THE ANATOMICAL RECORD 228:306-314 (1990)

Functional, Physical, and Ultrastructural Localization of CD15 Antigens to the Human Polymorphonuclear Leukocyte Secondary Granule E.S. BUESCHER, S.A. LIVESEY, J.G. LINNER, K.M. SKUBITZ, AND S.M. McILHERAN Department of Pediatrics, University of Texas Medical School at Houston, Houston, Texas 77030 (E.S.B., S.M.M.); University of Texas Cryobiology Research Center, The Woodlands, Texas 77381 (S.A.L.,L.G.L.); Department of Medicine, University of Minnesota Medical School, and the Masonic Cancer Center Minneapolis, Minnesota 55455 (K.M.S.)

ABSTRACT A murine monoclonal IgM antibody, M3, which interferes with both polymorphonuclear leukocyte (PMN) phagocytosis and bactericidal activity, was used to examine the subcellular location of antigens bearing 3-fucosyllactosamine (CD15 antigens) within this cell type. Percoll gradient-separated secondary granule fractions were rich in CD15 antigens, with at least seven antigens recognizable in SDS-PAGE/electroblot studies. Sonicatiodsedimentation experiments using secondary granule fractions showed that both soluble and sedimentable CD15 antigens were present. Exposure of purified PMN to the secondary granule secretagogue phorbol myristate acetate caused extracellular release of two or three CD15 antigens, which could be purified by immunoprecipitation using antibody M3. Triton X-114 phase-partition experiments showed that secondary granule fraction CD15 antigens could be partitioned into hydrophilic (aqueous phase) and hydrophobic (detergent phase) antigens, suggesting that several of these antigens were integral secondary granule membrane components. Ultrastructurally, PMN intracellular granules showed two patterns of CD15 expression, localization over both granule matrixlgranule membrane and localization to only granule membrane. Colocalization studies showed that lactoferrin and CD15 antigens were both present in a subset of intracellular granules, confirming a secondary granule location for these antigens. Among hematopoietic cells, CD15 antibodies are thought to be specific for polymorphonuclear leukocytes (PMN) (Tetteroo et al., 1984a). The epitope recognized by these antibodies is the trisaccharide 3-fucosyllactosamine (3-FL) (Tetteroo et al., 1984b), which is present in the pentasaccharide lacto-N-fucopentaose I11 and in the blood group antigen X-hapten (Umeda et al., 1986a). On the surface membrane of the PMN, at least five major CD15 antigens (105,135,165,185, and 220 kDa) can be radiolabeled and immunoprecipitated using CD15 antibody (Skubitz and August, 1985).Certain of these surface antigens are thought to be a relatively protease-resistant subgroup of the LFA-1/Macl/p150,95 antigen family (the 105, 165, and 185 kDa antigens) and the CR-1 receptor (the 220 kDa antigen) (Skubitz and Snook, 1987). Selected surface CD15 antigens are phosphorylated by a n ectoprotein kinase activity (Skubitz et al., 1988).Functionally, CD15 antibodies have been reported to interfere with phagocytosis of serum opsonized particles (Skubitz et al., 1985) as well as to suppress particulate stimulated oxygen consumption (Nauseef et al., 1983). One CD15 antibody, PMN 7C3, causes transient rises in cytosolic calcium (Apfeldorf et al., 1985), causes PMN shape changes and leukoagglutination, and is both chemotactic and chemokinetic (Melnick et al., 1986). At the subcellular level, fluorescence studies suggest t h a t 0 1990 WILEY-LISS, INC

