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Distinct granule populations in human neutrophils and lysosomal organelles identified by immuno-electron microscopy
Journal of Immunological Methods 232 Ž1999. 153–168 www.elsevier.nlrlocaterjim
Distinct granule populations in human neutrophils and lysosomal organelles identified by immuno-electron microscopy Dorothy F. Bainton
)
Department of Pathology, UniÕersity of California-San Francisco, Rm. S-115, 513 Parnassus AÕenue, San Francisco, CA 94143-0400, USA
Abstract In this paper, we illustrate the fine structural localization of distinct marker proteins in the organelles of human neutrophils and outline our preferred methods for processing ultrathin cryosections for use with immunoelectron microscopy. Previous work has determined the subcellular localization of certain marker proteins within intact polymorphonuclear neutrophilic leukocytes ŽPMN. and PMN fractions. These are as follows: myeloperoxidase ŽMPO. for azurophilic granules, lactoferrin for specificrsecondary granules, gelatinase for gelatinasertertiary granules, albumin for the secretory vesicles, and HLA class I and L-selectin for the plasma membrane. In addition to analyzing the heterogeneity of the PMN granule populations, new information on the lysosomal system of this cell is reviewed and extended by the localization of the lysosome-associated membrane proteins ŽLAMPs. and the cation-independent mannose 6-phosphate receptor ŽCI-M6PR.. LAMPs were absent in all identified granule populations, but were found in the membranes of vesicles, multivesicular bodies ŽMVB., and multilaminar compartments ŽMLC.. We show here that MVB contain CI-M6PR whereas MLC do not. Furthermore, since MLC contain LAMPs but not the receptor, they probably correspond to the late endosome. By current criteria, the true lysosomes of the resting PMN are MVB and MLC. Finally, although azurophil granules contain acid hydrolases their membranes do not contain LAMPs and they cannot be classified as lysosomes, but rather are more similar to regulated secretory granules. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Double-labeling; Granules; Lysosomes; Marker proteins
1. Introduction The ability of each type of leukocyte to conduct its special activities depends on its content of diverse AbbreÕiations: CI-M6PR, cation-independent mannose 6phosphate receptor; LAMPS, lysosome-associated membrane proteins; Man 6-P GP, mannose 6-phosphate containing glycoproteins; MVB, multivesicular bodies; MLC, multilaminar compartments; MPO, myeloperoxidase; PMN, polymorphonuclear neutrophilic leukocyte ) Corresponding author. Tel.: q1-415-476-4451; fax: q1-415476-0816; e-mail: [email protected]
chemical substances stored in concentrated form in membrane-bound cytoplasmic granules. The relationship between leukocyte granule content and cell function was first clearly documented in neutrophils by Cohn and Hirsch Ž1960., who successfully isolated PMN granules from rabbit peritoneal exudates and found that they contained a number of lysosomal enzymes as well as substances with antibacterial activity. These investigators further demonstrated that when microorganisms are engulfed by PMN, the cells degranulate, i.e., the storage granules discharge their contents into the digestive vacuole ŽZucker-
0022-1759r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 1 7 5 9 Ž 9 9 . 0 0 1 7 3 - 8
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Franklin and Hirsch, 1964.; subsequently, the organisms are killed and digested. These cytoplasmic granules are formed early in development, during the differentiation and proliferation of the immature cells in the bone marrow. Additional work by numerous investigators using enzyme cytochemical studies on PMN granules in situ and biochemical assays on granule fractions established that there were two basic chemically distinct granules types: MPO-positive azurophilic granules, which appear early in maturation during the promyelocyte stage, and MPO-negative specific granules, which are formed later during the myelocyte stage ŽFig. 1.. The MPO-positive azurophil Žprimary. granules are formed only during the promyelocyte stage and are reduced in number by mitosis. It was recently shown that the MPO-negative granules constitute a continuum from early appearing granules Žmyelocyte stage. that contain lactoferrin but no gelatinase, to granules that contain both lactoferrin and gelatinase Žmetamyelocyte stage., to granules that contain gelatinase but no lactoferrin Žband cell stage.. The aforementioned granules that contain lactoferrin are referred to as specific Žsecondary. granules while the granules that
contain gelatinase but not lactoferrin are referred to as gelatinase Žtertiary. granules. Thus, the mature circulating neutrophil contains the three major granule types Žextensively reviewed by Borregaard and Cowland, 1997; Bainton, 1999.. Within the framework of this well-known setting, this paper concentrates on relatively recent data that have been gleamed by using immunocytochemistry on developing and mature PMN. Although technically more difficult than enzyme cytochemistry, many more reagents Žantibodies and soluble receptors. are available to be applied with immunocytochemistry and a more complete picture of granule content and heterogeneity can be appreciated and new organelles identified ŽTable 1..
2. Materials and methods Our laboratory has explored many procedures for the study of the distribution of antigens within neutrophils and platelets ŽStenberg et al., 1984.. This paper will concentrate on those particular techniques that we consider optimal for leukocytes and does not intend to provide a comprehensive review, as a
Fig. 1. Diagrammatic representation of PMN life-span and stages of maturation. For discussion, see text. Out of every 100 nucleated cells in marrow, 0.5% are myeloblasts, 5% promyelocytes, 12% myelocytes, 22% metamyelocytes and bands, 20% are maturing and mature neutrophilic cells, yielding a total of ; 60% developing neutrophils in normal human marrow. The azurophilic Žprimary. granules, which are MPO-positive, are shown as solid black dots and the other granules are shown as open dots. Basically, the MPO-negative granules may be divided into specificrsecondary granules and gelatinasertertiary granules based on their relative content of lactoferrin and gelatinase. Modified from Bainton et al., 1971.
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Table 1 Specific markers for neutrophil compartments Granules
Content Markers
Membrane Markers
Azurophilic Žprimary.
Myeloperoxidase, lysosomal enzymes, M6P-glycoproteins Lactoferrin Gelatinase b
CD63, CD68
Specific Žsecondary. Gelatinase Žtertiary. Other compartments Secretory vesicles Multivesicular bodies ŽMVB. Multilaminar compartments ŽMLC. Plasma membrane
Albumin M6P-glycoproteins M6P-glycoproteins
number of excellent examples of such are already available ŽGriffiths, 1993; Griffiths et al., 1983; Tokuyasu, 1984; McCaffery and Farquhar, 1995; Liou et al., 1996; Peters, 1999.. With the exception of Fig. 7Ža–c., detailed methods have been previously published as noted in the references in the legends. For the localization of the CI-M6PR receptor in Fig. 7, the affinity-purified rabbit antibody made against bovine CI-M6PR ŽPL 603CI. was used. This was obtained from Dr. Peter Lobel, Center for Advanced Technology, Piscataway, NJ, 08854-5638.
CD11b, Cytochrome b CD11b, Cytochrome b
Alkaline phosphatase CD11B, Cytochrome b LAMP-2rLAMP-1 CI-M6P receptor LAMP-2rLAMP-1 HLA-1, L-selectin
Ž2. 2% paraformaldehyde, 0.05% glutaraldehyde. In general, this fixative has been the most satisfactory. If the glutaraldehyde is lower than 0.05%, there is no improvement in morphology. Ž3. Occasionally we have been able to use 0.5–2% glutaraldehyde, particularly if we need to stabilize the contents of granules of lower molecular weight and prevent ‘‘spillage’’ from the matrix of the granules Ždiscussed later.. 2.2. Immunogold labeling of ultrathin cryosections (Figs. 1–8)
2.1. FixatiÕes The most important step in immunocytochemical localization is the choice of fixative. The objective of fixation is to immobilize the antigens while retaining their antigenicity and preserving acceptable ultrastructure ŽGriffiths, 1993; McCaffery and Farquhar, 1995.. Glutaraldehyde is usually the superior fixative for preservation of cell structure, but frequently results in loss of antigenicity. Paraformaldehyde, particularly the periodic–lysine–paraformaldehyde ŽPLP. described by McLean and Nakane Ž1974., usually retains more antigenicity and gives acceptable, though sometimes not optimal organelle morphology. We have been pleased also with a double fixative of 2% paraformaldehyde and 0.05% glutaraldehyde Žsee Section 2.2.7.. We usually proceed as follows. Ž1. Initially, we try PLP fixation. If antigenicity is satisfactory, we next try to add glutaradehyde to improve the morphology, as outlined below.
2.2.1. Single labeling (examples, Fig. 3aFigs. 6–8) Ž1. Fixed leukocytes Žwithout washing. are cryoprotected by immersion in 2.1 M sucrose containing polyvinylpyrolidone Žsee Section 2.2.7. overnight at 48C and then frozen in liquid N2 ŽTokuyasu, 1989; Peters, 1999.. Ž2. Thin sections are cut on ultracut ultramicrotome with cryo-attachment equipment at y1158C and placed on Formvar and carbon-coated nickel EM grids. All the incubations and washes are carried out by floating the grid with section side down on droplets. Care should be taken to avoid wetting the back of the grid. All incubations are performed at room temperature unless otherwise indicated. All the phosphate-buffered saline ŽPBS. steps for washing contain 0.1% bovine serum albumin ŽBSA.. Water used in all the preparation is double-distilled deionized water. Ž3. After sectioning, grids are placed on PBS containing 0.2% glycine.
