IgG in B6.Sle2c2 mice in a dose-dependent manner (left), but had little effect in B6 mice (right). Five mice per strain per treatment were used. (B) Anti-dsDNA and anti-chromatin IgG found at d21 in the combined 120 × 1 and 12 × 2 cohorts. (C) Representative ANA IgG staining of Hep-2 cells incubated with sera collected at 21 d in each of the four treatment groups in the 120 × 1 and 12 × 2 cohorts and average staining intensity of 10 to 20 randomly selected cells per sample. Means and standard error of the mean with the significance value from the t-test for comparison of treated mice and controls (dex) are shown for each strain for a given time point in A, and from Dunnett's multiple comparison test between groups as indicated in B and C (*P < 0.05; **P < 0.01; ***P < 0.001).
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Figure 4: Exogenous human granulocyte-colony stimulation factor (huG-CSF) restores autoantibody (autoAb) production in B6.Sle2c2 mice after induction of chronic graft vs host disease (cGVHD). (A) huG-CSF-treatment induced anti-dsDNA IgG in B6.Sle2c2 mice in a dose-dependent manner (left), but had little effect in B6 mice (right). Five mice per strain per treatment were used. (B) Anti-dsDNA and anti-chromatin IgG found at d21 in the combined 120 × 1 and 12 × 2 cohorts. (C) Representative ANA IgG staining of Hep-2 cells incubated with sera collected at 21 d in each of the four treatment groups in the 120 × 1 and 12 × 2 cohorts and average staining intensity of 10 to 20 randomly selected cells per sample. Means and standard error of the mean with the significance value from the t-test for comparison of treated mice and controls (dex) are shown for each strain for a given time point in A, and from Dunnett's multiple comparison test between groups as indicated in B and C (*P < 0.05; **P < 0.01; ***P < 0.001).

Mentions: If the S378N mutation in the G-CSFR is responsible for the cGVHD resistance in B6.Sle2c2 mice, we postulated that huG-CSF treatment should eliminate the difference in cGVHD response between B6 and B6.Sle2c2 mice. If resistance in B6.Sle2c2 mice is due to a G-CSFR loss of function, huG-CSF treatment should restore cGVHD response in B6.Sle2c2 mice. On the other hand, if resistance is due to a G-CSFR gain of function, cGVHD resistance would be expected in huG-CSF-treated B6 mice. Based on our time course analysis showing a maximal effect at d4 after injection that was eliminated at d7, we tested a combination of treatment protocols in which the dose (1.2, 12.0, or 120.0 ug) and the frequency (one-, two-, or three-weekly injections starting at cGVHD induction) varied. Two treatment protocols (120 × 1 and 12 × 2) raised the production of anti-dsDNA IgG in B6.Sle2c2 mice significantly above the dextrose-treated controls (Figure 4A left), and to a level similar to the B6 controls, especially in the 12 × 2 cohort. For the 120 × 1 cohort, a drop in the third week was observed, possibly because the pharmacological effect of the single huG-CSF treatment was diminishing. With these two treatment regimens combined, the amount of anti-dsDNA and anti-chromatin IgG at d21 was significantly higher in the B6.Sle2c2 mice treated with huG-SCF than in controls (Figure 4B). Moreover, the autoAbs produced by treated B6.Sle2c2 mice reached equivalent levels as in treated B6 mice. cGVHD induced autoAbs in B6 mice with strong Hep-2 staining that combined cytoplasmic and nuclear patterns (Figure 4C). The 12 × 2 treatment induced Hep-2-staining in B6.Sle2c2 mice similar in intensity and pattern as in B6 mice (Figure 4C). Similar results were obtained with the 120 × 1 treatment, although with a more variable intensity (data not shown). The 1.2 × 3 treatment had no effect, indicating that the dose was too low. The 12 × 3 treatment had an intermediate effect in B6.Sle2c2 mice, and interestingly was the only regimen that induced a significant decrease in the B6 anti-dsDNA IgG response (Figure 4A right). The other treatments showed a trend in lowering autoAbs produced by B6 mice, but the difference was not significant. Overall, these results demonstrate that exogenous G-CSF eliminates the resistance to cGVHD induction in B6.Sle2c2 mice in a dose-dependent manner, with no significant effect in B6 mice at the same dose. This suggests that an impaired G-CSFR response mediates the cGVHD resistance in the B6.Sle2c2 strain, which can be compensated by an excess G-CSF to drive equilibrium binding or signaling to the receptor.


