Detection of cytoplasmic CD antigens within normal human peripheral blood leucocytes

Abstract

SUMMARYPolymorphonuclear neutrophils (PMNs) are capable of synthesizing various pro-inflammatory cytokines which may indirectly influence specific immune responses. PMNs may also have the capacity to present foreign peptides to helper T cells (Th cells). In support of this hypothesis, recent studies have shown that neutrophils, when activated by the correct combination of cytokines, can be induced to express cell surface major histocompatibility complex (MHC) Class II (DR) antigen, CD80 (B7.1) and CD86 (B7.2): molecules required for antigen presentation and subsequent T-cell activation. In this study we have used normal 'resting' human peripheral blood neutrophils and demonstrated, using a mild fixation and permeabilization protocol, significant cytoplasmic 'stores' of these molecules known to be important in antigen presentation. Cytoplasmic MHC Class II antigen was found with two out of 20 normal donors tested whereas cytoplasmic CD80 and CD86 were found to a variable extent within all normal donors. Surprisingly, we also found several other neutrophil cytoplasmic CD antigens more commonly associated with B cells, i.e. CD20, CD21 (CR2/EBV-R) and CD22 (BL-CAM). All of these antigens were confined to the 'resting' cell cytoplasm and were never found to be expressed on the cell surface. To exclude the possibility that these antigens were absorbed from plasma and to provide evidence for active synthesis, we used a novel whole blood in situ hybridization flow cytometry assay method to detect mRNA specific for these antigens within normal PMNs. We also conducted real-time polymerase chain reactions to confirm these findings using CD22 as a good example of an 'inappropriately expressed' CD antigen. These observations therefore provide support for the hypothesis that human PMNs have the potential to express molecules required for antigen presentation and cell signalling.

Cross‐linking of neutrophil CD11b results in rapid cell surface expression of molecules required for antigen presentation and T‐cell activation

“…Following fixation and permeabilization, normal human peripheral blood neutrophils were previously shown, at the protein and mRNA level, to express a variety of antigens normally associated with B‐lymphocytes and antigen presenting cells 8 . These observations suggest that neutrophils have the capacity to actively synthesize these molecules, thus supporting the hypothesis that activated neutrophils have the potential to function as APCs 2 .…”

“…6,7 In support of this hypothesis we have previously demonstrated, at the protein and mRNA level, that normal resting peripheral blood neutrophils appear to have significant cytoplasmic stores of these molecules. 8 This observation suggests that activated neutrophils have the capacity to actively synthesize the molecules required for antigen presentation. However this mechanism is slow requiring up to 48 hr for full expression of the molecules on the neutrophil surface.…”

“…Translocation assay using purified neutrophils. Neutrophils were isolated from whole blood using a combination of Ficoll‐Hypaque density centrifugation, dextran sedimentation and hypotonic lysis as described previously 8 . Cells were then adjusted to a final concentration of 1 × 10 6 cells per 100 µl Iscove's medium supplemented with 20% fetal calf serum.…”

Circumvention of Normal Constraints on Granule Protein Gene Expression in Peripheral Blood Neutrophils and Monocytes of Patients with Antineutrophil Cytoplasmic Autoantibody–Associated Glomerulonephritis

“…For in situ hybridization-flow cytometric assays, whole blood (2 ml) was incubated with FACS lysing solution (Becton Dickinson, San Jose, CA; 20 ml) at room temperature overnight. Hybridizations were performed with 5 g/ml probe for 3 h at 37°C, and positives were detected using ImmunoPure FITC-conjugated streptavidin (Pierce, Rockford, IL) (23). For flow cytometric quantification, PR3 mRNA levels are expressed as percentage of fluorescence intensity above glyceraldehyde-3-phosphate dehydrogenase control.…”

Neutrophile et immunité

“…Le neutrophile activé en présence d'IFN-γ, GM-CSF, TNF-α, IL-4 va se modifier et exprimer à sa surface le CMH de classe II, CD83, CD40 et CCR6 mais, de plus, les molécules de co-stimulation CD80 (B7-1) et CD86 (B7-2), critères définissant une cellule dendritique [22][23][24]. Certains neutrophiles au repos possèdent déjà, dans leur cytoplasme, ces molécules pré-stockées ; ils en possèdent éga-lement d'autres comme CD20, 21, 22, marqueurs de lymphocytes B [25]. La présentation antigénique par le neutrophile est efficace puisqu'il peut alors activer les lymphocytes T mis en présence du peptide antigénique par ce neutrophile, que ce soit un peptide conventionnel ou un superantigène [26][27][28].…”