ARTICLE |
Correspondence to: Fusayoshi Murata, Dept. of Anatomy, Faculty of Medicine, Kagoshima Univ., 8-35-1 Sakuragaoka, Kagoshima 890-8520, Japan.
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Summary |
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Using a high electron resolution staining method, cationic colloidal gold (CCG, pH 1.0) staining, we studied the fine structural localization of sulfated glycosaminoglycans (GAGs) in various maturational stages of guinea pig neutrophils. Azurophil and specific granules of neutrophils reacted positively to CCG, with variety in labeling according to maturation. All immature azurophil and specific granules were labeled selectively. Mature granules lost their affinity with CCG. CCG-positive labeling was also observed in the trans to trans-most Golgi apparatus of promyelocytes and myelocytes. Prior absorption with poly-L-lysine prevented CCG labeling of tissue sections. Mild methylation of ultrathin sections at 37C did not alter CCG labeling, whereas CCG labeling disappeared after active methylation at 60C. Treatment with chondroitinase ABC or heparinase I abolished the majority of CCG labeling. These findings suggest the existence of sulfated GAGs not only in immature azurophil but also in immature specific granules of neutrophils. Sulfation of GAGs occurs in the trans- to trans-most Golgi apparatus of neutrophil granulocytes. A possible correlation between accumulation of sulfated GAGs and maturation of specific granules in neutrophils is also discussed. (J Histochem Cytochem 47:881887, 1999)
Key Words: neutrophil, sulfated glycosaminoglycan, cationic colloidal gold, azurophil granule, specific granule
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Introduction |
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The existence of sulfated glycosaminoglycans (GAGs) in azurophil granules of neutrophils had been reported after the use of staining methods by 35S radioautography (
By using cationic colloidal gold (CCG) at pH 1.0, a recently developed method for demonstrating sulfated glycoconjugates with high electron resolution (
Several studies have revealed that specific granules of neutrophils also contain many kinds of basic substances such as lysozyme and lactoferrin (
To demonstrate sulfated GAGs in granules of neutrophils, we used Lowicryl K4M-embedded guinea pig bone marrow and the CCG (pH 1.0) staining method in this study. We observed CCG-positive labeling in immature azurophil granules, immature specific granules, and trans- to trans-most Golgi lamellae of granulocytes of premitotic maturational stages. Sulfated GAGs in the cell surface and bone marrow matrix were also revealed. By adding GAGs degrading enzymes and using other control experiments, we tried to analyze the nature of sulfated GAGs of neutrophils. A possible correlation between accumulation of sulfated GAGs and maturation of granules in neutrophils is also discussed.
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Materials and Methods |
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Preparation of Specimens
Three young guinea pigs weighing about 400 g each were used. The animals were anesthetized with ether. Bone marrow was removed from the femur, fixed in half-strength Karnovsky's fixative, and embedded in Lowicryl K4M (
Ultracytochemical Labeling Using CCG at pH 1.0
Cationic colloidal gold (CCG 10 nm) was purchased from British BioCell International (Cardiff, UK). Ultrathin sections designed for CCG labeling were pretreated with CCG buffer (potassium chloride-HCl buffer, pH 1.0) for 20 min and then labeled with 10 x CCG solution (diluted in the above buffer at pH 1.0) at room temperature (RT) overnight (
Control Experiments
Preabsorption with Poly-l-lysine.
Sections were incubated with 10 mg/ml poly-L-lysine (MW 682,600; Sigma, St Louis, MO) solution (dissolved in potassium chloride-HCl buffer, pH 1.0) for 4 hr at RT before labeling with CCG at pH 1.0.
Mild and Active Methylation. CCG labeling was performed after methylation of ultrathin sections with 0.01 N HClmethanol for 4 hr at 37C (mild methylation) and at 60C (active methylation).
Enzyme Degradation. Sections were incubated in either of the following enzymes: (a) 1 U/ml heparinase I (Flavobacterium heparinum, EC 4.2.2.8; Sigma) dissolved in Tris-buffered saline (TBS), pH 7.0, containing 0.04 M calcium chloride, or (b) 1 U/ml protease-free chondroitinase ABC (Proteus vulgaris, EC 4.2.2.4; Seikagakukogyo, Tokyo, Japan) dissolved in Tris-HCl buffer, pH 8.0. Both incubations were carried out in a moisture chamber at 37C overnight. After incubation, sections were washed three times with the same buffer for 5 min and then with distilled water. Finally, sections were labeled with CCG as previously described. All sections were observed with a Hitachi H7100 electron microscope at 75 kV accelerating voltage.