The granulocyte colony stimulating factor pathway regulates autoantibody production in a murine induced model of systemic lupus erythematosus.

Lantow M, Sivakumar R, Zeumer L, Wasserfall C, Zheng YY, Atkinson MA, Morel L - Arthritis Res. Ther. (2013)

Exogenous human granulocyte-colony stimulation factor (huG-CSF) restores autoantibody (autoAb) production in B6.Sle2c2 mice after induction of chronic graft vs host disease (cGVHD). (A) huG-CSF-treatment induced anti-dsDNA IgG in B6.Sle2c2 mice in a dose-dependent manner (left), but had little effect in B6 mice (right). Five mice per strain per treatment were used. (B) Anti-dsDNA and anti-chromatin IgG found at d21 in the combined 120 × 1 and 12 × 2 cohorts. (C) Representative ANA IgG staining of Hep-2 cells incubated with sera collected at 21 d in each of the four treatment groups in the 120 × 1 and 12 × 2 cohorts and average staining intensity of 10 to 20 randomly selected cells per sample. Means and standard error of the mean with the significance value from the t-test for comparison of treated mice and controls (dex) are shown for each strain for a given time point in A, and from Dunnett's multiple comparison test between groups as indicated in B and C (*P < 0.05; **P < 0.01; ***P < 0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: Exogenous human granulocyte-colony stimulation factor (huG-CSF) restores autoantibody (autoAb) production in B6.Sle2c2 mice after induction of chronic graft vs host disease (cGVHD). (A) huG-CSF-treatment induced anti-dsDNA IgG in B6.Sle2c2 mice in a dose-dependent manner (left), but had little effect in B6 mice (right). Five mice per strain per treatment were used. (B) Anti-dsDNA and anti-chromatin IgG found at d21 in the combined 120 × 1 and 12 × 2 cohorts. (C) Representative ANA IgG staining of Hep-2 cells incubated with sera collected at 21 d in each of the four treatment groups in the 120 × 1 and 12 × 2 cohorts and average staining intensity of 10 to 20 randomly selected cells per sample. Means and standard error of the mean with the significance value from the t-test for comparison of treated mice and controls (dex) are shown for each strain for a given time point in A, and from Dunnett's multiple comparison test between groups as indicated in B and C (*P < 0.05; **P < 0.01; ***P < 0.001).
Mentions: If the S378N mutation in the G-CSFR is responsible for the cGVHD resistance in B6.Sle2c2 mice, we postulated that huG-CSF treatment should eliminate the difference in cGVHD response between B6 and B6.Sle2c2 mice. If resistance in B6.Sle2c2 mice is due to a G-CSFR loss of function, huG-CSF treatment should restore cGVHD response in B6.Sle2c2 mice. On the other hand, if resistance is due to a G-CSFR gain of function, cGVHD resistance would be expected in huG-CSF-treated B6 mice. Based on our time course analysis showing a maximal effect at d4 after injection that was eliminated at d7, we tested a combination of treatment protocols in which the dose (1.2, 12.0, or 120.0 ug) and the frequency (one-, two-, or three-weekly injections starting at cGVHD induction) varied. Two treatment protocols (120 × 1 and 12 × 2) raised the production of anti-dsDNA IgG in B6.Sle2c2 mice significantly above the dextrose-treated controls (Figure 4A left), and to a level similar to the B6 controls, especially in the 12 × 2 cohort. For the 120 × 1 cohort, a drop in the third week was observed, possibly because the pharmacological effect of the single huG-CSF treatment was diminishing. With these two treatment regimens combined, the amount of anti-dsDNA and anti-chromatin IgG at d21 was significantly higher in the B6.Sle2c2 mice treated with huG-SCF than in controls (Figure 4B). Moreover, the autoAbs produced by treated B6.Sle2c2 mice reached equivalent levels as in treated B6 mice. cGVHD induced autoAbs in B6 mice with strong Hep-2 staining that combined cytoplasmic and nuclear patterns (Figure 4C). The 12 × 2 treatment induced Hep-2-staining in B6.Sle2c2 mice similar in intensity and pattern as in B6 mice (Figure 4C). Similar results were obtained with the 120 × 1 treatment, although with a more variable intensity (data not shown). The 1.2 × 3 treatment had no effect, indicating that the dose was too low. The 12 × 3 treatment had an intermediate effect in B6.Sle2c2 mice, and interestingly was the only regimen that induced a significant decrease in the B6 anti-dsDNA IgG response (Figure 4A right). The other treatments showed a trend in lowering autoAbs produced by B6 mice, but the difference was not significant. Overall, these results demonstrate that exogenous G-CSF eliminates the resistance to cGVHD induction in B6.Sle2c2 mice in a dose-dependent manner, with no significant effect in B6 mice at the same dose. This suggests that an impaired G-CSFR response mediates the cGVHD resistance in the B6.Sle2c2 strain, which can be compensated by an excess G-CSF to drive equilibrium binding or signaling to the receptor.

