Howard B. Fleit

Identification of FcγRI, II and III on normal human brain ramified microglia and on microglia in senile plaques in Alzheimer's disease

Abstract Using monoclonal antibodies to the three known human leukocyte IgG receptors, FcγR, we examined the expression of FcγR in normal brains and in Alzheimers disease. We found FcγRI, II and III immunoreactivity in senile plaques and on ramified microglia throughout the cortex and white matter of normal and Alzheimers disease brains. FcγRI expression was independently confirmed by a murine isotype binding study. These findings suggest that intrinsic FcγR may play an important role in normal and disordered immune-related processes in the brain. The support the idea that microglia are brain macrophages.

Fcγ Receptor Mediated Phagocytosis by Human Neutrophil Cytoplasts

Neutrophils utilize Fcγ Receptors (FcγR) to bind and internalize antibody coated cells or immune complexes. We have compared ultrastructurally FcγR-mediated phagocytosis by neutrophil cytoplasts (enucleated and granule-free neutrophils) prepared either by treatment with cytochalasin B (CB-cytoplasts) followed by ultracentrifugation or by brief heating at 45°C in suspension followed by ultracentrifugation. The phagocytosis of antibody-coated erythrocytes (EIgG) or the internalization of crosslinked IgG complexes by cytoplasts prepared by brief heating was comparable to that of untreated neutrophils. In contrast, CB-cytoplasts bound but failed to internalize EIgG or crosslinked IgG complexes. Comparison of these two methods for the preparation of neutrophil cytoplasts may assist in clarifying the signal transduction requirements involved following ligand binding to FcγR which initiate phagocytosis.

In vivo effect of recombinant human granulocyte colony-stimulating factor on phagocytic function and oxidative burst activity in septic neutropenic neonates.

Neutrophil dysfunction may contribute to an increased risk of sepsis in very-low-birth-weight (VLBW) neonates. The current study was designed to determine whether recombinant human granulocyte colony-stimulating factor (rhG-CSF) affects absolute neutrophil count (ANC), phagocytic function, and oxidative burst in neutropenic VLBW neonates. Fourteen ventilated VLBW neonates were treated with rhG-CSF (10 µg/kg/day × 3 days i.v.). Phagocytic activity and oxidative burst were assessed before and after treatment with rhG-CSF using flow cytometry and fluorescence labeled opsonized Staphylococcus aureus. Control (nonseptic, nonneutropenic, n = 4), preeclamptic neutropenic (PET; nonseptic, n = 5), and septic neutropenic (n = 5) neonates with a gestational age ranging from 24 to 30 weeks were studied. In both PET and septic neonates, posttreatment phagocytosis more than doubled, but did not achieve matching control levels, whereas rhG-CSF treatment maintained the level of the phagocytic activity in the control group. The oxidative burst increased in all groups, but, again, PET and septic groups did not achieve matching control values. These effects occurred independent of a 2- to 12-fold increase in ANC. These results suggest that other disease-specific factors delay full functional recovery even after rhG-CSF treatment. We speculate that PET and septic neonates may remain susceptible to infection due to deficient neutrophil-killing capacity, even though their ANC returns to normal ranges. Augmenting immune function beyond the immediate period of ANC recovery suggests that prophylaxis with rhG-CSF may be an important risk reduction strategy for susceptible VLBW neonates.

Interaction between Borrelia burgdorferi and Polymorphonuclear Leukocytes Phagocytosis and the Induction of the Respiratory Burst

The ability of polymorphonuclear leukocytes (PMN) to engulf and kill bacteria is of major importance in host defense against bacterial infection. An important microbicidal mechanism of phagocytic cells is the formation of toxic oxygen metabolites following the stimulation of the respiratory burst. The phagocytosis of Borrelia burgdorjeri by phagocytic cells has been described.’” The ability of the spirochetes to induce the respiratory burst in PMN was also examined using a chemiluminescence assay.’ Both of these studies described the phagocytosis of spirochetes in the presence and absence of immune serum, however little is known about the mechanism of uptake of these spirochetes in the absence of serum opsonins. In the present studies the early events in the phagocytosis of unopsonized Borrelia burgdorferi were examined ultrastructurally. In addition, the induction of the respiratory burst in PMN by opsonized and unopsonized spirochetes was compared using the nitroblue tetrazolium (NBT) reduction assay.3

Characterization of two distinct transglutaminases of murine bone marrow-derived macrophages: effects of exposure of viable cells to cigarette smoke on enzyme activity.

The present study examines the effects of water soluble extracts of gas‐phase cigarette smoke on intracellular transglutaminase activities of intact, murine, bone marrow‐derived macrophages maintained in culture. Western blotting of cell lysates utilizing non‐cross‐reactive antisera indicate that mouse bone marrow‐derived macrophages contain both tissue‐type transglutaminase and factor Xlll‐associated transglutaminase. This finding is also supported by data indicating that the intracellular transglutaminase activity of these cells contains thrombin‐dependent and ‐independent components. Macrophages incubated with cigarette smoke solutions for 15 minutes at 37° display a dose‐dependent decrease (maximum inhibition = 55%, p < .001) in tissue‐type (thrombin‐independent) transglutaminase activity, as compared to control cells incubated with phosphate‐buffered saline. Factor XIII (zymogen) is not inactivated following incubation of macrophages with smoke extracts. Smoke exposure under the conditions employed has no effect on either cell viability or adherence. Incubation with 2 μM retinoic acid for 24 hours leads to a modest (2‐fold) induction of tissue transglutaminase, but does not induce factor XIII; in contrast, incubation with 10% homologous serum for 24 hours results in a decrease in factor XIII, but does not affect tissue transglutaminase. These data indicate that: 1) bone marrow‐derived macrophages contain factor XIII as well as tissue‐type transglutaminase; and 2) gas‐phase cigarette smoke can inactivate tissue transglutaminase within viable murine bone marrow‐derived macrophages, but cannot inactivate zymogenic factor XIII.

