Comparison of proton channel, phagocyte oxidase, and respiratory burst levels between human eosinophil and neutrophil granulocytes.

Robust production of reactive oxygen species (ROS) by phagocyte NADPH oxidase (phox) during the respiratory burst (RB) is a characteristic feature of eosinophil and neutrophil granulocytes. In these cells the voltage-gated proton channel (Hv1) is now considered as an ancillary subunit of the phox needed for intense ROS production. Multiple sources reported that the expression of phox subunits and RB is more intensive in eosinophils than in neutrophils. In most of these studies the eosinophils were not isolated from healthy individuals, and a comparative analysis of Hv1 expression had never been carried out. We performed a systematic comparison of the levels of essential phox subunits, Hv1 expression and ROS producing capacity between eosinophils and neutrophils of healthy individuals. The expression of phox components was similar, whereas the amount of Hv1 was approximately 10-fold greater in eosinophils. Furthermore, Hv1 expression correlated with Nox2 expression only in eosinophils. Additionally, in confocal microscopy experiments co-accumulation of Hv1 and Nox2 at the cell periphery was observed in resting eosinophils but not in neutrophils. While phorbol-12-myristate-13-acetate-induced peak extracellular ROS release was approximately 1.7-fold greater in eosinophils, oxygen consumption studies indicated that the maximal intensity of the RB is only approximately 1.4-fold greater in eosinophils. Our data reinforce that eosinophils, unlike neutrophils, generate ROS predominantly extracellularly. In contrast to previous works we have found that the two granulocyte types display very similar phox subunit expression and RB capacity. The large difference in Hv1 expression suggests that its support to intense ROS production is more important at the cell surface

Reactive oxygen species in phagocytic leukocytes

Phagocytic leukocytes consume oxygen and generate reactive oxygen species in response to appropriate stimuli. The phagocyte NADPH oxidase, a multiprotein complex, existing in the dissociated state in resting cells becomes assembled into the functional oxidase complex upon stimulation and then generates superoxide anions. Biochemical aspects of the NADPH oxidase are briefly discussed in this review; however, the major focus relates to the contributions of various modes of microscopy to our understanding of the NADPH oxidase and the cell biology of phagocytic leukocytes

Intracellular localization of glycosyl-phosphatidylinositol-anchored CD67 and FcRIII (CD16) in affected neutrophil granulocytes of patients with paroxysmal nocturnal hemoglobinuria

Immunoelectron microscopical studies performed in healthy human neutrophils showed the presence of glycosyl-phosphatidylinositol (GPI)- linked CD67 in granules. The use of immunogold double-labeling of CD67 and lactoferrin (LF; as marker for specific granules) or CD67 and myeloperoxidase (MPO; as marker for azurophilic granules) showed that CD67 occurred only in the specific granules. Furthermore, flow cytometry showed that CD67 has a low level of expression on the plasma membrane of these cells. In paroxsymal nocturnal hemoglobinuria (PNH)- affected neutrophils, CD67 was not detected in any intracellular compartment by immunoelectron microscopy, and flow cytometry showed no CD67 on the plasma membrane. In earlier studies, FcRIII was found on the plasma membrane, in electron-lucent vesicles, and in the Golgi complex of healthy neutrophils, and in the Golgi complex of some of the PNH-affected neutrophils. Here we have studied FcRIII in PNH-affected cells of three other patients and found, by immunoelectron microscopy, that the receptor can not be detected in these cells. However, flow cytometry showed that FcRIII was not completely absent on the plasma membrane of the affected cells, but that the level of expression on these cells was low. Thus, PNH patients can differ from one another with respect to the occurrence of affected neutrophils that have a detectable level of FcRIII in the Golgi complex. In summary, these findings show not only that the expression of the two GPI-linked proteins, CD67 and FcRIII, is markedly lower on the plasma membrane, but also that neither occurred in any of the intracellular compartments of affected neutrophils of the PNH patients examined in this study.</jats:p

Localization of the low-Mr subunit of cytochrome b558 in human blood phagocytes by immunoelectron microscopy

Cytochrome b558 is a membrane-bound component of the NADPH-oxidase system in phagocytes and consists of a low-Mr subunit of 22 to 23 Kd and a high-Mr subunit of 75 to 90 Kd. The present study on the subcellular localization of the low Mr subunit of cytochrome b558 (p22- phox) in resting human peripheral blood phagocytes was based on immunogold labeling with monoclonal antibody (MoAb) 449, recently characterized. In post-embedding labeled neutrophils, this subunit was found mainly in the membrane of the specific granules. This conclusion was supported by a quantitative analysis of the results obtained in immunogold double-labeled sections with a polyclonal antiserum against lactoferrin (LF) as a marker for specific granules and a polyclonal antiserum against myeloperoxidase (MPO) used to identify azurophil granules. No labeling of the plasma membrane was observed, because of limited penetration of the antibody into the cryosections, preventing the detection of low antigen concentrations. Pre-embedding labeling of digitonin-permeabilized neutrophils, which has the advantage of a better penetration of the antibody into the cells, showed intense immunoreactivity on the cytoplasmic side of intact granules and low labeling on the inner surface of the plasma membrane. These complementary findings indicate that in resting neutrophils the epitope of p22-phox, recognized by MoAb 449, is present on the cytoplasmic side of the membrane of specific granules and the plasma membrane. Similar observations were made in eosinophils, where MoAb 449 reacted strongly with the cytoplasmic side of numerous small granules, and a low level of labeling was observed on the inner surface of the plasma membrane. In monocytes, MoAb 449 labeling also occurred on the inner surface of plasma membrane, of endocytotic compartments, and the outer surface of relatively small granules differing from peroxidase-containing lysosomes, as shown by immunogold double-labeling with MPO.</jats:p

