Granule Release by Polymorphonuclear Leukocytes Treated with the lonophore A231 87 PHILIP L. SANNES, HARVEY L. BANK, PATRICK L. MOORE ' A N D SAMUEL S. SPICER Department of Pathology, Medical Uniuersity of South Carolina, Charleston, South Carolina 29401
ABSTRACT Polymorphonuclear leukocytes (PMN's) incubated three t o eight minutes a t 37OC in medium containing 1 x M of the ionophore antibiotic A23187 released their cytoplasmic granules into the extracellular medium. Transmission electron microscopy of treated cells showed microfilament bundles extending between adjacent granules within the cytoplasm and between granules and the plasma membrane. Tiny dense projections (beads) 8-12 nm in diameter were observed along segments of the cytoplasmic surface of the plasma membrane with a periodicity of 20-30 nm. These beads were observed on the plasma membrane only in the vicinity of intra- or extracytoplasmic granules. The structural relationships of the beads with the plasma membrane microfilaments suggest they play a role in the process of ionophore-induced granule release from polymorphonuclear leukocytes. The ionophore antibiotic A23187, extracted from cultures of Streptomyces chartreusensis, has been shown to alter the permeability of biological membranes to calcium (Reed and Lardy, '72). Such phenomena as lymphocyte mitogenesis (Hovi e t al., '76; Luckasen e t al., '74), salivary gland secretion (Prince et al., '731, leukocyte chemotaxis (Estensen et al., '76; Wilkinson '751, lymphocyte agglutination (Poste and Nicholson, '76) and capping (Poste and Nicholson, '76; Schreiner and Unanue, '76) have been shown to be initiated, or otherwise affected by the changes in intracellular calcium levels induced by this ionophore. Recent biochemical evidence has indicated that this ionophore enhanced secretion of the specific granule enzyme, lysozyme, from human neutrophils, thus implicating calcium in the induction of its release (Estensen et al., '76; Goldstein et al., '74). In the present report, transmission and scanning electron microscopy were employed t o assess the structural changes that occur during granule release in rabbit PMN's exposed to the ionophore A23187. MATERIALS AND METHODS
Polymorphonuclear leukocytes (PMNs) were obtained from 1-2 kg New Zealand rabbits by intraperitoneal injection of 200 ml ANAT. REC.. 189: 177-186.
sterile PBS (pH 7.2) containing 200 mg glycogen (Type 11, Sigma Chemical Co., St. Louis, Missouri) as previously described (Moore et al., '76). The final concentration of cells was adjusted to 3 X lo6 celldm1 and the exudate separated into 15 ml aliquots. The cells were then centrifuged a t 500 g, (3 minutes), resuspended in one of the test or control media (table 1) a t the original cell concentration, and incubated a t 37OC for three to eight minutes on coverslips for SEM (scanning electron microscopy) or in test tubes for TEM (transmission electron microscopy). Cells for both TEM and SEM were fixed one hour with 2% glutaraldehyde in 0.1 M Na-cacodylateHC1 buffer, pH 7.2, 380 f 2OmOsm, and postfixed one hour in 2% osmium tetroxide in pH 7.2 Na-cacodylate-HC1 buffer. For TEM, cells were dehydrated either utilizing ethanol or dimethoxypropane (Miiller and Jacks, '75) and embedded in low viscosity resin (Spurr, '69). Thin sections were cut on a Porter-Blum MT-1 and stained with uranyl acetate and lead citrate and examined with either a Hitachi HS-8 or HU-12 electron microscope. Cells for SEM were treated with thiocarbohydrazide and osmium tetroxide (Kelley et Received Feb. 17. '77. Accepted Apr. 11, "77. ' Present address: Department of Biology, Yale University. New Haven, Connecticut.
177
178
P. L. SANNES, H. L. BANK, P L. MOORE AND S. S. SPICER TABLE 1 ~
lncuhation media
-
1 Minimal Essential Medium (MEM) 2 25mM Hepes buffer 150 mM NaCl (Hepes) 3 MEM 3 mM CaCl, 4 Hepes + 5 mM CaC1, 5 MEM DMSO 6 Hepes +DMSO 7 MEM+3 mM CaCl,+DMSO 8 Hepes 5 rnM CaC1, DMSO 9 MEM+lX 1 0 - f i M A23187i~DMSO 10 Hepes + 1 X 10-6M A23167 + DMSO 11 Hepes+5mMCaCl,+lX 1Ow6M A23167 + DMSO
+
+
+
'
+
Granule release
'
Negative Negative Negative Negative Negative Negative Negative Negative Positive Positive Positive
' Over half the cells show some degree of granule release Le.. cells depleted of granules or large numbers of extracellular granules were observed, fig. 1). Eagles, F-12. Grand Island B i d Co.. New York (contains 1.26m M CaCI,). Polysciences. Inc., Warrington. Pennsylvania. DMSO. a solvent for the ionophore, never exceeded concentrations of 0.lX (vol/voll in any of the media al., '751, dehydrated, critically point dried, and gold coated as previously described (Moore et al. '76). Specimens were viewed with a Coates and Welter model no. 106 Field Emission Scanning Electron Microscope. OBSERVATIONS
The addition of 1 X M of A23187 to any of the media containing calcium produced profound and unique morphological changes in rabbit PMN leukocytes. After a 3-minute incubation a majority of the viable PMN's released many primary, secondary and tertiary granules extracellularly as demonstrated by fixation and examination with TEM and SEM. In thin sections, some cells were devoid of granules (fig. l), while others were observed to have granules in various stages of release (fig. 2). PMN's which were releasing granules (exocytosis) often had irregular surface conformations and numerous small villar-like projections. Generally, granules were observed in the vicinity of the plasma membrane, both inside and outside the cell. The distribution of groups or bundles of microfilaments was confined to four locations: (1) between adjacent granules with the cytoplasm (fig. 3); (2) between intracellular granules and the plasma membrane (fig. 4); (3) between extracellular granules and the plasma membrane (fig. 5); (4) and between granules on opposite side of the plasma membrane (fig. 6).
