Influence of a deficiency of the second component of complement on the bactericidal activity of neutrophils in vitro.

Serum from three patients with a complete, selective deficiency of the second component of complement (C2) did not promote optimal killing of Staphylococcus aureus, 502A by neutrophilic polymorphonuclear leukocytes (PMN) in vitro. The addition of C2 reagent or the presence of heat-stable opsonin in the C2-deficient serum corrected the defective killing of S. aureus that was observed with patient or control PMN. PMN from the patients or control subjects killed bacteria with equal efficiency under conditions of optimal opsonization (normal pooled serum). However, twice-washed control PMN were better than patient PMN in killing S. aureus under circumstances of suboptimal opsonization (C2-deficient serum, heated C2-deficient serum, heated normal pooled serum, or no replacement of serum). The latter finding was due to residual C2 on the surface of twice-washed control cells. As repeated washing of control PMN progressively removed cell-associated C2, the staphylocidal effectiveness of the control RMN decreased to the level of patient PMN. In contrast to the findings with S. aureus, triply-washed PMN from patients or controls killed normal numbers of Escherichia coli, ON2, in C2-deficient serum.

Effects of phorbol myristate acetate on the metabolism and ultrastructure of neutrophils in chronic granulomatous disease.

Previous investigations have demonstrated that phorbol myristate acetate (PMA), the active principle of croton oil, stimulates alterations in normal polymorphonuclear leukocytes (PMN) that resemble closely the changes that develop in the cells after phagocytosis of bacteria. The present study has compared the effects of PMA and heat-killed bacteria on the oxygen uptake, glucose oxidation, nitroblue tetrazolium (NBT) reduction, and ultrastructure of normal neutrophils and PMN from six patients with chronic granulomatous disease (CGD). PMA stimulated oxygen consumption, hexose monophosphate shunt activity, and NBT reduction in normal cells but failed to produce similar effects in CGD neutrophils. However, PMA did induce formation of cytoplasmic vacuoles in the CGD cells similar to those observed in normal neutrophils. The results indicate that PMA is a useful nonparticulate agent for distinguishing between normal and CGD neutrophils and for studying basic mechanisms of phagocytosis in normal and abnormal PMN.

Size and filterability of human and hamster pulmonary macrophages exposed to cigarette smoke.

Pulmonary alveolar macrophages (PAM) obtained from healthy human cigarette smokers generated greater pressure in filtration through small cylindrical pores than PAM from nonsmoking controls. A well standardized hamster smoking model was employed to study the structural basis for the impaired PAM filtration accompanying cigarette smoke exposure. Hamsters also demonstrated increased PAM filtration pressure through cylindrical pores less than half a cell diameter in size after inhalation of cigarette smoke, but not after exposure to vapor phase smoke that had been strained through a particle filter. Cigarette smoker PAM were equally filterable as control through larger diameter channels, and showed comparable improvements in filterability with cytochalasin B treatment. Altered PAM filtration was associated with the formation of characteristic cytoplasmic smoker inclusions, an increase in cell size, and loss of redundant, ruffled surface membrane. The study of smoker and control PAM separated into different sizes on the basis of variations in cell density suggested that discrepancies in cell size were insufficient to explain the filtration disparity. A loss of availability of surface membrane for deformation appeared to be the most important factor responsible for the impaired filtration.

Opsonin-independent phagocytosis of surface-adherent bacteria by human alveolar macrophages.

Opsonin-independent mechanisms of phagocytosis may be important in host defense of certain body sites such as the lung. In this study, one such mechanism, "surface phagocytosis," was investigated by measuring the uptake of unopsonized [3H]-labeled Staphylococcus aureus and Pseudomonas aeruginosa adherent to a plastic surface by human alveolar macrophages (AM) and peripheral blood polymorphonuclear leukocytes (PMN). Efficient uptake of unopsonized bacteria by both cell types was observed. Electron microscopic studies suggested that the manner in which these cell types encounter adherent bacteria is different. While AM appear to gather in organisms at their periphery as they spread out upon the underlying substrate, PMN seem to sweep bacteria up as they move along the plastic surface. Bacterial killing determined by a fluorochrome microassay was decreased by AM compared to PMN. Although the precise mechanism whereby phagocytes recognize unopsonized bacteria adherent to a surface remains to be determined, this aspect of phagocytic cell function may prove to have clinical relevance.

Phagocytosis by polymorphonuclear leukocytes of Staphylococcus aureus and Pseudomonas aeruginosa adherent to plastic, agar, or glass.