Quantitation of CCG Labeling
The CCG labeling density of neutrophil granules was calculated by using a Macintosh computer. Electron micrographic negatives (x10,000) were scanned into the computer with a Nikon Film Scanner. For each experiment and at each stage, at least five neutrophil granulocytes with 3050 granules were used. After the scale was set, the granule areas (Ai) were measured by NIH Image software. The number of gold particles (Ni) was counted manually. The mean density of gold particles was calculated as =Ni/
Ai (number/µm2). One-way analysis of variance (ANOVA) was used to analyze the labeling difference between granulocytes with or without treatment with enzymes. Student's t-test was used to analyze the labeling difference between promyelocytes and myelocytes. A p value of less than 0.05 was considered significant.
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Results |
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We divided the development of neutrophil granulocytes into promyelocyte, myelocyte, band form (including metamyelocyte), and segmented neutrophils according to the methods of earlier investigators (
Promyelocytes
Promyelocytes are the largest cells of the neutrophil series and represent the first recognizable stage in development. The nucleus shows a dispersed euchromatin pattern and a small peripheral zone of moderately condensed heterochromatin (Figure 1 and Figure 2). Sometimes one or two nucleoli can be observed in an ultrathin section (Figure 2). The Golgi complex is situated at the concave side of the nucleus (Figure 1 and Figure 2). Azurophil granules at different developmental stages varied in size and electron density.
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CCG labeling was observed in the trans- to trans-most Golgi apparatus, small vesicles in the Golgi area, and immature azurophil granules (Figure 1 and Figure 2). The immature azurophil granules were electron-lucent. They were spherical or ellipsoid and were present around the Golgi area and elsewhere in the cytoplasm (Figure 2). Mature azurophil granules were highly electron-dense. They were present throughout the cytoplasm and showed decreased or absent labeling of CCG (Figure 1 and Figure 2). Sulfated GAGs in the cell surface and bone marrow matrix were also labeled with CCG (Figure 1 and Figure 2).
Myelocytes
Myelocytes were smaller than promyelocytes, with a round or elongated shape. The nucleus contained clumped and peripherally condensed heterochromatin. Both azurophil and specific granules were present at this stage. Immature azurophil granules had an electron lucent periphery with a somewhat dense core, whereas mature ones were electron-dense (Figure 3). Immature specific granules showed irregular flocculated contents, whereas mature ones were electron-lucent with a moderately electron-dense periphery (Figure 4). The Golgi apparatus had some stacks of cisternae. Small vesicles arising from the convex face of the Golgi apparatus indicated active specific granulogenesis. At this maturational stage, CCG positively labeled the trans- to trans-most Golgi apparatus, small vesicles in the Golgi area, immature azurophils, and immature specific granules (Figure 3 and Figure 4).
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Band Form (Including Metamyelocytes)
Because it is difficult to differentiate metamyelocytes and band form neutrophils in ultrathin sections, they are described together. These cells possessed variably shaped nuclei with peripherally condensed heterochromatin. The Golgi apparatus was relatively small and no immature, aggregating granules were associated with it. Negligible CCG labeling was observed at this stage (Figure 5). Both azurophil and specific granules were present, the latter outnumbering the former.
Segmented Neutrophils
The segmented neutrophil is the smallest cell of the neutrophil series. It is characterized by a segmented nucleus. The Golgi apparatus is small, with no granulogenesis. No CCG labeling was observed at this stage.
Control Experiments
Preabsorption with poly-L-lysine abolished CCG labeling at all stages of neutrophils. CCG labeling remained unchanged after mild methylation at 37C but disappeared after active methylation at 60C (Figure 6). Treatment with chondroitinase ABC reduced CCG binding by about 92%, and treatment with heparinase I reduced CCG binding by about 97% in cells at all maturational stages (Figure 7 and Figure 8; Table 1). The number of CCG labeled gold particles was greater in promyelocytes than that in myelocytes in sections not treated with GAG-degrading enzymes (Table 1).
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Discussion |
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In the past few decades, histochemical and other cytochemical studies pointed out that sulfated GAGs exist in the azurophil granules and Golgi apparatus but not in specific granules of neutrophils (
It is fascinating to note the positive CCG labeling in the immature specific granules of neutrophils. This indicates that the content of specific granules is negatively charged, probably due to the presence of sulfated GAGs. This result agrees with reports that suggest that GAGs are present in smaller quantities in secondary lysosomal granules (
GAGs in azurophil granules function by complexing with certain basic components and holding them inactive to facilitate granule storage (
Previous biochemical studies have determined that the major GAGs of neutrophils are mainly chondroitin-4-sulfate (
The existence of GAGs in specific granules of neutrophils is a new finding. It has not been previously reported, probably owing to the limitations of methodology in previous studies. Further studies, e.g., double staining by using CCG and immunogold with monoclonal antibodies against GAGs or lactoferrin, will provide more interesting evidence for this idea.
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