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(Journal of Leukocyte Biology. 2002;72:492-502.)
© 2002 by Society for Leukocyte Biology

The role of ceramide of human macrophage gangliosides in activation of human macrophages

Charles S. Berenson, Melissa A. Gallery, Jane M. Smigiera and Robin H. Rasp

Infectious Diseases Section, Department of Veterans Affairs Western New York Healthcare System, State University of New York at Buffalo, School of Medicine

Correspondence: Charles S. Berenson, Division of Infectious Diseases (151), VA Western NY Healthcare System, 3495 Bailey Avenue, Buffalo, NY 14215. E-mail: berenson{at}acsu.buffalo.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Gangliosides of macrophages have immunoregulatory and structural attributes, distinct from neural gangliosides. We previously produced a monoclonal antibody to human macrophage gangliosides (HMG; mAb25F4), which inhibited macrophage migration and recognized a surface-accessible epitope. We investigated expanded immunoregulatory properties and molecular domains for antibody recognition. mAb25F4 directly induced human macrophage production of proinflammatory cytokines, interleukin-1ß, and tumor necrosis factor {alpha}. Conditions were established for selective, reversible depletion of HMG with D-threo-(R,R)-1-phenyl-2-decanoyl-amino-3-morpholine-1-propa-nol. mAb25F4 had diminished recognition for ganglioside-depleted macrophages, which was restored with regeneration of gangliosides. Although desialylation of HMG did not impair mAb25F4 recognition, enzymatic cleavage of ceramide abolished antibody binding. Antibody recognition was specific for the ceramide fraction, with preferential recognition for ceramide of HMG and murine macrophage gangliosides and limited recognition for neural tissue ceramide and gangliosides. This study underscores the importance of structurally distinct ceramide of macrophage gangliosides as a critical domain for ganglioside-mediated activation of human macrophages.

Key Words: glycosphingolipids • mononuclear phagocytes • cytokines • PDMP


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Gangliosides (nomenclature according to ref. [1 ]) are sialylated glycosphingolipids (GSL), which exist in the cell membranes of virtually all eukaryotic cells and possess a diverse array of biological activities, including mediation of cell differentiation [2 , 3 ] and intracellular signal transduction [4 ]. The immunologic and structural attributes of gangliosides of macrophages are of particular importance and have been the subject of ongoing studies. The focus on macrophage gangliosides stemmed from the induction of striking diversity in macrophage ganglioside expression in response to lipopolysaccharide (LPS) [5 ]. Later studies demonstrated that macrophage gangliosides exerted a reversible down-regulation on T cell proliferation, which was distinct from the biologic activity of gangliosides of neural tissue. Specifically, macrophage gangliosides were 100-fold more potent and exerted their effect at the cell membrane level, and gangliosides of neural tissue acted extracellularly [6 ]. However, the structural basis for the immune properties of macrophage gangliosides remained obscure.

Conspicuous structural differences of gangliosides of macrophages have been recently appreciated and may define their immunologic properties. Macrophages of LPS-hyporesponsive mice are deficient in the sialidase-accessible form of GM1 (GM1b) compared with macrophages of their congenic, LPS-responsive counterparts [7 ]. The ceramide component of gangliosides of macrophages also differs structurally from ceramide of gangliosides of neural origin. Although ceramide species of both possess C18 sphingosine chains, the fatty acyl chain of ceramide of murine macrophage gangliosides (MMG) is predominantly comprised of C16 and C24 fatty acids, and ceramide of gangliosides of neural tissue contains mostly C18 fatty acids [7 , 8 ]. Recent studies confirmed that the same structurally distinct ceramide of gangliosides of murine-immune cells is also a constituent of gangliosides of human mononuclear cells [9 ]. These marked, structural distinctions may explain the potent, immunologic effects of macrophage gangliosides.

To better study the immunologic effects of macrophage gangliosides, we produced a monoclonal antibody (mAb25F4) to human macrophage gangliosides (HMG), which we have previously reported inhibits migration of human macrophages [10 ]. mAb25F4 further recognizes a cell surface epitope on human macrophages, which although present, is inaccessible to surface molecules in adults with advanced HIV infection [11 ]. Earlier studies indicated common recognition by mAb25F4 of the three major gangliosides of human macrophages, identified as GM3, sialosylparagloboside, and an extended monosialosylhexosylceramide [10 , 12 ]. Although this suggested that the critical molecular domain for 25F4 recognition was a molecular structure common to all three major HMG, the specific domain was not known. Further, the range of immunomodulatory properties of mAb25F4, beyond macrophage migration inhibition, was not known.

We theorized that ganglioside-mediated macrophage activation by mAb25F4 is not limited to migration inhibition and is mediated by specific recognition of a critical domain of the ganglioside molecule, and we performed studies to test this hypothesis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Solvents were of standard analytical, high-performance liquid chromatographic grade (Baker Chemical Co., Phillipsburg, NJ). Culture media and fetal bovine serum were purchased from Gibco Laboratories (Grand Island, NY) and BioWhittaker (Walkersville, MD). Endotoxin-free, human AB-positive serum was purchased from Nabi (Miami, FL). High-performance silica gel 60 thin-layer chromatography (TLC) plates were purchased from E. Merck (Darmstadt, Germany). Murine brain gangliosides (MBG) were used as standards and were prepared as previously described [13 ].

Monoclonal antibodies 25F4 [immunoglobulin G (IgG)2a] and 21C8 were produced by immunization of Balb/c mice with purified HMG and purification by affinity chromatography, as previously detailed [10 ]. Hybridomas were products of fusion of splenocytes and SP-2 myeloma cells. mAb5F3, generously donated by Dr. Timothy Murphy (SUNY, Buffalo, NY), recognizes an epitope of the P6 outer membrane protein of nontypeable Haemophilus influenzae and served as an irrelevant IgG2a. All antibodies were tested for endotoxin content by Limulus amebocyte lysate assay (Sigma Chemical Co., St. Louis, MO), which detected <10 pg/ml.