Tiny dense projections, termed beads, were found associated with segments of the inner surface of the plasma membrane of PMN leukocytes in which granule release was observed (figs. 7-11). These beads had dimensions of 81 2 nm, and were discrete, electron dense, and spaced regularly a t 20-30 nm intervals. These beads were often seen associated with microfilaments (5-7 nm in diameter) which appeared to be attached to cytoplasmic granules (figs. 7, 8). Plasma membrane associated beads were also observed adjacent to granules which were in intimate contact with the plasma membrane. These contact areas were characterized by a single dense lamina rather than two opposing trilaminar structures. Cells not releasing granules or those having released all their granules (fig. 1) had no plasma membrane associated beads. Longitudinally sectioned microtubules (18 nm) were only rarely observed in cells treated with A23187. Microtubule-like structures were occasionally seen associated with granule membranes in some PMN's (fig. 12). Microfilament bundles were observed between adjacent granules, but never between granules and plasma membrane in control PMN's, or the few non-reactive cells treated with ionophore. In addition, plasma membrane associated beads were not seen in control cells. In rabbit PMN's releasing granules, beads were not always observed in cross sections of granules in proximity to the plasma membrane. When present, however, the beads were always in close association with a granule (intra-or extracellular) filament bundle, or both (figs. 7-10). PMNs treated with medium containing ionophore but no calcium as well as those not containing ionophore showed comparatively little granule release as judged by the relative number of both cytoplasmic granules remaining within the cells and extracellular granules (table 1).Increased extracellular calcium alone did not result in appreciable granule release. Similar results were obtained with different buffer systems or low DMSO concentrations (
ABSTRACT Polymorphonuclear leukocytes (PMN's) incubated three t o eight minutes a t 37OC in medium containing 1 x M of the ionophore antibiotic A23187 released their cytoplasmic granules into the extracellular medium. Transmission electron microscopy of treated cells showed microfilament bundles extending between adjacent granules within the cytoplasm and between granules and the plasma membrane. Tiny dense projections (beads) 8-12 nm in diameter were observed along segments of the cytoplasmic surface of the plasma membrane with a periodicity of 20-30 nm. These beads were observed on the plasma membrane only in the vicinity of intra- or extracytoplasmic granules. The structural relationships of the beads with the plasma membrane microfilaments suggest they play a role in the process of ionophore-induced granule release from polymorphonuclear leukocytes. The ionophore antibiotic A23187, extracted from cultures of Streptomyces chartreusensis, has been shown to alter the permeability of biological membranes to calcium (Reed and Lardy, '72). Such phenomena as lymphocyte mitogenesis (Hovi e t al., '76; Luckasen e t al., '74), salivary gland secretion (Prince et al., '731, leukocyte chemotaxis (Estensen et al., '76; Wilkinson '751, lymphocyte agglutination (Poste and Nicholson, '76) and capping (Poste and Nicholson, '76; Schreiner and Unanue, '76) have been shown to be initiated, or otherwise affected by the changes in intracellular calcium levels induced by this ionophore. Recent biochemical evidence has indicated that this ionophore enhanced secretion of the specific granule enzyme, lysozyme, from human neutrophils, thus implicating calcium in the induction of its release (Estensen et al., '76; Goldstein et al., '74). In the present report, transmission and scanning electron microscopy were employed t o assess the structural changes that occur during granule release in rabbit PMN's exposed to the ionophore A23187. MATERIALS AND METHODS
Polymorphonuclear leukocytes (PMNs) were obtained from 1-2 kg New Zealand rabbits by intraperitoneal injection of 200 ml ANAT. REC.. 189: 177-186.