Bottom Line: G-CSF binding by B6.Sle2c2 leukocytes was reduced as compared to B6, which was associated with a reduced expansion in response to in vivo G-CSF treatment.G-CSF in vivo treatment also failed to mobilize bone-marrow B6.Sle2c2 neutrophils as it did for B6 neutrophils.This result was corroborated by the increased anti-dsDNA IgG production in G-CSF-treated B6.TC mice, which also carry the Sle2c2 locus.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Introduction: An NZB-derived genetic locus (Sle2c2) that suppresses autoantibody production in a mouse model of induced systemic lupus erythematosus contains a polymorphism in the gene encoding the G-CSF receptor. This study was designed to test the hypothesis that the Sle2c2 suppression is associated with an impaired G-CSF receptor function that can be overcome by exogenous G-CSF.

Methods: Leukocytes from B6.Sle2c2 and B6 congenic mice, which carry a different allele of the G-CSF receptor, were compared for their responses to G-CSF. Autoantibody production was induced with the chronic graft-versus-host-disease (cGVHD) model by adoptive transfer of B6.bm12 splenocytes. Different treatment regimens varying the amount and frequency of G-CSF (Neulasta®) or carrier control were tested on cGVHD outcomes. Autoantibody production, immune cell activation, and reactive oxygen species (ROS) production were compared between the two strains with the various treatments. In addition, the effect of G-CSF treatment was examined on the production autoantibodies in the B6.Sle1.Sle2.Sle3 (B6.TC) spontaneous model of lupus.

Results: B6.Sle2c2 and B6 leukocytes responded differently to G-CSF. G-CSF binding by B6.Sle2c2 leukocytes was reduced as compared to B6, which was associated with a reduced expansion in response to in vivo G-CSF treatment. G-CSF in vivo treatment also failed to mobilize bone-marrow B6.Sle2c2 neutrophils as it did for B6 neutrophils. In contrast, the expression of G-CSF responsive genes indicated a higher G-CSF receptor signaling in B6.Sle2c2 cells. G-CSF treatment restored the ability of B6.Sle2c2 mice to produce autoantibodies in a dose-dependent manner upon cGVHD induction, which correlated with restored CD4+ T cells activation, as well as dendritic cell and granulocyte expansion. Steady-state ROS production was higher in B6.Sle2c2 than in B6 mice. cGVHD induction resulted in a larger increase in ROS production in B6 than in B6.Sle2c2 mice, and this difference was eliminated with G-CSF treatment. Finally, a low dose G-CSF treatment accelerated the production of anti-dsDNA IgG in young B6.TC mice.

Conclusion: The different in vivo and in vitro responses of B6.Sle2c2 leukocytes are consistent with the mutation in the G-CSFR having functional consequences. The elimination of Sle2c2 suppression of autoantibody production by exogenous G-CSF indicates that Sle2c2 corresponds to a loss of function of G-CSF receptor. This result was corroborated by the increased anti-dsDNA IgG production in G-CSF-treated B6.TC mice, which also carry the Sle2c2 locus. Overall, these results suggest that the G-CSF pathway regulates the production of autoantibodies in murine models of lupus.

Show MeSH
Related in: MedlinePlus