Purification of human macrophages
Human mononuclear phagocytes were purified from buffy coat suspensions, obtained from healthy, HIV-seronegative volunteers from the Red Cross of Western New York. Cells for individual experiments were from single donors and were not pooled unless specified. Mononuclear cells were further purified by Ficoll-Hypaque density centrifugation and seeded onto 100-mm glass petri dishes (5x106 cells/ml) in RPMI 1640 supplemented with 10% heat-inactivated human AB-positive serum. After incubation at 5% CO2, 95% humidity, 37°C for 7 days, nonadherent cells were removed with serial rinses of warm (37°C) phosphate-buffered saline (PBS). Remaining monocyte-derived macrophages were incubated in RPMI 1640 with 10% fetal calf serum and were consistently 98–100% esterase-positive [10 ].

Purification of murine macrophages
Murine peritoneal macrophages were obtained from 6- to 8-week-old female C3N/HeN mice, as previously described [13 ]. Mice were killed four days following intraperitoneal injection of 1 ml 10% thioglycollate broth per mouse. Cells were recovered by peritoneal lavage with Hanks’ balanced salt solution. Cell suspensions were centrifuged at 10°C at 1000 RPM (200 g) for 10 min and resuspended. Approximately 25 x 106 cells in 10 ml were incubated on glass petri dishes for 90 min at 37°C with 5% CO2 and 95% humidity to permit adherence of macrophages. Cellular lipids were extracted following removal of nonadherent cells and rinse of adherent cells with 0.31 M pentaerythritol to remove excess salts.

Purification of gangliosides
The ganglioside fraction was purified from the total lipid extract as previously detailed [13 ]. Lipid extracts were filtered through a scintered glass filter funnel overlaid with a glass fiber mat and applied to a column of diethylaminoethyl-Sephadex A-25 (Sigma Chemical Co.). Neutral lipids were eluted with chloroform:methanol:water (30:60:8 v/v/v), and the ganglioside-containing acidic lipid fraction was eluted with chloroform:methanol:0.8 M sodium acetate (30:60:8 v/v/v). Gangliosides were further purified by serial chromatographic steps as previously described [14 ].

Cytokine assays
Adherent human macrophages derived from 106 cells/well were treated with mAb25F4, mAb21C8, or mAb5F3 (25 µg/ml). Cellular supernatants were assayed by sandwich-type, solid-phase immunoassay for interleukin (IL)-1ß (R&D Systems, Inc., Minneapolis, MN) or tumor necrosis factor {alpha} (TNF-{alpha}; Biosource International, Inc., Camarillo, CA). Supernatants of treated cells were harvested at appropriate timepoints, centrifuged, and removed from pelleted cells before being frozen. Standards and samples (200 µl) were added to individual wells precoated with mAb to IL-1ß and incubated at room temperature for 2 h. After washing three times, 200 µl polyclonal antibody to IL-1ß (or to TNF-{alpha}), conjugated to horseradish peroxidase, was incubated in each well for 20 min at room temperature. Acidic "stop" solution was added, followed by developer, and optical density (OD) of each well was measured at 450 nm within 30 min by enzyme-linked immunosorbent assay (ELISA) reader. All experiments included separate wells treated with Eschericha coli K235 LPS (1 µg/ml; Sigma Chemical Co.) or with purified SP-2 supernatants as additional controls and were repeated with cells from three separate donors. All conditions were performed in quadruplicate.

Ganglioside depletion
Depletion of cellular GSL was achieved with D-threo-(R,R)-1-phenyl-2-decanoylamino-3-morpholine-1-propanol (PDMP; Matreya, Inc., Pleasant Gap, PA), which competitively inhibits synthesis of glycosylceramide from ceramide and UDP-glc [15 , 16 ]. For each analysis, 800–900 x 106 total peripheral blood mononuclear cells (PBMC) were used for each condition. Of these, 5–10% are monocytes, which differentiate into monocyte-derived macrophages [10 ]. Time and dose kinetics for selective depletion of macrophage gangliosides, without depletion of neutral lipids, were determined by treating cells with 0–100 µM PDMP for 1–5 days. Neutral lipid and ganglioside fractions were independently collected from purified cellular lipids. Neutral lipids were placed on TLC, run in solvent of chloroform:methanol:water (65:35:8 v/v/v) for 42 min, and visualized with orcinol spray [17 ]. Gangliosides were also placed on chromatograms, run in solvent of chloroform:methanol:0.25% KCl (50:45:10 v/v/v) for 45 min, and visualized by resorcinol spraying [10 ]. The major neutral lipid and the major ganglioside, previously identified as paragloboside (nLc4) and GM3, respectively [11 ], were quantitated by scanning densitometry (Molecular Dynamics, Sunnyvale CA). As previously described, volumes of peaks were quantitatively determined by densitometry, based on area and light absorbance. We have determined that relative quantity maintains a linear relationship with OD at least from 0 to 20 µg sialic acid [18 ]. Although GM3 and nLc4 were measured as representatives of their respective lipid fractions, careful note was made of all minor GSL to be certain of consistency. Results are expressed as (volume of PDMP-treated GSL/volume of untreated GSL) x 100 and were repeated with cells from three separate donors.

In selected experiments, PDMP-containing media was removed from treated macrophages and replaced with PDMP-free media. Cells were then maintained in culture for 3–5 days before harvesting lipids. Gangliosides of untreated cells, PDMP-treated cells, and cells cultured for an additional 3–5 days post-removal of PDMP were quantitated.

Immunofluorescent surface-labeling of PDMP-treated human macrophages
Human PBMC (2x106/well) were grown in Lab-Tek chamber slides (Nunc Inc., Naperville, IL) for 10 days. Adherent macrophages were incubated with PDMP as described above. Cells were then treated with 6% nonfat dry milk for 30 min at 37°C, rinsed, and incubated with mAb25F4 (40 µg/ml) for 60 min at room temperature. After removal of excess antibody with vigorous washes, cells were incubated with fluorescein isothiocyanate-F(ab')2 fragments of goat anti-mouse IgG (1:50; Zymed Laboratories, San Francisco, CA) for 30 min at room temperature in a dark container. Cells were washed and evaluated by immunofluorescent microscopy. In all experiments, separate wells of the same donor’s cells, cultured on the same chamber slide, were treated with protein G-purified SP-2 cell supernatant or with an irrelevant IgG2a. Both negative controls have had identical background immunofluorescence to one another in previous studies [10 ].