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Ž4. Grids are then incubated for 10 min in 1% each of skim milk, BSA, fish gelatin and ovualbumin in PBS, followed by two 2-min washes in PBS. Ž5. The primary antibody is then incubated for 60–120 min. It is diluted in PBS containing an additional 500 mM of NaCl. Multiple dilutions are tested. Ž6. Grids are washed in PBS three times for 5 min each followed by three more washes of the BSArPBS buffer with a higher pH Ž8.1.. Ž7. Grids are then incubated for 1 h with goldlabeled secondary antibody, which is diluted in 0.1% BSArPBS ŽpH 8.1. and used at the dilution recommended by the supplier. The most commonly used dilutions are 1:50 or 1:20, of the goat anti-rabbit ŽGAR. or goat anti-mouse ŽGAM. conjugated with 5 or 10 nm gold particles, for polyclonal or monoclonal primary antibody, respectively. Ž8. Grids are washed in PBS at least three times for 5 min each to remove the unbound immunogold. Ž9. Grids are refixed in 0.5% glutaraldehyderPBS for 5 min followed by three changes of H 2 O washing in 5 min. Ž10. Grids are floated on neutral oxalate uranyl acetate Žsee Section 2.2.7. for 5 min to stabilize the membranes, followed by three changes of H 2 O. Ž11. Finally, grids are embedded and stained with a mixture of methyl cellulose and uranyl acetate Žsee Section 2.2.7. for two changes, 5 min each, on a cold block at 48C. Grids are then picked up on loops. As much of the excess fluid as possible is removed and the grids are allowed to air dry. Ž12. Grids are examined in a transmission electron microscope at 60 or 80 KV. Ž13. Controls consist of replacing the primary antibody with normal rabbit or mouse serum. 2.2.2. Applying both enzyme cytochemistry (prefreezing) and imunocytochemistry on frozen thin sections (examples, Figs. 2 and 3b) Fixed suspended leukocytes were washed in 0.1 M phosphate buffer with 5% sucrose. The method of Graham and Karnovsky Ž1966. was used for the demonstration of MPO in the azurophil granules. The cells were resuspended and preincubated for 10 min at room temperature in 5 mg DAB Ž3,3-diaminobenzidine, Sigma Grade II. dissolved in 10 ml 0.05 M Tris buffer pH 7.6. Then 0.1 ml of 1% H 2 O 2
was added to DAB–Tris solution and incubated for 60 min at room temperature Žstock solution of H 2 O 2 should be replaced frequently.. The cells were rinsed three times with 0.05 M Tris buffer with 5% sucrose. The cells were then pelleted and the cell pellet was embedded in 2.1 M sucrose, frozen, and sectioned as described above. 2.2.3. Double-labeling with monoclonal and polyclonal antibodies and different sizes of gold (example, Fig. 5) Double-labeling experiments to localize a monoclonal antibody and a polyclonal antibody can be performed by combining them in the same drops in the primary antibody incubation previously described in Section 2.2.1. After washing, the smaller gold for GAR-5 is applied first, then the larger gold, GAM-10, is applied in subsequent steps. An example of this method is illustrated in Fig. 5, where the monoclonal antibody for LAMP-2, H 4 B 4 , was labeled with GAM-10 and the polyclonal antibody for albumin was labeled with GAR-5. 2.2.4. Double-labeling with two polyclonal antibodies and two sizes of protein a-gold as described by Slot and Geuze (1984) (example, Fig. 4) A double-labeling experiment to localize gelatinase and lactoferrin was performed using protein A. The probes protein A-10 and A-20 Žobtained from Dr. G. Posthuma, Utrecht, Holland. were used at a 1:50 dilution. Rabbit antibody against gelatinase was applied and labeled with protein A-10. Before the second antibody was applied, 0.5% glutaraldehyderPBS was applied, followed by free protein A Ž0.05 mgrml.. Subsequently, an antibody to lactoferrin ŽAccurate Chemical, Westbury, NY. was applied, followed by protein A-20. Sections were also labeled in reverse, using the large gold label to label gelatinase and the small gold to label lactoferrin. Non-immune-purified rabbit lgG was used as a control. 2.2.5. Labeling with biotinylated antibody and probing with streptaÕidin gold (example, inset Fig. 4) The primary antibody can be biotinylated and detected with a secondary application of streptavidin gold ŽKjeldsen et al., 1993.. We used the same process as previously described except we omitted the skim milk and ovualbumin in the blocking steps.
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Fig. 2. Portion of a PMN stained for MPO to mark the azurophil granules Žag., then immunolabeled for lactoferrin. Many of the MPO-negative specific granules have gold label within their matrix Žarrows., but several contain no label ŽU .. =81,000. Modified from Bainton et al., 1987.
2.2.6. Labeling with a biotinylated soluble receptor and probing with streptaÕidin-gold for the receptor’s ligand Man 6P-containing proteins can be localized by the sCI-MPR probe ŽValenzano et al., 1993.. This probe is a biotinylated derivative of a soluble form of the CI-MPR isolated from fetal bovine serum. The probe binds Man 6-P-containing proteins ŽMan 6-P GP. with nanomolar affinity, and is used in much the same manner as an antibody is to detect a specific substrate. The biotinylated receptor binds its ligand; subsequently the biotin moiety binds with streptavidin-gold for detection at the fine structural level ŽCieutat et al., 1998.. Ultrathin cryosections were incubated for 2 h at 48C, in 1% BSA, 0.2% Tween-20
and PBS: and then incubated overnight with the biotinylated sCI-MRP Ž1 mgr100 ml. in PBS plus 1% BSA and 5 mmolrl b-glycerolphosphate. The phosphorylated enzymes were detected the next day by streptavidin-gold, 5 or 10, with O.D. at 520 nm of approximately 5.0 ŽSigma. diluted to 1:20. The sections were then further processed as described above. For double-labeling, the antibody was applied first, after washing. The biotinylated sCl-MPR was then applied and allowed to incubate overnight. For controls, the sections were preincubated with 10 mmolrl of man 6-P for 1 h before applying the biotinylated sCl-MPR. The grids were then further processed as described above. Using this technique, Cieutat et al. Ž1998., were able to determine that phosphorylated
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forms of the Man 6-P GP were present in large amounts in azurophil granules and smaller amounts in multivesicular bodies ŽMVB. and in multilaminar compartments ŽMLC..
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Žc. 40 ml water Žd. pH to 7.4 with HCl Že. 1 g sucrose Žf. add water to make 100 ml Žg. cells are fixed for 1 h at 228C
2.2.7. Reagents 2.2.7.1. McLean and Nakane’s PLP fixatiÕe. ŽA. 0.1 M lysiner0.05 M sodium phosphate buffer, pH 7.4. Dissolve 1.827 g lysine HCl in 50 ml of water, adjust pH to 7.4 with 0.1 M Na 2 HPO4 . Add 0.1 M phosphate buffer, pH 7.4, to make final volume of 100 ml. Store at 48C. ŽB. Preparation of 16% paraformaldehyde stock: Mix 16 g of paraformaldehyde in 100 ml of water. Heat in water at 60–708C stirring occasionally to depolymerize. Slowly add 2–5 drops of 1 N NaOH until solution is clear, about 30 min, filter before use. The solution will last about 2 weeks if stored at 48C. Just before use, combine three parts of ŽA. to one part ŽB., and then add sodium meta-periodate to a final concentration of 0.01 M Ž21.4 mg of sodium meta-periodate to 10 ml of fixative.. Cells are fixed for 4 h at 228C. 2.2.7.2. 2% paraformaldehyder 0.05% glutaraldehyde r 0.1 M phosphate buffer, pH 7.4. Mix the components, as follows: Ža. 12.5 ml 16% paraformaldehyde Žb. 0.2 ml 25% glutaraldehyde Žc. 50 ml 0.2 M phosphate buffer Žd. pH to 7.4, add water to make 100 ml Že. cells are fixed for 4 h at 228C
2.2.7.4. Solutions for frozen thin sections. 1. Polyvinylpyrolidone ŽPVP.rsucrose solution. Mix 13 g of PVP-10 and 2.6 ml 1.1 M Na 2 CO 3 Žin water, adjust pH to 7.4. to a paste, then add 2.3 M sucrose Žin PBS.. Mix well, store at 48C. The solution can last for a long time. 2. Neutral oxalate uranyl acetate. Mix equal amounts of 4% aqueous uranyl acetate and 0.3 M aqueous oxalic acid, pH to 7.4, with 10% ammonium hydroxide. Test with pH paper. 3. Stock of 1.8% methyl cellulose: 0.9 g methyl cellulose, 50 ml water. Mix in stirrer in ice bath for at least 4 h, store overnight at 48C. Centrifuge for 1–4 h at 48C. Divide into aliquots and store at 48C. Will be good for about a month. Older stock yields precipitates on the grids. 4. 4% Uranyl acetate stock: 2 g uranyl acetate, 50 ml water. Mix until dissolved, store in brown bottle at 48C. 5. Methyl cellulose and Uranyl acetate mixture Žsee Peters, 1999.: 1 ml 1.8% methyl cellulose, 0.1 ml 4% uranyl acetate.
3. Results and discussion 3.1. Marker enzymes
2.2.7.3. Preparation of 1.5% glutaraldehyde solution in 0.067 M sodium cacodylate buffer, pH 7.4, with 1% sucrose. Mix components, as follows: Ža. 4.2 ml 25% glutaraldehyde Žb. 50 ml 0.134 M sodium cacodylate
In addition to the presence or absence of MPO, it is now recognized that granules can be further subdivided on the basis of other granular and membrane proteins. Table 1 lists the specific markers of human
Fig. 3. Ža. Frozen thin section of normal resting PMN labeled with D545, as the primary antibody and GAM-10 nm gold as the secondary antibody to demonstrate the presence of CD63 along the membranes of large extracted granules, characteristic of azurophilic granules Žag.. Specific granules Žsg. were not labeled Ž=72,000. Žmodified from Cham et al., 1994.. Žb. Human PMN were stained for MPO before being frozen. Then, cryosections were made and incubated with rabbit polyclonal antibody to a leukocyte adhesion receptor Žb 2 ., and labeled with GAR-5. The MPO-positive azurophil granules Žag. contain dense reaction product, whereas the lighter, specific granules are MPO-negative. Most of the gold grains outline the membranes of the specific granules Žlarge arrows., although some are present in the matrix of these granules. Because GAR-5 labels in clumps of two to six particles, label for an antibody site on the membrane may appear both on the membrane and in the matrix. =75,000. Modified from Bainton et al., 1987.
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neutrophilic granules and other organelles as analyzed by both cytochemical and fractionation procedures ŽBainton et al., 1971; Baggiolini, 1978; Cramer et al., 1985; Borregaard and Cowland, 1997; Ganz and Lehrer, 1998..
3.2. Azurophilic granules In addition to myeloperoxidase ŽFig. 2., the azurophilic granule contains numerous lysosomal enzymes or acid hydrolases. Also elastase ŽDamiano et
Fig. 4. Colocalization of gelatinase and lactoferrin in human PMN. PMNs were fixed, cryosectioned, and double-labeled Žwith antibodies against gelatinase and lactoferrin. as described in Section 2. For labeling of gelatinase, protein A-10 gold particles were used. For labeling of lactoferrin, protein A-20 was used. L, lactoferrin granule; DL, double-labeled granules; G, gelatinase granules. Note that some small and large vesicles Žv. do not contain label. In inset, biotinylated antigelatinase antibodies were used and streptavidin gold-10. N, nucleus =52,500; =70,000. Reprinted with permission from Kjeldsen et al., 1993 and Blood.
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al., 1988; Calafat et al., 1990., proteinase 3 ŽCalafat et al., 1990; Csernok et al., 1990., and a-1 antitrypsin ŽMason et al., 1991. colocalize with some MPO-positive granules. Bactericidal factors such as defensins ŽGanz et al., 1985; Rice et al., 1987; Ganz and Lehrer, 1998., azurophil-derived bactericidal factors ŽGabay et al., 1986., and bactericidal permeability-increasing proteins ŽWeiss and Olsson, 1987., which were previously called cationic proteins, have been found in some azurophilic granules ŽGabay et al., 1989.. Lysozyme has been found in both
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azurophilic and specific granules ŽCramer and Breton-Gorius, 1987; Mutasa, 1989.. Of the 10 antimicrobial proteins of known sequence in the human azurophil granules ŽCampanelli et al., 1990., two have unique primary structures Žlysozyme and bactericidal permeability-increasing protein., while the remaining eight fall into two families of four members each: the defensins Žwhich compromise 30–50% of granule proteins. on one hand, and cathepsin G, elastase, proteinase-3, and azurocidin on the other. These latter four proteins
Fig. 5. Portions of human neutrophils double-labeled for LAMP-2 with a monoclonal antibody and rabbit polyclonal antibody against human albumin to identify various organelles. The large gold ŽGAM-10. labels the location of LAMP-2 in vesicles Žarrows., whereas the small gold ŽGAR-5. Žarrowheads. detects albumin. Albumin Žalb. ŽGAR-5. Žarrowheads. is found in different vesicular structures from LAMP-2 ŽGAM-10., Žarrows.. =110,000.
