©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Ligand Binding and Phagocytosis by CD16 (Fc Receptor III) Isoforms
PHAGOCYTIC SIGNALING BY ASSOCIATED AND SUBUNITS IN CHINESE HAMSTER OVARY CELLS (*)

(Received for publication, July 17, 1995)

Shanmugam Nagarajan (1)(§) Scott Chesla (3) Lisa Cobern (1) Paul Anderson (2) Cheng Zhu (3) Periasamy Selvaraj (1)(¶)

From the  (1)Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia 30322, the (2)Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, and the (3)School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

CD16, the low affinity Fc receptor III for IgG (FcRIII), exists as a polypeptide-anchored form (FcRIIIA or CD16A) in human natural killer cells and macrophages and as a glycosylphosphatidylinositol-anchored form (FcRIIIB or CD16B) in neutrophils. CD16A requires association of the subunit of FcRI or the subunit of the TCR-CD3 complex for cell surface expression. The CD16B is polymorphic and the two alleles are termed NA1 and NA2. In this study, CD16A and the two alleles of CD16B have been expressed in Chinese hamster ovary (CHO) cells and their ligand binding and phagocytic properties analyzed. The two allelic forms of CD16B showed a similar affinity toward human IgG1. However, the NA1 allele showed approximately 2-fold higher affinity for the IgG3 than the NA2 allele. Although all three forms of CD16 efficiently bound rabbit IgG-coated erythrocytes (EA), only CD16A coexpressed with the subunit phagocytosed EA. The phagocytosis mediated by CD16A expressed on CHO cells was independent of divalent cations but dependent on intact microfilaments. CHO cells expressing CD16A- and CD16A- chimeras also phagocytosed EA. The phagocytosis was specifically inhibited by tyrphostin-23, a tyrosine kinase inhibitor. In summary, our results demonstrate that glycosylphosphatidylinositol-anchored CD16B alleles differ from CD16A in their ability to mediate phagocytosis. Furthermore, since studies with other FcRs have shown that CHO cells lack the phagocytic pathway mediated by the cytoplasmic domain of FcRs, the phagocytosis of EA by CHO cells stably transfected with CD16A and CD16A-subunit chimera provides an ideal system to dissect the phagocytic signaling pathways mediated by these FcR-associated subunits.


INTRODUCTION

The binding of the Fc region of IgG to specific receptors (FcRs) (^1)expressed on hematopoietic cells results in a wide array of cellular responses that include release of inflammatory mediators, lymphokine production, cytotoxic triggering, cell activation, regulation of antibody production, and phagocytosis of antibody coated particles(1, 2, 3, 4, 5, 6) . Thus FcR interaction with IgG bridges the cellular and humoral immune responses. Three major distinct types of FcRs have been described. They are FcRI (CD64), FcRII (CD32), and FcRIII (CD16). CD64 is a high affinity receptor for monomeric IgG expressed on monocytes and activated neutrophils. CD32 is a low affinity receptor expressed on B cells, monocytes, neutrophils, and nonhematopoietic cells such as epithelial cells. CD16 is also a low affinity receptor for monomeric IgG and is expressed on neutrophils, macrophages, NK cells, activated endothelial cells(7) , and placental trophoblasts(8) . Thus many cells coexpress more than one type of FcRs. Moreover, each type of FcRs is polymorphic and expressed in different structural forms.

In humans, CD16 is expressed as two distinct (CD16A and CD16B) forms (9, 10, 11, 12, 13, 14, 15) which are products of two different highly homologous genes. CD16B is expressed on neutrophils in a glycosylphosphatidylinositol (GPI)-anchored form, whereas CD16A is expressed on NK cells, macrophages, and placental trophoblasts as a polypeptide-anchored transmembrane protein(8, 16, 17, 18) . The GPI-anchored CD16B exists as two allelic forms termed NA1 (CD16B) and NA2 (CD16B)(19) . The polypeptide-anchored CD16A expressed on NK cells and macrophages is associated with subunits such as the chain of the TCR-CD3 complex (20, 21) or the chain of FcRI(22, 23, 24) . The NA1 and NA2 alleles of CD16B are 95% homologous to each other and are 95-97% homologous to CD16A in their extracellular domain(12) . The functional significance of the existence of membrane isoforms and the polymorphism of CD16 are not clear. However, some studies have shown that CD16A differs from CD16B by triggering killing of tumor targets and signaling for IL-2 production (16, 25, 26) .

We established CHO cell lines expressing isoforms of CD16 and determined their ligand binding and phagocytic properties. The results show that the polypeptide-anchored CD16A is able to mediate phagocytosis of antibody-coated target cells, whereas under similar conditions the NA1 and NA2 alleles of GPI-anchored CD16B are not. Moreover, chimeric molecules created by replacing the cytoplasmic domain of CD16A with that of or chains also delivered signal for phagocytosis in CHO cells. These results show that the membrane anchor and associated subunits have a profound influence on the biological properties of cell surface receptors.


MATERIALS AND METHODS

Reagents

Human transferrin and rabbit anti-human transferrin IgG were purchased from Boehringer Mannheim and sheep erythrocytes (SE) from Colorado Serum Co. (Denver, CO). Hygromycin B was obtained from Calbiochem. Phosphatidylinositol-specific phospholipase C (PIPLC) was kindly provided by Dr. M. G. Low (Columbia University, New York). Human IgG subtypes, rabbit anti-DNP IgG, tyrphostin-23, and Me(2)SO were bought from Sigma.

