Ultrastructural localization of lactoferrin and myeloperoxidase in human neutrophils by immunogold

Abstract

Colloidal gold was used as a marker for immunoelectron microscopy to localize lactoferrin (LF) and myeloperoxidase (MPO) in human peripheral blood neutrophils. Cells were reacted with monospecific antibodies against LF or MPO and then with gold-labeled antiglobulin. MPO cytochemistry was also associated with immunologic detection of LF. Immunologic labeling of thin sections after embedding in glycol methacrylate gave good ultrastructural morphology and specific localization of both proteins. MPO was detected in the large azurophil granules, whereas LF was consistently localized in the matrix of another population of morphologically distinct granules, smaller and more numerous than azurophil granules. When cytochemical detection of MPO was coupled with immunologic detection of LF, LF was observed in the population of MPO-negative granules, which were identified as specific. This was confirmed on cells that were permeabilized with saponin and stained for LF and MPO before embedding. No other neutrophil organelles displayed labeling for LF; other blood cells also were unreactive for LF. In the bone marrow, myeloblast and promyelocyte granulations were not stained and LF-containing granules appeared at the myelocyte stage. In conclusion, we confirm previous biochemical and light microscopic studies by ultrastructural demonstration of LF and MPO in two categories of granules, the specific and azurophil granules, respectively. The method described in this article avoids disruption caused by cell fractionation procedures. In the future, other intragranular proteins can be localized by a similar approach.

The individual regulation of granule protein mRNA levels during neutrophil maturation explains the heterogeneity of neutrophil granules

“…The cells were analyzed for the presence of transcripts for matrix proteins found exclusively in azurophil granules (MPO [28], proteinase-3 [29], neutrophil elastase [30], and defensin (HNP-3) [31], Fig. 1), in specific and gelatinase granules (lactoferrin [28], hCAP-18 [32], NGAL [3], SGP28 [33], transcobalamin-I [34], and gelatinase [9,35], Fig. 2) or in all three types of granules (serglycin [36,37] and lysozyme [38], Fig.…”

Neutrophil-activating potential of antineutrophil cytoplasm autoantibodies

“…For this reason, in the past few years, several investigators analyzed subcellular localization of ANCA antigens in the neutrophil and its consequences ( Table 1). The presence of MPO is restricted to the azurophilic granules and the cell membrane only [9,47,70], whereas subcellular distribution of PR3 is more complicated. Originally, PR3 was found to be stored in azurophil granules of the neutrophil [48,49,55].…”

“…Compared with the effect on PR3 expression, the effect of TNF-␣ on the level of MPO on the neutrophil surface is less pronounced [9, 90 -92]. The reason for this difference in sensitivity to up-regulation by TNF-␣ might be a difference in the intracellular localization of PR3 and MPO, where MPO localizes only in azurophil granules [70], which require a strong stimulus to degranulate [105], whereas PR3 is also present in the easily mobilizable-specific granules and secretory vesicles [67]. Moreover, in contrast to PR3, MPO does not display a bimodal expression pattern.…”

Serum amyloid A is an activator of PMN antimicrobial functions: induction of degranulation, phagocytosis, and enhancement of anti-Candida activity

“…Specific granules are known to represent a storage site not only for adhesion molecules but also for antimicrobial proteins such as lactoferrin [30]. Therefore, we investigated whether SAA induced secretion of lactoferrin.…”