Phagocytic cells may encounter bacteria in vivo that are stationary or adherent to a surface. In this study, recently developed in vitro techniques were adapted to evaluate the interaction of polymorphonuclear leukocytes (PMN) with adherent Staphylococcus aureus and Pseudomonas aeruginosa. By measuring the uptake of radiolabeled bacteria, we found that normal human PMN readily phagocytize these organisms when they are attached to plastic or when they are grown on the surface of nutrient agar. Bacteria adherent to glass elicited a chemiluminescent response, and such organisms were phagocytized and killed by PMN. Opsonization of S. aureus and P. aeruginosa was not required for surface phagocytosis, chemiluminescence, or killing. These new methods should allow evaluation of certain biological and clinical aspects of surface phagocytosis in host defense.

Surface morphology of polyvinyl chloride and silicone elastomer umbilical artery catheters by scanning electron microscopy.

The surface features of a new silicone elastomer umbilical artery catheter have been defined by scanning electron microscopy and compared with those of a standard polyvinyl chloride catheter. Little difference was noted in the surfaces of the catheter materials themselves, but the radiopaque material used to render the catheters visible by x-ray resulted in a considerable difference in the overall smoothness. The polyvinyl chloride catheter's marker was in the form of a wide, rough band extending the length of the tubing whereas the marker particles of the silicone elastomer were dispersed, more uniform in size, and resulted in less overall irregularity of the surface.

Sputum peroxidase activity correlates with the severity of lung disease in cystic fibrosis.

Patients with cystic fibrosis (CF) of the same age differ significantly in their degree of pulmonary disease. Based on preliminary observations, we postulated that the activity of myeloperoxidase would be significantly increased in patients with greater structural lung damage than in those with less lung damage. Acid extracts of weighed sputum samples were assayed for lactoferrin concentrations by ELISA. Activities of peroxidase, cathespsin G, and elastase (with and without proteinase 3) were determined by kinetic analysis using chromogenic substrates. The patients were divided into quartiles based on their Brasfield chest-radiograph score. Patients in the first quartile (least amount of structural lung abnormality) were compared to those in the fourth quartile. The concentration of lactoferrin, a specific (secondary) granule protein of neutrophils, did not differ between the two patient groups. However, the activities of the neutrophil primary granule proteins, peroxidase, elastase, and elastase plus proteinase 3, were significantly elevated in the group with the most structural lung abnormality. Sputum albumin concentration was used to estimate leakages of plasma proteins into the airways. Peroxidase activity, but not the activity of cathepsin G, of elastase, or of elastase plus proteinase 3, correlated significantly with albumin/g sputum in both quartile groups. To confirm the association of sputum peroxidase activity with differences in lung structure and to test its correlation with lung function, spirometry was performed in a second group of patients during the week prior to the time of sputum sampling. In this second group, increased sputum peroxidase activity was associated with worse Brasfield scores and with decreased percent-predicted forced expiratory volume in 1 sec.(ABSTRACT TRUNCATED AT 250 WORDS)

Proteinase-free myeloperoxidase increases airway epithelial permeability in a whole trachea model.

In cystic fibrosis the bronchiectatic conducting airways have large numbers of neutrophils in their walls and in their luminal contents. The neutrophil's primary granule enzyme activities of elastase and peroxidase are increased in the sputum of these patients. It has been postulated that these enzymes--together or individually--act to damage the airway epithelium. However, only peroxidase activity has consistently correlated with the degree of structural and functional airway disease in these patients with leakage of plasma protein into the airway lumen (Regelmann et al., Pediatr Pulmonol, 1995; 19:1-9). The present study was designed to test whether human neutrophil-derived myeloperoxidase can independently produce bronchial epithelial damage without the presence of proteases, as measured by increased permeability of the airway epithelium. Human peripheral blood neutrophils were purified, their primary granules isolated, and their peroxidase purified using affinity and ion exchange column chromatography. Activity of the proteinase-free peroxidase was measured using a chromogenic substrate. The effect of this peroxidase on the permeability of excised rat tracheas was measured using radioactive and fluorescent-labeled non-ionic molecules of varying molecular weight. Rat tracheas exposed to 15 minute treatments with either 130 U of peroxidase or hydrogen peroxide (10(-5) M) did not show a significant increase in the permeability of the epithelium to [3H]inulin, [14C]sucrose, and fluorescein isothiocyanate dextran 20 compared with control tracheas. However, those tracheas exposed to 130 U peroxidase followed by 10(-5) M hydrogen peroxide showed an increased permeability to each of the three test solutes. We conclude that proteinase-free myeloperoxidase, in the presence of non-toxic concentrations of its substrates, hydrogen peroxide and halide, produced increases in permeability to non-ionic molecules in the rat trachea within 15 minutes.

