Organization and mobility of CD11b/CD18 and targeting of superoxide on the surface of degranulated human neutrophils.

A monoclonal IgM, specifically recognizing both CD11b and CD18 of human neutrophils, was used to examine the organization and mobility of CD11b/CD18 in the plasma membrane of human neutrophils degranulated by dihydrocytochalasin B (dhCB) treatment and fMet-Leu-Phe (fMLF) stimulation. Subcellular fractionation analysis of untreated or dhCB-treated control neutrophils indicated that 20% of CD11b/CD18 cosedimented with plasma membrane and the remainder with specific granules. In contrast, fMLF stimulation of dhCB-treated cells caused a major reorganization of CD11b/CD18, in which 60-70% of CD11b/CD18 sedimented in dense plasma membrane fractions that were also enriched in superoxide-generating NADPH oxidase activity. Similarly pretreated neutrophils were fixed, immunogold labeled, and examined by scanning electron microscopy. Immunogold particles were distributed uniformly over the symmetrically ruffled surface of unstimulated neutrophils. On dhCB-treated cells, immunogold was mostly uniformly distributed on a smooth membrane with a small percentage of particles lining up into linear arrays. After fMLF + dhCB stimulation, CD11b/CD18 gold label was more abundant on the cell surface and formed large aggregates on polarized membrane protrusions. However, when cells were adhered to an albumin-coated quartz surface and stimulated with fMLF in the presence of dhCB, immunogold was excluded on the articulated and rounded cell body but concentrated on the periphery of adherent lamellae. Fluorescence photobleaching recovery indicated that in unstimulated cells 38 +/- 3% of CD11b/CD18 was mobile (R) with a diffusion constant D of 3.1 +/- 0.3 x 10(-10) cm2/s. Treatment with dhCB raised R and D 24 and 74%, respectively. Stimulation using 1 microM fMLF with dhCB lowered D and R to near control levels. Since NADPH oxidase and CD11b/CD18 cosediment in high-density plasma membrane domains after fMLF + dhCB stimulation, we speculate that a stimulus-induced reorganization of CD11b/CD18 and NADPH oxidase to common membrane domains may occur in fMLF + dhCB-degranulated neutrophils.

Immunocytochemical detection of lipid peroxidation in phagosomes of human neutrophils: correlation with expression of flavocytochrome b.

Oxidants generated by the NADPH oxidase of activated neutrophils can react with a number of tissue targets to form toxic metabolites such as 4-hydroxynonenal (4-HNE). 4-HNE is a lipid peroxidation product generated by free radical attack on omega-6 polyunsaturated fatty acids and is a marker for membrane lipid peroxidation. In this study, we examined the accumulation of 4-HNE-protein adducts in phagosomes of neutrophils obtained from a male patient with homozygous X-linked, flavocytochrome b-deficient chronic granulomatous disease (CGD), his heterozygous mother, and his normal father. Specific polyclonal antibodies recognizing 4-HNE-protein adducts and gp91-phox (flavocytochrome b large subunit) were prepared and used to immunocytochemically detect these antigens in cryofixed, molecular distillation-dried neutrophils. No 4-HNE-protein adducts were detected in flavocytochrome b-deficit cells from the homozygous patient or from the heterozygous CGD carrier. However, in gp91-phox-positive cells from both the normal and heterozygous CGD carrier, significant 4-HNE-protein adduct labeling was observed, primarily in the phagosomes. When data from single- and double-labeled cells were combined, the frequency distribution of the labels in phagosomes supported this observation, showing that neutrophils from the heterozygous CGD carrier were 71% 4-HNE-protein adduct-positive and 56% gp91-phox-positive, while cells from the normal father were > 97% positive for both 4-HNE-protein adducts and gp91-phox. These results confirmed the nitroblue tetrazolium tests of 100%, 60 +/- 2%, and 0% positive for the father's, mother's, and son's cells, respectively, and demonstrated that 4-HNE-protein adduct antibodies are useful and accurate probes of the occurrence of lipid peroxidation in vivo. We conclude that 4-HNE and resulting 4-HNE-protein adducts are generated as a result of NADPH oxidase activity in the phagosomes of human neutrophils and that these lipid peroxidation products may contribute to microbial killing and/or damage of neutrophil phagolysosomal proteins.