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can be termed ‘‘serprocidins’’ to denote that they are closely related to serine proteases with microbicidal activity ŽCampanelli et al., 1990.. Very little is known about the limiting membrane of azurophilic granules, but CD63 ŽFig. 3a. ŽKuijpers et al., 1991; Cham et
al., 1994. and CD68 ŽSaito et al., 1991. are present. We had anticipated that the lysosomal-associated membrane proteins ŽLAMPs. would be found there, but such was not the case ŽBainton and August, 1988; Cieutat et al., 1998.. Rather, LAMPs were
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absent in all identified granule populations, but were consistently found in the membranes of vesicles, ŽMVBs., and ŽMLCs., which are identified by their concentric arrays of internal membranes ŽCieutat et al., 1998.. These organelles will be illustrated later. 3.3. Specific granules The specific or secondary granule, which by definition does not contain MPO, contains lactoferrin ŽFig. 2., lysozyme, B12 binding proteins, and other proteins ŽBorregaard et al., 1993; Borregaard and Cowland, 1997.. These MPO-negative granules vary greatly in size, shape, electron-lucency, isopycnic density, and granule content. However, they can be loosely categorized by the distribution of two proteins, lactoferrin and gelatinase. Approximately 16% of the MPO-negative granules contain only lactoferrin and 24% contain only gelatinase, but 60% contain both marker enzymes. The common leukocyte adhesion receptor B 2 was found on the membrane of specific granules, representing CD 11b ŽFig. 3b.. 3.4. Gelatinase granules By ultrastructure alone, three types of MPO-negative granules can be identified: peroxidase-negative granules containing gelatinase but no lactoferrin, peroxidase-negative granules containing lactoferrin but no gelatinase, and peroxidase-negative granules containing both lactoferrin and gelatinase Žsee Fig. 4. ŽKjeldsen et al., 1993.. This heterogeneity may be a result of overlapping synthesis and packaging of different granule proteins during granulopoiesis and is functionally significant, inflammatory mediators cause the gelatinase-containing granules to be re-
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leased from the cells more readily than those containing lactoferrin ŽBorregaard and Cowland, 1997.. 3.5. Secretory Õesicles Secretory vesicles have recently been discovered within neutrophils. These vesicles are distinct from the azurophilic, specific, and gelatinase granules and have been defined as intracellular organelles that contain CD35 and latent alkaline phosphatase ŽBorregaard and Cowland, 1997; Borregaard et al., 1992, 1993; Kjeldsen et al., 1993.. The latter enzyme is located on the luminal side of the vesicle membrane and can, therefore, be identified in the presence of detergent as latent alkaline phosphatase. This localization was also demonstrated by enzyme cytochemistry ŽRobinson and Kobayashi, 1991.. This work helped to clarify the location of alkaline phosphatase in resting and stimulated PMN. Further, these secretory vesicles contain plasma proteins such as albumin Žsee Fig. 5., which are not synthesized by the cells but are endocytosed from plasma. These represent a specialized form of endocytic vesicle. Secretory vesicles are transported to the cell surface after the stimulations by formyl methionyl-leucyl phenylalanine or certain cytokines ŽBorregaard et al., 1993.. 3.6. Localization of LAMPs and the M 6-P GP recognition marker During granulocyte differentiation in the bone marrow, neutrophilic leukocyte precursors synthesize large amounts of lysosomal enzymes. These enzymes are sequestered into azurophilic storage granules until used days later for digesting of phagocytized microorganisms following leukocyte emigration to
Fig. 6. Electron micrograph of a frozen thin section of myelocytes immunolabeled for LAMP-2. Large vesicles ŽV. and the MLC are labeled with GAM-10 Žarrow.. Note the absence of labeling in large azurophil granules as well as in the specific granules and plasma membrane ŽPM.. In inset, higher magnification of another MLC positive for LAMP-2. =52,000, inset, =86,000. Reprinted with permission from Cieutat et al., 1998 and Blood. Fig. 7. Ža,b,c. Immunolabeled cryosection labeled with rabbit anti-bovine polyclonal antibody against CI-M6PR and labeled with GAR-5. The receptor was found in the multivesicular bodies ŽMVB. but absent in the multi-lamellated compartments ŽMLC. Žsee a and c.. Occasionally, peroxidase-negative granule membranes Žarrows. appeared labeled as well as the plasma membrane. Magnification Ža–b. =63,000, Žc. =60,450.
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inflamed tissues. This azurophil granule population has previously been defined as a primary lysosome, i.e., a membrane-bound organelle containing acid hydrolases that have not entered into a digestive event ŽDeDuve and Wattiaux, 1966.. In the study by Cieutat et al. Ž1998., azurophil granules were purified and shown to contain large amounts of Man 6-P GP but little LAMP. In addition, the fine structural localization of Man 6-P GP and LAMP was investigated at various stages of maturation in human bone marrow and blood. Man 6-P GPs Žnot illustrated. were present within the azurophilic granules at all stages of maturation, in typical MVBs and in MLCs, LAMP was absent in all identified granule populations, but was consistently found in the membranes of vesicles, MVB, and MLC ŽFig. 6, see inset.. The
latter compartment has not been previously described in this cell type. In conclusion, the azurophilic granules, which contain an abundance of lysosomal enzymes and Man 6-P GP, lack the LAMP glycoproteins. By current criteria, therefore, they cannot be classified as lysosomes, but rather may have the functional characteristics of a regulated secretory granule. The true lysosomes of the resting neutrophil are probably the MVBs and MLC. Finally, the typical ‘‘dense bodies’’ or mature lysosomes described in other cells in vivo are not present in resting neutrophils. Although not illustrated in this paper, one additional point should be made about the utility of applying immunocytochemistry to frozen thin sections of the fractions themselves. The subcellular distribution of LAMPs and Man 6-P GP in fractions
Fig. 8. Phagocytizing human PMN labeled with GAM-10 for LAMP-2. Note that the plasma membrane ŽPM. and bacteria ŽB. are not labeled, nor is the early endocytic vacuole ŽE. on the right. The large phagocytic vacuole or secondary lysosome ŽL. contains degenerating bacteria ŽB.. The membrane of the phagocytic vacuole Žarrows. is labeled with gold. Magnification =52,000.
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of mature PMNs ŽCieutat et al., 1998. revealed that Man 6-P GP colocalized with MPO in the most dense fraction while LAMPs were found in the less dense fractions that contain lactoferrin and gelatinase and in small amounts in the lighter membrane fractions. However, direct examination of these fractions revealed that LAMPs were not in specific granules but were in MLC and MVB just as they were in intact cells ŽCieutat et al., 1998.. 3.7. Localization of cation-independent mannose 6phosphate receptor (CI-M6PR) Recently, we used the affinity-purified rabbit polyclonal antibody developed by Peter Lobel’s laboratory against the bovine CI-M6PR. Our preliminary data, illustrated in Fig. 7Ža,b,c., demonstrate that CI-M6PR is found in MVB but is absent in the MLC. This adds further credence to the suggestion that MLCs are late endosomes since they are LAMP-positive and receptor-negative Žsee review by Geuze, 1998..
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3.8. LAMPs are present in secondary lysosomes Finally, by studying human PMNs that have phagocytized bacteria ŽFig. 8., we now can demonstrate that the phagocytic vacuole contains LAMPs, whereas the plasma membrane and early phagosome does not. This means that MVB andror MLC must fuse with the newly formed endosome. 3.9. Surface labeling of plasma membrane proteins Two membrane adhesion proteins have been found on the tips of neutrophil microvilli in resting neutrophils, L-selectin ŽFig. 9. and P-selectin glycoprotein-1 ŽBorregaard et al., 1994; Moore et al., 1995; Bruehl et al., 1996, 1997.. Here the immunocytochemistry was performed by surface labeling the intact cells in suspension followed by embedding in Epon and sectioning rather than by frozen thin sectioning. This is suitable when it can be anticipated that the antigen is primarily on the external surface
Fig. 9. High magnification of human PMN plasma membrane showing the positioning of L-selectin on microvilli. A typical segment of PMN plasma membrane showing six microvilli with groups of gold particles labeling L-selectin localized at the tips Žarrows.. Note that the number of particles per microvillus is independent of microvillus length. Gold particles on the planar cell surface can also be seen Žarrowheads.. About 90% of the label can be seen on the microvilli. =42,000 Žmodified from Bruehl et al., 1996..
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of the plasma membrane. In quantitative studies on the ectoenzyme, 5-nucleotidase ŽHowell et al., 1987. showed that labeling on frozen thin sections with a polyclonal antibody is only of 15% efficiency. Labeling the cells in suspension as was done in Fig. 9 resulted in a fourfold increase in labeling efficiency.
tion of the manuscript. This work was supported by DK-10486 and HLB-31610 from the National Institutes of Health.
References
3.10. Limitations and artifacts Whereas the artifactual redistribution of lead phosphate reaction product has been reported during enzyme cytochemistry ŽCramer et al., 1988., relatively few artifacts have been seen with immunocytochemistry, particularly if the cells are appropriately fixed and the dilutions of the primary antibodies and immunogold are optimized. In areas near azurophilic granules with high labeling for Man 6-P GP, we frequently found ‘‘spillage’’ of the antigen in the nearby cytoplasm, ŽCieutat et al., 1998.. This artifact has been previously observed with the lowmolecular-weight defensins, ŽGanz et al., 1985. and proteinase 3, ŽCsernok et al., 1990.. A possible explanation is that the membranous compartments are cut open by the cryostat method, and some of the granule contents become soluble and may partially relocate during the subsequent incubation procedures. A word of caution about the use of Protein A in PMNs fixed with formaldehyde fixative alone. Occasionally, but inconsistently, controls were faintly labeled with Protein A gold, mainly in large clear vesicles. The addition of a fixative with glutaraldehyde yielded cleaner controls. For this reason, we preferred not to use Protein A on PMN, and if we did, we confirmed the observations using biotinylated primary antibodies ŽKjeldsen et al., 1993.. Finally, the major limitation is that one is only able to visualize about 15% of the antigen present ŽHowell et al., 1987.. Therefore, proteins present in low amounts may not be seen, and interpretations of negative data must be made with caution.