CD15 antigens are spread diffusely over the surface of the PMN, and accumulate over the uropod in the motile cell (Melnick et al., 1985). Based on cellular fractionation studies, multiple CD15 antigens have been reported to be present in both the primary and secondary granule fractions of human PMN (Melnick et al., 1985), although secretory stimuli do not consistently cause increased expression of CD15 antigens on the PMN surface (Tetteroo et al., 1984b). The specific ultrastructural localization of PMN CD15 antigens has not been demonstrated, however. Over the course of studies examining surface expression of CD15 antigens on PMN, i t appeared that surface expression might be dynamic, suggesting a need for intracellular stores of these antigens. We therefore hypothesized that CD15 antigens were likely to be present in the intracellular “secondary” (secretory) granules of the PMN. The following studies were performed to test this hypothesis. MATERIALS AND METHODS PMN Purification

Heparinized (l/unit/ml) human blood was obtained from adult volunteer donors by venipuncture, and Received October 20, 1989; accepted February 6, 1990.

CD15 ANTIGENS IN PMN SECONDARY GRANULE

307

PMN were purified by Hypaque-Ficoll density gradient separation, dextran sedimentation, and hypotonic lysis (Boyum, 1968).

poorly). Phagocytosis was expressed as the percent of cells containing staphylococci.

Monoclonal Antibodies

A standard assay was performed as described by Gallin (1974), with the exception that freshly grown logphase s. aureus were preopsonized with fresh serum and washed before use, and heat-inactivated serum rather than fresh serum was used in the assay. Hybridoma culture supernates (final dilution 1:2 in the assay) were the source of monoclonal antibody.

Balb/c mice were immunized five times (over 10 weeks) with alum-precipitated human PMN cytoplasts (Roos e t al., 1983). Spleen cell fusion was performed by standard methods (Goding, 1980), and immunoglobulin-secreting hybridoma clones were identified by ELISA. These cell lines were cloned three times by limiting dilution, and clones producing PMN-reactive antibody (by fluorescence-activated cell sorter) were selected and further propagated. Two murine IgM antibodies (M3 and M6) were selected; both antibodies recognize 3-FL by ELISA (Kimura et al., 1989). Monoclonal antibodies used as controls in these studies were 1H12E7, a murine IgM antibody directed against a rat liver lysosomal antigen (a gift from Linda Raab, University of Texas Medical School); MPM-1, a murine IgM antibody directed against mitotic spindle antigens (a gift from Frances Davis, M.D. Anderson Cancer Center); a commercially purchased, purified IgM (Coulter clone IgM, Coulter Electronics, Hialeah, FL), or MOPC 104E, a n IgM myeloma protein (Sigma Chemical Co., St. Louis). Antibody M3 was affinity purified using p c h a i n specific anti-IgM linked to CNBr-sepharose. Culture supernate and/or ascites fluid containing antibody M3 (diluted 1:lO with 0.05 M Tris, 0.15 M NaC1, pH 8.5) was loaded onto the anti-IgM column over 18 hours. The column was washed and bound antibody was eluted with pH 2.5 glycine 0.05 M/NaCl 0.15 M buffer. The pH was rapidly brought to 8.0, and the antibody was concentrated to 1-3 mg/ml using a n Amicon filter system. The purified antibody was dialyzed against phosphate-buffered saline for 24-48 hours a t 4"C, and aliquots were conjugated with either fluorescein isothiocyanate (FITC-M3) or tetramethylrhodamine isothiocyanate (TRITC). Both labeled and unlabeled antibody was frozen in aliquots at -70°C in 5 mg/ml human serum albumin until used. PMN Functional Studies Phagocytosis assay

A laboratory strain of Staphylococcus aureus, previously grown to log phase in trypticase-soy broth, washed and boiled for 10 minutes, was incubated in autologous fresh serum for 30 minutes at 37°C. After two washes, the staphylococci were resuspended in heat-inactivated, autologous serum at 109/ml. Purified PMN (lo6) in 0.4 ml heat-inactivated, pooled serum, 0.1 ml of S. aureus, and 0.5 ml of hybridoma culture supernate containing antibody M3 were combined and tumbled at 37°C for 30 minutes. At intervals, 0.25 ml aliquots were removed and combined with 20 units of lysostaphin (Sigma), and these aliquots were then incubated for 30 minutes at 37°C. After incubation, samples of each aliquot were applied to glass slides using a cytospin and were stained with Diff-Quick Stain (Scientific Products, McGaw Park, IL). The slides were examined microscopically, and 100 cells were graded for whether they contained one or more darkly stained staphylococci (as a result of lysostaphin digestion, extracellular, nonphagocytosed staphylococci stained very