In selected experiments, PDMP-containing media was removed from treated macrophages and replaced with PDMP-free media. Cells were then maintained in culture for 3–5 days before immunostaining with mAb25F4, as described above. Gangliosides of untreated cells, PDMP-treated cells, and cells cultured for an additional 3–5 days post-removal of PDMP were evaluated by immunofluorescent microscopy.

Radioisotope labeling and detection of glycolipids of PDMP-treated macrophages
Single-donor macrophages were isolated by density gradient centrifugation and adherence, as described earlier. Equal numbers of cells in culture were treated with 50 µM PDMP or with PDMP-free medium for 5 days. On day 3 of PDMP incubation, all cells were incubated with 14C-galactose (American Radiolabel Chemicals, St. Louis, MO; specific activity, 55 mCi/mmol) at 5 µCi/20 x 106 cells for 48 h before extracting cellular glycolipids [19 ]. Purified neutral lipid and ganglioside fractions were isolated. Five percent of each fraction was analyzed by scintillation counting, and identical percentages of each sample were plated on TLCs and run in chloroform:methanol:water (65:35:8 v/v/v) for 42 min for neutral lipids and chloroform:methanol:0.25% KCl (50:45:10 v/v/v) for 45 min for gangliosides. 14C-galactose-labeled glycolipids were visualized by autoradiography and created by exposure of chromatograms to hypersensitized XAR-5 film (Eastman Kodak Co., Rochester, NY) for 1 week at -70°C. X-ray film was hypersensitized by exposure to 7% hydrogen/93% nitrogen at 48°C for 16 h, as previously described [20 ]. Major glycolipids were localized by spraying TLC plates with resorcinol for gangliosides and orcinol for neutral lipids. Autoradiographic bands were then aligned by overlay with visible bands on each TLC plate, which included known standards.

Sialidase treatment of macrophage gangliosides
HMG containing 5–7 µg sialic acid were incubated with Arthrobacter ureafaciens sialidase (200 mU/ml; EY Laboratories, San Mateo, CA) in 1 ml 100 mM sodium acetate buffer, pH 4.8, at 37°C for 18 h, following the method of Saito et al. [21 ]. A duplicate sample of equal quantity of gangliosides was incubated in buffer alone. Reactions were terminated with addition of 0.1 M NaOH and neutralized with 0.1 M HCl. Solutions were desalted on SepPak (Waters Assoc., Milford, MA) columns, tested for purity on TLCs, and run in chloroform:methanol:0.25% KCl (50:45:10) by resorcinol-spraying. Volume was determined by quantitation of resorcinol-positive intensity with scanning densitometry, and the percent desialylation was expressed as 1 - [volume of (resorcinol-positive) sialidase-treated sample/volume of (resorcinol-positive) untreated sample] x 100.

Ceramide-glycanase treatment of macrophage gangliosides
HMG (5–7 µg) were incubated in 0.2 U Marobdella decora ceramide glycanase [22 , 23 ] (V-Labs, Covington, LA) in 200 µl 50 mM sodium acetate buffer, pH 5.0, containing 0.75 µg/µl sodium cholate at 37°C for 16 h. The reaction was terminated by addition of 5 vol chloroform:methanol (2:1 v/v). Cleaved products were separated into lower (ceramide-containing) organic and upper (carbohydrate-containing) aqueous phases by gentle centrifugation. Each phase was dried separately. After separation, the aqueous phase was additionally rinsed with chloroform:methanol (2:1) to optimize purity.

Released oligosaccharide was detected by resorcinol spraying on TLC run in n-butanol:acetic acid:H2O (2:1:1, v/v/v) for 80 min. Ceramides were independently assayed on TLC run in chloroform:methanol (9:1 v/v) for 20 min and developed with Coomassie blue. To verify absence of uncleaved, sialylated substrate, the organic (ceramide-containing) phase was also run on TLC in chloroform:methanol (9:1) and developed with resorcinol spray.

ELISA
Each well of a 96-well microtiter plate (Immunlon-1, Dynatech Laboratories, Inc., Chantilly, VA) was coated with substrate dissolved in 100% ethanol and left to dry for 16 h. Substrates included HMG; mixed neural-tissue ceramides; purified GM3, GM1, GD3, GD1b, GD1a all from neural tissue (Matreya, Inc.); sialidase-treated HMG; organic and aqueous phases of ceramide glycanase-treated HMG; MBG; and thioglycollate-elicited peritoneal MMG.

Glycolipid or oligosaccharide substrates were coated onto wells at 0.3 µg/well. Cleaved fractions of enzyme-treated HMG were coated in equimolar ratios of original product. Wells were rinsed with PBS/0.05% Tween 20 and incubated with 3% bovine serum albumin at room temperature for 1 h. After vigorous rinses with PBS/0.05% Tween 20, 50 µl purified mAb (10 µg/ml) was added and incubated for 3 h at 37°C. Wells were again rinsed, and 50 µl (1:1000) goat anti-mouse IgG peroxidase (Kierkegaard and Perry, Gaithersburg, MD) diluted in PBS was added for 1 h at room temperature. After rinsing, 50 µl 3,4,5-trimethoxybenzoic acid-developing buffer (0.1 µg/ml) was added. Reaction was stopped after 5–10 min with 4 N H2SO4 and read on a 96-well microplate reader (BioRad Instruments, Hercules, CA) at 450 nm. A negative control was performed and included substitution of SP-2 tissue culture supernatant for the primary antibody. Known positive controls were also run simultaneously. For all comparative ELISAs, results are expressed as [(OD substrate-OD background)/(OD macrophage gangliosides-OD background)] x 100. Background was determined by including empty wells with no substrate in every experiment.