Antibodies and cDNAs

Anti-CD16 (3G8 and CLBFcgran1) and anti-FcRII (IV.3) monoclonal antibodies (mAbs) were described before (9, 16) . mAbs specific for NA1 (CLBFcgran11) and NA2 (GRM1) allotypes of the GPI-anchored form of CD16 were kindly provided by Drs. T. Huizinga (Amsterdam, Netherlands) and F. Garrido, (Virgen de las Nieves AVD, Grenada, Spain), respectively. Peroxidase-conjugated mouse anti-human k light chain mAb HP 6156 (IgG1) was purchased from Kirkkegard & Perry Laboratories, Gaithersburg, MD.

cDNAs encoding NA1 and NA2 alleles of CD16B in a CDM8 vector were provided by Dr. B. Seed (Massachusetts General Hospital, Boston, MA). cDNAs encoding CD16A in a pSVL vector and the subunit of rat FcRI also in a pSVL vector were kind gifts from Drs. J. Ravetch (Sloan-Kettering Institute for Cancer Research, New York) and J. P. Kinet (National Institutes of Health, Bethesda, MD), respectively. Hygromycin resistance gene in a plasmid pSV2 vector was kindly provided by Dr. C. L. Saxe III (Emory University, Atlanta, GA).

Cell Lines and DNA Transfections

Transfections of CHO-K1 cells were carried out by the calcium phosphate method(27) . Briefly, CHO cells were plated at 3 times 10^5 cells in 100-mm Petri dishes and cultured overnight in RPMI 1640, 10% Nuserum, gentamycin. Plasmid DNAs corresponding to CD16B (10 µg) were coprecipitated with a hygromycin-resistant gene (200 ng) in 0.5 ml of 2 times N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, pH 7.0, and 0.5 ml of 0.25 M CaCl(2). CHO cell transfectants expressing CD16A were established by cotransfecting plasmid DNA corresponding to CD16A (10 µg), subunit of FcRI (10 µg), and hygromycin-resistant (200 ng) genes. After 24 h, cells were washed in PBS and cultured overnight in RPMI 1640, 10% Nuserum, gentamicin. Forty-eight hours after transfection, cells were cultured in RPMI 1640, 10% FBS containing 400 µg/ml hygromycin B. Cultures were fed with medium containing hygromycin every 4 days. The hygromycin B-resistant clones were expanded and selected for CD16-positive cells.

CD16 cells were selected for their ability to form strong rosettes with rabbit anti-DNP IgG-opsonized TNP-coated SE. Briefly, SE (2% packed cell volume) were washed in PBS, 5 mM EDTA, resuspended in 10 ml of trinitrobenzene sulfonic acid (6 mg/ml) in 50 mM cacodylate buffer, pH 6.9, and incubated for 30 min at room temperature with constant slow rotation. TNP-E was washed once in HBSS, incubated for 5 min at room temperature in HBSS with 40 mM glycine, and washed three times in HBSS. Finally, the TNP-E were resuspended in 10 ml of RPMI 1640, 10% FBS. TNP-E (1.5 ml) were incubated with a subagglutinating concentration of rabbit anti-DNP IgG for 1 h at room temperature. After washing the cells in RPMI 1640, 10% FBS twice, the TNP-E treated with rabbit anti-DNP IgG was used on the same day.

The opsonized EA were incubated with CD16 transfected CHO cells in 1 ml of HBSS, 5 mM EDTA, 1% FBS (100:1 ratio) at 25 °C for 30 min. Rosetted CHO cells were separated from unrosetted CHO cells by Histopaque-1077 density gradient centrifugation. The heavily rosetted cells pelleted with free E were washed in RPMI, 10% FBS, and the surface-bound E were removed by hypotonic lysis in distilled H(2)O for 20 s. CD16+ cells were expanded, and this selection procedure was repeated four more times to get cells expressing higher level of CD16. CHO cell transfectants were maintained in CHO-S-SFM II serum-free medium (Life Technologies, Inc.) supplemented with 0.1% heatinactivated IgG low FBS (Life Technologies, Inc.) and 200 µg/ml hygromycin B.

CHO cell lines expressing CD16A- and CD16A- chimeric molecules described previously (28) were maintained in CHO-S-SFM II, 0.1% IgG free FBS with 0.4 mg/ml neomycin analog, Geneticin (Life Technologies, Inc.). For the purpose of clarity, the CD16A expressed in CHO cells after cotransfection of the CD16A gene with the subunit gene will be referred as CD16A, and the CD16A chimeric molecules made with either the subunit or the subunit will be referred as CD16A- and CD16A-, respectively.

PIPLC Treatment and Flow Cytometric Analysis

PIPLC treatment of cells was carried out as described earlier(9) . After PIPLC treatment, the cells were then washed, stained with anti-CD16 mAbs followed by fluorescein isothiocyanate-conjugated F(ab`)2 goat anti-mouse IgG and analyzed using FACScan (Becton Dickinson, San Jose, CA) flow cytometry.

Cell Surface Labeling and Immunoprecipitation

CHO cell transfectants, granulocytes, and NK cells isolated from peripheral blood of healthy individuals (9) were used for immunoprecipitation. Cells (1 times 10^7) were iodinated with 1 mCi of NaI (Amersham Corp.) using IODO-GEN (Pierce) (29) . Cells were washed and lysed in 50 mM Tris-HCl, pH 8.0, 1 mM phenylmethylsulfonyl fluoride, 5 mM iodoacetamide, 1 mM diisopropyl fluorophosphate, 1% aprotinin, 1% bovine hemoglobin with either 1% Triton X-100 (for granulocytes, NK cells, and CHO cells expressing CD16A) or 50 mMn-octyl beta-glucoside (for CHO cells expressing CD16B). An aliquot of the cell lysates was immunoprecipitated with CLBFcgran1-Sepharose. N-Glycanase digestion of immunoprecipitates was performed as described earlier (16) and separated on 10% SDS-PAGE under reducing conditions.

