(LSM; Litton Bionetics,. Kensington,. MD), and washed twice in Dulbecco's .... 100 (2 milL solution). The percentage release of myeloper- oxidase, lysozyme,.
CLIN.CHEM.31/9, 1444-1448 (1985)
Fc Receptorsfor lgG on HumanNeutrophils:Analysisof Structureand Functionby UsingMonoclonalAntibodyProbes Jonathan S. Rosenberg, Meryle J Melnicoff, and Peter Wilding Structural and functional characteristics of Fc receptors for IgG (Fcy) on human neutrophilswere examined with two monoclonal antibody probes specific for the FCV receptors, Leu 11b and 3G8. To determine the distribution, density, and membrane mobility of the Fcy receptor, we used immunogold
(Mabs)-Leu lib and 3G8-directed toward the Fey receptor, we have probed both the physical and functional aspects of this receptor. This series of experiments has not only provided useful information elucidating the interaction of biochemical mechanisms through which a neutrophil responds to various stimuli by a single receptor, it has also indicated whether detection of the presence and density of Fey receptor can be useful in the clinical analysis of disease or cellular functional capacity.
staining techniques, flow cytometry analysis, and fluorescence microscopy.Both 3G8 and Leu 11b inhibited several cellfunctions,therebydepictingthe regulatoryroleof the Fcy receptor in mediatingneutrophilactivities.Among the functionsstudiedwere release of lysosomalenzymes, releaseof superoxideanion (Ok), and Fc-dependentrosette formation and phagocytosis.The densitiesof Fcy determinantsrecognizedby Leu 11b and 3G8 on cells from a patient with chronic myelogenous leukemia were less than the density of epitopes on neutrophilsfrom a normalindividual.Taken together, the detailedanalysisof physicaland functionalaspectsof the Fcy receptoron neutrophilsdescribedin this studyserve as a model for further assessment of the use of Fcy phenotypingof cells as a diagnostictool.
Monoclonal antibodies specific for T helper cells (Leu 3a) or for Fc receptors for IgG (Leu lib) were obtained from Becton Dickinson, Mountain View, CA. Mab 3G8, also specific for Fe receptor, was obtained from New England Nuclear, Boston, MA. The control reagent consisted of IgG in purified supernates or ascites fluids from NS-1 myeloma cells (CooperBiomedical, Malvern, PA).
AddItIonalKeyphrases:chronic myelogenousleukemia
Procedures
munogold staining
py
imfluorescence microscoimmunology cell-surface markers
flow cytometry
immunefunction
.
Receptors for the Fe portion of IgG (Fcy) on human neutrophils modulate several of the activities exhibited by this cell type in its mediation of host defense.’ The best studied of these activities include chemotaxis, the directed migration in response to cellular or soluble invaders or lymphokines (1, 2), as well as the microbicidal capacity of neutrophils (3). Other recent evidence has shown that neutrophils may serve as cytotoxic effectors, which lyse cells sensitized with antibodies (4). In this phenomenon, called antibody-dependent cell-mediated cytotoxicity, the final lysis of either microbial or eukaryotic cells may be effected, in part, by reactive oxygen species such as superoxide anion (O) or hydrogen peroxide, which are generated in the oxidative burst the neutrophil undergoes upon contact with a foreign cell or an antigen (5, 6). Besides these short-lived oxygen metabolites, other mediators of cell damage or destruction include lysosomal enzymes such as lysozyme or myeloperoxidase, released from specific or azurophiic granules, respectively, in a process called degranulation (7). These enzymes and reactive oxygen species may be released intracellularly into phagocytic vacuoles as well as into the extracellular milieu. Neither the structure of the Fey receptor, nor the functions it regulates, has been fully characterized in normal and leukemic cells. Using specific monoclonal antibodies Research and Development Department, Geometric Data/Division of SnuthKline Beckman, 999W. Valley Rd., Wayne, PA 19087. 1Nonstandsrd abbreviations: Fcy, Fe portion of human IgG; Mab,
monoclonal antibody; D-PBS-BSA, Dulbecco’s phosphate-buffered saline containing bovine serum albumin (10 g/L); HBSS, Hank’s Balanced Salt Solution; FMLP, N-formyl-methionyl-leucyl-phenylalanine; PMA, phorbol myristyl acetate. Received May 9, 1985; accepted June 17, 1985. 1444 CLINICALCHEMISTRY, Vol. 31, No.9, 1985
Materials and Methods Reagents
