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H B Fleit

Publications and source records attributed to H B Fleit.

45 records · Page 3Linked to original sources

Culture and recovery of macrophages and cell lines from tissue culture-treated and -untreated plastic dishes.

Macrophages can be separated from other cell types by their ability to readily attach and spread on glass or on plastic surfaces which are treated for optimal growth of cultured cells (tissue culture-treated plastic). To detach macrophages from these surfaces, techniques must be used which require prior preparation of special flasks or vessels, utilize expensive equipment, are time-consuming and almost uniformly require that the macrophages be exposed to various chemicals. We now report that macrophages can be enriched and recovered efficiently after attachment to disposable polystyrene bacteriologic petri dishes simply by gentle scraping with a rubber policeman. In this paper we compare this method to others currently in use in which resident peritoneal cells, peritoneal exudate cells or cells from bone marrow-derived cultures are detached from treated dishes using cold shock, chelating agents and lidocaine. In all studies, advantages were noted when cells were incubated in untreated dishes and detached by gentle scraping. In addition, untreated dishes supported the growth of adherent cell lines IC-21 and L929B and yielded large numbers of cells, with high viability, which were easily harvested.

Animals↗

Interactions of phagocytes with the Lyme disease spirochete: role of the Fc receptor.

The phagocytic capacity of murine and human mononuclear and polymorphonuclear phagocytes (including peripheral blood monocytes and neutrophils), rabbit and murine peritoneal exudate cells, and the murine macrophage cell line P388D1 against the Lyme disease spirochete was studied. All of these cells were capable of phagocytosing the spirochete; phagocytosis was measured by the uptake of radiolabeled spirochetes, the appearance of immunofluorescent bodies in phagocytic cells, and electron microscopy. Both opsonized and nonopsonized organisms were phagocytosed. The uptake of opsonized organisms by neutrophils was blocked by a monoclonal antibody specific for the Fc receptor and by immune complexes; these findings suggested that most phagocytosis is mediated by the Fc receptor. Similarly, the uptake of opsonized organisms by human monocytes was inhibited by human monomeric IgG1 and by immune complexes. These results illustrate the role of immune phagocytosis of spirochetes in host defense against Lyme disease.

Animals↗

Ontogeny of Fc receptors and complement receptor (CR3) during human myeloid differentiation.

Two different Fc receptors for IgG (Fc gamma R) have been identified on human leukocytes: a high avidity receptor (Fc gamma Rhi) present on monocytes but not on neutrophils, and a low avidity receptor (Fc gamma Rlo) present on neutrophils but not on monocytes. Fc gamma Rlo can be inhibited and the receptor precipitated by monoclonal antibody 3G8. We have used this monoclonal antibody to study the course of Fc gamma Rlo appearance on bone marrow cells, leukocytes of patients with chronic myelogenous leukemia (CML), and HL-60 and U937 cells induced to differentiate with agents such as dimethyl sulfoxide (DMSO), retinoic acid, phorbol myristate acetate, and lymphokine. We report that Fc gamma Rlo is a late differentiation antigen, first expressed at the metamyelocyte stage. Since precursors to metamyelocytes bear Fc gamma R, and the promyelocyte line HL-60 bears Fc gamma Rhi, there must be a progressive loss of Fc gamma Rhi during myeloid differentiation and the reciprocal expression of Fc gamma Rlo. Results of immunoprecipitation and polyacrylamide gel analysis of the proteins are consistent with these results. We have also studied the receptor for the C3bi complement component (CR3), which is blocked and immunoprecipitated by monoclonal antibody OKM10. During DMSO-driven differentiation of HL-60 cells, we find that CR3 is induced on all cells, whereas Fc gamma Rlo is induced on only 24% of cells, suggesting that CR3 appears earlier during differentiation than Fc gamma Rlo does.

Antibodies, Monoclonal↗

Nocodazole inhibits polyI:C induction of interferon by mouse macrophages.

Mouse bone marrow-derived macrophages respond to both viral and nonviral inducers of interferon (IFN). The effect of nocodazole, an antitubulin drug, on the induction of IFN by either polyinosinic:polycytidylic acid, or Newcastle disease virus was studied in mouse macrophages. Nocodazole, 0.5 microM and 15 microM, maximally inhibited polyI:C induction of IFN in bone marrow-derived macrophages and resident peritoneal macrophages, respectively, but had no effect on Newcastle disease virus (NDV) induction of IFN.