To assess adherence of oligosaccharides and ceramides to ELISA wells and to determine if potential differential configurations would affect binding, TLC immunostaining was also done with mAb25F4 and 21C8 on the oligosaccharide and ceramide fractions derived from treatment of macrophage gangliosides with ceramide glycanase. Oligosaccharide fractions were run in butanol:acetic acid:H2O (2:1:1 v/v/v), and ceramides were run in chloroform:methanol (9:1 v/v) as detailed earlier. Immunostaining on TLCs was performed as described previously to confirm ELISA results [10 ].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
mAb25F4 induces IL-1ß production from human macrophages
We previously showed that mAb25F4 recognizes surface-accessible gangliosides of human macrophages and inhibits macrophage migration [10 ]. To determine if mAb25F4 is also capable of inducing human macrophages to produce proinflammatory cytokines, human monocyte-derived macrophages were treated with mAb25F4 (25 µg/ml) or with an irrelevant IgG2a (5F3), and supernatants were assayed over time for IL-1ß. Peak IL-1ß production was seen at 24–48 h (249.1±31.1 pg/ml) in cells from three separate donors (Fig. 1 ). Measurements were taken from quadruplicate samples (mean±SE) of each donor. IL-1ß induction in response to mAb21C8, mAb5F3, or purified SP-2 supernatant was negligible. In all experiments, macrophages in separate wells also produced IL-1ß in response to incubation with E. coli K235 LPS (1 µg/ml).



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  		Figure 1. mAb25F4-induced IL-1ß release from human macrophages. Peak responses for antibody 25F4 ({blacksquare}; 25 µg/ml) occurred at 24–48 h. Negligible responses were seen with mAb21C8 ({circ}), mAb5F3 ({square}), or with purified SP-2 supernatant. Each time point represents means of three separate measurements ± SE and was reproducible for cells from three separate donors. To confirm macrophage reactivity, macrophages from each donor were also treated with LPS ({blacktriangleup}; 1 µg/ml) at each time point.

 
mAb25F4 induces TNF-{alpha} production from human macrophages
To determine if mAb25F4-induced cytokine release was specific for IL-1ß, human macrophages were treated with mAb25F4, and supernatants were assayed for TNF-{alpha} (Fig. 2 ). Peak levels of 25F4-induced TNF-{alpha} levels were seen at 24 h (1321.8±48.3 pg/ml) with cells from three donors. As with IL-1ß production, measurements (mean±SE) were again taken from quadruplicate samples of each donor. Treatment with mAb21C8, mAb5F3, or SP-2 supernatant had a minimal effect on TNF-{alpha} production. Macrophages also produced TNF-{alpha} in response to LPS (1 µg/ml) in all experiments.



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  		Figure 2. mAb25F4-induced TNF-{alpha} release from human macrophages. As with IL-1ß production, peak TNF-{alpha} release with 25F4 ({blacksquare}) occurred at 24 h. Negligible responses were seen with mAb21C8 ({circ}), mAb5F3 ({square}), or with purified SP-2 supernatant. Each time point represents means of three separate measurements ± SE and was reproducible for cells from three separate donors. LPS-treated controls (1 µg/ml) are also represented ({blacktriangleup}).

 
HMG can be selectively depleted with PDMP
To determine optimal conditions for selective depletion of gangliosides, without depleting neutral lipids, human macrophages were incubated with PDMP (0–100 µM) for 1–7 days. After harvesting lipids, neutral lipid and ganglioside fractions were purified and visualized on TLCs with orcinol or resorcinol spraying, respectively. For each analysis, GSL from untreated and from PDMP-treated macrophages, derived from 800–900 x 106 PBMC, were extracted from an identical number of macrophages from the same donor to eliminate the potential of inter-donor variability. Paragloboside (nLc4) and GM3, which are, respectively, the major neutral lipid and ganglioside of human macrophages, were quantitated as representative of each group. Treatment with 50 µM PDMP for 5 days resulted in near-total depletion of macrophage gangliosides to 9.8 ± 5.6% of the content of untreated controls without depletion of neutral lipids (Fig. 3 ). Treatment with 10 µM PDMP effectively depleted 38.8% of GM3 at 3 days but was less consistent. Conversely, neutral lipids could be selectively depleted to 23.7 ± 14.3% of the content of untreated controls without depletion of gangliosides by treatment with 10 µM PDMP for 5 days (Fig. 4 ). Neutral lipid content was most severely impaired at 3 days with 50 µM PDMP but returned to the levels of untreated controls by 5 days. Toxicity as a result of 50 µM PDMP was ruled out by trypan blue exclusion. However, 100 µM PDMP resulted in visible cell death, confirmed by trypan blue staining. In all experiments, neutral lipids and gangliosides from untreated cells from the same donor were left in culture and concurrently evaluated for accurate comparison. All experiments were repeated with cells from three separate donors. These results permitted us to establish conditions (50 µM PDMP for 5 days) to selectively deplete gangliosides without depleting neutral GSL in human macrophages.



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  		Figure 3. Selective depletion of macrophage gangliosides with PDMP. Depletion of macrophage gangliosides (GM3) over time is shown (A) with 10 µM ({blacksquare}), 50 µM ({blacktriangleup}), and 100 µM PDMP ({circ}). Depletion of GM3 with increasing concentrations of PDMP (C) is shown for 3 days ({blacksquare}) and 5 days ({blacktriangleup}) of incubation. Optimal depletion occurred in macrophages incubated in 50 µM PDMP for 5 days. Ganglioside depletion is represented by quantitation of the major component GM3. Results are means ± SE for three separate measurements. TLC results of one experiment are shown (B). Blurring of bands (GM3) is a result of intentional overloading of the TLC to permit maximal detection (B). Samples on TLC were incubated with PDMP for 5 days. Lanes are bracketed in pairs based on the concentration of PDMP (0, 10, and 50 µM) with which cells were treated. Lanes: 1, 0 µM PDMP; 2, 0 µM PDMP plus 5 days incubation post-removal; 3, 10 µM PDMP; 4, 10 µM PDMP plus 5 days incubation post-removal; 5, 50 µM PDMP; 6, 50 µM PDMP plus 5 days incubation post-removal. Position of ganglioside GM3 standard is shown at right.