Remodeling of the plasma membrane after stimulation of neutrophils with f-Met-Leu-Phe and dihydrocytochalasin B: identification of membrane subdomains containing NADPH oxidase activity.

Superoxide (O2-) production by neutrophils stimulated with chemotactic peptides [e.g., formylmethionyl-leucyl-phenylalanine (fMLP)] is transient but increases in rate and duration after pretreatment of the cells with dihydrocytochalasin B (dhCB), suggesting a possible role for the plasma membrane and membrane skeleton in the regulation of the O2- generating system. Analysis of plasma membrane isolated from these cells by isopycnic sucrose density gradient sedimentation showed that there were no significant variation in the distribution of plasma membrane markers between control and dhCB-treated cells, whereas a significant redistribution of plasma membrane markers was observed in dhCB + fMLP-treated cells. Instead of sedimenting to 31-35% sucrose, as in the former two groups, plasma membrane markers were broadly distributed over 25-50% sucrose in the dhCB + fMLP-treated cells. In addition, approximately 80% degranulation was achieved in these cells, whereas little granule release (< 5%) was observed in control and dhCB-treated cells. Analysis of the gradient fractions for membrane skeletal (actin and fodrin) and NADPH oxidase (cytochrome b and p47-phox) components in dhCB + fMLP-treated cells demonstrated that the distribution of fodrin, actin, cytochrome b, and p47-phox followed the broad distribution of plasma membrane markers, with an overall eightfold increase in membrane-associated actin. Despite the broad redistribution of plasma membrane markers, the distribution of O2- generating activity remained confined to a narrower peak at approximately 50% sucrose. These results demonstrate that a heterogeneous surface membrane of higher density with a differential distribution of proteins and O2- generating activity are created after dhCB + fMLP treatment; however, domain structure is conserved in the new membrane, with only a subfraction of the reorganized plasma membrane containing all of the components necessary for active O2- generation. Our results support a role for plasma membrane lateral organization and participation of the membrane skeleton in the regulation of the O2- generating system.

Subcellular distribution of the Rap1A protein in human neutrophils: colocalization and cotranslocation with cytochrome b559.

Rap1A, a low molecular weight guanosine triphosphate-binding protein (LMWG), has been shown previously by us to be associated with purified cytochrome b from stimulated human neutrophils. In the present studies, we show that Rap1A is also associated with affinity-purified cytochrome b from unstimulated neutrophils and use specific anti-Rap1 peptide antibodies to biochemically and immunocytochemically determine the subcellular distribution of Rap1A in resting and activated human neutrophils. Analysis of the subcellular fractionation of unstimulated cells by Western blotting of isopycnic sucrose density gradient fractions with anti-Rap1 peptide antibodies indicated that Rap1A colocalized with cytochrome b in the plasma membrane as well as in the specific granule membranes and that it was translocated, along with cytochrome b, to the plasma membrane when the cells were stimulated with phorbol myristate acetate (PMA). No evidence for a cytosolic localization of Rap1A was found in our studies; however, if the cells were disrupted by sonication, rather than N2 cavitation, a fraction of the Rap1A was released from the membrane. Electron microscopy of thin sections of cryofixed, molecular-distillation dried neutrophils labeled with anti-Rap1 antibody alone or double-labeled with anti-Rap1 and anti-cytochrome b peptide antibodies confirmed our biochemical localization, and quantitation showed that more than half of the specific granule-associated Rap1A was translocated to the plasma membrane in PMA-stimulated cells. Ultrastructural analysis of neutrophils phagocytosing Staphylococcus aureus also demonstrated the translocation of Rap1A with cytochrome b. Approximately 70% of the total Rap1A labeling was associated with the phagolysosomal membrane, the site of assembly of the superoxide-generating system. The colocalization and cotranslocation of Rap1A with cytochrome b in resting and activated neutrophils is consistent with a functional association of these two molecules in the intact cell and provides further evidence for a role of this LMWG in the structure or function of the neutrophil superoxide-generating system.