Binding of Immune Complex

Human transferrin was iodinated with I using IODO-GEN(29) . Immune complex was prepared by mixing I-transferrin (10 µg/ml) with rabbit anti-human transferrin IgG (10 µg/ml) for 4 h at 4 °C. The complex was centrifuged at 15,000 rpm for 30 min, and the supernatant was used for an I-IC binding assay. No visible pellet was seen after centrifugation of the IC. Cells (5 times 10^5/well) were preincubated with indicated mAbs (50 µl) for 30 min at 4 °C. I-IC (50 µl of 10 µg/ml) was then added and incubation continued for 45 min at 4 °C. After washing the cells with cold HBSS, 1% FBS, the I-IC bound to cells was counted by a counter.

Rosette Assay

Human IgG subtypes were coupled to SE by the chromium chloride method(30) . CHO cell transfectants in HBSS, 5 mM EDTA, 1% IgG low FBS at 5 times 10^5 cells/well were incubated with 2 times 10^7 cells/well of SE coupled to human IgG subtypes in the presence and absence of specified reagents in a microtiter plate for 4 h at 4 °C. A minimum of 200 CHO cells were examined, and samples were counted in duplicate for rosette formation using a light microscopy. CHO cells rosetting with a minimum of four EA were scored as rosettes.

Human IgG Binding Studies

Human IgG1(kappa) and IgG3(kappa) were separately incubated overnight with peroxidase-conjugated mouse anti-human k light chain mAb HP6156 at a molar ratio of 1:0.5 at 4 °C. The immune complex was centrifuged at 15,000 rpm for 30 min, and soluble material was used for binding studies. No precipitate was observed after centrifugation. Monomeric IgGs were also centrifuged at 100,000 times g for 1 h before use in the competition assay.

CHO cell transfectants (50 µl of 5 times 10^6 cells/ml in RPMI 1640, 5% heat-inactivated IgG low FBS, Hepes, pH 7.4) were incubated with 50 µl of IC (5 µg/ml final concentration) in the presence and absence of 50 µl of IgG1 () or IgG3 () for 1 h at 4 °C with constant gentle shaking in a microtiter plate. After washing, the cells were resuspended in peroxidase assay buffer (0.1 M citrate buffer, pH 4.0, with 0.1% Triton X-100). Peroxidase activity was developed using ABTS as substrate with H(2)O(2) (0.03%) in 0.1 M citrate buffer, pH 4.0. The color produced was read in an ELISA reader (Bio-Rad) at 405 nm.

Phagocytosis Assays

Phagocytosis of EA was assayed as described(31) . Briefly, CHO cells (1 times 10^6) suspended in 100 µl of RPMI 1640, 2% IgG low FBS, 10 mM Hepes, pH 7.3, were incubated on ice with 50 µl of 80 times 10^6 rabbit anti-DNP IgG-opsonized DNP-coated SE (1:80) for 45 min in duplicate tubes. One set of tubes was transferred to 37 °C and incubated further for 90 min with occasional shaking. Unopsonized DNP-coated SE mixed with CD16 transfectants were used as controls. Surface-bound and unbound SE were removed by hypotonic lysis in H(2)O for 20 s. The cells were washed three times in cold PBS and resuspended in 200 µl of 10 mM phosphate buffer, pH 6.0, containing 0.1% SDS and 0.1% Triton X-100. Pseudoperoxidase activity of hemoglobin from the ingested E was assayed as described before(31) , using o-toluidine (50 µg/ml) in 50 mM acetate buffer, pH 5.5, in the presence of 0.12% H(2)O(2). The color developed was read at 405 nm in a Bio-Rad ELISA plate reader.

Determination of Phagocytosis Using a Micropipette Technique

The micropipette apparatus utilized has been described (32) . The methods used to observe phagocytosis at the single cell level are illustrated by the sequential photomicrographs in Fig. 6. Briefly, micropipettes with tips of 3 µm (for red cells) and 10 µm (for CHO cells) inner diameters were prepared using a pipette puller (Kopf, Tujunga, CA) and a microforge (built in-house). The wide end of each pipette was connected to a hydraulic pressure regulation system (built in-house). The movement of each pipette was controlled by a hydraulic micromanipulator (Narishige, Tokyo). Human E opsonized with Fab fragments of CLBFcgan1, an anti-CD16 mAb, were mixed with CHO cells expressing CD16A in the cell chamber (PBS, 1% bovine serum albumin) mounted on the stage of an inverted microscope (model Axiovert 100, Carl Zeiss, Oberkochan, Germany) which was placed on an anti-vibration table (Kinetic Systems, Boston, MA). A CHO cell with an adherent EA was aspirated by the micropipette with a suction pressure of a few millimeters of water height and held stationary for the period of observation. The phagocytic process was viewed through the bottom of the cell chamber with a 100times objective (numerical aperture of 1.25, oil immersion) and a 20times eyepiece. For brightfield microscopy (Fig. 6, A-C, and Fig. 7E), the images were recorded through a charge-coupled device (CCD) camera (Dage MTI, Michigan City, IN) mounted on the top camera port of the microscope and a super-VHS video cassette recorder (Mitsubishi, Cypress, CA). A video monitor (Panasonic, Secaucus, NJ) displayed the image at a final magnification of approximately times 2,500 as calibrated by a stage micrometer. For very low light epi-fluorescence microscopy (Fig. 7, B, D, and F), an ultrasensitive liquid nitrogen cooled CCD camera (Princeton Instruments, Trenton, NJ) mounted on the side camera port of the microscope was used. The images were obtained digitally using the image acquisition hardware and software equipped with the cooled CCD camera. On some occasions, the cooled CCD camera was also used to record the bright field images (Fig. 7, A and C).