 


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  		Figure 4. Selective depletion of macrophage neutral GSL with PDMP. Depletion of the neutral GSL fraction of human macrophages over time (A) is shown with 10 µM ({blacksquare}), 50 µM ({blacktriangleup}), and 100 µM PDMP ({circ}). Neutral lipid depletion with increasing concentrations of PDMP (C) is shown for 3 days ({blacksquare}) and 5 days ({blacktriangleup}) of incubation. Neutral lipid depletion is represented by quantitation of the major component, nLc4. Maximal depletion occurred in macrophages incubated in 10 µM PDMP for 5 days. Results are means ± SE for three separate measurements. TLC results of one experiment are shown (B). Blurring of bands (nLc4) is a result of intentional overloading of the TLC to permit maximal detection (B). Conditions of samples on lanes 1–6 are identical to those in Figure 3 . Position of paragloboside (nLc4) standard is shown at right.

 
To further confirm absence of toxicity, macrophages were treated with 50 µM PDMP for 5 days and then were left in PDMP-free medium for 3–5 more days before harvesting GSL. This resulted in restoration of ganglioside content, determined by quantitation of GM3 to 75% of previous baseline (Fig. 5 ). In all experiments, untreated and PDMP-treated, ganglioside-depleted cells from the same donor were concurrently evaluated for accurate comparison. All experiments were repeated with cells from three separate donors.



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  		Figure 5. Restoration of macrophage gangliosides with removal of PDMP from cell culture. Macrophages were depleted of gangliosides by incubation with 50 µM PDMP for 5 days and then incubated in PDMP-free medium for an additional 3 (left) or 5 (right) days. Quantitation of GM3 by scanning densitometry of baseline, untreated sample (solid bars), of PDMP-treated sample (dark-gray bars), and of PDMP-treated sample 5 days post-removal of PDMP (light-gray bars) is shown. Results are expressed as a quantitative percentage of ganglioside (GM3) of the untreated sample. Each bar represents mean ± SEM of samples from three separate donors. Samples treated for 5 days correspond with TLC samples of Figure 3 . Results are given as a quantitative percentage of ganglioside of untreated macrophages and were reproducible in three separate experiments.

 
Detection of glycolipids of 14C-galactose-labeled, PDMP-treated macrophages
To further assess relative depletion of gangliosides compared with neutral lipids, glycolipids of PDMP-treated (50 µMx5 days) and untreated macrophages were radiolabeled with 14C-galactose. The purified, neutral-lipid fractions and ganglioside fractions of each were evaluated by autoradiography of chromatograms (Fig. 6 ). GSL from untreated and from PDMP-treated macrophages were extracted from an identical number of macrophages from the same donor to eliminate the potential of inter-donor variability. Paragloboside (nLc4) and GM3 were again quantitated as major representatives of each group.



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  		Figure 6. Selective depletion of macrophage gangliosides with PDMP detected by 14C-galactose-labeled GSL. Selective depletion of gangliosides relative to major neutral lipids was confirmed with GSL of 14C-galactose-labeled macrophages in culture for autoradiographic comparison. Identical volumetric percentages of neutral lipids (left) and of gangliosides (right) were taken of macrophages from the same donor. Material in lane A of each panel was purified from untreated macrophages; material in lane B of each panel was purified from PDMP-treated (50 µM) macrophages. As in Figures 3 and 4 , a comparison was made between the major ganglioside (GM3) and the major neutral lipid (nLc4) components.

 
The purified 14C-galactose-labeled ganglioside fractions contained 7.0 x 104 cpm and 2.9 x 104 cpm for PDMP-treated and untreated samples, respectively. In comparison, purified 14C-galactose-labeled neutral lipid fractions of the same cells contained 25.4 x 105 cpm and 19.2 x 105 cpm for PDMP-treated and untreated samples, respectively. Relative depletion of gangliosides compared with neutral lipids was confirmed by autoradiography of identical percentages using 3% of each ganglioside fraction and 1% of each neutral lipid fraction (Fig. 6) . Each autoradiographic band was quantitated by densitometry, and the relative quantitative shift of each GSL was expressed as (volume of PDMP-treated GSL/volume of untreated GSL) x 100.

Results confirm a modest increase of the major neutral lipid (nLc4) to 124.9% compared with depletion of the major ganglioside (GM3) to 14.4% of baseline quantity in PDMP-treated macrophages. Thus, an alternative method to measure glycolipid content confirmed that treatment with 50 µM PDMP for 5 days resulted in relative depletion of gangliosides compared with neutral lipids in human macrophages.

Ganglioside-depleted human macrophages have diminished surface recognition by mAb25F4
To confirm relative specificity of mAb25F4 for gangliosides in intact human macrophages, ganglioside-depleted cells were immunostained with mAb25F4 (25 µg/ml) as previously described [10 ]. In all experiments, concurrent immunostaining with an irrelevant IgG2a was performed on same-donor cells to confirm absence of nonspecific binding (not shown). Macrophage gangliosides and neutral GSL from each donor’s cells used for these studies were simultaneously analyzed by TLC (described above) to confirm selective depletion of gangliosides. In all experiments, ganglioside depletion resulted in diminished immunofluorescent surface-labeling with mAb25F4 (Fig. 7 ). The presence of adherent cells was always confirmed by light microscopy.



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  		Figure 7. Immunofluorescent surface-labeling of human macrophages with mAb25F4. Macrophages of the same donor were immunostained with mAb25F4. Figure includes macrophages that were untreated (A), ganglioside-depleted with 50 µM PDMP for 5 days (B), or had PDMP removed and demonstrated regeneration of gangliosides (C), as described in Figures 3 and 5 . Images correspond with quantitative ganglioside data shown in Figure 5 . Results shown are from one donor and were reproducible with macrophages of three separate donors.