Bactericidal assay

Chemotaxis assay

An under-agarose chemotaxis assay, modified as described by Buescher et al. (1988), was used for these studies. The leading front distance was the locomotive parameter examined. Cells were incubated in antibody containing culture supernate (1:2 final) for 15 minutes at 25°C before assay and were not washed prior to addition to the assay. lmmunofluorescence studies

Purified PMN were labeled with FITC-M3 (80 pg/ml in HBSS without Ca2+ and M 2 + , 30 minutes, 4°C) in the presence of 5 pg/ml cytochalasin B (Sigma). The cells were washed twice in 5 pg/ml cytochalasin B and then warmed to 37°C in the presence of lop6 M f-metleu-phe (Sigma) plus 5 pg/ml cytochalasin B. After 15 minutes, the cells were fixed with 0.1% glutaraldehyde (Sigma) for 15 minutes, washed twice, and restained with 80 pg/ml TRITC-M3 (30 minutes, 4°C). The cells were washed twice and examined by fluorescence microscopy. In control experiments, exposure of purified PMN to antibody AHN-1 (a reference CD15 antibody [Skubitz and August, 19851) before fixation and labeling with TRITC-M3 eliminated TRITC-M3 binding, whereas exposure to MOPC 104E antibody prior to fixation and labeling had no effect on TRITC-M3 binding. This demonstrated that TRITC-M3 surface labeling of PMN was specific for CD15 antigens. PMN granule fractionation

PMN granule fractions were isolated in one experiment on continuous sucrose gradients a s described by West et al. (1984). In all other experiments, PMN granules were isolated on discontinuous Percoll gradients (Borregaard et al., 1983). Prior to granule isolation, purified PMN (1-2 x lo8) were treated with 5 mM diisopropylfluorophosphate (30 minutes, at 4"C), washed twice, and then processed. SDS-PAGE/electroblot studies

Specimens for SDS-PAGE were boiled in solubilization buffer and then separated on 10% polyacrylamide gels (Laemmli, 1970). Molecular weight standards were run with each experiment. SDS-PAGE separated specimens and molecular weight standards were then electrophoretically blotted onto nitrocellulose paper (Bittner et al., 1980). Blotted antigens were detected by incubating the nitrocellulose paper in either hybridoma culture supernate or polyclonal antibody (rabbitantihuman lactoferrin [Cappel Laboratories, Malvern, PA], rabbit-antihuman myeloperoxidase [Calbiochem, San Diego, CAI, rabbit-antihuman lysozyme [Dako Corp, Santa Barbara, CAI, rabbit-antihuman @,-micro-

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Fig. 1. Effects of C15 monoclonal antibody on PMN phagocytosis of S. aureus. Data shown are the mean 2 SE percent of phagocytic PMN a t the times shown on the abscissa. PMN were incubated with either antibody M3 (CD15) or 1H12E7, an irrelevant IgM monoclonal antibody; n = 4 for both antibodies. *P < 0.01 (Student’s t test).

globulin [Boehringer-Mannheim, Indianapolis, IN]), followed by peroxidase-conjugated, species-specific secondary antibody and then developed with 4-chloro-lnaphthol (BioRad). Blotted molecular weight standards were stained for 5 minutes with Coomassie blue (Sigma) and then destained with methanol/acetic acid. Surface iodination/immunoprecipitation studies Diisopropylfluoro hosphate-treated PMN were surface labeled with l2PI using lactoperoxidase (Skubitz et al., 1983). Surface-labeled cells were solubilized, immunoprecipitated (using monoclonal antibody, antimouse Ig, and protein A-bearing S. aureus), separated by SDS-PAGE, and examined by autoradiography.