Figure 6: Sequential photomicrographs of phagocytosis of EA by a CHO cell expressing CD16A. The human erythrocytes were opsonized with CLBFcgran1, an anti-CD16 mAb, and allowed to adhere to CHO cells in a micromanipulation chamber. A CHO cell with a bound EA (indicated by arrow) was captured by a micropipette (A) and the entire phagocytic process (B and C) was viewed with brightfield microscopy, and the images were recorded using a closed circuit video system.




Figure 7: Brightfield (A, C, and E) and fluorescent (B, D, and F) images of a human EA (A and B), a CD16A-positive CHO cell (C and D), and a CD16A-positive CHO cell that has engulfed an EA (E and F). The fluorescent images were obtained using an ultrasensitive liquid nitrogen-cooled CCD camera. No fluorescent dyes were used; therefore, the images seen were from autofluorescence of the cells. The fluorescent images shown in B, D, and F were the same cells as those brightfield images shown in A, C, and E. It can be seen that the originally nonfluorescent CHO cell (D) became fluorescent after engulfing the autofluorescent EA (F). For comparative purposes Fig. 6C is reproduced as E.




RESULTS

Characterization of CHO Cell Transfectants Expressing CD16 Isoforms

The specificity of CD16 isoforms expressed on CHO cell lines were analyzed by flow cytometry (Fig. 1). All three isoforms of CD16 expressed on CHO cells (CD16B, CD16B, and CD16A) bound to nonpolymorphic mAb, CLBFcgran1 and 3G8. CD16B and CD16B bound to NA1-specific mAb, CLBgran11, and the NA2-specific mAb, GRM1, respectively. GRM1 also showed reactivity to CD16A. PIPLC treatment completely released the GPI-anchored NA1 and NA2 alleles of CD16B, whereas CD16A was resistant (Fig. 1). The CD16 isoforms bound to all of the fourth international leukocyte typing workshop mAbs against CD16 (33) with expected specificities (data not shown). CD16B isoforms did not require the cotransfection of the subunit for surface expression. However, as reported previously(28) , there was no surface expression of CD16A achieved in CHO cells without cotransfection of the subunit.


Figure 1: Immunofluorescent flow cytometry analysis of CHO cell transfectants expressing CD16 isoforms. CHO cell stable transfectants expressing CD16B, CD16B, and CD16A isoforms were stained with the indicated mAbs followed by goat (Fab`)(2) anti-mouse IgG. CLBFcgran1, an anti-CD16 mAb, binds to all isoforms, whereas CLBgran11 (GRAN11) and GRM1 are specific for CD16B and CD16B, respectively. X63 is a nonbinding mouse myeloma IgG1. Dashed lines represent the staining after PIPLC treatment.



The cell surface expression of CD16 isoforms on stable cell lines was further confirmed by cell surface labeling with I and immunoprecipitation. The CD16 isoforms moved as a broad band on SDS-PAGE with a molecular mass of 40-60 kDa (Fig. 2). The mobility of CD16B, CD16B, and CD16A from CHO stable cell lines was slightly faster than that of CD16 from NA1/NA1, NA2/NA2 homozygous neutrophils, and NK cells, respectively. However after N-glycanase digestion, the mobility of CD16 isoforms expressed on CHO cells was identical to that of CD16 isoforms expressed on neutrophils and NK cells, suggesting that the difference in mobility may be due to differences in cell-specific glycosylation. Since the most of the subunit resides on the cytoplasmic side of the membrane surface radiolabeling was not possible. Therefore, the expression of the subunit in CD16A transfectants was confirmed by analyzing mRNA specific for the subunit. Oligonucleotide primers specific for the 5` end (23-40 base pairs) and 3` end (263-280 base pairs) of subunit (34) were synthesized, and reverse transcription polymerase chain reaction was performed using Life Technologies, Inc. superscript kit as per the manufacturer's instructions. The results showed that a single product of 260 base pairs could be obtained from CD16A CHO cell transfectants that comigrated with the polymerase chain reaction product obtained using pSVL vector containing subunit cDNA (data not shown). Under identical conditions no reverse transcription polymerase chain reaction product was obtained using mRNA from CHO cells expressing CD16B. This suggests that the subunit was expressed in CD16A but not in CD16B CHO cell transfectants.


Figure 2: SDS-PAGE analysis of CD16 isoforms. CD16 was immunoprecipitated from lysate of I-surface-labeled granulocytes from a CD16B homozygous donor (lane 1), CD16B homozygous donor (lane 2), NK cells (lane 3) from peripheral blood and CHO cell stable transfectants expressing CD16B (lane 4), CD16B (lane 5), and CD16A (lane 6) isoforms. CD16 was immunoprecipitated from the lysates using CLBFcgran1-Sepharose 4B and treated with or without N-glycanase and run on SDS-PAGE under nonreducing conditions. Molecular mass markers are indicated. Lane 1 was obtained from a different gel run under identical conditions.



The stable cell lines expressing CD16 isoforms were analyzed for their functional ability to bind to IC. Radiolabeled, soluble transferrin/rabbit anti-transferrin IgG IC was used for binding studies. As shown in Fig. 3, I-IC bound to CHO cells expressing CD16 isoforms, but not to untransfected CHO cells. This binding was completely inhibited in the presence of anti-CD16 mAbs, 3G8 and CLBFcgran1, but not by the nonbinding mouse myeloma IgG1, X63, and IV.3, a FcRII mAb.