Functional, physical, and ultrastructural localization of CD15 antigens to the human polymorphonuclear leukocyte secondary granule.

A murine monoclonal IgM antibody, M3, which interferes with both polymorphonuclear leukocyte (PMN) phagocytosis and bactericidal activity, was used to examine the subcellular location of antigens bearing 3-fucosyllactosamine (CD15 antigens) within this cell type. Percoll gradient-separated secondary granule fractions were rich in CD15 antigens, with at least seven antigens recognizable in SDS-PAGE/electroblot studies. Sonication/sedimentation experiments using secondary granule fractions showed that both soluble and sedimentable CD15 antigens were present. Exposure of purified PMN to the secondary granule secretagogue phorbol myristate acetate caused extracellular release of two or three CD15 antigens, which could be purified by immunoprecipitation using antibody M3. Triton X-114 phase-partition experiments showed that secondary granule fraction CD15 antigens could be partitioned into hydrophilic (aqueous phase) and hydrophobic (detergent phase) antigens, suggesting that several of these antigens were integral secondary granule membrane components. Ultrastructurally, PMN intracellular granules showed two patterns of CD15 expression, localization over both granule matrix/granule membrane and localization to only granule membrane. Colocalization studies showed that lactoferrin and CD15 antigens were both present in a subset of intracellular granules, confirming a secondary granule location for these antigens.

Human neutrophil granule heterogeneity: immunolocalization studies using cryofixed, dried and embedded specimens.

The heterogeneity in human neutrophil granules was examined by the ultrastructural localization of a series of antigens which have been previously identified with neutrophil granules by either physical separation or biochemical/biological techniques. All samples were prepared by cryofixation and molecular distillation drying (LifeCell Process), a two-step physical method that achieves cryofixation by metal mirror freezing and drying by the controlled, incremental heating of cryofixed samples in an ultrahigh vacuum. After drying, the samples were either exposed to vapor-phase osmium followed by embedment in Spurr resin, or they were exposed to formaldehyde vapor followed by embedment in Araldite resin. An indirect streptavidincolloidal gold procedure was used for immunoelectron microscopy on ultrathin sections. Subcellular ultrastructural morphology of neutrophils prepared by this method was good compared to standard electron microscopic techniques and superior compared to comparable, published electron microscopic cryomethods applied to neutrophils. Immunogold localization of myeloperoxidase, cathepsin G, lysozyme, lactoferrin, beta 2-microglobulin, and CD-15 antigens showed high intensity and specificity of labeling in the intracellular granules. Patterns of labeling varied from antigen to antigen, demonstrating granule heterogeneity both within and among neutrophils. This methodology is useful in the exploration and definition of granule heterogeneity and function.

A new technique for removal of amorphous phase tissue water without ice crystal damage: a preparative method for ultrastructural analysis and immunoelectron microscopy.

An apparatus has been produced that can remove amorphous phase tissue water via molecular distillation without devitrification or rehydration. This method represents a fundamental advance in tissue preparation, making possible for the first time ultrastructural localization of soluble molecular entities without the problems of alteration, re-distribution, and loss which have plagued conventional techniques. Fresh slices of rat brain, liver, or kidney, and monkey retinal tissue were cryofixed by bounce-free, metal mirror cooling on copper bars immersed in liquid nitrogen (LN2). Tissue transferred under LN2 was then placed in a precooled copper specimen block, which was subsequently lowered into a LN2-cooled stainless steel chamber. After rough pumping at 1 X 10(-3) mbar with a mechanical pump to remove LN2, the chamber was evacuated with a cryopump or turbomolecular pump to achieve a hydrocarbon-free, ultra-high vacuum of 1 X 10(-8) mbar. Equilibrium temperature in the chamber before the drying cycle was -192 degrees C. The copper specimen block was equipped with a thermocouple and a programmable feedback-controlled heating circuit. Tissue was dried by increasing the specimen block temperature 1 degree C/hr during the critical drying phase while monitoring the rate of water removal with a partial pressure analyzer. Results obtained indicate that drying is complete below the devitrification temperature of amorphous phase tissue water. Dried tissue was fixed with osmium tetroxide vapor, vacuum-embedded in a low-viscosity epoxy resin, sectioned, stained, and viewed with the electron microscope. Processed tissue exhibits excellent morphological preservation without the use of pre-fixation or cryoprotective agents. Thin sections of this tissue are excellent for immunocytochemical staining and electron microprobe analysis.