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.

Detection of tumor necrosis factor induced nuclear damage with p-phenylenediamine

A simple technique has been used to observe the effects of rTNF alpha on nuclear morphology. Using the intercalating dye p-phenylenediamine to stain nuclei, we detected TNF alpha-induced nuclear alterations which characteristically occur during apoptosis in the TNF alpha sensitive U937 cell line. Nuclear alterations were visible prior to the loss of plasma membrane integrity and subsequent cell death. A subclone of U937 cells was isolated in which TNF alpha failed to alter either cell viability or nuclear morphology. TNF alpha resistant U937 cells, however, retained the ability to bind, internalize and degrade TNF alpha. These results suggest that nuclear damage induced by TNF alpha in sensitive U937 cells occurs early and precedes cell death as measured by dye exclusion assays. Staining cells with p-phenylenediamine and visualization with a 520 nm fluorescence filter provides a rapid and simple method to monitor apoptotic cell death.

Identification of Nonadherent Mononuclear Cells in Human Cord Blood That Differentiate into Macrophages

We describe a population of nonadherent cells in neonatal cord blood that, upon in vitro cultivation, develop into monocyte‐macrophages. These cells initially are negative for nonspecific esterase cytoplasmic activity, lack the monocyte marker MO.2, fall into smaller, nonmonocytic cell size areas, as determined by fluorescence‐activated cell sorter (FACS)‐assisted size analysis, and differentiate into macrophages under nonstimulatory culture conditions (in the absence of exogenous colony stimulating factors, <0.1 ng/ml endotoxin, and growth in suspension). In contrast to the adherent, committed macrophage precursors in cord blood, which differentiate into macrophages after 2‐3 days of culture, the nonadherent precursor does not acquire monocyte‐macrophage characteristics until day 14 of culture. Earlier induction is achieved by adding the monocyte‐activating agents lipopolysaccharide or 1,25 dihydroxyvitamin D3 to cultures.

Functional analysis of monocyte-macrophages derived from nonadherent cord blood progenitor cells: correlation with the ontogeny of cell surface proteins.

In this study we examine some of the phenotypic and functional characteristics that accompany the differentiation of monocyte‐macrophages from nonadherent precursors present in cord blood. Class II major histocompatibility complex (MHC) molecules identified by monoclonal antibody (mAb) L243 were the earliest monocyte‐macrophage‐associated antigens to be expressed. CD14 molecules, identified by mAb MO.2, and the transferrin receptor, identified by mAb OKT9, increased linearly over the 21‐day culture period. In contrast, FcγR were not expressed on these cells until 14 days of culture. FcγRI was present on approximately 80% of the cells, whereas FcγRII and FcγRIII were present on 30% and 20% of the cells, respectively.

Monoclonal antibodies to human neutrophil FcγRIII (CD16) identify polypeptide epitopes

Abstract Human neutrophils constitutively express two low-affinity FcγR, FcγRII (CD32) and FcγRIII (CD16). Eleven monoclonal antibodies (mAb) to CD16 were used to identify antigenic differences among FcγRIII-bearing cells, to define functional epitopes of FcγRIII on neutrophils, and to characterize biochemically the epitopes identified by some of these mAb. Flow cytometry demonstrated that 9 of the 11 mAb reacted with neutrophils, 10 of the 11 reacted with natural killer cells, and 9 of 11 reacted with monocytes and monocyte-derived macrophages. These mAb reacted with CD16 positive cells with varying fluorescence intensities. The ability of anti-CD16 mAb to block the binding of 125 I-labeled immune complexes to neutrophils was examined. Four monoclonal antibodies strongly inhibited (87–96%) the binding to neutrophils of 125 I-labeled immune complexes. Competitive binding assays were performed to determine whether any other anti-CD16 mAb identify the epitope identified by mAb 3G8. Two other mAb, CLBFCGRAN 1 and CLBGRAN 11, blocked binding of 125 I-3G8 IgG to neutrophils. Six of the anti-CD16 mAb efficiently immunoprecipitated polypeptides of broad mobility ranging from 45 to 84 kDa from 125 I-labeled neutrophils. When FcγRIII, a complex sialoglycoprotein consisting of almost 50% oligosaccharides, was immunoprecipitated from neutrophils with 3G8 Fab Sepharose and subsequently digested with N -glycanase, 5 of the 6 mAb were capable of immunoprecipitating a deglycosylated polypeptide migrating at 29 kDa. These results demonstrate that these 5 mAb identify polypeptide epitopes of FcγRIII, whereas 1 mAb, YFC120.5, may react with a glycosyl moiety or a determinant whose conformation is dependent on the presence of oligosaccharides.