Figure 3: Binding of I-labeled immune complex by CHO cell transfectants expressing CD16 isoforms. The untransfected CHO cells (A) or CHO cell stable transfectants expressing CD16B (B), CD16B (C), or CD16A (D) were used for IC binding. I-transferrin rabbit anti-transferrin IgG IC was formed as described under ``Materials and Methods.'' Cells (5 times 10^5) were incubated in medium with I-IC in the absence or presence of anti-CD16 mAbs, CLBFcgran1 (CLB), 3G8, or anti-FcRII mAb IV.3. X63, a nonbinding mouse myeloma IgG1, served as control. Data are represented as mean ± S.D. from triplicates.



Human IgG Subtype Binding to CD16 Isoforms Expressed on CHO Cells

We analyzed the binding of human IgG subtypes to CD16 isoforms using IgG subtypes coupled to SE. The level of IgG subtypes conjugated to SE was similar, as analyzed by flow cytometry using fluorescent-conjugated goat anti-human IgG (data not shown). Rosetting of SE coated with human IgG subtypes was quantitated by counting the rosette formed between CHO cells and SE. CHO cell transfectants expressing CD16B, CD16B, and CD16A rosetted strongly with E-IgG1 and E-IgG3 and weakly with E-IgG2. CD16A formed very few rosettes with E-IgG4 (Table 1); whereas, CD16B did not rosette with E-IgG4. No rosetting was observed with untransfected or vector-transfected CHO cells (data not shown). Complete inhibition of rosette formation was observed in the presence of anti-CD16 mAbs 3G8 or CLBFcgran1, showing that rosetting was specifically mediated by CD16.



The binding of human IgG1 and IgG3 to CD16B allelic forms was further investigated to determine the consequences of CD16B polymorphism on the relative binding affinity of the human IgG subclasses. IgG1(kappa) or IgG3(kappa) was complexed with mAb HP 6156, a peroxidase-conjugated mouse anti-human Ig kappa chain mAb, as described under ``Materials and Methods.'' Either human IgG1 () or IgG3 () was used for competition studies since neither cross-reacts with the anti-human IgG kappa chain-specific mAb used for preparing IC.

IgG1 or IgG3 complexes bound to stable cell lines expressing CD16B allelic forms and were specifically inhibited with anti-CD16 mAb. Competitive inhibition of IgG1(kappa) IC binding by various concentration of monomeric IgG1() showed that CD16B and CD16B follow a similar inhibition pattern, suggesting that CD16B and CD16B have a similar affinity toward IgG1 (Fig. 4A). At 100 µg/ml of monomeric IgG1, nearly 84 and 72% of IgG1 IC inhibition was observed for CD16B and CD16B, respectively. In contrast, the inhibition studies with IgG3 showed a striking difference, with 100 µg/ml of monomeric IgG3() inhibiting IgG3(kappa) IC nearly 98% for CD16B and 50% for CD16B (Fig. 4B). The IC for monomeric IgG3 inhibition of IgG3(kappa) complex binding was 300 nM (45 µg/ml) for CD16B and 667 nM (100 µg/ml) for CD16B, suggesting that CD16B has at least 2-fold higher affinity for IgG3 than CD16B. However, the results from rosetting studies described above showed that CD16 isoforms rosetted less with E-IgG3 than E-IgG1. This could be due to a higher susceptibility of IgG3 than IgG1 for chemical coupling procedures.


Figure 4: Binding of CD16 isoforms to IgG1 or IgG3 complexes. Human IgG1(kappa) or IgG3(kappa) were incubated with peroxidase-conjugated mouse anti-human Ig kappa light chain mAb HP6156 to obtain a soluble complex as described under ``Materials and Methods.'' CHO cell transfectants expressing CD16B (box) or CD16B () were allowed to bind the IgG1 complex in the presence of monomeric IgG1 (A). Similarly, binding of the IgG3 complex to CD16 isoforms was done in the presence of monomeric IgG3 (B). The binding of IgG1 or IgG3 complexes was quantitated by assaying the peroxidase activity associated with the bound complex. The values are mean ± S.D. of triplicates.



Phagocytosis of IgG-opsonized Erythrocytes by CD16-transfected CHO Cells

CHO cells expressing the three isoforms were examined for phagocytic ability to ingest DNP-conjugated SE opsonized with rabbit anti-DNP IgG (EA). Phagocytosis of EA by CD16 transfectants was determined by assaying the pseudoperoxidase activity of hemoglobin from the ingested E as described(31) . Suspension of CHO cell transfectants incubated at 4 °C with EA showed 65, 56, and 55% rosette formation for CD16B, CD16B, and CD16A, respectively. The surface-bound EA was phagocytosed at 37 °C by CHO cells expressing CD16A. However, no phagocytosis was observed by CHO cells expressing CD16B and CD16B (Fig. 5). Phagocytosis of EA by CD16A-expressing CHO cell transfectant was completely inhibited in the presence of either anti-CD16 mAb, CLBFcgran1, or cytochalasin D (Fig. 5). During each phagocytosis assay standards of known quantities of lysed EA were run to determine the number of EA phagocytosed. From data of four separate experiments it was calculated that approximately one EA was ingested per 3.5 CHO cells. Assuming that one EA was ingested per CHO cell, nearly 29% of the CHO cell population phagocytosed EA. However the phagocytosis assay employed did not demonstrate single or multiple ingestion. This phagocytosis assay originally was used to measure the phagocytosis of EA by macrophages (31) and gave extremely low background with CHO cells as can be seen in the Fig. 5and 9.