Immunogold staining. Peripheral blood was collected into EDTA-containing Vacutainer Tubes (Becton Dickinson, Rutherford, NJ). Bufiy-coat cells were obtained after centrifugation (15 mm, 480 x g), and the leukocyte-enriched “huffy coat” was collected. Cells were stained with the “Immutrak Cell Labeling System” (Geometric Data, Wayne, PA), according to the manufacturer’s directions, then examined by light microscopy. Positive cells were easily distinguished by the presence of blue-black gold aggregates on the cell surface (8). Cells were considered to be tagged when five or more surface granules were observed. Immunofluorescent staining. Peripheral blood specimens were partitioned into leukocytes, and mononuclear cells or granulocytes. Whole leukocyte populations were obtained by incubating 100-ML aliquots of whole blood with erythrocyte-lysing buffer (pH 7.2-7.4, containing 3.7 mg of disodiurn EDTA, 0.1 g of KHCO3 and 0.83 g of NH4C1 in 100 mL of H20), then washing in Dulbecco’s phosphate-buffered saline (Gibco, Grand Island, NY) containing 10 g of bovine serum albumin per liter (D-PBS-BSA). Mononuclear cells were collected from the interface after centrifligation over FicollHypaque gradients (LSM; Litton Bionetics, Kensington, MD), and washed twice in Dulbecco’s phosphate-buffered saline (D-PBS). Granulocytes were recovered from the gradient pellets after lysis of the erythrocytes and washed twice with D-PBS; more than 95% of these cells were granulocytes. Leukocyte pellets; mononuclear cells, i07 cells/mL; or granulocytes, 10 cellsfmL, in 100-L aliquots of D-PBSBSA, were incubated with 5 iL of Mab in 12 x 75 mm polystyrene tubes for 30 mm at 4#{176}C. After two washes, the cells were then reacted for 30 mm at 4#{176}C with fluoresceinlabeled F(ab’)2 goat anti-mouse IgG (CooperBiomedical). The labeled cells were washed and then either (a) resuspended to 1 x 106 to 2 x 106/mL in D-PBS-BSA containing 10 g of paraformaldehyde per liter, for analysis by flow
cytometry (EPICS V; Coulter Electronics, Hialeah, FL) with automated enzyme immunoassay reader (Biotek, Burlinggated analysis; or (b) prepared for immunofluorescence by ton, VT). The reagent blank consisted of cells plus cytofixation in 7 g/L paraformaldehyde for 5 to 7 mn#{236}, followed chrome c plus superoxide dismutase (EC 1.15.1.1), 300 U/L, which metabolizesOjto H202. The percent of Ojrelease was by washing, resuspension of the cell pellet in buffered glycerol (glycerol/PBS, 30/70 by vol), then mounted onto calculated as (iA plus stimulantIM without stimuslides and examined by fluorescence microscopy with a Leitz lant) x 100. The percentage inhibition of release was “Orthoplan” microscope (8). calculated as described below for enzyme secretion. Membrane mobility capping. Neutrophils at a density of 2 Enzyme assays. The ability of Mabs to inhibit degranulax 106/mL were pre-equilibrated with 25 zg/L coichicine tion was measured by their effect on induced release of lysosomal enzymes by the chemotactic tripeptide FMLP. solution, to inhibit microtubule elongation, or 2 ug/L cytochalasin D, to prevent microfilament gelation, in a total of Neutrophils resuspended to a concentration of 107/mL in 500 L of D-PBS for 30 miii at 37#{176}C. Cells were washed, HBSS with 2.5 g of bovine serum albumin per liter were resuspended in D-PBS-BSA with the appropriate drug, incubated in duplicate 200-pL aliquots (2 x 106 cells) with incubated with 5 1L of 3G8 or Leu lib for 30 miii at 4#{176}C, 10 zL of Mab for 30 miii at 4#{176}C, washed, incubated with then reacted with fluoresceun-labeled F(ab’)2 goat anticytochalasin B (5 g/mL) (Sigma) for 5 miii at 37#{176}C, then with FMLP (10 pmol/L) for 15 miii, at 37#{176}C. After centrifumouse IgG for 30 mm at 4#{176}C. After pelleting, the cells were resuspended in 0.5 mL of prewarmed (37 #{176}C) D-PBS-BSA gation, the enzyme content of the cell-free supernate was containing the appropriate drug, fixed at various intervals analyzed and compared with the total cell-associated en(from 0 to 30 mm) in 7 g/L paraformaldehyde at 4#{176}C,zyme present in samples treated with 1.0 mL of Triton Xwashed twice, resuspended in buffered glycerol, and then 100 (2 milL solution). The percentage release of myelopermounted onto slides. The number of fluorescent cells and the oxidase, lysozyme, and lactate dehydrogenase were meanumber of labeled cells that were “capped” were examined sured as indicators of azurophilic granule release, specific by fluorescent microscopy. A cell was considered capped if granule release, and cell viability, respectively, according to one-third or less of its surface membrane was labeled. The established procedures (11). The percent inhibition of renumber of capped cells was calculated as (the total number lease was calculated as follows: of capped cells/the total number of fluorescent cells) x 100. Rosette formation and antibody-dependent phagocytosis. [1-(% release with Mab/% release without Mab)] X 100 Rosetteassays were slightly modified from published procedures (9). Bovine erythrocytes (Hazieton Labs, Hazieton, PA) were