 
To determine whether restoration of gangliosides, achieved with removal of PDMP, would result in concomitant restoration of mAb25F4 surface recognition, ganglioside-depleted macrophages were cultured in PDMP-free medium for 5 days and then immunostained with mAb25F4. In all experiments, macrophages with restored ganglioside content, after removal of PDMP, had concomitant restoration of immunofluorescent surface-labeling with mAb25F4 (Fig. 7) . As before, macrophage gangliosides and neutral GSL from each donor’s cells used for these studies were simultaneously analyzed by TLC to confirm selective depletion and restoration of ganglioside content. All experiments were repeated with cells from three separate donors.

Recognition of desialylated macrophage gangliosides by mAb25F4
To determine if sialic acid is a critical component for mAb25F4 recognition, HMG were treated with A. ureafaciens sialidase [24 ]. Before performing ELISA studies, it was crucial to determine purity of desialylated preparations. Equimolar volumes of sialidase-treated and untreated samples, prepared on TLC and sprayed with resorcinol, confirmed that 98% of sialic acid residues were effectively removed by sialidase treatment (Fig. 8 ). Concomitant incubation of gangliosides with buffer alone did not cause degradation. Treatment of control samples showed that GM3 was no longer detectable with resorcinol spray, and GM1a and GM2 remained resorcinol-positive.



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  		Figure 8. A. ureafaciens sialidase-treated HMG. Conditions were established for removal of 98% of sialic acid residues, determined by densitometric quantitation of resorcinol-sprayed TLC. A sialidase-treated sample is compared with untreated sample of equal concentration. Lanes: 1, Sialidase-treated sample; 2, buffer-treated sample; 3, MBG standard. Ganglioside standards are indicated along the right margin as GM3 (M3), GM1 (M1), GD1a (D), and GT1b (T).

 
Desialylated products were then tested in ELISA for recognition by mAb25F4. Results indicated no impairment of recognition by mAb25F4 of desialylated macrophage gangliosides compared with untreated gangliosides (Fig. 9 ). Wells containing untreated HMG were run with each experiment, and results are expressed as a percentage of the OD450 for equimolar amounts of HMG run on the same ELISA plate. All results were reproducible in three separate experiments.



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  		Figure 9. ELISA recognition by mAb25F4 of desialylated macrophage gangliosides. Desialylated macrophage gangliosides were produced as in Figure 8 . Equimolar concentrations of desialylated macrophage gangliosides were placed in ELISA wells and were tested for recognition by mAb25F4 (10 µg/ml). Results are expressed as a percentage of the OD450 of HMG for each substrate and are given as means ± SE of three separate experiments. Each value was calculated with controls from the same experiment.

 
Our earlier results indicated no cross-recognition between mAb25F4 and macrophage glycoproteins [10 ]. In related studies, human macrophage membrane proteins were extracted in HEPES buffer (pH 7.4), solubilized, and run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis [25 ]. Lysates were included in ELISA and were also transferred to nitrocellulose membranes (2–4 µg) for incubation (14 h, 27°C) with mAb25F4 (1–50 µg/ml) [26 ]. In each instance, no evidence of antibody binding to macrophage proteins was detected (not shown).

Recognition of ceramide-glycanase degradation products of HMG by mAb25F4
To independently determine if mAb25F4 has preferential recognition of the ceramide portion or of the oligosaccharide portion, HMG were treated with M. decora ceramide glycanase. Purity and completeness of enzymatic cleavage of enzyme-treated fractions were confirmed by TLC. The aqueous (carbohydrate-containing) fraction was analyzed on resorcinol-sprayed TLCs and consisted predominantly of a single band with identical chromatographic mobility to the sialyllactose standard (Fig. 10 ). Untreated (nondegraded) HMG were run on the same TLC to determine chromatographic position and confirmed absence of any residual, uncleaved substrate in the enzyme-treated samples.



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  		Figure 10. M. decora ceramide glycanase-treated HMG. Conditions were established for optimal cleavage of macrophage gangliosides into ceramide-containing (A) and carbohydrate-containing (B) fractions. TLC of ceramide fraction (left) includes lane 1, ceramide (organic fraction) of ceramide glycanase-treated HMG; 2, HMG incubated with buffer; 3, HMG standard; and 4, ceramide of bovine neural tissue. TLC of carbohydrate fraction (right) includes lane 5, carbohydrate fraction of ceramide glycanase-treated HMG; 6, aqueous fraction of HMG incubated with buffer alone; 7, HMG standard; and 8, sialyllactose standard. Solvents for each are given in Materials and Methods. Labels to the right indicate positions of GM3 and of sialyllactose (SL) standards. HMG standards were included on all TLCs to determine chromatographic position to verify absence of uncleaved substrate.

 
The organic (ceramide-containing) fraction was analyzed on Coomassie blue-stained TLCs and displayed four separate bands (Fig. 10) . Untreated (nondegraded) macrophage gangliosides were again included on the same TLCs to determine chromatographic position and confirmed absence of any residual, uncleaved substrate in enzyme-treated samples. Ceramide of bovine neural tissue, run on the same TLC, had chromatographic mobilities that were distinct from those of ceramide of HMG in this solvent system (Fig. 10) . Incubation of gangliosides with buffer alone caused no detectable degradation in components of either fraction. In separate experiments, the organic (ceramide-containing) fraction was independently run and developed on resorcinol-sprayed TLC with standards. Minimal (5–7% by densitometry) cross-contamination of the ceramide fraction with sialyllactose was detected, but no uncleaved macrophage gangliosides were detectable based on chromatographic mobilities (not shown).

Equimolar amounts of each enzymatically cleaved fraction were then tested in ELISA for recognition by mAb25F4. Results indicated no binding by mAb25F4 to components of the carbohydrate-containing fraction. In contrast, ELISA recognition was exclusively retained by the ceramide-containing fraction (Fig. 11 ).