Ultrastructural localization of LH-RH-immunoreactive synapses in the hamster accessory olfactory bulb.

Electron microscopic immunocytochemistry was used to localize luteinizing hormone-releasing (LH-RH) immunoreactivity within the male golden hamster accessory olfactory bulb. Two LH-RH-immunoreactive fiber populations were identified in the accessory olfactory bulb. A superficial system of immunoreactive axons was localized to the vomeronasal nerve and glomerular layers, and a periventricular system appeared in granule cell and periventricular layers. LH-RH-immunoreactive varicosities were observed to contain large reactive vesicles (80-120 nm) as well as a variable degree of cytoplasmic reaction product. Additionally, small vesicles with unreactive lumens and mitochondria were often present. Intravaricose segments of immunoreactive fibers invariably displayed fewer reactive vesicles than did varicosities. Within both glomerular and periventricular layers, some LH-RH-immunoreactive varicosities were observed to form asymmetric contacts characterized by prominent postjunctional densities. In the glomerular layer, these junctions could be identified as synaptic by several features. The presence of LH-RH-immunoreactivity in presynaptic elements supports a neuromodulatory role for LH-RH. As the accessory olfactory system is critically involved in the initiation of mating behavior of the male golden hamster, LH-RH-immunoreactive synapses in the accessory olfactory bulb may function to regulate reproductive behavior.

Ultrastructure of the subfornical organ of the chicken (Gallus domesticus).

The SFO of the chicken is divided in half by a large central blood sinus; ventrally it is covered by a thin layer of ependyma (including tanycytes, dendrites, and axons) which connects the two lateral halves and protrudes as a midsagittal crest into the lumen of the third ventricle. The ependyma consists predominantly of tanycytes with long basal processes which terminate upon perivascular spaces. These cells have an extensive Golgi apparatus and abundant lysosomes; their cellular apices containing polyribosomes and a few vesicles frequently protrude into the ventricle. In addition to astrocytes, oligodendrocytes, and microglial cells, there is another glial cell population that is distinguished by the presence of parallel stacks or spherical to ovoid conglomerates of rough ER and their unique location, i.e., limited to areas ventral and ventral-lateral to the large blood sinus. Two types of neurons are present: neurons in which there is a paucity of granulated vesicles and occasional vacuoles in both the cytoplasm and nuclei, the second type of neuron elaborates many granulated vesicles. Numerous puncta adhaerentia are observed between adjacent neuronal perikarya and between glial processes and neuronal perikarya. Diverse axon types are found within the chicken SFO. Axo-dendritic and axo-somatic axon terminals and presynaptic axon dilations contain assorted combinations of electron-lucent and granulated vesicles of different maximal diameters. Based on the morphology of these axons, cholinergic, peptidergic, and serotoninergic fibers are described. There are two additional groups of axons whose classification awaits further investigation. The chicken SFO differs from the mammalian SFO in several respects: it possesses an ependyma with secretory and/or absorptive tanycytes predominating; it is divided midsagittally by a central blood sinus; its lateral and dorsal limits are nebulous; a previously undescribed peculiar type of glial cell is found in a limited portion of the organ; supraependymal neurons are lacking.