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Abstract: FR-PO1045

Transcriptional Dysregulation in Low Density Granulocytes from Patients with ANCA Vasculitis

Session Information

Category: Glomerular Diseases

  • 1202 Glomerular Diseases: Immunology and Inflammation

Authors

  • Ciavatta, Dominic J., University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States
  • Jones, Britta E., University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States
  • Bass, William, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States
  • Herrera, Carolina A., University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States
  • Agosto-Burgos, Christian, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States
  • Poulton, Caroline J., University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States
  • Starmer, Joshua, Univesity of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States
  • Free, Meghan E., University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States
  • Jennette, J. Charles, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States
  • Falk, Ronald J., University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States
Background

ANCA vasculitis is characterized by elevated expression of autoantigen genes, MPO and PRTN3; however, the source and implications of the elevated expression are unresolved.

Methods

We isolated monocytes, neutrophils, and an enriched fraction of PBMC from 30 healthy controls (HC) and 75 patients. PBMC from 8 HC and 18 patients were sorted for CD15+ and CD15+/CD10+ cells. Low density granulocytes (LDG) were enriched from PBMC of 28 HC and 67 patients. Gene expression was measured by quantitative RT-PCR. LDG were immunophenotyped by flow cytometry for cell surface markers.

Results

Expression of MPO and PRTN3 was significantly elevated in active patients compared to controls in neutrophils (2.6 and 3.6-fold) and in an enriched PBMC fraction (7 and 15.8-fold). Based on the greater fold-change expression in the PBMC fraction we sorted PBMC into CD15+ and CD15+/CD10+ cells. The nuclear morphology of the CD15+ cells is consistent with immature progenitor granulocytes, while the CD15+/CD10+ cells had segmented nuclei of mature neutrophils. We purified LDGs containing both CD15+ and CD15+/CD10+ cells. MPO and PRTN3 expression in LDGs were elevated in active patients compared to controls (9.3 and 16.5-fold), and correlated with expression in total leukocytes (WBC). LDG heterogeneity was analyzed by flow cytometry and the percentages of cell populations were compared to autoantigen gene expression. The percent of LDGs in PBMC and the percent of CD15+ cells in the LDG population showed weak correlations with MPO and PRTN3 expression (r2<0.09) (Figure).

Conclusion

The frequency of LDGs or CD15+ cells explains less than 10% of the variation in MPO and PRTN3 expression suggesting transcriptional dysregulation, not an increase in neutrophil progenitors, drives increased expression.

Heatmap represents the correlation coefficients (color scale shown at right) for pairwise comparisons.

Funding

  • NIDDK Support