Acknowledgements Dr. Bainton thanks Ivy Hsieh and Yvonne Jacques for their excellent technical assistance, David Geller for his help in editing, and Silvia Molina for prepara-
Baggiolini, M., 1978. The neutrophil: function and clinical disorders. In: Klebanoff, S.J., Clark, R.A. ŽEds... Elsevier, North Holland, p. 556. Bainton, D.F., 1999. Developmental biology of neutrophils and eosinophils. In: Gallin, J.I., Snyderman, R. ŽEds.., Inflammation: Basic Principles and Clinical Correlates, 3rd edn. Lippincott Williams & Wilkins, Philadelphia, pp. 13–34. Bainton, D.F., August, J.T., 1988. Multivesicular bodies of human neutrophils ŽPMN. not granules, immunolable with the two major lysosomal membrane glycoproteins hLAMP-1 and hLAMP-2. J. Histochem. Cytochem. 36, 953. Bainton, D.F., Ullyot, J.L., Farquhar, M.G., 1971. The development of neutrophilic polymorphonuclear leukocytes in human bone marrow: origin and content of azurophil and specific granules. J. Exp. Med. 134, 907. Bainton, D.F., Miller, L.J., Kishimoto, T.K., Springer, T.A., 1987. Leukocyte adhesion receptors are stored in peroxidase-negative granules of human neutrophils. J. Exp. Med. 166, 1641. Borregaard, N., Cowland, B.J., 1997. Granules of the human neutrophilic polymorphonuclear leukocyte. Blood 89, 3503. Borregaard, N., Kjeldsen, L., Rygaard, K., Bastholm, L., Nielsen, M.H., Sengelov, H., Bjerrum, O.W., Johnsen, A.H., 1992. Stimulus-dependent secretion of plasma proteins from human neutrophils. J. Clin. Invest. 90, 86. Borregaard, N., Lollike, K., Kjeldsen, L., Sengelov, H., Bastholm, L., Nielsen, M.H., Bainton, D.F., 1993. Human neutrophil granules and secretory vesicles. Eur. J. Haematol. 51, 187. Borregaard, N., Kjeldsen, L., Sengelov, H., Diamond, M.S., Springer, T.A., Anderson, H.C., Kishimoto, T.K., Bainton, D.F., 1994. Changes in subcellular localization and surface expression of L-selectin, alkaline phosphatase, and Mac-1 in human neutrophils during stimulation with inflammatory mediators. J. Leukoc. Biol. 56, 80. Bruehl, R.E., Springer, T.A., Bainton, D.F., 1996. Quantitation of l-selectin distribution on human leukocyte microvilli by immunogold labeling and electron microscopy. J. Histochem. Cytochem. 44, 835. Bruehl, R.E., Moore, K.L., Lorant, D.E., Borregaard, N., Zimmerman, G.A., McEver, R.P., Bainton, D.F., 1997. Leukocyte activation induces surface redistribution of P-selectin glycoprotein ligand-1. J. Leukoc. Biol. 61, 489. Calafat, J., Goldschmeding, R., Ringeling, P.L., Janssen, H., van der Schoot, C.E., 1990. In situ localization by double-labeling immunoelectron microscopy of anti-neutrophil cytoplasmic autoantibodies in neutrophils and monocytes. Blood 75, 242. Campanelli, D., Detmers, P.A., Nathan, C.F., Gabay, J.E., 1990. Azurocidin and a homologous serine protease from neutrophils. J. Clin. Invest. 85, 904.
D.F. Baintonr Journal of Immunological Methods 232 (1999) 153–168 Cham, B.P., Gerrard, J.M., Bainton, D.F., 1994. Granulophysin is located in the membranes of azurophilic granules in human neutrophil and mobilizes to the plasma membrane following cell stimulation. Am. J. Pathol., 1369. Cieutat, A.-M., Lobel, P., August, J.T., Kjeldsen, L., Sengelov, H., Borregaard, N., Bainton, D.F., 1998. Azurophilic granules of human neutrophilic leukocytes are deficient in lysosome-associated membrane proteins but retain the mannose 6-phosphate recognition. Blood 91, 1044. Cohn, Z.A., Hirsch, J.G., 1960. The isolation and properties of the specific cytoplasmic granules of rabbit polymorphonuclear leucocytes. J. Exp. Med. 112, 983. Cramer, E.M., Breton-Gorius, J., 1987. Ultrastructural localization of lysozyme in human neutrophils by immunogold. J. Leukoc. Biol. 41, 242. Cramer, E., Pryzwansky, K.B., Villeval, J.-L., Testa, U., BretonGorius, J., 1985. Ultrastructural localization of lactoferrin and myeloperoxidase in human neutrophils by immunogold. Blood 65, 423. X Cramer, E.M., Werb, Z., Bainton, D.F., 1988. Pitfalls in ecto-5 nucleotidase enzyme cytochemistry as demonstrated by the immunogold-labelling technique on macrophages. Histochem. J. 20, 108. Csernok, E., Ludemann, J., Gross, W.L., Bainton, D.F., 1990. ¨ Ultrastructural localization of proteinase 3, the target antigen of anti-cytoplasmic antibodies circulating in Wegener’s granulomatosis. Am. J. Pathol. 137, 1113. Damiano, V.V., Kucich, U., Murer, E., Laudenslager, N., Weinbaum, G., 1988. Ultrastructural quantitation of peroxidase- and elastase-containing granules in human neutrophils. Am. J. Pathol. 131, 235. DeDuve, C., Wattiaux, R., 1966. Functions of lysosomes. Annu. Rev. Physiol. 28, 435. Gabay, J.E., Heiple, J.M., Cohn, Z.A., Nathan, C.F., 1986. Subcellular location and properties of bactericidal factors from human neutrophils. J. Exp. Med. 164, 1407. Gabay, J.E., Scott, R.W., Campanelli, D., 1989. Antibiotic proteins of human polymorphonuclear leukocytes. Proc. Natl. Acad. Sci. U. S. A. 86, 5610. Ganz, T., Lehrer, R.I., 1998. Antimicrobial peptides of vertebrates. Curr. Opin. Immunol. 10 Ž1., 41. Ganz, T., Selsted, M.E., Szklarek, D., Harwig, S.S.L., Daher, K., 1985. Defensins: natural peptide antibiotics of human neutrophils. J. Clin. Invest. 76, 1427. Geuze, H.J., 1998. The role of endosomes and lysosomes in MHC class II functioning. Immunol. Today 19, 282. Graham, R.C., Karnovsky, M.J., 1966. The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem. 14, 291. Griffiths, G., 1993. Fine Structure Immunocytochemistry. Springer, Berlin, Chap. 3, pp. 26–80. Griffiths, G., Simmons, K., Warren, G.W., Tokuyasu, K.T., 1983. Immunoelectron microscopy using thin, frozen sections: application to studies of the intracellular transport of semliki forest virus spike glycoproteins. Methods Enzymol. 96, 466. Howell, K.E., Reuter-Carlson, U., Devaney, E., Luzio, J.P., Fuller,
167
S.D., 1987. One antigen or gold? A quantitative analysis of X immunogold labeling of plasma membrane 5 -nucleotidase in frozen thin sections. Eur. J. Cell Biol. 44, 318. Kjeldsen, L., Bainton, D.F., Sengelov, H., Borregaard, N., 1993. Structural and functional heterogeneity among peroxidase negative granules in human neutrophils: identification of a distinct gelatinase containing granule subset by combined immunocytochemistry and subcellular fractionation. Blood 82, 3183. Kuijpers, T.W., Tool, A.T.J., van der Schoot, D.E., Ginsel, L.A., Onderwater, J.J.M., Roos, D., Verhoeven, A.J., 1991. Membrane surface antigen expression on neutrophils: a reappraisal of the use of surface markers for neutrophil activation. Blood 78, 1105. Liou, W., Geuze, H.J., Slot, J.W., 1996. Improving structural integrity of cryosections for immunogold labeling. Histochem. Cell Biol. 106, 41. Mason, D.Y., Cramer, E.M., Masse, ´ J.-M., Crystal, R., Bassot, J.-M., Breton-Gorius, J., 1991. Alpha1-antitrypsin is present within the primary granules of human polymorphonuclear leukocytes. Am. J. Pathol. 139, 623. McCaffery, J.M., Farquhar, M.G., 1995. Localization of GTPases by indirect immunofluorescence and immunoelectron microscopy. Methods Enzymol. 257, 259. McLean, I.W., Nakane, P.K., 1974. Periodate–lysine–paraformaldehyde fixative for immunoelectron microscopy. J. Histochem. Cytochem. 22, 1077. Moore, K.L., Patel, K.D., Bruehl, R.E., Fugang, L., Johnson, D.A., Lichenstein, H.S., Cummings, R.D., Bainton, D.F., McEver, R.P., 1995. P-selectin glycoprotein ligand-1 mediates rolling of human neutrophils on P-selectin. J. Cell Biol. 128 Ž4., 661. Mutasa, H.C., 1989. Combination of diaminobenzidine staining and immunogold labeling: a novel technical approach to identify lysozyme in human neutrophil cells. Eur. J. Cell Biol. 49, 319. Peters, P.J., 1999. Cryo-immunogold electron microscopy. Current Protocols in Cell Biology, 4.7.1–4.7.12. Rice, W.G., Ganz, T., Kinkade, J.M. Jr., Selsted, M.E., Lehrer, R.I., Parmley, R.T., 1987. Defensin-rich dense granules of human neutrophils. Blood 70, 757. Robinson, J.M., Kobayashi, T.A., 1991. Novel intracellular compartment with unusual secretory properties in human neutrophils. J. Cell Biol. 113, 743. Saito, N., Pulford, K.A.F., Breton-Gorius, J., Masse, ´ J.-M., Mason, D.Y., Cramer, E.M., 1991. Ultrastructural localization of the CD68 macrophage-associated antigen in human blood neutrophils and monocytes. Am. J. Pathol. 139, 1053. Slot, J.W., Geuze, H.J., 1984. Gold markers for single and double immunolabeling of cryosections. In: Polak, J.M., Varndell, I.M. ŽEds... Immunolabeling for Electron Microscopy. Elsevier, Oxford, UK, p. 129. Stenberg, P.E., Shuman, M.A., Levine, S.P., Bainton, D.F., 1984. Optimal techniques for the immunocytochemical demonstration of b-thromboglobulin, platelet factor 4, and fibrinogen in the alpha granules of unstimulated platelets. Histochem. J. 16, 983. Tokuyasu, K.T., 1984. Immuno-cryoultramicrotomy. In: Polak,
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J.M., Varndell, I.M. ŽEds.., Immunolabelling for Electron Microscopy. Elsevier, Amsterdam, pp. 71–82. Tokuyasu, K.T., 1989. Use of polyŽvinylpyrrolidone. and polyŽvinyl alcohol. for cryoultramicrotomy. Histochem. J. 21, 163. Valenzano, K.J., Kallay, L.M., Lobel, P., 1993. An assay to detect glycoproteins that contain mannose 6-phosphate. Anal. Biochem. 209, 156.
Weiss, J., Olsson, I., 1987. Cellular and subcellular localization of the bactericidalrpermeability-increasing protein of neutrophils. Blood 69, 652. Zucker-Franklin, D., Hirsch, J.G., 1964. Electron microscope studies on the degranulation of rabbit peritoneal leukocytes during phagocytosis. J. Exp. Med. 120, 569.
