Receptor modulation by Fc gamma RI-specific fusion proteins is dependent on receptor number and modified by IgG.

The high-affinity IgG receptor, FcgammaRI (CD64), is constitutively expressed exclusively on professional APCs. Human FcgammaRI binds monomeric IgG with high affinity and is, therefore, saturated in vivo. The binding of IgG to FcgammaRI causes receptor recycling, while Abs that cross-link FcgammaRI cause rapid down-modulation of surface FcgammaRI. Because studies performed in the absence of ligand may not be representative of FcgammaRI modulation in vivo, we investigated the ability of FcgammaRI-cross-linking Abs and non-cross-linking derivatives to modulate FcgammaRI in the presence and absence of ligand. In the absence of ligand mAb H22 and wH22xeGFP, an enhanced green fluorescent protein (eGFP)-labeled fusion protein of H22, cross-linked and rapidly down-modulated surface FcgammaRI on the human myeloid cell line, U937, and its high FcgammaRI-expressing subclone, 10.6. This effect was dependent on the concentration of fusion protein and the level of FcgammaRI expression and correlated with internalization of both wH22xeGFP and FcgammaRI, itself, as assessed by confocal microscopy. A single-chain Fv version, sFv22xeGFP, which does not cross-link FcgammaRI, was unable to modulate FcgammaRI in the absence of IgG. However, if ligand was present, treatment with either monovalent or cross-linking fusion protein led to intracellular receptor accumulation. These findings suggest at least two alternate mechanisms of internalization that are influenced by ligand and demonstrate the physiologic potential of FcgammaRI to transport a large antigenic load into APCs for processing. These studies may lead to the development of better FcgammaRI-targeted vaccines, as well as therapies to down-modulate FcR involved in autoimmune diseases.

Colocalization of Fc gamma RI-targeted antigen with class I MHC: implications for antigen processing.

The high-affinity receptor for IgG (CD64 or FcgammaRI) is constitutively expressed exclusively on professional APCs (monocytes, macrophages, and dendritic cells). When Ag is targeted specifically to FcgammaRI, Ag presentation is markedly enhanced, although the mechanism of this enhancement is unknown. In an effort to elucidate the pathways involved in FcgammaRI targeting, we developed a model targeted Ag using enhanced green fluorescent protein (eGFP). This molecule, wH22xeGFP, consists of the entire humanized anti-FcgammaRI mAb H22 with eGFP genetically fused to the C-terminal end of each CH3 domain. wH22xeGFP binds within the ligand-binding region by its Fc end, as well as outside the ligand-binding region by its Fab ends, thereby cross-linking FcgammaRI. Confocal microscopy studies revealed that wH22xeGFP was rapidly internalized by the high-FcgammaRI-expressing cell line U937 10.6, but did not associate with intracellular proteins Rab4, Rab5a, or Lamp-1, suggesting that the targeted fusion protein was not localized in early endosomes, recycling vesicles, or lysosomes. Interestingly, wH22xeGFP was found colocalized with intracellular MHC class I, suggesting that FcgammaRI-targeted Ags may converge upon a class I processing pathway. These data are in agreement with studies in the mouse showing that FcgammaRI targeting can lead to Ag-specific activation of cytotoxic T cells. Data obtained from these studies should lead to a better understanding of how Ags targeted to FcgammaRI are processed and under what conditions they lead to presentation of antigenic peptides in MHC class I, as a foundation for the use of FcgammaRI-targeted Ags as vaccines.

Characterization of expression, cytokine regulation, and effector function of the high affinity IgG receptor Fc gamma RI (CD64) expressed on human blood dendritic cells.

The mechanisms responsible for efficient sequestration of Ag by cells of the dendritic cell (DC) lineage remain incompletely characterized. One pathway, internalization of Ag-IgG complexes via CD32 (the type II IgG FcR, Fc gamma RII) enhances Ag presentation 100-fold over noncomplexed Ag. Blood leukocytes differentially express two additional IgG FcR, Fc gamma RI (CD64) and Fc gamma RIII (CD16), which may also participate in leukocyte functions such as phagocytosis, Ab-dependent cellular cytotoxicity (ADCC), release of oxygen intermediates, and enhancement of Ag presentation. A phagocytically active form of CD64 was recently demonstrated on human blood DC, but complete functional potential of CD64 on the DC lineage remains undefined. Therefore, highly purified human blood DC (CD33(2)+, CD14-, CD11c2+, HLA-DR3+, CD64+ (CD83+ after overnight culture)) and monocytes (CD33(2)+, CD14(3)+, CD11c2+, HLA-DR+, CD64(2)+, CD83-) were compared for cytokine modulation and effector functions of CD64. Both DC and monocyte CD64 expression was increased by IFN-gamma and IL-10, but while monocyte CD64 was decreased by IL-4, DC CD64 remained unchanged. FcR-mediated functional differences were also evident between the DC and the monocytes. Monocytes generated robust Fc gamma R-dependent superoxide anion release and ADCC activity, while DC failed to release reactive oxygen intermediates and demonstrated minimal ADCC activity, despite apparently normal expression of the gamma-chain subunit and the signaling molecule Syk. In contrast, DC were more efficient than monocytes with respect to T cell activation when Ag was targeted specifically to CD64. These new findings suggest a previously unappreciated potential for CD64 to shape the immune response by dramatically increasing the efficiency with which DC sequester Ag prior to achieving full T cell stimulatory potential.