coated with rabbit IgG specific for these cells Results (CooperBiomedical) by incubating 0.25 mL (2 x iO erythrocytes) in Hank’s Balanced Salt Solution (HBSS; Gibco) with Distribution and Density of Fcy on Neutrophils 0.25 mL of a sub-agglutinating dilution (1/50) of antibody The percentages of neutrophils bearing Fey receptors for 1 h at room temperature. Cells were then washed and were determined by immunogold staining and flow cytomeresuspended to a 10 g/L stock solution. We suspended try. Almost all neutrophils were positively labeled with Leu neutrophils, 2 x 107/mL, in RPMI 1640 plus 100 mL/L fetal lib or 3G8 (Table 1); an example of Leu lib-positive calf serum (both from Gibco), centrifuged at 50 x g for 1-2 neutrophils labeled by the immunogold staining technique miii at room temperature, then incubated 25-jzL aliquots for is shown in Figure 1. 30 mm at 4#{176}C with 5 pL of Leu lib or 3G8, 25 1zL of PBS, The intensity of neutrophil staining by Lou lib or 3G8 and 50 1zL of the erythrocyte stock solution. We then was determined by flow cytometry. Using forward and determined the proportion of 200 neutrophils binding five or right-angle light scatter to identify the neutrophil populamore erythrocytes (rosette-positive cells) in samples contion, then analyzing the fluorescence properties of the cells taining Mab (A) and in the control samples (B, no Mab), and within the neutrophil “window,” we obtained an indication calculated the percent inhibition of rosette formation by of the receptor density. The histograms in Figure 2 repreMab: (A/B) x 100. sent the intensity of Lou lib or 3G8 staining. As shown, the To determine antibody-dependent phagocytosis we incupeak channels of fluorescence for Leu llb or 3G8 are almost bated samples for 30 mm at 37 #{176}C, washed them, and the same around channels 190-200, indicating that both subjected them to hypotonic lysis buffer to remove nounMabs recognize Fey determinants present in similar density gested erythrocytes. Phagocytes that had ingested two or on the cell surface. We estimated the binding sites of each more erythrocytes were considered positive. The effect of Mab per cell by linear proportion. Previous studies having Mab on phagocytosis was calculated as above. shown that antigen density on lymphocytes positively Superoxide anion (0) production. We suspended neutrostained by Lou 3a (a Mab specific for helper T cells) was phils in HBSS containing 2.5 g of bovine serum albumin per relatively invariant in both normal and diseased states, we liter, then incubated 300 pL of the cell suspensions with 15 pL of Mab for 30 mm, at 4#{176}C, washed, and resuspended these to 3 x 106 cells/mL in PBS. The ( release was Table 1. DistributIon of Fc2-Positive Neutrophils measured by a modification of the method of Pick and Mizel As Determined by immunogold Staining and Flow (10). We added 100-FL aliquots (3 X iO cells) to each well of Cytometry 96-well flat-bottomed microtiter plates, plus 100 jzL of 200 pmolJL cytochrome c (Type ifi, from horse heart; Sigma Mean% of positive cells (and ranse) Chemical Co., St. Louis, MO) in HBSS, in the presence or lmmunogoldb Flow cytometry#{176} Meb absence of one of three compounds: N-formyl-methionyl3.9 (2.2-4.5) 3.5 (2-5) Control leucyl-phenylalanine (FMLP) (2); phorbol myristyl acetate 95.3 (91.3-97.4) 99 (98-100) 3G8 (PMA), an oxidative metabolite stimulant; or Zymosan A 95.5 (93.8-97.3) 99.5 (99-100) Leulib (from Saccharomyces cerevisiae; Sigma Chemical Co.). a Resultsrepresent the mean of three to five separate experiments. #{176}Per. Each sample was run in triplicate. The plates were centage of 200 neutrophils counted for each determination. cpementage of incubated for 1 h at 37 #{176}C and centrifuged at 50 x g for 1-2 10 000 neutrophils analyzed for eachdetermination. mm. The change in absorbance at 550 nm was read on an CLINICALCHEMISTRY, Vol. 31, No. 9, 1985
1445
Table 2. Calculation of the Number of Mab Binding Sites Mab
Leu3a Leu11b 3G8
EstImated no. of bindIng sites per cell
Meanfluorescent channelno. (and 144.8 (136-1 52)
70000
94000 100000 a.fl* mean channel numberof peak fluorescence(log scale) from 10 separateexperimentsin which a total of 10000 neutrophllswere examined In each experiment.
FIg.1.Pttotomlcrograph of neutrophils labeled by the lmmutrak’ Cell Labeling SystemwithLeu 11b Positivecellsare clearlydetectableby the darkblue-blackgranulesoverthe cell. Mao shownIs a lymphocytethat was not labeledwith this Mab
194.8(182-201)
205.8(182-211)
Table 3. Analysis of Variance of Data Obtained by Analysis of Patients’ Specimens by Various Methodsa Source of variation Patients Methods Patients x methods Wlthin-dupbcateerror
d.f.
M.S.
F
p
52
1.674 4.1393
156 212
60.08
0.0689
6.96