Platelet interaction with bacteria. 3. Ultrastructure.

The interaction of several common strains of bacteria with rabbit or human platelets in vitro has been examined sequentially with scanning and transmission electron microscopy. Bacteria were added to platelets in their native plasma or to washed platelets in a balanced salt solution at ratios of about 1:1 or at low bacteria to platelet ratios (down to 1:100). The platelet-bacterial interaction (PBI) was studied with recording nephelometry. Matched samples were fixed for microscopy at various points in the aggregation response. The results support these conclusions: a) Bacteria stimulate platelet aggregation by direct contact and adhesion with the platelet surface. b) Adhesion between the two cell types requires divalent cations, occurs through fusion of normal cell-surface coats and appears identical in the presence or absence of extracellular plasma protein. c) The morphologic transformation of platelets during PBI is identical to that produced by collagen. d) During PBI the bacteria are incorporated into the forming platelet aggregates and reside predominantly intercellularly. e) Phagocytosis of bacteria by a single platelet is very rare. f) Bacteria which have resided within platelet aggregates for one hour are unaltered morphologically. g) PBI occurs even at very low bacterial numbers and produces platelet-bacterial aggregates in small numbers without stimulating generalized platelet aggregation. Methods for concentration of thrombocytopenic plasma and washing human platelets are presented.

The surface-connected canalicular system of blood platelets--a fenestrated membrane system.

Recent reports have suggested that channels of the surface-connected or open canalicular system (OCS) in discoid blood platelets represent a reservoir of membrane that can be evaginated following activation and shape change and contribute to an increased ratio of surface area to volume. The present study has used electron cytochemistry and freeze-fracture to examine the organization of the OCS in unaltered platelets. Results of the investigation indicate that channels of the OCS are seldom if ever single tubular invaginations of the surface membrane. Each channel joins with other canaliculi of the OCS to form an anastomosing network of fenestrated conduits spreading throughout the cytoplasm from one side of the platelet to the other. The multiple connections of the interlocked channels to different sites on the platelet surface, their association in a continuous labyrinth, and participation with elements of the dense tubular system to form membrane complexes suggest that the OCS would have to be torn apart or undergo radical rearrangement before it could be evaginated and contribute to an increased surface area on activated cells.

Development of giant granules in platelets during prolonged storage.

Giant granules are uncommon in normal human platelets but are frequently observed in platelets from patients with the Chédiak-Higashi syndrome, some case of May-Hegglin anomaly, and individuals with chronic refractory anemia (preleukemia) or myelomonocytic leukema. However, the numbers of platelets containing the giant granule anomaly are so low that it has been impossible to isolate them in sufficient quantity to identify the mechanisms involved in their formation. Recently conditions were found that foster the long-term preservation of functional platelets in vitro. Ultrastructural examination of samples of C-PRP stored for 15--21 days revealed the presence of giant granules in 10--20% of the cells. Although factors involved in large organelle development were not determined in the present study, the new in vitro system for platelet storage may provide a useful approach for investigation of giant granule formation in normal and abnormal platelets.

Effects of small latex particle uptake on the surface connected canalicular system of blood platelets: a freeze-fracture and cytochemical study.

Recent investigations have suggested that channels of the surface connected open canalicular system are the major source of membrane contributing to increased surface area of platelets following activation or uptake of particulates. However, study of large latex particle phagocytosis suggested that channels filled with spherules become dilated and lose connections with the outer surface, but do not become extruded onto the platelet membrane. The present study has employed electron dense tracers, freeze-fracture and transmission electron microscopy to follow the uptake of small latex spherules by blood platelets. Small particles were rapidly incorporated into the numerous channels without causing significant changes in surface or internal morphology. Intramembranous particles on both E and P faces in replicas of freeze-fractured platelets were unaffected by ingestion of latex spherules. Prolonged exposure to small latex particles caused platelets to lose their discoid form. The irregular spheres contained two or three dilated vacuoles filled with latex and a reduced number of single channels. Results of this and previous studies suggest that channels of the open canalicular system are internalized in platelets after phagocytosis, and are not extruded onto the platelet surface.