Distinct granule populations in human neutrophils and lysosomal organelles identified by immuno-electron microscopy Dorothy F. Bainton
)
Department of Pathology, UniÕersity of California-San Francisco, Rm. S-115, 513 Parnassus AÕenue, San Francisco, CA 94143-0400, USA
Abstract In this paper, we illustrate the fine structural localization of distinct marker proteins in the organelles of human neutrophils and outline our preferred methods for processing ultrathin cryosections for use with immunoelectron microscopy. Previous work has determined the subcellular localization of certain marker proteins within intact polymorphonuclear neutrophilic leukocytes ŽPMN. and PMN fractions. These are as follows: myeloperoxidase ŽMPO. for azurophilic granules, lactoferrin for specificrsecondary granules, gelatinase for gelatinasertertiary granules, albumin for the secretory vesicles, and HLA class I and L-selectin for the plasma membrane. In addition to analyzing the heterogeneity of the PMN granule populations, new information on the lysosomal system of this cell is reviewed and extended by the localization of the lysosome-associated membrane proteins ŽLAMPs. and the cation-independent mannose 6-phosphate receptor ŽCI-M6PR.. LAMPs were absent in all identified granule populations, but were found in the membranes of vesicles, multivesicular bodies ŽMVB., and multilaminar compartments ŽMLC.. We show here that MVB contain CI-M6PR whereas MLC do not. Furthermore, since MLC contain LAMPs but not the receptor, they probably correspond to the late endosome. By current criteria, the true lysosomes of the resting PMN are MVB and MLC. Finally, although azurophil granules contain acid hydrolases their membranes do not contain LAMPs and they cannot be classified as lysosomes, but rather are more similar to regulated secretory granules. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Double-labeling; Granules; Lysosomes; Marker proteins
1. Introduction The ability of each type of leukocyte to conduct its special activities depends on its content of diverse AbbreÕiations: CI-M6PR, cation-independent mannose 6phosphate receptor; LAMPS, lysosome-associated membrane proteins; Man 6-P GP, mannose 6-phosphate containing glycoproteins; MVB, multivesicular bodies; MLC, multilaminar compartments; MPO, myeloperoxidase; PMN, polymorphonuclear neutrophilic leukocyte ) Corresponding author. Tel.: q1-415-476-4451; fax: q1-415476-0816; e-mail: [email protected]
chemical substances stored in concentrated form in membrane-bound cytoplasmic granules. The relationship between leukocyte granule content and cell function was first clearly documented in neutrophils by Cohn and Hirsch Ž1960., who successfully isolated PMN granules from rabbit peritoneal exudates and found that they contained a number of lysosomal enzymes as well as substances with antibacterial activity. These investigators further demonstrated that when microorganisms are engulfed by PMN, the cells degranulate, i.e., the storage granules discharge their contents into the digestive vacuole ŽZucker-
0022-1759r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 1 7 5 9 Ž 9 9 . 0 0 1 7 3 - 8
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Franklin and Hirsch, 1964.; subsequently, the organisms are killed and digested. These cytoplasmic granules are formed early in development, during the differentiation and proliferation of the immature cells in the bone marrow. Additional work by numerous investigators using enzyme cytochemical studies on PMN granules in situ and biochemical assays on granule fractions established that there were two basic chemically distinct granules types: MPO-positive azurophilic granules, which appear early in maturation during the promyelocyte stage, and MPO-negative specific granules, which are formed later during the myelocyte stage ŽFig. 1.. The MPO-positive azurophil Žprimary. granules are formed only during the promyelocyte stage and are reduced in number by mitosis. It was recently shown that the MPO-negative granules constitute a continuum from early appearing granules Žmyelocyte stage. that contain lactoferrin but no gelatinase, to granules that contain both lactoferrin and gelatinase Žmetamyelocyte stage., to granules that contain gelatinase but no lactoferrin Žband cell stage.. The aforementioned granules that contain lactoferrin are referred to as specific Žsecondary. granules while the granules that
contain gelatinase but not lactoferrin are referred to as gelatinase Žtertiary. granules. Thus, the mature circulating neutrophil contains the three major granule types Žextensively reviewed by Borregaard and Cowland, 1997; Bainton, 1999.. Within the framework of this well-known setting, this paper concentrates on relatively recent data that have been gleamed by using immunocytochemistry on developing and mature PMN. Although technically more difficult than enzyme cytochemistry, many more reagents Žantibodies and soluble receptors. are available to be applied with immunocytochemistry and a more complete picture of granule content and heterogeneity can be appreciated and new organelles identified ŽTable 1..
2. Materials and methods Our laboratory has explored many procedures for the study of the distribution of antigens within neutrophils and platelets ŽStenberg et al., 1984.. This paper will concentrate on those particular techniques that we consider optimal for leukocytes and does not intend to provide a comprehensive review, as a
Fig. 1. Diagrammatic representation of PMN life-span and stages of maturation. For discussion, see text. Out of every 100 nucleated cells in marrow, 0.5% are myeloblasts, 5% promyelocytes, 12% myelocytes, 22% metamyelocytes and bands, 20% are maturing and mature neutrophilic cells, yielding a total of ; 60% developing neutrophils in normal human marrow. The azurophilic Žprimary. granules, which are MPO-positive, are shown as solid black dots and the other granules are shown as open dots. Basically, the MPO-negative granules may be divided into specificrsecondary granules and gelatinasertertiary granules based on their relative content of lactoferrin and gelatinase. Modified from Bainton et al., 1971.
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Table 1 Specific markers for neutrophil compartments Granules
Content Markers
Membrane Markers
Azurophilic Žprimary.
Myeloperoxidase, lysosomal enzymes, M6P-glycoproteins Lactoferrin Gelatinase b
CD63, CD68
Specific Žsecondary. Gelatinase Žtertiary. Other compartments Secretory vesicles Multivesicular bodies ŽMVB. Multilaminar compartments ŽMLC. Plasma membrane
Albumin M6P-glycoproteins M6P-glycoproteins
number of excellent examples of such are already available ŽGriffiths, 1993; Griffiths et al., 1983; Tokuyasu, 1984; McCaffery and Farquhar, 1995; Liou et al., 1996; Peters, 1999.. With the exception of Fig. 7Ža–c., detailed methods have been previously published as noted in the references in the legends. For the localization of the CI-M6PR receptor in Fig. 7, the affinity-purified rabbit antibody made against bovine CI-M6PR ŽPL 603CI. was used. This was obtained from Dr. Peter Lobel, Center for Advanced Technology, Piscataway, NJ, 08854-5638.
CD11b, Cytochrome b CD11b, Cytochrome b
Alkaline phosphatase CD11B, Cytochrome b LAMP-2rLAMP-1 CI-M6P receptor LAMP-2rLAMP-1 HLA-1, L-selectin
Ž2. 2% paraformaldehyde, 0.05% glutaraldehyde. In general, this fixative has been the most satisfactory. If the glutaraldehyde is lower than 0.05%, there is no improvement in morphology. Ž3. Occasionally we have been able to use 0.5–2% glutaraldehyde, particularly if we need to stabilize the contents of granules of lower molecular weight and prevent ‘‘spillage’’ from the matrix of the granules Ždiscussed later.. 2.2. Immunogold labeling of ultrathin cryosections (Figs. 1–8)
2.1. FixatiÕes The most important step in immunocytochemical localization is the choice of fixative. The objective of fixation is to immobilize the antigens while retaining their antigenicity and preserving acceptable ultrastructure ŽGriffiths, 1993; McCaffery and Farquhar, 1995.. Glutaraldehyde is usually the superior fixative for preservation of cell structure, but frequently results in loss of antigenicity. Paraformaldehyde, particularly the periodic–lysine–paraformaldehyde ŽPLP. described by McLean and Nakane Ž1974., usually retains more antigenicity and gives acceptable, though sometimes not optimal organelle morphology. We have been pleased also with a double fixative of 2% paraformaldehyde and 0.05% glutaraldehyde Žsee Section 2.2.7.. We usually proceed as follows. Ž1. Initially, we try PLP fixation. If antigenicity is satisfactory, we next try to add glutaradehyde to improve the morphology, as outlined below.
2.2.1. Single labeling (examples, Fig. 3aFigs. 6–8) Ž1. Fixed leukocytes Žwithout washing. are cryoprotected by immersion in 2.1 M sucrose containing polyvinylpyrolidone Žsee Section 2.2.7. overnight at 48C and then frozen in liquid N2 ŽTokuyasu, 1989; Peters, 1999.. Ž2. Thin sections are cut on ultracut ultramicrotome with cryo-attachment equipment at y1158C and placed on Formvar and carbon-coated nickel EM grids. All the incubations and washes are carried out by floating the grid with section side down on droplets. Care should be taken to avoid wetting the back of the grid. All incubations are performed at room temperature unless otherwise indicated. All the phosphate-buffered saline ŽPBS. steps for washing contain 0.1% bovine serum albumin ŽBSA.. Water used in all the preparation is double-distilled deionized water. Ž3. After sectioning, grids are placed on PBS containing 0.2% glycine.