sterile PBS (pH 7.2) containing 200 mg glycogen (Type 11, Sigma Chemical Co., St. Louis, Missouri) as previously described (Moore et al., '76). The final concentration of cells was adjusted to 3 X lo6 celldm1 and the exudate separated into 15 ml aliquots. The cells were then centrifuged a t 500 g, (3 minutes), resuspended in one of the test or control media (table 1) a t the original cell concentration, and incubated a t 37OC for three to eight minutes on coverslips for SEM (scanning electron microscopy) or in test tubes for TEM (transmission electron microscopy). Cells for both TEM and SEM were fixed one hour with 2% glutaraldehyde in 0.1 M Na-cacodylateHC1 buffer, pH 7.2, 380 f 2OmOsm, and postfixed one hour in 2% osmium tetroxide in pH 7.2 Na-cacodylate-HC1 buffer. For TEM, cells were dehydrated either utilizing ethanol or dimethoxypropane (Miiller and Jacks, '75) and embedded in low viscosity resin (Spurr, '69). Thin sections were cut on a Porter-Blum MT-1 and stained with uranyl acetate and lead citrate and examined with either a Hitachi HS-8 or HU-12 electron microscope. Cells for SEM were treated with thiocarbohydrazide and osmium tetroxide (Kelley et Received Feb. 17. '77. Accepted Apr. 11, "77. ' Present address: Department of Biology, Yale University. New Haven, Connecticut.
177
178
P. L. SANNES, H. L. BANK, P L. MOORE AND S. S. SPICER TABLE 1 ~
lncuhation media
-
1 Minimal Essential Medium (MEM) 2 25mM Hepes buffer 150 mM NaCl (Hepes) 3 MEM 3 mM CaCl, 4 Hepes + 5 mM CaC1, 5 MEM DMSO 6 Hepes +DMSO 7 MEM+3 mM CaCl,+DMSO 8 Hepes 5 rnM CaC1, DMSO 9 MEM+lX 1 0 - f i M A23187i~DMSO 10 Hepes + 1 X 10-6M A23167 + DMSO 11 Hepes+5mMCaCl,+lX 1Ow6M A23167 + DMSO
+
+
+
'
+
Granule release
'
Negative Negative Negative Negative Negative Negative Negative Negative Positive Positive Positive
' Over half the cells show some degree of granule release Le.. cells depleted of granules or large numbers of extracellular granules were observed, fig. 1). Eagles, F-12. Grand Island B i d Co.. New York (contains 1.26m M CaCI,). Polysciences. Inc., Warrington. Pennsylvania. DMSO. a solvent for the ionophore, never exceeded concentrations of 0.lX (vol/voll in any of the media al., '751, dehydrated, critically point dried, and gold coated as previously described (Moore et al. '76). Specimens were viewed with a Coates and Welter model no. 106 Field Emission Scanning Electron Microscope. OBSERVATIONS
The addition of 1 X M of A23187 to any of the media containing calcium produced profound and unique morphological changes in rabbit PMN leukocytes. After a 3-minute incubation a majority of the viable PMN's released many primary, secondary and tertiary granules extracellularly as demonstrated by fixation and examination with TEM and SEM. In thin sections, some cells were devoid of granules (fig. l), while others were observed to have granules in various stages of release (fig. 2). PMN's which were releasing granules (exocytosis) often had irregular surface conformations and numerous small villar-like projections. Generally, granules were observed in the vicinity of the plasma membrane, both inside and outside the cell. The distribution of groups or bundles of microfilaments was confined to four locations: (1) between adjacent granules with the cytoplasm (fig. 3); (2) between intracellular granules and the plasma membrane (fig. 4); (3) between extracellular granules and the plasma membrane (fig. 5); (4) and between granules on opposite side of the plasma membrane (fig. 6).
Tiny dense projections, termed beads, were found associated with segments of the inner surface of the plasma membrane of PMN leukocytes in which granule release was observed (figs. 7-11). These beads had dimensions of 81 2 nm, and were discrete, electron dense, and spaced regularly a t 20-30 nm intervals. These beads were often seen associated with microfilaments (5-7 nm in diameter) which appeared to be attached to cytoplasmic granules (figs. 7, 8). Plasma membrane associated beads were also observed adjacent to granules which were in intimate contact with the plasma membrane. These contact areas were characterized by a single dense lamina rather than two opposing trilaminar structures. Cells not releasing granules or those having released all their granules (fig. 1) had no plasma membrane associated beads. Longitudinally sectioned microtubules (18 nm) were only rarely observed in cells treated with A23187. Microtubule-like structures were occasionally seen associated with granule membranes in some PMN's (fig. 12). Microfilament bundles were observed between adjacent granules, but never between granules and plasma membrane in control PMN's, or the few non-reactive cells treated with ionophore. In addition, plasma membrane associated beads were not seen in control cells. In rabbit PMN's releasing granules, beads were not always observed in cross sections of granules in proximity to the plasma membrane. When present, however, the beads were always in close association with a granule (intra-or extracellular) filament bundle, or both (figs. 7-10). PMNs treated with medium containing ionophore but no calcium as well as those not containing ionophore showed comparatively little granule release as judged by the relative number of both cytoplasmic granules remaining within the cells and extracellular granules (table 1).Increased extracellular calcium alone did not result in appreciable granule release. Similar results were obtained with different buffer systems or low DMSO concentrations (
