Flow Cytometry to Identify Cell Types to Which Enzymes Bind EFFECT OF LACTIC DEHYDROGENASE VIRUS ON ENZYME BINDING*

Flow cytometry was used to measure the binding of enzymes (i.e. lactate dehydrogenases 1 and 5, malate dehydrogenase, and asparaginase) to cells. Of the four enzymes studied, asparaginase showed the greatest binding. Single color analysis revealed that asparagi- nase bound best to preparations enriched in macro- phages, and dual color analysis showed that the binding was to macrophages. Studies on continuous cell lines revealed that asparaginase bound to one mouse mac- rophage line, but not to another or to murine fibro- blasts. Inoculation of mice with lactic dehydrogenase virus, a virus that infects macrophages, decreased the in vivo clearance of asparaginase from the circulation and the in vitro binding of asparaginase to peritoneal macrophages. It is concluded that flow cytometry can be used to study the binding of enzymes to cells, to identify the cell type to which the enzyme binds, and to measure changes in the capacity of cells to bind enzymes.

Flow cytometry was used to measure the binding of enzymes (i.e. lactate dehydrogenases 1 and 5, malate dehydrogenase, and asparaginase) to cells. Of the four enzymes studied, asparaginase showed the greatest binding.
Single color analysis revealed that asparaginase bound best to preparations enriched in macrophages, and dual color analysis showed that the binding was to macrophages.
Studies on continuous cell lines revealed that asparaginase bound to one mouse macrophage line, but not to another or to murine fibroblasts. Inoculation of mice with lactic dehydrogenase virus, a virus that infects macrophages, decreased the in vivo clearance of asparaginase from the circulation and the in vitro binding of asparaginase to peritoneal macrophages.
It is concluded that flow cytometry can be used to study the binding of enzymes to cells, to identify the cell type to which the enzyme binds, and to measure changes in the capacity of cells to bind enzymes.
Alterations of enzyme levels in the blood are important for the diagnosis of certain diseases (Zimmerman and Henry, 1984). Enzyme elevation is generally attributed to release of enzymes from damaged tissue; but, in fact, the enzyme level in the blood represents the balance between enzyme influx and enzyme clearance (Hayashi et al., 1988;Notkins, 1965). Some enzymes are thought to be cleared by cells of the reticuloendothelial system (RES)' (Bijsterbosch et al., , 1982DeJong et al., 1981;Sinke et al., 1979). Factors which affect RES (Hayashi et al., 1988;Mahy et al., 1967;Notkins, 1965) function have been shown to influence enzyme clearance. For example, mice inoculated with lactic dehydrogenase virus (LDV), which infects macrophages, show a lifelong 5lo-fold increase in the level of several plasma enzymes and a marked decrease in enzyme clearance (Hayashi et al., 1988;Mahy et al., 1964;Notkins, 1965;Notkins and Scheele, 1964). Agents which inhibit macrophage function, such as silica, also can produce long-term impairment of enzyme clearance and increase plasma enzyme levels (Hayashi et al., 1988).
Current methods for studying enzyme clearance in animals involve injecting a known amount of enzyme intraperitoneally or intravenously and monitoring, over time, the disappearance of the enzyme from the circulation. Currently, there is no good in vitro correlate, and information on the specific cell types involved is limited. This study, which utilizes flow cytometry, was undertaken: 1) to analyze the first step in enzyme clearance, the binding of enzymes to cells; 2) to identify the specific cell type(s) involved; and 3) to examine factors which affect the binding of enzymes to cells. In Viva Asparaginase Clearance-Mice were infected intraperitoneally with lo7 IDS0 of LDV. Seven days later, infected and uninfected mice were injected intravenously with 100 units of asparaginase in 0.2 ml of phosphate-buffered saline. Immediately after the asparaginase injection, mice were bled by the retro-orbital bleeding technique. Blood samples were taken 2,4,6, and 10 h later. Asparaginase activity in plasma was measured (Wriston, 1970) and clearance was calculated by methods described elsewhere (Notkins and Scheele, 1964). Cell Analyses-Cells (-2 X 10') were suspended in 0.

Fluorocytometric Analysis of Enzyme Binding to Peritoneal
Macrophoges-Biotinylated lactate dehydrogenases 1 and 5, malate dehydrogenase, and asparaginase were incubated with peritoneal macrophages, and the binding of the enzymes to macrophages was analyzed by flow cytometry. As seen in Fig.  1, lactate dehydrogenase 1 showed no binding, lactate dehydrogenase 5 minimal binding, and malate dehydrogenase moderate binding, whereas asparaginase bound to over 80% of the cells. Asparaginase was used in all subsequent experiments.