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Ž4. Grids are then incubated for 10 min in 1% each of skim milk, BSA, fish gelatin and ovualbumin in PBS, followed by two 2-min washes in PBS. Ž5. The primary antibody is then incubated for 60–120 min. It is diluted in PBS containing an additional 500 mM of NaCl. Multiple dilutions are tested. Ž6. Grids are washed in PBS three times for 5 min each followed by three more washes of the BSArPBS buffer with a higher pH Ž8.1.. Ž7. Grids are then incubated for 1 h with goldlabeled secondary antibody, which is diluted in 0.1% BSArPBS ŽpH 8.1. and used at the dilution recommended by the supplier. The most commonly used dilutions are 1:50 or 1:20, of the goat anti-rabbit ŽGAR. or goat anti-mouse ŽGAM. conjugated with 5 or 10 nm gold particles, for polyclonal or monoclonal primary antibody, respectively. Ž8. Grids are washed in PBS at least three times for 5 min each to remove the unbound immunogold. Ž9. Grids are refixed in 0.5% glutaraldehyderPBS for 5 min followed by three changes of H 2 O washing in 5 min. Ž10. Grids are floated on neutral oxalate uranyl acetate Žsee Section 2.2.7. for 5 min to stabilize the membranes, followed by three changes of H 2 O. Ž11. Finally, grids are embedded and stained with a mixture of methyl cellulose and uranyl acetate Žsee Section 2.2.7. for two changes, 5 min each, on a cold block at 48C. Grids are then picked up on loops. As much of the excess fluid as possible is removed and the grids are allowed to air dry. Ž12. Grids are examined in a transmission electron microscope at 60 or 80 KV. Ž13. Controls consist of replacing the primary antibody with normal rabbit or mouse serum. 2.2.2. Applying both enzyme cytochemistry (prefreezing) and imunocytochemistry on frozen thin sections (examples, Figs. 2 and 3b) Fixed suspended leukocytes were washed in 0.1 M phosphate buffer with 5% sucrose. The method of Graham and Karnovsky Ž1966. was used for the demonstration of MPO in the azurophil granules. The cells were resuspended and preincubated for 10 min at room temperature in 5 mg DAB Ž3,3-diaminobenzidine, Sigma Grade II. dissolved in 10 ml 0.05 M Tris buffer pH 7.6. Then 0.1 ml of 1% H 2 O 2
was added to DAB–Tris solution and incubated for 60 min at room temperature Žstock solution of H 2 O 2 should be replaced frequently.. The cells were rinsed three times with 0.05 M Tris buffer with 5% sucrose. The cells were then pelleted and the cell pellet was embedded in 2.1 M sucrose, frozen, and sectioned as described above. 2.2.3. Double-labeling with monoclonal and polyclonal antibodies and different sizes of gold (example, Fig. 5) Double-labeling experiments to localize a monoclonal antibody and a polyclonal antibody can be performed by combining them in the same drops in the primary antibody incubation previously described in Section 2.2.1. After washing, the smaller gold for GAR-5 is applied first, then the larger gold, GAM-10, is applied in subsequent steps. An example of this method is illustrated in Fig. 5, where the monoclonal antibody for LAMP-2, H 4 B 4 , was labeled with GAM-10 and the polyclonal antibody for albumin was labeled with GAR-5. 2.2.4. Double-labeling with two polyclonal antibodies and two sizes of protein a-gold as described by Slot and Geuze (1984) (example, Fig. 4) A double-labeling experiment to localize gelatinase and lactoferrin was performed using protein A. The probes protein A-10 and A-20 Žobtained from Dr. G. Posthuma, Utrecht, Holland. were used at a 1:50 dilution. Rabbit antibody against gelatinase was applied and labeled with protein A-10. Before the second antibody was applied, 0.5% glutaraldehyderPBS was applied, followed by free protein A Ž0.05 mgrml.. Subsequently, an antibody to lactoferrin ŽAccurate Chemical, Westbury, NY. was applied, followed by protein A-20. Sections were also labeled in reverse, using the large gold label to label gelatinase and the small gold to label lactoferrin. Non-immune-purified rabbit lgG was used as a control. 2.2.5. Labeling with biotinylated antibody and probing with streptaÕidin gold (example, inset Fig. 4) The primary antibody can be biotinylated and detected with a secondary application of streptavidin gold ŽKjeldsen et al., 1993.. We used the same process as previously described except we omitted the skim milk and ovualbumin in the blocking steps.
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Fig. 2. Portion of a PMN stained for MPO to mark the azurophil granules Žag., then immunolabeled for lactoferrin. Many of the MPO-negative specific granules have gold label within their matrix Žarrows., but several contain no label ŽU .. =81,000. Modified from Bainton et al., 1987.
2.2.6. Labeling with a biotinylated soluble receptor and probing with streptaÕidin-gold for the receptor’s ligand Man 6P-containing proteins can be localized by the sCI-MPR probe ŽValenzano et al., 1993.. This probe is a biotinylated derivative of a soluble form of the CI-MPR isolated from fetal bovine serum. The probe binds Man 6-P-containing proteins ŽMan 6-P GP. with nanomolar affinity, and is used in much the same manner as an antibody is to detect a specific substrate. The biotinylated receptor binds its ligand; subsequently the biotin moiety binds with streptavidin-gold for detection at the fine structural level ŽCieutat et al., 1998.. Ultrathin cryosections were incubated for 2 h at 48C, in 1% BSA, 0.2% Tween-20
and PBS: and then incubated overnight with the biotinylated sCI-MRP Ž1 mgr100 ml. in PBS plus 1% BSA and 5 mmolrl b-glycerolphosphate. The phosphorylated enzymes were detected the next day by streptavidin-gold, 5 or 10, with O.D. at 520 nm of approximately 5.0 ŽSigma. diluted to 1:20. The sections were then further processed as described above. For double-labeling, the antibody was applied first, after washing. The biotinylated sCl-MPR was then applied and allowed to incubate overnight. For controls, the sections were preincubated with 10 mmolrl of man 6-P for 1 h before applying the biotinylated sCl-MPR. The grids were then further processed as described above. Using this technique, Cieutat et al. Ž1998., were able to determine that phosphorylated
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forms of the Man 6-P GP were present in large amounts in azurophil granules and smaller amounts in multivesicular bodies ŽMVB. and in multilaminar compartments ŽMLC..
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Žc. 40 ml water Žd. pH to 7.4 with HCl Že. 1 g sucrose Žf. add water to make 100 ml Žg. cells are fixed for 1 h at 228C
2.2.7. Reagents 2.2.7.1. McLean and Nakane’s PLP fixatiÕe. ŽA. 0.1 M lysiner0.05 M sodium phosphate buffer, pH 7.4. Dissolve 1.827 g lysine HCl in 50 ml of water, adjust pH to 7.4 with 0.1 M Na 2 HPO4 . Add 0.1 M phosphate buffer, pH 7.4, to make final volume of 100 ml. Store at 48C. ŽB. Preparation of 16% paraformaldehyde stock: Mix 16 g of paraformaldehyde in 100 ml of water. Heat in water at 60–708C stirring occasionally to depolymerize. Slowly add 2–5 drops of 1 N NaOH until solution is clear, about 30 min, filter before use. The solution will last about 2 weeks if stored at 48C. Just before use, combine three parts of ŽA. to one part ŽB., and then add sodium meta-periodate to a final concentration of 0.01 M Ž21.4 mg of sodium meta-periodate to 10 ml of fixative.. Cells are fixed for 4 h at 228C. 2.2.7.2. 2% paraformaldehyder 0.05% glutaraldehyde r 0.1 M phosphate buffer, pH 7.4. Mix the components, as follows: Ža. 12.5 ml 16% paraformaldehyde Žb. 0.2 ml 25% glutaraldehyde Žc. 50 ml 0.2 M phosphate buffer Žd. pH to 7.4, add water to make 100 ml Že. cells are fixed for 4 h at 228C
2.2.7.4. Solutions for frozen thin sections. 1. Polyvinylpyrolidone ŽPVP.rsucrose solution. Mix 13 g of PVP-10 and 2.6 ml 1.1 M Na 2 CO 3 Žin water, adjust pH to 7.4. to a paste, then add 2.3 M sucrose Žin PBS.. Mix well, store at 48C. The solution can last for a long time. 2. Neutral oxalate uranyl acetate. Mix equal amounts of 4% aqueous uranyl acetate and 0.3 M aqueous oxalic acid, pH to 7.4, with 10% ammonium hydroxide. Test with pH paper. 3. Stock of 1.8% methyl cellulose: 0.9 g methyl cellulose, 50 ml water. Mix in stirrer in ice bath for at least 4 h, store overnight at 48C. Centrifuge for 1–4 h at 48C. Divide into aliquots and store at 48C. Will be good for about a month. Older stock yields precipitates on the grids. 4. 4% Uranyl acetate stock: 2 g uranyl acetate, 50 ml water. Mix until dissolved, store in brown bottle at 48C. 5. Methyl cellulose and Uranyl acetate mixture Žsee Peters, 1999.: 1 ml 1.8% methyl cellulose, 0.1 ml 4% uranyl acetate.
3. Results and discussion 3.1. Marker enzymes
2.2.7.3. Preparation of 1.5% glutaraldehyde solution in 0.067 M sodium cacodylate buffer, pH 7.4, with 1% sucrose. Mix components, as follows: Ža. 4.2 ml 25% glutaraldehyde Žb. 50 ml 0.134 M sodium cacodylate
In addition to the presence or absence of MPO, it is now recognized that granules can be further subdivided on the basis of other granular and membrane proteins. Table 1 lists the specific markers of human
Fig. 3. Ža. Frozen thin section of normal resting PMN labeled with D545, as the primary antibody and GAM-10 nm gold as the secondary antibody to demonstrate the presence of CD63 along the membranes of large extracted granules, characteristic of azurophilic granules Žag.. Specific granules Žsg. were not labeled Ž=72,000. Žmodified from Cham et al., 1994.. Žb. Human PMN were stained for MPO before being frozen. Then, cryosections were made and incubated with rabbit polyclonal antibody to a leukocyte adhesion receptor Žb 2 ., and labeled with GAR-5. The MPO-positive azurophil granules Žag. contain dense reaction product, whereas the lighter, specific granules are MPO-negative. Most of the gold grains outline the membranes of the specific granules Žlarge arrows., although some are present in the matrix of these granules. Because GAR-5 labels in clumps of two to six particles, label for an antibody site on the membrane may appear both on the membrane and in the matrix. =75,000. Modified from Bainton et al., 1987.
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neutrophilic granules and other organelles as analyzed by both cytochemical and fractionation procedures ŽBainton et al., 1971; Baggiolini, 1978; Cramer et al., 1985; Borregaard and Cowland, 1997; Ganz and Lehrer, 1998..
3.2. Azurophilic granules In addition to myeloperoxidase ŽFig. 2., the azurophilic granule contains numerous lysosomal enzymes or acid hydrolases. Also elastase ŽDamiano et
Fig. 4. Colocalization of gelatinase and lactoferrin in human PMN. PMNs were fixed, cryosectioned, and double-labeled Žwith antibodies against gelatinase and lactoferrin. as described in Section 2. For labeling of gelatinase, protein A-10 gold particles were used. For labeling of lactoferrin, protein A-20 was used. L, lactoferrin granule; DL, double-labeled granules; G, gelatinase granules. Note that some small and large vesicles Žv. do not contain label. In inset, biotinylated antigelatinase antibodies were used and streptavidin gold-10. N, nucleus =52,500; =70,000. Reprinted with permission from Kjeldsen et al., 1993 and Blood.
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al., 1988; Calafat et al., 1990., proteinase 3 ŽCalafat et al., 1990; Csernok et al., 1990., and a-1 antitrypsin ŽMason et al., 1991. colocalize with some MPO-positive granules. Bactericidal factors such as defensins ŽGanz et al., 1985; Rice et al., 1987; Ganz and Lehrer, 1998., azurophil-derived bactericidal factors ŽGabay et al., 1986., and bactericidal permeability-increasing proteins ŽWeiss and Olsson, 1987., which were previously called cationic proteins, have been found in some azurophilic granules ŽGabay et al., 1989.. Lysozyme has been found in both
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azurophilic and specific granules ŽCramer and Breton-Gorius, 1987; Mutasa, 1989.. Of the 10 antimicrobial proteins of known sequence in the human azurophil granules ŽCampanelli et al., 1990., two have unique primary structures Žlysozyme and bactericidal permeability-increasing protein., while the remaining eight fall into two families of four members each: the defensins Žwhich compromise 30–50% of granule proteins. on one hand, and cathepsin G, elastase, proteinase-3, and azurocidin on the other. These latter four proteins
Fig. 5. Portions of human neutrophils double-labeled for LAMP-2 with a monoclonal antibody and rabbit polyclonal antibody against human albumin to identify various organelles. The large gold ŽGAM-10. labels the location of LAMP-2 in vesicles Žarrows., whereas the small gold ŽGAR-5. Žarrowheads. detects albumin. Albumin Žalb. ŽGAR-5. Žarrowheads. is found in different vesicular structures from LAMP-2 ŽGAM-10., Žarrows.. =110,000.