Figure 5: Phagocytosis of rabbit IgG-opsonized SE by CD16 isoforms. CHO cell stable transfectants expressing CD16B, CD16B, or CD16A were assayed for phagocytosis of EA. CHO cells (1 times 10^6) in RPMI 1640, 2% IgG low FBS, 10 mM Hepes, pH 7.3, were incubated at 4 °C for 45 min with rabbit anti-DNP IgG-opsonized DNP-coated E (80 times 10^6 in 50 µl) in the absence or presence of CLBFcgran1 or cytochalasin D (2 µg/ml). The phagocytosis assay was performed as described under ``Materials and Methods.'' The pseudoperoxidase activity of hemoglobin from ingested E was assayed and read in an ELISA reader.



To confirm that the methodology we used to measure the phagocytosis by CHO cells truly reflects the internalization of antibody-coated particles, we have analyzed the phagocytosis of fluorescein isothiocyanate-labeled Candida albicans coated with rabbit antibody using fluorescent microscopy. The fluorescence from uninternalized C. albicans was quenched by trypan blue. CHO cells expressing CD16A phagocytosed the C. albicans, whereas no phagocytosis was observed by CHO cells expressing either CD16B or CD16B No phagocytosis was also observed by CD16A-expressing CHO cells kept at 4 °C or treated with cytochalasin D. Microscopic observations further showed that the number of C. albicans phagocytosed by CHO cells varied; most of the cells phagocytosed more than one C. albicans.

We have also directly visualized the phagocytosis of EA by CHO cells at the single cell level using micropipette manipulation. This technique has been successfully used to observe and manipulate phagocytosis of yeast by granulocytes (35) and macrophages. (^2)CHO cells were added to the test chamber in PBS containing 1% bovine serum albumin and allowed to spontaneously rosette with EA. As shown in Fig. 6A, a single conjugated pair of cells, CHO cell and EA, was captured and held by a micropipette to enable close observation of the phagocytic process in detail. After 5 min the CHO cell began to engulf the attached EA (Fig. 6B). In an additional 6 min the phagocytic process was completed, and ruffling of CHO cell membrane was observed (Fig. 6C). To further confirm the ingestion of the EA by the CHO cell, an ultrasensitive liquid nitrogen-cooled CCD camera was used to image the cells under epi-fluorescence illumination. When examined separately, the EA was autofluorescent (Fig. 7B), whereas the CHO cell was not (Fig. 7D). After engulfing the EA, however, the CHO cell became fluorescent, as can be seen in Fig. 7F. These results demonstrate that CD16A-expressing CHO cells can phagocytose EA and confirm the phagocytosis data obtained using colorimetric and fluorescence methods described above.

Integrins are known to cooperate with FcRs in neutrophil and macrophage phagocytosis(36, 37, 38, 39) . Integrins require divalent cations such as Ca and Mg for ligand binding. To determine the requirement of extracellular divalent cations for CHO cell phagocytosis, the phagocytosis assay was carried out in a Ca2- and Mg2-free medium, in the presence of Ca or Mg or both. As shown in Fig. 8the phagocytosis mediated by CD16A on CHO cells is independent of extracellular Ca and Mg concentration, suggesting that integrins are not involved in CHO cell phagocytosis.


Figure 8: Effect of divalent cations on phagocytosis of EA by CHO cells expressing CD16A. Phagocytosis of rabbit anti-DNP IgG-opsonized DNP-E by CHO cells expressing CD16A was performed in one of the following buffers: HBSS with Ca/Mg, HBSS without Ca/Mg (buffer A), buffer A with 5 mM EDTA, or buffer A with 5 mM EGTA. Phagocytosis was determined by assaying the pseudoperoxidase activity of hemoglobin from ingested E as described under ``Materials and Methods.''



Phagocytosis of EA by CD16A Chimeras

CD16A expressed in macrophages and NK cells was associated with the subunit of FcRI and/or the subunit of the CD3 complex(20, 21, 22, 23) . The surface expression of CD16A in CHO cell transfectant was achieved by cotransfecting with the subunit of FcRI. Both the subunit of FcRI and/or the subunit of the CD3 complex are signaling molecules. Therefore, we examined the importance of associated and subunits in mediating phagocytosis by using chimeric molecules. The CD16A- and CD16A- chimeras were created by replacing the CD16 transmembrane and cytoplasmic domains with that of or subunits, respectively(28) . We examined the phagocytosis of EA by CHO cells transfected with CD16A- and CD16A- chimeras. The CHO cells expressing the CD16A- or CD16A- chimera ingested the EA (Fig. 9). The phagocytic ability of CHO cells expressing CD16A-, CD16A-, or CD16A cotransfected with the subunit were comparable. The phagocytosis was completely inhibited by anti-CD16 mAb, CLBFcgran1 (Fig. 9).


Figure 9: Phagocytosis of rabbit IgG-sensitized DNP-E by CD16A- and CD16A- chimeras. CHO cell transfectants expressing CD16B, CD16B, CD16A cotransfected with the subunit of FcRI, and CD16A- and CD16A- chimeras were used in the phagocytosis assay. CD16.3 represents a control CD16A CHO cell transfectant with no significant surface expression of CD16A.



Inhibition of Phagocytosis by Tyrphostin-23

The CHO cell transfectants expressing CD16A and CD16A- and CD16A- chimeras were incubated with tyrphostin-23, a protein tyrosine kinase inhibitor (40) , and the phagocytosis assay was carried out. As shown in Table 2, 0.4 mM tyrphostin-23 efficiently inhibited the CD16A-mediated phagocytosis in CHO cells. Both CD16A- and CD16A- chimera-mediated phagocytosis was inhibited by tyrphostin-23. Interestingly, the phagocytosis mediated by CD16A coexpressed with the chain required higher concentrations of tyrphostin-23 than the CD16A- or the CD16A- chimera. 0.4% Me(2)SO, which was used to solubilize tyrphostin-23, inhibited the CD16A--mediated phagocytosis but had little effect on the phagocytosis mediated by CD16A and CD16A-.