Animals↗

Human neutrophil Fc gamma receptor distribution and structure.

Fc receptors (FcR) for IgG on human cells were analyzed with two monoclonal antibodies, 3G8 and 4F7. Fab fragments of both hybridoma IgGs inhibited binding to neutrophils of soluble rabbit IgG complexes and sheep erythrocytes (E) coated with rabbit IgG. The number of sites for 3G8 Fab was 135,000 per neutrophil, roughly equivalent to the number of sites for rabbit IgG in immune complexes (112,000 per cell). We did not observe human IgG1 binding sites on neutrophils, although binding of IgG1 to FcR-bearing lines U937 and HL-60 was readily demonstrated. Other cell types bearing 3G8 binding sites were mature chronic myelogenous leukemia cells, eosinophils, 6% of E-rosetting lymphocytes, and 15% of Ig-bearing peripheral lymphocytes. No binding of 3G8 Fab was observed on the FcR-bearing cell lines U937, HL-60 Raji, Daudi, and K562 or on blood monocytes. However, 15% of monocytes cultured for 7 days and 60% of lung macrophages expressed 3G8 antigen. When analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the neutrophil FcR immunoprecipitated with 3G8 or 4F7 Fab-Sepharose displayed a broad band extending from Mr 73,000 to 51,000; in many experiments this band was resolved into two poorly separated components, centered at Mr 66,000 and 53,000. These results show that human neutrophil FcR for IgG is different from that on monocytes, with respect to both antigenic composition and binding of monomeric IgG1.

Antibodies, Monoclonal↗

Interferon induction in marrow-derived macrophages: regulation by L cell conditioned medium.

Mouse bone marrow cells grown in medium enriched with L cell conditioned medium (LCM) as a source of colony stimulating factor (CSF) yield populations of adherent macrophages which are quite sensitive to induction of interferon (IFN) by viral and nonviral inducers. We examined the role of LCM in the sensitivity of marrow macrophage cultures to IFN induction. Removal of LCM from the cultures for as little as 3 hours markedly reduced the IFN titers induced by a double stranded ribopolynucleotide (poly I:C) or a lipopolysaccharide (LPS), while induction by Newcastle disease virus (NDV) was unaffected. Addition of anti-CSF serum to LCM medium also reduced IFN titers in response to polyI:C but had no effect on NDV induction. The inhibitory effect of anit-CSF indicates that the LCM requirement is at least partially related to the colony stimulating activity of the medium. We postulate that CSF regulates the initial interaction of macrophages with polyI:C or LPS rather than the synthesis and secretion of interferon by the phagocytes. Nearly complete restoration of IFN induction with polyI:C was obtained when LCM deprived cultures were reincubated with LCM medium previously conditioned by marrow cultures.

Animals↗

Mononuclear phagocytes: responders to and producers of interferon.

We have provided evidence that in vivo-induced type I IF enhanced Fc-mediated particle uptake by mouse macrophages. Fc-mediated phagocytosis of opsonized erythrocytes by unelicited fresh or cultivated macrophages was stimulated by 4-8 hours of cultivation with 100 ohms/ml IF. A 1-hour pulse was sufficient when followed by incubation in If-free medium. Pactamycin, a protein synthesis inhibitor, and camptothecin, and RNA synthesis inhibitor, blocked the stimulation of phagocytosis, indicating a requirement for macromolecular synthesis. Binding by the macrophages of a radioiodinated monoclonal antibody with anti-Fc receptor II specificity indicated that the stimulation of phagocytosis did not result from an increase in the numbers of available Fc receptors. Inflammatory macrophages, while more phagocytic than resting cells, could be further stimulated by IF in vitro, as could macrophages stimulated with LPS. In contrast, macrophages obtained from animals treated with IF inducers could not be further stimulated by IF. LPS-prestimulated and normal macrophages showed similar time-course and dose-response curves to IF, indicating that the probable mechanism of stimulation is similar in both types of cells. Cultivated bone marrow-derived macrophages found to be sensitive to IF induction by LPS, poly I.C., or NDV. The induction of IF by either LPS or poly I.C. was greater at 26 degrees C than at 37 degrees C, while no such difference was found using NDV. A 2-hour pulse of LPS was sufficient to induce IF in marrow-derived macrophages. The induced IF activity was shown to be type I IF.

Animals↗