Binding of Asparaginase
to Different Cell Types-The binding of asparaginase to different cell types is illustrated in Fig.  2. Asparaginase bound extensively to both peritoneal and splenic macrophages. The enzyme also bound to one macrophage line (RAW 264.7), but not to another macrophage line (IC21 (BALB/3T3) showed essentially no binding. Proof that the asparaginase was binding to macrophages in the peritoneal exudate population comes from analysis by dual color flow cytometry. As seen in Fig. 3, a rat anti-mouse macrophage antibqdy labeled to give red fluorescence identified the macrophage population (Fig. 3C), and biotin-labeled asparaginase incubated with FITC-avidin identified the cell population to which the asparaginase bound (Fig. 3B). Dual fluorescence showed that the asparaginase bound to macrophages (Fig. 30). LDV-infected (a) and uninfected control (0) mice were injected intravenously with 100 units of asparaginase. Plasma samples were taken at different times after injection, and the percent asparaginase cleared was calculated (A). Values represent mean + S.E. of five mice. Peritoneal macrophages infected with LDV in uiuo (B) or in vitro (C) were incubated with biotinylated asparaginase and FITC-avidin. Peritoneal macrophages from uninfected mice incubated with biotinylated asparaginase and FITC-avidin served as positive controls, and infected macrophages incubated with FITC-avidin alone served as negative controls.

Inhibition
Asparaginuse by LDV Infection-LDV infects macrophages and raises enzyme levels in the circulation by impairing enzyme clearance. This is illustrated in Fig. 4A, which shows that the rate of asparaginase clearance in LDV-infected animals is about one-half that in uninfected controls. To see if this impairment of enzyme clearance might be related to a decrease in enzyme binding, the binding of asparaginase to LDV-infected macrophages was determined.
As seen in Fig. 4 (B and C), both in viva and in vitro infected macrophages bound less asparaginase than did uninfected macrophages.

DISCUSSION
Flow cytometry has been used to measure the binding of a variety of molecules to cells. In this study, we use flow cytometry to measure the binding of several enzymes to cells. It is clear from the results in Fig. 1 that there is a substantial difference in the binding of different enzymes to the same cell type (peritoneal macrophages), ranging from no binding of lactate dehydrogenase 1 to high binding of asparaginase. Moreover, asparaginase binds to only certain cell types. These observations raise the possibility that there might be a specific receptor on the cell surface for asparaginase. In fact, a variety of receptors having different biological properties have been found on the surface of RES cells (Ashwell and Harford, 1982;Goldstein et al., 1979;Vlassara et al, 1986). However, competition experiments' with a lo-fold excess of unlabeled asparaginase did not inhibit the binding of labeled asparaginase to macrophages. If a specific receptor for asparaginase exists on the surface of macrophages, it is present in great excess.
These experiments also show that the specific cell type (i.e. macrophages) in a mixed cell population to which asparaginase binds can be identified by use of appropriate cell-surface markers and dual immunofluorescence.
This suggests that, in some cases, in uitro enzyme binding studies might serve as a correlate of in vivo enzyme clearance. LDV, which infects 'H. Nakayama, T. Hayashi, K. F. Salata, and A. L. Notkins, unpublished data.

RES cells, dramatically
impairs the clearance of circulating asparaginase and reduces the binding of asparaginase to both in vivo and in vitro infected macrophages.
This decrease in enzyme binding (Fig. 4) is substantial, considering that less than 20% of macrophages become infected with LDV (Oldstone et al., 1974;Tong et al., 1977). Perhaps lymphokines produced by LDV-infected cells responsible for the downregulation of enzyme binding. Moreover, macrophages are only one of the several cell types that make up the RES and may not necessarily be the major cell type responsible for the clearance of a particular enzyme. For example, cultured hepatic Kupffer cells have been shown to take up considerable lactate dehydrogenase 5 (Smit et al., 1987), which suggests that these cells, and not peritoneal macrophages, may be the principal cell type that removes lactate dehydrogenase 5 from the circulation.
Recent studies in mice showed that genetic factors determine whether an individual is a slow or fast enzyme clearer (Hayashi et al., 1988). Thus, the enzyme level in the blood following acute tissue damage may reflect, in part, differences among individuals in enzyme clearance. Whether sensitive flow cytometry enzyme binding studies will make it possible to distinguish slow from fast enzyme clearers and whether this technique can be applied to human peripheral blood leukocytes remain to be determined.
Further studies on the binding of enzymes to subsets of RES cells should reveal new information about the properties of these cells and perhaps uncover specific dysfunctions in various disease states.