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Antineutrophil cytoplasmic autoantibodies interact with primary granule constituents on the surface of apoptotic neutrophils in the absence of neutrophil priming.

Gilligan HM, Bredy B, Brady HR, Hébert MJ, Slayter HS, Xu Y, Rauch J, Shia MA, Koh JS, Levine JS - J. Exp. Med. (1996)

Bottom Line: We show by electron microscopy that apoptosis of unprimed PMN is also associated with the translocation of cytoplasmic granules to the cell surface and alignment just beneath an intact cell membrane.Immunofluorescent microscopy and FACS analysis demonstrate reactivity of ANCA-positive sera and antimyeloperoxidase antibodies with apoptotic PMN, but not with viable PMN.Moreover, we show that apoptotic PMN may be divided into two subsets, based on the presence or absence of granular translocation, and that surface immunogold labeling of myeloperoxidase occurs only in the subset of PMN showing translocation.

View Article: PubMed Central - PubMed

Affiliation: Renal Section, Evans Memorial Department of Clinical Research, Boston, Massachusetts, USA.

ABSTRACT
The pathogenic role of antineutrophil cytoplasmic autoantibodies (ANCA) remains controversial because of the difficulty in explaining how extracellular ANCA can interact with intracellular primary granule constituents. It has been postulated that cytokine priming of neutrophils (PMN), as may occur during a prodromal infection, is an important trigger for mobilization of granules to the cell surface, where they may interact with ANCA. We show by electron microscopy that apoptosis of unprimed PMN is also associated with the translocation of cytoplasmic granules to the cell surface and alignment just beneath an intact cell membrane. Immunofluorescent microscopy and FACS analysis demonstrate reactivity of ANCA-positive sera and antimyeloperoxidase antibodies with apoptotic PMN, but not with viable PMN. Moreover, we show that apoptotic PMN may be divided into two subsets, based on the presence or absence of granular translocation, and that surface immunogold labeling of myeloperoxidase occurs only in the subset of PMN showing translocation. These results provide a novel mechanism that is independent of priming, by which ANCA may gain access to PMN granule components during ANCA-associated vasculitis.

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PMN apoptosis leads to decreased cellular content of ANCA  Ag. PMN were cultured overnight, treated with P-ANCA (a) or C-ANCA  (b) sera, and then analyzed by FACS® for PI and FITC staining. All PMN  were included.
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Figure 7: PMN apoptosis leads to decreased cellular content of ANCA Ag. PMN were cultured overnight, treated with P-ANCA (a) or C-ANCA (b) sera, and then analyzed by FACS® for PI and FITC staining. All PMN were included.

Mentions: Given these morphologic features, we hypothesized that translocated primary granules may be shed from the cell surface in the form of membrane-enclosed apoptotic bodies. Thus, when apoptosis has progressed to the stage of nuclear disintegration and loss of membrane integrity, there should be diminished numbers of primary granules within the cytoplasm. To test this, we regated selected FACS® analyses to include all apoptotic PMN, irrespective of PI entry, and assessed FITC staining in relation to PI staining (Fig. 7 a and b). Three distinct populations were observed: (a) a PI-negative and low FITC population comprised of viable PMN and apoptotic PMN whose primary granules have not yet undergone translocation; (b) a PI-negative and high FITC population comprised of apoptotic PMN with granular translocation; and (c) a PI-positive and intermediate FITC population (consistent with loss of primary granules) containing apoptotic PMN that have progressed to postapoptosis (see Fig. 9). This result is noteworthy in that FITC staining within the PI-positive population reflects the full complement of primary granules, both surface and cytoplasmic, whereas FITC staining within the PI-negative population represents only those granules that have undergone translocation.


Antineutrophil cytoplasmic autoantibodies interact with primary granule constituents on the surface of apoptotic neutrophils in the absence of neutrophil priming.

Gilligan HM, Bredy B, Brady HR, Hébert MJ, Slayter HS, Xu Y, Rauch J, Shia MA, Koh JS, Levine JS - J. Exp. Med. (1996)

PMN apoptosis leads to decreased cellular content of ANCA  Ag. PMN were cultured overnight, treated with P-ANCA (a) or C-ANCA  (b) sera, and then analyzed by FACS® for PI and FITC staining. All PMN  were included.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196384&req=5

Figure 7: PMN apoptosis leads to decreased cellular content of ANCA Ag. PMN were cultured overnight, treated with P-ANCA (a) or C-ANCA (b) sera, and then analyzed by FACS® for PI and FITC staining. All PMN were included.
Mentions: Given these morphologic features, we hypothesized that translocated primary granules may be shed from the cell surface in the form of membrane-enclosed apoptotic bodies. Thus, when apoptosis has progressed to the stage of nuclear disintegration and loss of membrane integrity, there should be diminished numbers of primary granules within the cytoplasm. To test this, we regated selected FACS® analyses to include all apoptotic PMN, irrespective of PI entry, and assessed FITC staining in relation to PI staining (Fig. 7 a and b). Three distinct populations were observed: (a) a PI-negative and low FITC population comprised of viable PMN and apoptotic PMN whose primary granules have not yet undergone translocation; (b) a PI-negative and high FITC population comprised of apoptotic PMN with granular translocation; and (c) a PI-positive and intermediate FITC population (consistent with loss of primary granules) containing apoptotic PMN that have progressed to postapoptosis (see Fig. 9). This result is noteworthy in that FITC staining within the PI-positive population reflects the full complement of primary granules, both surface and cytoplasmic, whereas FITC staining within the PI-negative population represents only those granules that have undergone translocation.

Bottom Line: We show by electron microscopy that apoptosis of unprimed PMN is also associated with the translocation of cytoplasmic granules to the cell surface and alignment just beneath an intact cell membrane.Immunofluorescent microscopy and FACS analysis demonstrate reactivity of ANCA-positive sera and antimyeloperoxidase antibodies with apoptotic PMN, but not with viable PMN.Moreover, we show that apoptotic PMN may be divided into two subsets, based on the presence or absence of granular translocation, and that surface immunogold labeling of myeloperoxidase occurs only in the subset of PMN showing translocation.

View Article: PubMed Central - PubMed

Affiliation: Renal Section, Evans Memorial Department of Clinical Research, Boston, Massachusetts, USA.

ABSTRACT
The pathogenic role of antineutrophil cytoplasmic autoantibodies (ANCA) remains controversial because of the difficulty in explaining how extracellular ANCA can interact with intracellular primary granule constituents. It has been postulated that cytokine priming of neutrophils (PMN), as may occur during a prodromal infection, is an important trigger for mobilization of granules to the cell surface, where they may interact with ANCA. We show by electron microscopy that apoptosis of unprimed PMN is also associated with the translocation of cytoplasmic granules to the cell surface and alignment just beneath an intact cell membrane. Immunofluorescent microscopy and FACS analysis demonstrate reactivity of ANCA-positive sera and antimyeloperoxidase antibodies with apoptotic PMN, but not with viable PMN. Moreover, we show that apoptotic PMN may be divided into two subsets, based on the presence or absence of granular translocation, and that surface immunogold labeling of myeloperoxidase occurs only in the subset of PMN showing translocation. These results provide a novel mechanism that is independent of priming, by which ANCA may gain access to PMN granule components during ANCA-associated vasculitis.

Show MeSH
Related in: MedlinePlus