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  		Figure 11. ELISA recognition by mAb25F4 of ceramide glycanase-degraded fractions of macrophage gangliosides. Enzymatically degraded fractions of HMG were produced with ceramide glycanase, as shown in Figure 10 . Equimolar concentrations of the organic (HMG Ceramide Fraction) and of the aqueous carbohydrate-containing HMG CHO Fraction] components were placed in ELISA wells and tested for recognition by mAb25F4 (10 µg/ml). Substrates also included ceramide of bovine neural tissue, selected gangliosides of bovine neural origin, MBG, and MMG. Results are expressed as a percentage of the OD450 of HMG for each substrate and are given as mean ± SE of three separate experiments. Each value was calculated with controls from the same experiment.

 
To determine whether mAb25F4 had identical affinity for ceramide or gangliosides derived from neural tissue, we tested antibody recognition in ELISA for a variety of substrates of neural origin. In each experiment, mAb25F4 showed preferential recognition for ceramide of HMG compared with ceramides of bovine neural tissue origin. mAb25F4 also showed preferential recognition for HMG compared with gangliosides of bovine neural tissue, including GM1, GM3, GD1a, GD1b, and GD3 (Fig. 11) . To further test whether mAb25F4 had affinity for non-HMG possessing ceramide comprised of C16 and C24 fatty acyl chains, ELISA was performed with murine peritoneal macrophage gangliosides (MMG). Murine brain gangliosides (MBG) were also included as nonmacrophage representatives of the same species. Recognition of gangliosides purified from murine peritoneal macrophages was comparable with that of HMG. In fact, under these ELISA conditions, mAb25F4 binding was preferential for gangliosides derived from murine peritoneal macrophages and from human monocyte-derived blood macrophages. MBG demonstrated minimal antibody binding.

As before, wells containing untreated HMG were run with each experiment, and results are expressed as a percentage of the OD450 for equimolar amounts of HMG run on the same ELISA plate. All results were reproducible in three separate experiments. In all ELISAs, empty wells containing no substrate were included to correct for nonspecific background binding.

mAb21C8 also recognized macrophage gangliosides but did not elicit IL-1ß or TNF-{alpha} (Figs. 1 and 2) . To determine the molecular domain of mAb21C8, ELISA was also performed with this mAb toward oligosaccharide and ceramide fractions derived from ceramide-glycanase cleavage of HMG. In contrast to mAb25F4, mAb21C8 did not show recognition of the ceramide fraction, but displayed preferential binding to the oligosaccharide-containing fraction (Fig. 12 ). Further studies to more precisely localize the binding domain of mAb21C8 are in progress.



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  		Figure 12. ELISA recognition by mAb21C8 of ceramide glycanase-degraded fractions of macrophage gangliosides. Enzymatically degraded fractions of HMG were produced with ceramide glycanase, as shown in Figure 10 . Equimolar concentrations of the organic (HMG Ceramide Fraction), of the aqueous (HMG CHO Fraction), and of intact HMG were placed in ELISA wells and tested for recognition by mAb21C8 (30 µg/ml). Results are expressed as the OD450 for each substrate and are given as means ± SE of three separate experiments. Each value was calculated with controls from the same experiment.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Macrophage gangliosides possess unique, immunologic attributes compared with gangliosides of neural tissues, as demonstrated by several studies. Macrophage gangliosides inhibit T cell proliferation with far greater potency than do brain gangliosides and act at the level of the cell membrane, and the effect of brain gangliosides is primarily extracellular [4 ]. Murine peritoneal macrophages display dramatic, cytokine-mediated changes in expression of gangliosides in response to LPS [6 , 13 ]. All gangliosides of normal human macrophages are represented in macrophages of patients with AIDS but become progressively inaccessible to surface molecules with progression of HIV infection [11 ].

We pursued this investigation by eliciting immunologic responses of human macrophages with mAb produced expressly to gangliosides of human macrophages [10 ]. Our early studies indicated that the 25F4 epitope was surface-accessible and was absent in several nonmacrophage cell lines. Moreover, mAb25F4 was capable of inducing antibody-specific migration inhibition of human macrophages. These findings were intriguing because collective results of several earlier studies suggested that the receptor for macrophage migration inhibitory factor (MIF), a known inhibitor of macrophage migration, is a macrophage ganglioside [27 , 28 ]. More recently, MIF has been recognized as having far broader immunologic functions than were originally recognized [29 , 30 ]. In the current study, binding of mAb25F4 to surface-accessible macrophage gangliosides also induced macrophage cytokine production. Specificity was demonstrated through lack of response to irrelevant mAb. Thus, mAb25F4 also has broader immunoregulatory properties than were originally recognized. Immunologic activation via glycolipid receptors was first demonstrated in T cells with antibodies to GD3 and O-acetyl GD3 and represents a novel means of induction [31 , 32 ]. Our results indicate that endogenous gangliosides of human macrophages also possess receptor properties and further indicate that ceramide of macrophage gangliosides is critical to this function.

Our previous data indicated that mAb25F4 recognized an epitope common to each of the major gangliosides of human macrophages [12 ]. Although antibody cross-recognition of diverse ganglioside structures that differ in core carbohydrate composition, such as GM3 and sialosylparagloboside, has been described for an antimelanoma mAb [33 ], the specific molecular domain of ganglioside molecules recognized by mAb25F4 was not known. Our current studies indicate that 25F4 not only recognizes a surface epitope, but preferentially also recognizes the ceramide moiety. Further, this antibody preferentially recognizes gangliosides of macrophages of humans and mice and discriminates between these and gangliosides of neural tissue. Our conclusions are supported by preferential recognition of ceramide of HMG, which is structurally distinct from ceramide of neural origin; failure to abolish recognition with desialylation; absence of recognition of several gangliosides of neural origin; and interspecies cross-recognition of MMG, which share the ceramide structure of HMG [8 , 9 ].

Ceramides are heterogeneous molecules comprised of sphingosine and fatty acid chains. Ceramides of MMG are predominantly comprised of C18 sphingosine and C16 and C24 fatty acids [7 ]. The latter finding is remarkable because gangliosides of neural tissues are predominantly comprised of ceramide with alternative fatty acid constituents. Furthermore, ceramide comprised of C16 and C24 fatty acids is a shared, structural attribute of macrophage gangliosides of mice and of humans [9 ]. The structurally distinct ceramide moieties, shared by gangliosides of macrophages of different species, might be the structural basis for some of the immunologic properties of macrophage gangliosides. It is intriguing that ceramide comprised of C16 and C24 fatty acids is also present in B lymphocyte gangliosides [34 ]. Although our findings support the hypothesis that structural distinctions might explain some of the biologic attributes of gangliosides of immune cells, the immunologic properties of macrophage gangliosides may also be reliant on discrete, molecular conformations of ceramide of macrophages, which have not yet been determined.