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can be termed ‘‘serprocidins’’ to denote that they are closely related to serine proteases with microbicidal activity ŽCampanelli et al., 1990.. Very little is known about the limiting membrane of azurophilic granules, but CD63 ŽFig. 3a. ŽKuijpers et al., 1991; Cham et
al., 1994. and CD68 ŽSaito et al., 1991. are present. We had anticipated that the lysosomal-associated membrane proteins ŽLAMPs. would be found there, but such was not the case ŽBainton and August, 1988; Cieutat et al., 1998.. Rather, LAMPs were
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absent in all identified granule populations, but were consistently found in the membranes of vesicles, ŽMVBs., and ŽMLCs., which are identified by their concentric arrays of internal membranes ŽCieutat et al., 1998.. These organelles will be illustrated later. 3.3. Specific granules The specific or secondary granule, which by definition does not contain MPO, contains lactoferrin ŽFig. 2., lysozyme, B12 binding proteins, and other proteins ŽBorregaard et al., 1993; Borregaard and Cowland, 1997.. These MPO-negative granules vary greatly in size, shape, electron-lucency, isopycnic density, and granule content. However, they can be loosely categorized by the distribution of two proteins, lactoferrin and gelatinase. Approximately 16% of the MPO-negative granules contain only lactoferrin and 24% contain only gelatinase, but 60% contain both marker enzymes. The common leukocyte adhesion receptor B 2 was found on the membrane of specific granules, representing CD 11b ŽFig. 3b.. 3.4. Gelatinase granules By ultrastructure alone, three types of MPO-negative granules can be identified: peroxidase-negative granules containing gelatinase but no lactoferrin, peroxidase-negative granules containing lactoferrin but no gelatinase, and peroxidase-negative granules containing both lactoferrin and gelatinase Žsee Fig. 4. ŽKjeldsen et al., 1993.. This heterogeneity may be a result of overlapping synthesis and packaging of different granule proteins during granulopoiesis and is functionally significant, inflammatory mediators cause the gelatinase-containing granules to be re-
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leased from the cells more readily than those containing lactoferrin ŽBorregaard and Cowland, 1997.. 3.5. Secretory Õesicles Secretory vesicles have recently been discovered within neutrophils. These vesicles are distinct from the azurophilic, specific, and gelatinase granules and have been defined as intracellular organelles that contain CD35 and latent alkaline phosphatase ŽBorregaard and Cowland, 1997; Borregaard et al., 1992, 1993; Kjeldsen et al., 1993.. The latter enzyme is located on the luminal side of the vesicle membrane and can, therefore, be identified in the presence of detergent as latent alkaline phosphatase. This localization was also demonstrated by enzyme cytochemistry ŽRobinson and Kobayashi, 1991.. This work helped to clarify the location of alkaline phosphatase in resting and stimulated PMN. Further, these secretory vesicles contain plasma proteins such as albumin Žsee Fig. 5., which are not synthesized by the cells but are endocytosed from plasma. These represent a specialized form of endocytic vesicle. Secretory vesicles are transported to the cell surface after the stimulations by formyl methionyl-leucyl phenylalanine or certain cytokines ŽBorregaard et al., 1993.. 3.6. Localization of LAMPs and the M 6-P GP recognition marker During granulocyte differentiation in the bone marrow, neutrophilic leukocyte precursors synthesize large amounts of lysosomal enzymes. These enzymes are sequestered into azurophilic storage granules until used days later for digesting of phagocytized microorganisms following leukocyte emigration to
Fig. 6. Electron micrograph of a frozen thin section of myelocytes immunolabeled for LAMP-2. Large vesicles ŽV. and the MLC are labeled with GAM-10 Žarrow.. Note the absence of labeling in large azurophil granules as well as in the specific granules and plasma membrane ŽPM.. In inset, higher magnification of another MLC positive for LAMP-2. =52,000, inset, =86,000. Reprinted with permission from Cieutat et al., 1998 and Blood. Fig. 7. Ža,b,c. Immunolabeled cryosection labeled with rabbit anti-bovine polyclonal antibody against CI-M6PR and labeled with GAR-5. The receptor was found in the multivesicular bodies ŽMVB. but absent in the multi-lamellated compartments ŽMLC. Žsee a and c.. Occasionally, peroxidase-negative granule membranes Žarrows. appeared labeled as well as the plasma membrane. Magnification Ža–b. =63,000, Žc. =60,450.
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inflamed tissues. This azurophil granule population has previously been defined as a primary lysosome, i.e., a membrane-bound organelle containing acid hydrolases that have not entered into a digestive event ŽDeDuve and Wattiaux, 1966.. In the study by Cieutat et al. Ž1998., azurophil granules were purified and shown to contain large amounts of Man 6-P GP but little LAMP. In addition, the fine structural localization of Man 6-P GP and LAMP was investigated at various stages of maturation in human bone marrow and blood. Man 6-P GPs Žnot illustrated. were present within the azurophilic granules at all stages of maturation, in typical MVBs and in MLCs, LAMP was absent in all identified granule populations, but was consistently found in the membranes of vesicles, MVB, and MLC ŽFig. 6, see inset.. The
latter compartment has not been previously described in this cell type. In conclusion, the azurophilic granules, which contain an abundance of lysosomal enzymes and Man 6-P GP, lack the LAMP glycoproteins. By current criteria, therefore, they cannot be classified as lysosomes, but rather may have the functional characteristics of a regulated secretory granule. The true lysosomes of the resting neutrophil are probably the MVBs and MLC. Finally, the typical ‘‘dense bodies’’ or mature lysosomes described in other cells in vivo are not present in resting neutrophils. Although not illustrated in this paper, one additional point should be made about the utility of applying immunocytochemistry to frozen thin sections of the fractions themselves. The subcellular distribution of LAMPs and Man 6-P GP in fractions
Fig. 8. Phagocytizing human PMN labeled with GAM-10 for LAMP-2. Note that the plasma membrane ŽPM. and bacteria ŽB. are not labeled, nor is the early endocytic vacuole ŽE. on the right. The large phagocytic vacuole or secondary lysosome ŽL. contains degenerating bacteria ŽB.. The membrane of the phagocytic vacuole Žarrows. is labeled with gold. Magnification =52,000.
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of mature PMNs ŽCieutat et al., 1998. revealed that Man 6-P GP colocalized with MPO in the most dense fraction while LAMPs were found in the less dense fractions that contain lactoferrin and gelatinase and in small amounts in the lighter membrane fractions. However, direct examination of these fractions revealed that LAMPs were not in specific granules but were in MLC and MVB just as they were in intact cells ŽCieutat et al., 1998.. 3.7. Localization of cation-independent mannose 6phosphate receptor (CI-M6PR) Recently, we used the affinity-purified rabbit polyclonal antibody developed by Peter Lobel’s laboratory against the bovine CI-M6PR. Our preliminary data, illustrated in Fig. 7Ža,b,c., demonstrate that CI-M6PR is found in MVB but is absent in the MLC. This adds further credence to the suggestion that MLCs are late endosomes since they are LAMP-positive and receptor-negative Žsee review by Geuze, 1998..
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3.8. LAMPs are present in secondary lysosomes Finally, by studying human PMNs that have phagocytized bacteria ŽFig. 8., we now can demonstrate that the phagocytic vacuole contains LAMPs, whereas the plasma membrane and early phagosome does not. This means that MVB andror MLC must fuse with the newly formed endosome. 3.9. Surface labeling of plasma membrane proteins Two membrane adhesion proteins have been found on the tips of neutrophil microvilli in resting neutrophils, L-selectin ŽFig. 9. and P-selectin glycoprotein-1 ŽBorregaard et al., 1994; Moore et al., 1995; Bruehl et al., 1996, 1997.. Here the immunocytochemistry was performed by surface labeling the intact cells in suspension followed by embedding in Epon and sectioning rather than by frozen thin sectioning. This is suitable when it can be anticipated that the antigen is primarily on the external surface
Fig. 9. High magnification of human PMN plasma membrane showing the positioning of L-selectin on microvilli. A typical segment of PMN plasma membrane showing six microvilli with groups of gold particles labeling L-selectin localized at the tips Žarrows.. Note that the number of particles per microvillus is independent of microvillus length. Gold particles on the planar cell surface can also be seen Žarrowheads.. About 90% of the label can be seen on the microvilli. =42,000 Žmodified from Bruehl et al., 1996..
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of the plasma membrane. In quantitative studies on the ectoenzyme, 5-nucleotidase ŽHowell et al., 1987. showed that labeling on frozen thin sections with a polyclonal antibody is only of 15% efficiency. Labeling the cells in suspension as was done in Fig. 9 resulted in a fourfold increase in labeling efficiency.
tion of the manuscript. This work was supported by DK-10486 and HLB-31610 from the National Institutes of Health.
References
3.10. Limitations and artifacts Whereas the artifactual redistribution of lead phosphate reaction product has been reported during enzyme cytochemistry ŽCramer et al., 1988., relatively few artifacts have been seen with immunocytochemistry, particularly if the cells are appropriately fixed and the dilutions of the primary antibodies and immunogold are optimized. In areas near azurophilic granules with high labeling for Man 6-P GP, we frequently found ‘‘spillage’’ of the antigen in the nearby cytoplasm, ŽCieutat et al., 1998.. This artifact has been previously observed with the lowmolecular-weight defensins, ŽGanz et al., 1985. and proteinase 3, ŽCsernok et al., 1990.. A possible explanation is that the membranous compartments are cut open by the cryostat method, and some of the granule contents become soluble and may partially relocate during the subsequent incubation procedures. A word of caution about the use of Protein A in PMNs fixed with formaldehyde fixative alone. Occasionally, but inconsistently, controls were faintly labeled with Protein A gold, mainly in large clear vesicles. The addition of a fixative with glutaraldehyde yielded cleaner controls. For this reason, we preferred not to use Protein A on PMN, and if we did, we confirmed the observations using biotinylated primary antibodies ŽKjeldsen et al., 1993.. Finally, the major limitation is that one is only able to visualize about 15% of the antigen present ŽHowell et al., 1987.. Therefore, proteins present in low amounts may not be seen, and interpretations of negative data must be made with caution.