Fig. 2. Autoradiographs of lZ5I-labeled PMN surface antigens immunoprecipitated by antibodies AHN-1, M3, and M6. Control (CT) lane used mouse serum as the source of the primary antibody. Locations of specific molecular weight markers (in kDa) are shown at left. The arrows a t the right identify the five major PMN surface antigens immunoprecipitated by CD15 antibodies (see text).

Postsecretion supernate irnrnunoprecipitation studies Purified PMN (1 x lo8 in HBSS containing 1 mM PMSF) were exposed to 20 ng/ml PMA for 20 minutes at 37°C. The postsecretion supernate was collected by Triton X-114 separation studies centrifugation (5 minutes, 18,OOOg) and preadsorbed PMN granule fractions were solubilized in Triton X- with the pellet from 250 p1 of Pansorbin (Calbiochem) 114 (Sigma), phase partitioned as described by Steven- for 15 minutes (25°C). The Pansorbin was removed son et al., (1987), and then examined by SDS-PAGE/ (18,00Og, 5 minutes), and the supernate was divided into two parts. Each part was processed in parallel a s electroblot. follows. Supernate was combined with 200 pg of purified antibody M3 with 5 mg/ml human serum albumin Sonicationkedimentation studies or a n irrelevant IgM (MOPC 104E ascites [Sigma] or PMN granule fractions were sonicated on ice for 1 Coulter clone-purified IgM) with 5 mg/ml human seminute at maximum power, and then pelleted a t rum albumin and incubated at 25°C for 120 minutes. 108,OOOg for 60 minutes. Samples of the original ma- Pansorbin (the pellet from 250 pl of suspension) was terial and the postsedimentation supernate and pellet then added, and incubation was continued (30 minutes, were examined by SDS-PAGE/electroblot. 25°C). The Pansorbin was pelleted and washed three times in HBSS. The washed Pansorbin was resusGranule secretion studies pended in 3.5 M MgCl with frequent mixing (30 minPurified PMN (10-12 x lo7 per ml in HBSS contain- utes, 25°C) and again pelleted. The resulting supering 1 mM phenylmethylsulfonylf luoride [PMSF], 10 nates were examined by SDS-PAGE/electroblot. pg/ml aprotinin, and 0.1 mM leupeptin) were exposed to 20 ng/ml phorbol myristate acetate (PMA) at 37°C Ultrastructural studies Purified PMN were cryofixed on a CF-100 device for 20 minutes. The cells were pelleted (18,00Og, 3 minutes), and the postsecretion supernate was examined (Lifecell Corp., Woodlands, TX) and then dried by molecular distillation (Linner e t al., 1986). Dried speciby SDS-PAGE/electroblot.

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CD15 ANTIGENS IN PMN SECONDARY GRANULE

mens were vapor osmicated, embedded in Spurr's resin, and sectioned. I n some instances, sections were floated on saturated sodium metaperiodate for 5 minutes a t room temperature (to remove excess osmium) before they were stained using a primary antibody-biotinylated secondary antibody-steptavidin colloidal gold technique. Sections were counterstained with uranyl acetate and examined in a Philips CT-12 electron microscope. In double-labeling experiments, sections were floated on droplets (rather than immersed) to allow staining of one side of the section only. Staining for CD15 (using antibody M3) was performed on one side, followed by staining for lactoferrin on the other. This approach was necessary because lactoferrin immunolocalization was optimal in Na metaperiodate-treated specimens, while this treatment destroyed the carbohydrate epitope recognized by antibody M3. Statistical Analysis

Unless otherwise noted, all data shown are the mean

* SEM, and statistical significance was determined by

Student's t test, with P < 0.05 taken as a significant difference.

