Phagocytosis of Streptococcus pyogenes by all-trans retinoic acid-differentiated HL-60 cells: roles of azurophilic granules and NADPH oxidase.

BACKGROUND: New experimental approaches to the study of the neutrophil phagosome and bacterial killing prompted a reassessment of the usefulness of all-trans retinoic acid (ATRA)-differentiated HL-60 cells as a neutrophil model. HL-60 cells are special in that they possess azurophilic granules while lacking the specific granules with their associated oxidase components. The resulting inability to mount an effective intracellular respiratory burst makes these cells more dependent on other mechanisms when killing internalized bacteria. METHODOLOGY/PRINCIPAL FINDINGS: In this work phagocytosis and phagosome-related responses of ATRA-differentiated HL-60 cells were compared to those earlier described in human neutrophils. We show that intracellular survival of wild-type S. pyogenes bacteria in HL-60 cells is accompanied by inhibition of azurophilic granule-phagosome fusion. A mutant S. pyogenes bacterium, deficient in M-protein expression, is, on the other hand, rapidly killed in phagosomes that avidly fuse with azurophilic granules. CONCLUSIONS/SIGNIFICANCE: The current data extend our previous findings by showing that a system lacking in oxidase involvement also indicates a link between inhibition of azurophilic granule fusion and the intraphagosomal fate of S. pyogenes bacteria. We propose that differentiated HL-60 cells can be a useful tool to study certain aspects of neutrophil phagosome maturation, such as azurophilic granule fusion

The cytochrome b-558 molecules involved in the fibroblast and polymorphonuclear leucocyte superoxide-generating NADPH oxidase systems are structurally and genetically distinct.

We have demonstrated that human fibroblasts can release O2-. radicals by an NADPH oxidase system that appears to be functionally similar to the phagocytic system. Further analysis of these systems, however, with respect to the low-potential b-type cytochromes involved suggests that these two O2-.-generating systems are not structurally identical. Immunoblot analysis of fibroblast membranes with six different antibodies directed against both subunits of human neutrophil cytochrome b-558 indicated that the b-type cytochrome molecules involved in these systems were not identical. None of these anti-(neutrophil cytochrome b) antibodies recognized a similar cytochrome in fibroblast membranes, suggesting that the two cytochrome species are immunologically distinct. In addition, fibroblasts obtained from a patient suffering from X-linked chronic granulomatous disease (CGD) had a normal cytochrome b-558 content compared with control fibroblast membranes, whereas the cytochrome b-558 concentration in polymorphonuclear leucocytes (PMNs) from this patient was decreased to 10% of that found in PMNs from healthy controls. Likewise, the stimulated O2-. release in PMNs from this patient was less than 10% of that in control PMNs, whereas the fibroblasts showed stimulated O2-.-release rates that were indistinguishable from those of fibroblasts obtained from healthy persons. Since the genetic mutation responsible for this type of CGD results in the absence of cytochrome b-558 in PMNs, fibroblasts should be affected in the same way if both cytochrome species were identical. These results suggest therefore that the low-potential b-type cytochromes in PMNs and fibroblasts are structurally and genetically distinct

Kinetics of transfused neutrophils in peripheral blood and BAL fluid of a patient with variant X-linked chronic granulomatous disease.

Patients with chronic granulomatous disease (GCD), and uncommon inherited disorder of phagocytes resulting in the defective production of reactive oxygen intermediates, are prone to bacterial and fungal infections. In the case presented, therapeutic efforts including white cell transfusions, and amphotericin B and IFN gamma administration were undertaken to treat pneumonia caused by Aspergillus fumigatus. During a phase of artificial respiration, transfused white cells in peripheral blood and bronchoalveolar lavage fluid were monitored in order to examine their kinetics and functional activity. Using flowcytometrical methods, host-derived and transfused neutrophils could be distinguished by cytochrome b558 expression using the monoclonal antibody 7D5 for immunofluorescent staining as well as by production of reactive oxygen intermediates. Transfused PMN could be detected in both compartments and their kinetics could be followed up to 24 hours after transfusion. Using flowcytometry, eve n small numbers of transfused PM could be measured during episodes of extreme leukocytosis. Since functionally intact transfused PMN were found in the bronchioalveolar lavage fluid, white cell transfusions in combination with antibiotic and immunomodulating therapy should be considered a part of the therapeutic regimens for life-threatening infections in GCD patients

Detection, isolation and partial characterization of an immunostimulating glycoprotein from Rhodococcus fascians

In a search for novel immunostimulating substances we detected that culture supernatants of the gram-positive phytopathogenic bacterium, Rhodococcus fascians, were able to induce cytokine release (TNF alpha) from mouse peritoneal macrophages. Monoclonal antibodies were generated against the active principle, and were employed for its isolation and partial characterization as a high molecular (MW>100 kDa) glycoprotein. In addition, methods practicable for its biotechnological preparation and several ELISA variants for its determination were developed

Kinetics of human neutrophil surface marker expression and chemotaxis during granulocyte colony-stimulating factor treatment

We previously reported an altered surface marker expression and chemotaxis of G-CSF-induced neutrophils from patients with severe congenital neutropenia. However, effects of G-CSF and the influence of the underlying disease on neutrophils could not be distinguished from one another. In this study, we evaluated the effects of G-CSF on neutrophil phenotype and function in patients under chemotherapy and in healthy test subjects. We found a significantly enhanced expression of Fc(gamma)RI, CD14, and CD54 and a decrease in the level of Fc(gamma)RIII. In addition, motility of G-CSF-induced neutrophils was significantly decreased. Neutrophil phenotype and migration were altered only during G-CSF therapy. The effects were seen in patients under cytotoxic chemotherapy and in healthy test subjects. Surface marker alterations and neutrophil motility were affected by G-CSF in a dosedependent manner. (Abstract truncated

Validation of a modified 28-day rat study to evidence effects of test compounds of the immune system

The toxicology profile of new test compounds is ascertained in repeat-dose toxicity studies with exposure over 4 weeks or 3 months which yield a range between a high dose causing overt toxicity and a low dose with no observable (toxic) effect. In recent years, the immune system has been identified as a target of the direct toxic action of chemicals (Dean et al., 1989). A first step to the EU level to developed a test program to detected undesired effects on the immune system was undertaken in Luxembourg in 1984 (Berlin et al., 1987) . In consequence of this international activity, a number of collaborative studies were initiated. The aim of these studies was to select and validate available testing procedures in a tiered approach to find out their appropriateness of indicating adverse effects on the immune system after exposure to chemicals (Vos and vanLoveren, 1987; Hess et al., 1990). In a collaborative toxicity study initiated and coordinated by the Federal Institute for Health Prot ection of Consumers and Veterinary Medicine, four industrial laboratorys and one independent institute participated. The purpose of this study was to qualify and validate additional investigation (i.e., enhanced pathology, functional tests) within an ordinary subacute toxicity test protocol (Annex V, 67/548/EEC; OECD 407) to identify the effects of chemicals on the immune system during their regular safety evaluation