While the roles of carbohydrate moieties of GSL as molecular determinants of GSL-mediated biological activity are well described, increasing evidence implicates ceramide structures of gangliosides as important immunoregulatory components. As with our findings, Ladisch et al. [35 ] reported that fatty acyl ceramide length was also the principal determinant of immunosuppressive activity of neuroblastoma GD2 ganglioside. They further identified cross-species ceramide activity, dependent on fatty acyl chain length, in murine and human cellular gangliosides. The immunologic recognition of mAb25F4 is also directed at gangliosides of macrophages of human and murine origin. Verotoxin binding to globotriaosylceramide is similarly affected by heterogeneity in the ceramide fatty acyl chain length, and the greatest binding capacity is held by ceramides with longer fatty acyl chains [36 ]. Besides serving as lipid anchors for protruding alkyl chains, recent mechanistic paradigms also support direct interaction of ceramides with signaling proteins [37 ]. This may also occur in models of synthetic membranes in which long ceramide fatty acyl chains protruded further from the surface of the lipid bilayer, potentially availing themselves to surface molecules [38 ]. Surface access to relatively hydrophobic regions of gangliosides also occurs with ceramide glycanase and ceramidase in intact cells [39 40 41 ]. Enzymatic access to the lipid moiety by ceramide glycanase may be a result of the relative hydrophobicity of the enzyme itself, binding directly to more hydrophobic sphingosine moieties [42 ]. Finally, structural mimicry between gangliosides and bacterial lipooligosaccharides and between their respective ceramide and lipid A components has been previously appreciated [43 ]. Remarkably, lipid A of lipooligosaccharide of H. influenzae, also previously believed to be inaccessible to surface molecules, is clearly accessible to mAb in intact bacteria [44 ]. These results collectively suggest that the fluidity of cellular membranes permits greater surface accessibility of ganglioside-bound ceramide than has been traditionally considered.

Ceramide itself has numerous immunologic properties, including regulation of intracellular pathways for induction of apoptosis [45 ]. Free ceramide can be generated through sphingomyelinase hydrolysis of GSL and is involved in multiple signaling pathways [46 ]. Signal transduction through ceramide has, among its downstream targets, IL-2 and IL-6 and can therefore affect immune response directly or through cytokine induction [47 ]. Selected ceramide species also regulate differentiation of HL-60 cells and act as second messengers in signal transduction [48 , 49 ]. Ceramide-mediated, intracellular signaling can also regulate mRNA transcription of the c-myc protooncogene, as well as nuclear factor-{kappa}B expression [50 , 51 ].

Besides being a constituent of gangliosides, ceramide is also a component of nonsialylated (neutral) GSL. Therefore, it was critical to our studies to establish appropriate time and dose kinetics for selective depletion of gangliosides with PDMP. Metabolic inhibition of glucosyl-ceramide synthase has been a valuable tool for studying the roles of GSL in many cell systems [15 ]. In previous studies, refinements have permitted selective depletion of individual GSL components. Depletion of globo series GSL demonstrated decreased cellular adherence of P-fimbriated E. coli [52 ]. In fact, PDMP-mediated, metabolic inhibition in one study was so selectively regulated as to permit synthesis of neuroblastoma gangliosides yet to impair extracellular shedding of gangliosides [53 ]. Metabolic depletion of gangliosides and other GSL by ceramide-synthase inhibition in mice resulted most markedly in cellular depletion of lymphoid tissues, underscoring the importance of GSL in the biology of the immune system [54 ], prompting us to apply metabolic depletion of GSL to the study of human macrophages. By establishing conditions for selective depletion of macrophage gangliosides, our ganglioside-depletion experiments further support the immunologic importance of ganglioside-bound ceramide. This does not discount the potential immunologic significance of nonganglioside-bound ceramide, including ceramide of neutral GSL of macrophages, which will require independent verification.

The mechanism by which intracellular signaling is initiated by binding to macrophage gangliosides remains speculative. GSL-mediated cell signaling may originate from detergent-insoluble lipid rafts, acting as localized membrane sites for initiating signal transduction by selected pathways. These domains are enriched, not only in gangliosides but also in phospholipids and cholesterol. In fact, T lymphocyte activation is enhanced by clustering signaling molecules in membrane lipid domains [55 ]. Lipid rafts may be potent substrate pools for production of ceramide, which may further form microdomains and regulate downstream signaling [56 ]. Indeed, 25F4-epitope signaling may originate from stabilization of membrane lipid rafts, an aspect that will be the focus of ongoing experiments of our laboratory.

Studies of immunologic functions of GSL have often relied on exogenous, commercially available compounds, often derived from neural tissue. It is reasonable to presume that had we attempted these studies with mAb raised to gangliosides of other tissues, we would not have arrived at the same results. This investigation therefore supports the use of endogenous GSL of immune cells for studies of GSL immunoregulation and highlights the importance of ceramide of HMG as a critical domain for ganglioside-mediated activation of human macrophages.


   ACKNOWLEDGEMENTS
 
This work was supported by research grant 1RO1HL6654901 from the National Institutes of Health and by Merit Review funding from the Department of Veteran’s Affairs (C. S. B.). The authors are grateful for ongoing scientific advice offered by Herbert C. Yohe, Ph.D., and by Alan J. Lesse, M.D. We also appreciate the assistance of Timothy F. Murphy, M.D., for offering scientific advice, for the generous donation of mAb5F3, and for critically reading this manuscript and of Ms. Sonia Sierra for secretarial assistance in preparing this manuscript.

Received August 11, 2001; revised March 28, 2002; accepted April 17, 2002.


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 DISCUSSION
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