Acknowledgements Dr. Bainton thanks Ivy Hsieh and Yvonne Jacques for their excellent technical assistance, David Geller for his help in editing, and Silvia Molina for prepara-
Baggiolini, M., 1978. The neutrophil: function and clinical disorders. In: Klebanoff, S.J., Clark, R.A. ŽEds... Elsevier, North Holland, p. 556. Bainton, D.F., 1999. Developmental biology of neutrophils and eosinophils. In: Gallin, J.I., Snyderman, R. ŽEds.., Inflammation: Basic Principles and Clinical Correlates, 3rd edn. Lippincott Williams & Wilkins, Philadelphia, pp. 13–34. Bainton, D.F., August, J.T., 1988. Multivesicular bodies of human neutrophils ŽPMN. not granules, immunolable with the two major lysosomal membrane glycoproteins hLAMP-1 and hLAMP-2. J. Histochem. Cytochem. 36, 953. Bainton, D.F., Ullyot, J.L., Farquhar, M.G., 1971. The development of neutrophilic polymorphonuclear leukocytes in human bone marrow: origin and content of azurophil and specific granules. J. Exp. Med. 134, 907. Bainton, D.F., Miller, L.J., Kishimoto, T.K., Springer, T.A., 1987. Leukocyte adhesion receptors are stored in peroxidase-negative granules of human neutrophils. J. Exp. Med. 166, 1641. Borregaard, N., Cowland, B.J., 1997. Granules of the human neutrophilic polymorphonuclear leukocyte. Blood 89, 3503. Borregaard, N., Kjeldsen, L., Rygaard, K., Bastholm, L., Nielsen, M.H., Sengelov, H., Bjerrum, O.W., Johnsen, A.H., 1992. Stimulus-dependent secretion of plasma proteins from human neutrophils. J. Clin. Invest. 90, 86. Borregaard, N., Lollike, K., Kjeldsen, L., Sengelov, H., Bastholm, L., Nielsen, M.H., Bainton, D.F., 1993. Human neutrophil granules and secretory vesicles. Eur. J. Haematol. 51, 187. Borregaard, N., Kjeldsen, L., Sengelov, H., Diamond, M.S., Springer, T.A., Anderson, H.C., Kishimoto, T.K., Bainton, D.F., 1994. Changes in subcellular localization and surface expression of L-selectin, alkaline phosphatase, and Mac-1 in human neutrophils during stimulation with inflammatory mediators. J. Leukoc. Biol. 56, 80. Bruehl, R.E., Springer, T.A., Bainton, D.F., 1996. Quantitation of l-selectin distribution on human leukocyte microvilli by immunogold labeling and electron microscopy. J. Histochem. Cytochem. 44, 835. Bruehl, R.E., Moore, K.L., Lorant, D.E., Borregaard, N., Zimmerman, G.A., McEver, R.P., Bainton, D.F., 1997. Leukocyte activation induces surface redistribution of P-selectin glycoprotein ligand-1. J. Leukoc. Biol. 61, 489. Calafat, J., Goldschmeding, R., Ringeling, P.L., Janssen, H., van der Schoot, C.E., 1990. In situ localization by double-labeling immunoelectron microscopy of anti-neutrophil cytoplasmic autoantibodies in neutrophils and monocytes. Blood 75, 242. Campanelli, D., Detmers, P.A., Nathan, C.F., Gabay, J.E., 1990. Azurocidin and a homologous serine protease from neutrophils. J. Clin. Invest. 85, 904.
D.F. Baintonr Journal of Immunological Methods 232 (1999) 153–168 Cham, B.P., Gerrard, J.M., Bainton, D.F., 1994. Granulophysin is located in the membranes of azurophilic granules in human neutrophil and mobilizes to the plasma membrane following cell stimulation. Am. J. Pathol., 1369. Cieutat, A.-M., Lobel, P., August, J.T., Kjeldsen, L., Sengelov, H., Borregaard, N., Bainton, D.F., 1998. Azurophilic granules of human neutrophilic leukocytes are deficient in lysosome-associated membrane proteins but retain the mannose 6-phosphate recognition. Blood 91, 1044. Cohn, Z.A., Hirsch, J.G., 1960. The isolation and properties of the specific cytoplasmic granules of rabbit polymorphonuclear leucocytes. J. Exp. Med. 112, 983. Cramer, E.M., Breton-Gorius, J., 1987. Ultrastructural localization of lysozyme in human neutrophils by immunogold. J. Leukoc. Biol. 41, 242. Cramer, E., Pryzwansky, K.B., Villeval, J.-L., Testa, U., BretonGorius, J., 1985. Ultrastructural localization of lactoferrin and myeloperoxidase in human neutrophils by immunogold. Blood 65, 423. X Cramer, E.M., Werb, Z., Bainton, D.F., 1988. Pitfalls in ecto-5 nucleotidase enzyme cytochemistry as demonstrated by the immunogold-labelling technique on macrophages. Histochem. J. 20, 108. Csernok, E., Ludemann, J., Gross, W.L., Bainton, D.F., 1990. ¨ Ultrastructural localization of proteinase 3, the target antigen of anti-cytoplasmic antibodies circulating in Wegener’s granulomatosis. Am. J. Pathol. 137, 1113. Damiano, V.V., Kucich, U., Murer, E., Laudenslager, N., Weinbaum, G., 1988. Ultrastructural quantitation of peroxidase- and elastase-containing granules in human neutrophils. Am. J. Pathol. 131, 235. DeDuve, C., Wattiaux, R., 1966. Functions of lysosomes. Annu. Rev. Physiol. 28, 435. Gabay, J.E., Heiple, J.M., Cohn, Z.A., Nathan, C.F., 1986. Subcellular location and properties of bactericidal factors from human neutrophils. J. Exp. Med. 164, 1407. Gabay, J.E., Scott, R.W., Campanelli, D., 1989. Antibiotic proteins of human polymorphonuclear leukocytes. Proc. Natl. Acad. Sci. U. S. A. 86, 5610. Ganz, T., Lehrer, R.I., 1998. Antimicrobial peptides of vertebrates. Curr. Opin. Immunol. 10 Ž1., 41. Ganz, T., Selsted, M.E., Szklarek, D., Harwig, S.S.L., Daher, K., 1985. Defensins: natural peptide antibiotics of human neutrophils. J. Clin. Invest. 76, 1427. Geuze, H.J., 1998. The role of endosomes and lysosomes in MHC class II functioning. Immunol. Today 19, 282. Graham, R.C., Karnovsky, M.J., 1966. The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem. 14, 291. Griffiths, G., 1993. Fine Structure Immunocytochemistry. Springer, Berlin, Chap. 3, pp. 26–80. Griffiths, G., Simmons, K., Warren, G.W., Tokuyasu, K.T., 1983. Immunoelectron microscopy using thin, frozen sections: application to studies of the intracellular transport of semliki forest virus spike glycoproteins. Methods Enzymol. 96, 466. Howell, K.E., Reuter-Carlson, U., Devaney, E., Luzio, J.P., Fuller,
167
S.D., 1987. One antigen or gold? A quantitative analysis of X immunogold labeling of plasma membrane 5 -nucleotidase in frozen thin sections. Eur. J. Cell Biol. 44, 318. Kjeldsen, L., Bainton, D.F., Sengelov, H., Borregaard, N., 1993. Structural and functional heterogeneity among peroxidase negative granules in human neutrophils: identification of a distinct gelatinase containing granule subset by combined immunocytochemistry and subcellular fractionation. Blood 82, 3183. Kuijpers, T.W., Tool, A.T.J., van der Schoot, D.E., Ginsel, L.A., Onderwater, J.J.M., Roos, D., Verhoeven, A.J., 1991. Membrane surface antigen expression on neutrophils: a reappraisal of the use of surface markers for neutrophil activation. Blood 78, 1105. Liou, W., Geuze, H.J., Slot, J.W., 1996. Improving structural integrity of cryosections for immunogold labeling. Histochem. Cell Biol. 106, 41. Mason, D.Y., Cramer, E.M., Masse, ´ J.-M., Crystal, R., Bassot, J.-M., Breton-Gorius, J., 1991. Alpha1-antitrypsin is present within the primary granules of human polymorphonuclear leukocytes. Am. J. Pathol. 139, 623. McCaffery, J.M., Farquhar, M.G., 1995. Localization of GTPases by indirect immunofluorescence and immunoelectron microscopy. Methods Enzymol. 257, 259. McLean, I.W., Nakane, P.K., 1974. Periodate–lysine–paraformaldehyde fixative for immunoelectron microscopy. J. Histochem. Cytochem. 22, 1077. Moore, K.L., Patel, K.D., Bruehl, R.E., Fugang, L., Johnson, D.A., Lichenstein, H.S., Cummings, R.D., Bainton, D.F., McEver, R.P., 1995. P-selectin glycoprotein ligand-1 mediates rolling of human neutrophils on P-selectin. J. Cell Biol. 128 Ž4., 661. Mutasa, H.C., 1989. Combination of diaminobenzidine staining and immunogold labeling: a novel technical approach to identify lysozyme in human neutrophil cells. Eur. J. Cell Biol. 49, 319. Peters, P.J., 1999. Cryo-immunogold electron microscopy. Current Protocols in Cell Biology, 4.7.1–4.7.12. Rice, W.G., Ganz, T., Kinkade, J.M. Jr., Selsted, M.E., Lehrer, R.I., Parmley, R.T., 1987. Defensin-rich dense granules of human neutrophils. Blood 70, 757. Robinson, J.M., Kobayashi, T.A., 1991. Novel intracellular compartment with unusual secretory properties in human neutrophils. J. Cell Biol. 113, 743. Saito, N., Pulford, K.A.F., Breton-Gorius, J., Masse, ´ J.-M., Mason, D.Y., Cramer, E.M., 1991. Ultrastructural localization of the CD68 macrophage-associated antigen in human blood neutrophils and monocytes. Am. J. Pathol. 139, 1053. Slot, J.W., Geuze, H.J., 1984. Gold markers for single and double immunolabeling of cryosections. In: Polak, J.M., Varndell, I.M. ŽEds... Immunolabeling for Electron Microscopy. Elsevier, Oxford, UK, p. 129. Stenberg, P.E., Shuman, M.A., Levine, S.P., Bainton, D.F., 1984. Optimal techniques for the immunocytochemical demonstration of b-thromboglobulin, platelet factor 4, and fibrinogen in the alpha granules of unstimulated platelets. Histochem. J. 16, 983. Tokuyasu, K.T., 1984. Immuno-cryoultramicrotomy. In: Polak,
168
D.F. Baintonr Journal of Immunological Methods 232 (1999) 153–168
J.M., Varndell, I.M. ŽEds.., Immunolabelling for Electron Microscopy. Elsevier, Amsterdam, pp. 71–82. Tokuyasu, K.T., 1989. Use of polyŽvinylpyrrolidone. and polyŽvinyl alcohol. for cryoultramicrotomy. Histochem. J. 21, 163. Valenzano, K.J., Kallay, L.M., Lobel, P., 1993. An assay to detect glycoproteins that contain mannose 6-phosphate. Anal. Biochem. 209, 156.
Weiss, J., Olsson, I., 1987. Cellular and subcellular localization of the bactericidalrpermeability-increasing protein of neutrophils. Blood 69, 652. Zucker-Franklin, D., Hirsch, J.G., 1964. Electron microscope studies on the degranulation of rabbit peritoneal leukocytes during phagocytosis. J. Exp. Med. 120, 569.