FlTC

RESULTS Functional Effects of Antibodies M3 and M6 on PMN

Exposure of PMN to antibodies M3 or M6 during the bactericidal assay resulted in significant suppression of bactericidal activity at 90 minutes. With no antibody exposure, bacterial survival was 50% k 6% (n = 10) at 90 minutes. With control IgM antibody exposure, bacterial survival was 44% k 13%(n = 3). With exposure to antibody M3, bacterial survival was 99% & 13% (n = 5), and, with exposure to antibody M6, survival was 88% 7% (n = 5) (both P < 0.05 vs. IgM control). To examine whether the suppressive effect on PMN microbial killing seen with antibodies M3 and M6 might be due to suppression of phagocytosis, this function was examined directly with antibody M3. As shown in Figure 1, antibody M3 significantly suppressed phagocytosis of fresh serum opsonized s. aureus compared with control IgM. Similar results were observed with antibody M6 (Buescher and McIlheran, 1989).

TRITC

*

Surface Antigen Recognition by Antibodies M3 and M6

Fig. 3.Immunofluorescence patterns of CD15 antigens after stimulation of PMN with fMLP and cytochalasin B. Live cells, labeled with FITC-conjugated antibody M3, were stimulated and fixed with glutaraldehyde, then restained with TRITC-conjugated antibody M3 as described in the text. Upper panels show cells viewed by ordinary illumination; middle panels show the FITC fluorescence of the same cells. The lower panels show the TRITC fluorescence of the same cells. Note that FITC fluorescence appears centrally, whereas TRITC fluorescence is predominantly peripheral.

Antibodies M3 and M6 immunoprecipitated the same five major radiolabeled PMN surface antigens (105, 135, 165, 185, and 220 kDa) as antibody AHN-1 (Fig. 21, a reference CD15 IgM antibody (Skubitz and August, 1985).The multiple surface antigens recognized by antibodies M3 and M6 and their participation in phagocytosis prompted examination of whether surface expression of CD15 antigens was fixed or dynamic.

surface of PMN, suggesting that secretion mobilized fresh CD15 antigens to the PMN surface. No binding of second label was observed when PMN were labeled first a t 4"C, washed a t 4"C, fixed, and then stained with the second label. These observations prompted examination of the intracellular granules as possible internal pools of CD15.

Effects of Granule Secretion on Surface CD15 Antigen Expression

Localization of CD15 Antigens to PMN Secondary Granule Fractions

To evaluate whether surface CD15 antigen expression was fixed or changed with stimulation, doublelabel immunofluorescent examination of purified PMN before and after exposure to lop6M fMLP plus 5 p,g/ml cytochalasin B was performed (Fig. 3). Secretagogue exposure resulted in binding of the second label to the

Primary and secondary granule fractions formed in continuous sucrose gradients were examined in one experiment. Multiple CD15 antigens were present in both granule fractions as previously reported (Melnick et al., 1985). However, when PMN primary and secondary granule fractions were produced using discontinu-

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MOL. WGT:

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Fig. 4. Nitrocellulose electroblots of PMN primary (1")and secondary (2") granules separated by SDS-PAGE, then probed with antibodies against myeloperoxidase (MPO), lysozyme (LYSOZ), p,;microglobulin (P,M), lactoferrin (LACTOF), and CD15. The locations of respective molecular weight standards (in kDa) are shown by the dots to the left of each blot. The published sizes of each antigen's components (under reducing conditions) are shown below each blot. Three

different Percoll-separated granule preparations that were probed with antibody M3 are shown, to demonstrate the variation in CD15 antigens from preparation to preparation. CD15 antigens are localized to the same fractions as the secondary granule markers lactoferrin and &-microglobulin. Primary granule contamination of secondary granule fractions (as seen with MPO) are typical of the preparative method.

ous Percoll gradients, a distinctly different pattern was observed. Multiple CD15 antigens were present in the secondary granule fractions (Fig. 4), the same fractions that contained the secondary granule marker antigens lactoferrin and &-microglobulin. On rare occasions, several low-molecular-weight (