The Chédiak-Higashi syndrome; the nature of the giant neutrophil granules and their interactions with cytoplasm and foreign particulates. I. Progressive enlargement of the massive inclusions in mature neutrophils. II. Manifestations of cytoplasmic injury and sequestration. III. Interactions between giant organelles and foreign particulates.

Defective bactericidal functioning of polymorphonuclear leukocytes (PMNs) from patients with the Chédiak-Higashi syndrome (CHS) has been related in previous reports to a failure of the giant granules characteristics of the disorder to participate in degranulation after uptake of foreign particulates by neutrophils. However, the reason massive CHS inclusions do not fuse with and discharge their contents into phagocytic vacuoles has not been defined. The problem is particularly puzzling because it has been postulated that the hugh organelles in CHS neutophils originate by fusion of small azurophilic granules in promyelocytes and myelocytes. The present series of investigations into the cytopathology of the CHS has employed electron microscopy and ultrastructural cytochemistry to characterize the progressive enlargement of the hugh bodies in mature PMNs, their interaction with cytoplasmic constituents resulting in various manifestations of cell injury, and their response to foreign particulates. Each study clarifies abnormal features of the giant organelles essential to the understanding of their role in the defective bactericidal function of CHS neutrophils. The first report demonstrates that most of the hugh inclusions in PMNs are not primary lysosomes. The interaction and fusion of giant azurophilic granules with each other, with normal-sized primary and secondary granules, and with cytoplasmic components converts the massive primary granules into huge secondary lysosomes. Transformation to secondary hysosomes represents a critical alteration in the state of the giant granules that underlies their damaging influence on the cytoplasm and loss of reactivity wtih phagocytic vacuoles.

The Chediak-Higashi syndrome: microtubules in monocytes and lymphocytes.

Recent investigations of Chediak-Higashi syndrome (CHS) leukocytes have suggested that defective cell function and formation of giant granules may be due to an inability of the cells to assemble microtubules because of an abnormality in synthesis of cyclic 3',5'-guanosine monophosphate (cGMP). In the present study we have examined normal and CHS lymphocytes and monocytes for the presence and frequency of centriole-associated microtubules. No statistically significant differences between the mean numbers of centriole associated microtubules in normal and CHS mononuclear cells could be detected. Results of the study fail to support the hypothesis that microtubule assembly is a fundamental defect in all CHS leukocytes.

Chediak-Higashi syndrome: variable cytochemical reactivity of giant inclusions in polymorphonuclear leukocytes.

Neutrophils from the peripheral blood of patients with the Chediak-Higashi syndrome (CHS) contain large numbers of giant granules. Previous studies have demonstrated that the huge neutrophil organelles contain hydrolytic enzymes and are, therefore, massive lysosomes. The basis for the inability of giant granules to react with phagocytic vacuoles containing foreign particulates taken up by CHS neutrophils has not been determined. In the present investigation we have examined the ultrastructural cytochemistry of the huge inclusions in neutrophils from 3 patients. Electron-dense reaction product of the acid phosphatase reaction stained the giant inclusions and a few normal-sized azurophilic granules in the process of fusion with them. The amount of reaction product deposited in the huge organelles and its distribution, however, were extremely variable, even in the same cell. Myeloperoxidase staining revealed a similar variability in the intensity and distribution of its electron-dense reaction product. The findings suggest that giant primary granules in circulating CHS neutrophils retain characteristics similar to newly formed azurophilic lysosomes in promyelocytes and myelocytes. Dilution of enzyme reaction products and variability in their localization indicate that most of the giant primary granules undergo transformation or incorporation into huge secondary lysosomes, which are virtually unable to participate in the degranulation reaction after uptake of foreign particulates by CHS neutrophils.

Overview article: biostructure of blood platelets.

Advances in biochemistry, physiology, immunology, and cytochemistry, combined with a variety of new approaches for the evaluation of fine structure, have yielded new insights into the structural physiology and pathology of blood platelets. Subpopulations of platelet granules have been clearly defined; they include the catalase containing organelles referred to as peroxisomes; lysosomes enclosing hydrolytic enzymes; and the alpha-granules in which platelet factor 4, mitogenic factor, beta thromboglobulin, thrombin sensitive protein, fibrinogen, and coagulation factor V are localized. Features of platelet membrane systems have been particularly well-delineated, and recent evidence suggests that membrane complexes serve as the sarcoplasmic reticulum of platelets and the site of prostaglandin synthesis. Improved understanding of platelet biostructure resulting from these observations has made it possible to develop specific relationships between defects in structure and pathological behavior of the cells in vitro and in vivo.