DISCUSSION

Existence of more than one FcR on neutrophils has complicated the analysis of specificity and affinity of CD16B and CD16B for human IgG subtypes. Expression of cloned genes in FcR negative cell lines offers the advantage of analyzing the properties of individual isoforms of FcR. Our study provides a comparison of ligand binding by CD16B alleles in an isolated system. The results presented in this paper also demonstrate that the CD16A isoform differs from CD16B and CD16B in its phagocytic properties and that both the and subunits of CD16A can deliver signals for phagocytosis of IgG-coated particles in nonhematopoietic cells such as CHO cells.

The CD16 isoforms expressed on CHO cells bind IC complexes and IgG-coated cells. As expected, the NA1 and NA2 alleles of CD16B were susceptible to PIPLC treatment, whereas CD16A was not. Biochemical characterization of CD16 expressed on CHO cells showed that the mobility of CD16-expressed CHO cells is slightly faster than corresponding forms expressed on NK cells and neutrophils. However, the apparent size of the polypeptide was similar after N-glycanase treatment. This suggests that the difference in mobility is due to difference in cell-specific glycosylation of CD16A. A slight shift in electrophoretic mobility due to heterogeneity in glycosylation has also been observed for CD16A expressed on NK cells and macrophages(16, 17) .

It is well established that polymorphism leads to functional differences in various receptors, including FcRII(41) . However, only very limited comparative studies are available on the allelic forms of CD16B. We have compared the ligand binding and phagocytic properties of CD16 isoforms expressed on transfected CHO cell lines. Erythrocytes coated with human IgG1 and IgG3 subtypes efficiently formed rosettes, whereas IgG2-E mediated weak rosetting with CD16 isoforms. IgG4-E did not form rosettes with CD16B and consistently showed very few rosettes with CD16A. The relative affinity of CD16B allelic forms to IgG1 and IgG3 was further compared by analyzing the competitive inhibition of soluble IC with the respective monomeric IgG. These competition binding studies are an indirect measure of the relative affinity of CD16 for the IgG subtypes. The inhibition of IgG1 complex binding by monomeric IgG1 to CD16B and CD16B followed a similar pattern, suggesting that CD16B and CD16B have a similar affinity for IgG1. The inhibition pattern with IgG3 reveals that the NA1 allele has a 2-fold higher affinity for IgG3 than the NA2 allelic form of CD16B. Monomeric IgG3 inhibited IC more efficiently than monomeric IgG1, suggesting that CD16B alleles have a higher affinity for IgG3 than IgG1. Our results of the IgG subtype binding pattern of CD16B alleles expressed in CHO cells are similar to those reported specificities for CD16 on NK cells and neutrophils (42, 43, 44) .

The ability of the two allelic forms of GPI-anchored CD16B and CD16A expressed on CHO cell transfectants to mediate phagocytosis of EA was tested. CHO cells expressing CD16B and CD16B did not phagocytose EA, whereas under the same conditions CHO cells expressing CD16A ingested EA. The phagocytosis was specifically blocked by anti-CD16 mAb and was independent of extracellular divalent cations. Treatment of CHO cells with cytochalasin D also blocked CD16A-mediated phagocytosis, suggesting that intact microfilaments are necessary for the phagocytic process. These results demonstrate that GPI-anchored CD16B does not deliver a phagocytic signal, whereas the polypeptide-anchored CD16A that is coexpressed with the subunit can deliver a signal for phagocytosis of EA.

The signaling and phagocytosis by GPI-anchored CD16B are controversial. Salmon et el. (45, 46) have shown that neutrophil CD16B can mediate phagocytosis of E coated with concanavalin A. Moreover it has been observed (46) that neutrophils from NA2 homozygous individuals show depressed phagocytosis of E coated with IgG or concanavalin A when compared with NA1 homozygous individuals. On the other hand, studies using bispecific antibodies by Anderson et al.(47) have shown that CD16B expressed on neutrophils cannot mediate phagocytosis. These contradictory observations on the phagocytic ability of CD16B could be due to the difference in the type of ligand used or cell type in which CD16B is expressed. It is possible that neutrophils, unlike CHO cells, may have other proteins that under certain conditions can associate with GPI-anchored CD16B and provide phagocytic signals. The GPI-anchored CD16B expressed on neutrophils can deliver signals for lysosomal enzyme release, Ca mobilization, actin assembly, and antibody-dependent cellular cytotoxicity of chicken E(48, 49, 50, 51, 52) , whereas CD16B transfected into T cell lines does not signal for Ca mobilization (26) . Thus the cell-specific differences in signaling by CD16B further suggests that CD16B by itself cannot transduce signals from outside to inside the cell but may do so by associating with cell-specific components. Precedents for association of signaling molecules with GPI-anchored receptors such as CD59 and CD55 have been reported (53, 54, 55) .

