C. C. Clawson

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.

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.

Deficiency of the Second Component of Complement (C2) with Chronic Vasculitis

A patient had complete deficiency of the second component of complement associated with chronic vasculitis and increased susceptibility to infection. We discuss here results of the complement profile, histocompatibility typing, and studies of the functional properties of patient plasma or serum in chemotaxis and opsonization in relation to the disease entity and host susceptibility to infection.

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.

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.

Spectrum of function of neutrophils from carriers of sex-linked chronic granulomatous disease

Bactericidal and metabolic activities were compared for polymorphonuclear leukocytes from normal controls, patients with sex-linked chronic granulomatous disease, five obligate carriers, and six potential carriers of CGD. Bacteria surviving at one hour were quantitated in a standardized assay which employed 1.25 Staphylococcus aureus per neutrophil. Heat-killed bacteria or a chemical agent, phorbol myristate acetate, were used to stimulate increases in utilization of oxygen, oxidation of [1--14C] glucose, and reduction of neotetrazolium chloride by PMN. The results demonstrate that PMN from the individual obligate carriers of CGD have a broad spectrum of functional capabilities. Neutrophils from one obligate carrier performed in the above in vitro tests and others on a par with normal control cells, whereas the PMN of others displayed deficiencies nearly as profound as those of the affected CGD patients. The observations parallel the broad range of phenotypic expression observed in heterozygotic carriers of other sex-linked recessive disorders as a result of random inactivation of the X chromosome. Although predictable from the current concept of random X inactivation, the spectrum has not been previously demonstrated for carriers of sex-linked recessive CGD and thus has important implications for the detection and counseling of carriers of CGD.

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.

Primary Leucocyte Alkaline Phosphatase Deficiency in an Adult with Repeated Infections

SUMMARY. An individual with repeated bacterial infections, eczema, hyperimmuno‐globulin E, and a primary deficiency of leucocyte alkaline phosphatase is described. The LAP‐deficient neutrophils from this patient had marginally deficient bactericidal activity particularly when challenged with high ratios of bacteria per neutrophil. Leucotactic, metabolic and morphologic features of the neutrophils from the patient were normal. Evidence is presented which contrasts this patients condition with previously described primary or secondary deficiencies of LAP.

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.

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 neutrophils 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.