Intracluster restriction of Fc receptor gamma-chain tyrosine phosphorylation subverted by a protein-tyrosine phosphatase inhibitor.

This study shows that aggregation of U937 cell high affinity IgG Fc receptor (Fc gamma RI) results in the transient tyrosine phosphorylation of Fc gamma RI gamma-chain but not the phosphorylation of gamma-chains associated with nonaggregated IgA Fc receptors (Fc alpha R) on the same cells. Thus, normally, tyrosine phosphorylation of gamma-chains is limited to FcR in aggregates. In contrast, aggregation of Fc gamma RI in the presence of vanadate induced the sustained tyrosine phosphorylation of Fc gamma RI gamma-chains and the rapid and extensive phosphorylation of nonaggregated Fc alpha R gamma-chains and low affinity IgG Fc receptors (Fc gamma RII). This global phosphorylation of motifs on nonaggregated FcR was also detected upon aggregation of Fc alpha R or Fc gamma RII, which induced the phosphorylation of nonaggregated Fc gamma RI gamma-chains. Vanadate prevented dephosphorylation of proteins and increased kinase activity in stimulated cells. Evidence failed to support alternative explanations such as acquisition of phospho-gamma through subunit exchange or a coalescence of nonaggregated with aggregated FcR. It is likely, therefore, that activated kinases interacted with nonaggregated FcR in stimulated cells. Pervanadate induced the tyrosine phosphorylation of gamma-chains in the absence of FcR cross-linking, indicating that the kinases could be activated by phosphatase inhibition and could react with nonaggregated substrates. We conclude that under normal conditions there is a vanadate-sensitive mechanism that prevents tyrosine phosphorylation of nonaggregated FcR gamma-chain motifs in activated cells, restricting their phosphorylation to aggregates.

A novel role for IgG-Fc. Transductional potentiation for human high affinity Fc gamma receptor (Fc gamma RI) signaling.

Human type 1 Fc gamma receptors (Fc gamma RI) bind with high affinity (Kd = approximately 10(-9) M) Fc regions of monomeric IgG1 and IgG3. As demonstrated in this report, interaction of IgG-Fc with the ligand binding site on Fc gamma RI alters its capacity for aggregation-dependent signaling. This Fc-dependence was demonstrated in normal monocytes and U937-10.6 cells exposed to monomeric IgG and then to anti-Fc gamma RI F(ab')2 that cross-link the receptor. Using O2- production to measure cell signaling, we found that binding by high affinity IgGs of various species, as well as by murine hybrid IgGs containing only one high affinity heavy chain, resulted in a marked increase in Fc gamma RI-mediated signaling. Preaggregated Fc gamma RI/IgG had a ratio of one. IgG binding after aggregation of unligated Fc gamma RI did not restore signaling. Dose responses indicated that concentrations of IgG that saturated Fc gamma RI optimized transductional activity. The inclusion of unligated with ligated Fc gamma RI in aggregates depressed activity, indicating a lack of trans-activation of unligated Fc gamma RI. Significantly, IgG-binding markedly increased aggregation-dependent tyrosine phosphorylation of Fc gamma RI gamma-chains and the association of tyrosine phosphorylated Syk. Thus, the consequences of IgG-Fc binding were increases in aggregation-dependent phosphorylation of Fc gamma RI gamma-chains, recruitment of pp72Syk to Fc gamma RI, and signaling of the NADPH oxidase pathway.

Prospective analysis of coagulase-negative staphylococcal infection in hospitalized infants.

Coagulase-negative staphylococci are the major cause of late-onset nosocomial neonatal sepsis. We prospectively examined all infants less than 6 months of age hospitalized at Children's Hospital of Philadelphia from whom at least one of two or more blood cultures grew coagulase-negative staphylococci. We considered as infections only those episodes in which multiple blood cultures grew identical isolates. Among 59 episodes that yielded specimens meeting study criteria, 25 were considered infection and 34 contamination. Cultures from infected infants yielded significantly higher numbers of coagulase-negative staphylococci than cultures representing contamination (p = 0.001). Infected infants were more likely to have central venous lines (p = 0.009), and to have received any parenteral nutrition (p = 0.002) or lipids (0.017). Hematologic values were not helpful in distinguishing between infected and uninfected infants. Isolates representing true infection were not different from contaminants in the frequency of positivity for putative virulence phenotypes. Our data corroborate previous studies indicating risk factors and predictors of coagulase-negative staphylococcal infection.

Doxorubicin inhibits human DNA topoisomerase I.

Purified human DNA topoisomerase I was assayed quantitatively by enzyme titrations with supercoiled pHC624 DNA in the presence of 0-2.0 microM doxorubicin. Supercoiled and relaxed DNAs were resolved by agarose gel electrophoresis in the presence of ethidium bromide, and the percentage of conversion of supercoiled DNA to relaxed DNA was quantified by scanning microdensitometry. The inhibition of DNA topoisomerase I activity was measured at varying concentrations of doxorubicin. Doxorubicin inhibited enzyme activity at an IC50 value (the concentration required to inhibit 50% of the total activity) of 0.8 microM. Similar inhibition was observed for daunomycin, a structurally related anthracycline antitumor drug. These results indicate that anthracyclines inhibit human DNA topoisomerase I activity at concentrations that cause DNA damage and cytotoxicity in vivo.