In contrast to our observations on CD16A, the human FcRIIA expressed on CHO cell transfectants with intact transmembrane and cytoplasmic domains did not mediate phagocytosis(56) . However, under similar conditions the FcRIIA transfected into a macrophage cell line-mediated phagocytosis, suggesting that CHO cells lack the machinery for FcR mediated phagocytosis. The phagocytic signal delivered by FcRIIA was mapped to a distinct region in the cytoplasmic domain(56) . It should be noted that FcRIIA does not require any subunits for cell surface expression, whereas CD16A cannot be expressed on the cell surface without association of a or a subunit. Therefore, it is possible that the phagocytosis observed in CHO cell transfectants may be due to the CD16A-associated subunit. To address this we have analyzed the phagocytic ability of chimeric CD16A- and CD16A- molecules expressed on CHO cells. These chimeras were made by joining the extracellular domain of CD16A and transmembrane and cytoplasmic domains of a or chain(28) . Both chimeras mediated phagocytosis of EA as efficiently as CD16A expressed in association with the chain, demonstrating that both and chains can deliver a phagocytic signal in CHO cells. This suggests that CHO cells are capable of mediating phagocytosis if the phagocytic signal is delivered by and chains. Therefore, it may be possible that other types of FcRs can also mediate phagocytosis of IgG-coated particles in CHO cells if they are coexpressed with the or chain. Indeed, a recent study shows that FcRI expressed on COS cells requires coexpression of the subunit to mediate phagocytosis(57) .

The phagocytosis by CD16A-expressing CHO cell transfectants was blocked by tyrphostin-23, a protein tyrosine kinase inhibitor, suggesting that protein tyrosine phosphorylation plays an important role in the phagocytosis events initiated by CD16A-associated subunits. Studies by others have also shown that the phagocytosis mediated by FcRs associated with the or subunit can be blocked by tyrphostin-23 (57, 58) . Protein tyrosine phosphorylation, including the phosphorylation of the associated subunit, is an essential signaling pathway in FcR-dependent phagocytosis mediated by mouse macrophages(59, 60) . Triggering of cells via TCR or Fc receptors results in phosphorylation of associated or chain at tyrosine residues(26, 61, 62, 63, 64) . However, it was shown that CD16A cross-linking does not induce phosphorylation of or subunits in CHO cells(28) . This suggests that tyrphostin-23 may interfere with phosphorylation of proteins other than or subunits that are essential for the phagocytic process. However, although tyrphostin-23 is a more specific inhibitor of tyrosine kinases than genistein (40) and has been widely used in many signal transduction studies, our results did not rule out nonspecific inhibition of phagocytosis by tyrphostin-23 in CHO cells. Recently, Park et al.(58, 65) have shown that COS cells transiently cotransfected with CD16A and the or subunit can mediate phagocytosis of EA. They have further shown that conserved tyrosine residues on the or chain are necessary for both the phagocytosis and Ca mobilization in COS cells. Unlike CD16A expressed on COS cells(65) , CD16A expressed on CHO cells does not induce intracellular Ca mobilization(26, 28) , suggesting that the phagocytic events initiated by or subunits in CHO cells do not require intracellular Ca mobilization. Ca-dependent and Ca-independent phagocytosis has been described in human neutrophils and macrophages(66, 67, 68) . Therefore, it is possible that the phagocytosis mediated by CD16A and its chimeras in CHO cells may represent the Ca-independent phagocytic pathway observed in macrophages and neutrophils.

The results shown here with CD16A chimeras, in conjunction with the observations on FcRIIA(56) , suggest that at least two distinct components can deliver signals for FcR-mediated phagocytosis, i.e. one is the cytoplasmic domain of FcRs as in the case of FcRIIA, and the second is the FcR-associated and subunits. Depending upon the type of cells and FcRs, either one or both pathways may be involved in phagocytosis. At present it is not clear whether the FcR-associated subunits are obligatory components of the phagocytic machinery of all FcR-dependent phagocytosis mediated by cells such as macrophages and neutrophils. Recently, it has been shown that FcRI and FcRII associate with the chain, although they do not require subunit association for expression(69, 70) . Phagocytic cells such as neutrophils and macrophages constitutively express the chain that may associate with FcRs and thus provide a phagocytic signal(22, 71) . Very recently, Takai et al.(72) have shown that macrophages from chain-deficient mice are defective in Fc-R mediated phagocytosis, suggesting that the chain expression is crucial for FcR-mediated phagocytosis in macrophages. The phagocytosis mediated by the CD16A-subunit chimeras expressed on CHO cells described in this report provides an ideal model system to study the Ca-independent phagocytic signaling pathway initiated by FcR-associated subunits. Furthermore, the CHO cell system could also be used to determine whether other types of FcRs also use the or subunit-initiated signaling pathway to mediate phagocytosis, even though they do not require the subunits for surface expression.


FOOTNOTES

*
This work was supported by Grants AI30631 (to P. S.) and CA 53595 (to P. A.) from National Institutes of Health and by grants from the Emory/Georgia Tech Biomedical Technology Center (to P. S. and C. Z.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Institute of Pathology, Case Western Reserve University, Cleveland, OH 44106.

To whom correspondence and reprint request should be addressed. Tel.: 404-727-5929; Fax: 404-727-8540; pselvar@emory.edu.

(^1)
The abbreviations used are: FcR, Fc receptor; CCD, charge-coupled device; CHO, Chinese hamster ovary; SE, sheep erythrocytes; E, erythrocytes; EA, antibody- or IgG-opsonized erythrocytes; FcRI, Fc receptor; GPI, glycosylphosphatidylinositol; IC, immune complex(es); NA, neutrophil alloantigen; PIPLC, phosphatidylinositol-specific phospholipase C; TNP, trinitrophenyl; DNP, 2,4-dinitrophenyl; FBS, fetal bovine serum; HBSS, Hanks' balanced salt solution; ELISA, enzyme-linked immunosorbent assay; mAb, monoclonal antibody; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis.

(^2)
S. Chesla and C. Zhu, unpublished data.


ACKNOWLEDGEMENTS

We thank Nawaz Ahmed and Robin Gilmartin for technical assistance, Pat Keller for excellent secretarial assistance, and Dr. Peter Jensen, Rebecca McHugh, and Dr. V. Udhayakumar for their critical comments on the manuscript.


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