Localization of the low-Mr subunit of cytochrome b558 in human blood phagocytes by immunoelectron microscopy

Abstract Cytochrome b558 is a membrane-bound component of the NADPH-oxidase system in phagocytes and consists of a low-Mr subunit of 22 to 23 Kd and a high-Mr subunit of 75 to 90 Kd. The present study on the subcellular localization of the low Mr subunit of cytochrome b558 (p22- phox) in resting human peripheral blood phagocytes was based on immunogold labeling with monoclonal antibody (MoAb) 449, recently characterized. In post-embedding labeled neutrophils, this subunit was found mainly in the membrane of the specific granules. This conclusion was supported by a quantitative analysis of the results obtained in immunogold double-labeled sections with a polyclonal antiserum against lactoferrin (LF) as a marker for specific granules and a polyclonal antiserum against myeloperoxidase (MPO) used to identify azurophil granules. No labeling of the plasma membrane was observed, because of limited penetration of the antibody into the cryosections, preventing the detection of low antigen concentrations. Pre-embedding labeling of digitonin-permeabilized neutrophils, which has the advantage of a better penetration of the antibody into the cells, showed intense immunoreactivity on the cytoplasmic side of intact granules and low labeling on the inner surface of the plasma membrane. These complementary findings indicate that in resting neutrophils the epitope of p22-phox, recognized by MoAb 449, is present on the cytoplasmic side of the membrane of specific granules and the plasma membrane. Similar observations were made in eosinophils, where MoAb 449 reacted strongly with the cytoplasmic side of numerous small granules, and a low level of labeling was observed on the inner surface of the plasma membrane. In monocytes, MoAb 449 labeling also occurred on the inner surface of plasma membrane, of endocytotic compartments, and the outer surface of relatively small granules differing from peroxidase-containing lysosomes, as shown by immunogold double-labeling with MPO.</jats:p

Intracellular localization of glycosyl-phosphatidylinositol-anchored CD67 and FcRIII (CD16) in affected neutrophil granulocytes of patients with paroxysmal nocturnal hemoglobinuria

Abstract Immunoelectron microscopical studies performed in healthy human neutrophils showed the presence of glycosyl-phosphatidylinositol (GPI)- linked CD67 in granules. The use of immunogold double-labeling of CD67 and lactoferrin (LF; as marker for specific granules) or CD67 and myeloperoxidase (MPO; as marker for azurophilic granules) showed that CD67 occurred only in the specific granules. Furthermore, flow cytometry showed that CD67 has a low level of expression on the plasma membrane of these cells. In paroxsymal nocturnal hemoglobinuria (PNH)- affected neutrophils, CD67 was not detected in any intracellular compartment by immunoelectron microscopy, and flow cytometry showed no CD67 on the plasma membrane. In earlier studies, FcRIII was found on the plasma membrane, in electron-lucent vesicles, and in the Golgi complex of healthy neutrophils, and in the Golgi complex of some of the PNH-affected neutrophils. Here we have studied FcRIII in PNH-affected cells of three other patients and found, by immunoelectron microscopy, that the receptor can not be detected in these cells. However, flow cytometry showed that FcRIII was not completely absent on the plasma membrane of the affected cells, but that the level of expression on these cells was low. Thus, PNH patients can differ from one another with respect to the occurrence of affected neutrophils that have a detectable level of FcRIII in the Golgi complex. In summary, these findings show not only that the expression of the two GPI-linked proteins, CD67 and FcRIII, is markedly lower on the plasma membrane, but also that neither occurred in any of the intracellular compartments of affected neutrophils of the PNH patients examined in this study.</jats:p

Intracellular localization and de novo synthesis of FcRIII in human neutrophil granulocytes

Abstract Immunoelectron microscopic studies in human neutrophils showed that FcRIII was present on the plasma membrane, in the Golgi complex, and in many small vesicles (120 to 180 nm). FcRIII was not found in specific or azurophilic granules as shown by immunogold double-labeling experiments, visualizing both FcRIII and either lactoferrin (a marker of specific granules) or myeloperoxidase (a marker for azurophilic granules). Because the occurrence of FcRIII in the Golgi complex suggested that biosynthesis of this receptor occurs in these cells, metabolic labeling experiments were performed. Immunoprecipitation of FcRIII from NP-40 lysates of cells labeled with 35S-methionine showed a diffuse 50- to 70-Kd band corresponding with the band noted after immunoprecipitation of FcRIII from surface iodinated cells. These findings show that de novo synthesis of FcRIII occurs in neutrophils and suggest that at least some of the small vesicles containing FcRIII derive from the Golgi complex and thus are involved in transport of newly synthesized FcRIII to the plasma membrane.</jats:p

Intracellular localization and de novo synthesis of FcRIII in human neutrophil granulocytes

Immunoelectron microscopic studies in human neutrophils showed that FcRIII was present on the plasma membrane, in the Golgi complex, and in many small vesicles (120 to 180 nm). FcRIII was not found in specific or azurophilic granules as shown by immunogold double-labeling experiments, visualizing both FcRIII and either lactoferrin (a marker of specific granules) or myeloperoxidase (a marker for azurophilic granules). Because the occurrence of FcRIII in the Golgi complex suggested that biosynthesis of this receptor occurs in these cells, metabolic labeling experiments were performed. Immunoprecipitation of FcRIII from NP-40 lysates of cells labeled with 35S-methionine showed a diffuse 50- to 70-Kd band corresponding with the band noted after immunoprecipitation of FcRIII from surface iodinated cells. These findings show that de novo synthesis of FcRIII occurs in neutrophils and suggest that at least some of the small vesicles containing FcRIII derive from the Golgi complex and thus are involved in transport of newly synthesized FcRIII to the plasma membrane.</jats:p