Structural Elucidation and Monokine-inducing Activity of Two Biologically Active Zwitterionic Glycosphingolipids Derived from the Porcine Parasitic Nematode Ascaris suum *

 The isolated neutral glycosphingolipid fraction from the pig parasitic nematode,Ascaris suum, was fractionated by silica gel chromatography to yield a neutral and a zwitterionic glycosphingolipid fraction, the latter of which mainly contained two zwitterionic glycosphingolipids termed components A and C. Preliminary chemical characterization with hydrofluoric acid treatment and immunochemical characterization with a phosphocholine-specific monoclonal antibody indicated that both components contained phosphodiester substitutions: phosphocholine for component A, and phosphocholine and phosphoethanolamine for component C. Both components were biologically active in inducing human peripheral blood mononuclear cells to release the inflammatory monokines tumor necrosis factor α, interleukin 1, and interleukin 6. Component A was the more bioactive molecule, and its biological activity was abolished on removal of the phosphocholine substituent by hydrofluoric acid. The glycosphingolipid components were structurally analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, liquid secondary ion mass spectrometry, methylation analysis, 1H NMR spectroscopy, exoglycosidase cleavage, and ceramide analysis. Their chemical structures were elucidated to be (see Structure I below),           phosphocholine 6 − ‖ Component A                   Gal ( α 1 – 3 ) GalNAc ( β 1 – 4 ) GlcNAc ( β 1 – 3 ) Man ( β 1 – 4 ) Glc ( β 1 – 1 )   ceramide           phosphocholine 6   _ ‖                 ‖ _   6 phosphoethanolamine   Component C                   Gal ( α 1 – 3 ) GalNAc ( β 1 – 4 ) GlcNAc ( β 1 – 3 ) Man ( β 1 – 4 ) Glc ( β 1 – 1 )   ceramide Structure I  The carbohydrate moiety oligosaccharide core was characterized as belonging to the arthro series of protostomial glycosphingolipids. The ceramide moiety was distinguished by (R)-2-hydroxytetracosanoic acid as the dominant fatty acid species and by the C17 iso-branched sphingosine and sphinganine bases, 15-methylhexadecasphing-4-enine and 15-methylhexadecasphinganine, respectively.

in the extracts of numerous species of parasitic nematodes by immunological means (4 -9). Structurally, this moiety has been found bound to N-and O-linked glycans of glycoproteins, although the exact structure of the PC-oligosaccharide linkage is at present unknown (10). The biological significance of PC glycans in the host parasite relationship revolves around their immunomodulatory activity (11) such that the frequent observation of host T-cell hyporesponsiveness to filarial nematode infection (12) may involve PC because of its ability to block Tand B-cell antigen-specific proliferation (13,14).
Little is known as to the biological activity of glycolipids, in general, and parasitic helminth-derived glycolipids, in particular, as regards their putative modulation of the host immune response via the cytokine network. Gangliosides have been found to be inhibitory in terms of cytokine synthesis and release (15), whereas neutral glycosphingolipids of the cestode Echinococcus multilocularis inhibited the production of interleukin 2 (IL-2) (16). Because of the physico-chemical similarity between glycosphingolipids and lipopolysaccharides (LPS) of Gram-negative bacteria and the induction by the latter of bioactive protein mediators in the host, i.e. cytokines, responsible for the effects of endotoxemia (17), a comparative study was performed by Krziwon et al. (18) on the ability of the former to stimulate the production of inflammation-associated cytokines. An atypical, zwitterionic glycosphingolipid (as regards the linkage of the glucuronic acid residue to the ceramide moiety and the presence of nonacetylated glucosamine) from the LPS-negative, Gram-negative bacterium Sphingomonas paucimobilis induced the synthesis and release of the human mononuclear cell-derived, inflammation-associated cytokines tumor necrosis factor ␣ (TNF-␣), IL-1, and IL-6 but with approximately 10,000-fold less activity than LPS, in this respect.
We report here on the structures and biological activity of two immunoreactive, zwitterionic fraction glycosphingolipids from A. suum in terms of their ability to stimulate the production of the human mononuclear cell-derived, inflammationassociated cytokines TNF-␣, IL-1, and IL-6.

EXPERIMENTAL PROCEDURES
Materials-Undamaged, washed adult male and female worms were collected from the local abattoir and stored at Ϫ70°C until further use. LPS from Salmonella friedenau was kindly donated by H. Brade (Borstel Research Institute).
Bioassay Determination of Released Cytokines-The isolated Nz-glycosphingolipids, component A and C, and ceramide pentahexoside (CPH) derived from component A by HF treatment (see below) were subjected to sterile distilled water dialysis to remove potential cell culture-perturbing contaminants and traces of organic solvents. After Speed-Vac lyophilization, the glycosphingolipids were resuspended at 1 mg/ml in sterile distilled water, sonicated, and stored at Ϫ20°C until further use. As a positive control, S. friedenau-derived LPS was solubilized in pyrogen-free phosphate-buffered saline at 1 mg/ml, neutralized with triethylamine, sonicated, and stored at 4°C until further use.
Human peripheral blood mononuclear cells (PBMC) from healthy donors were isolated with Ficoll-Paque (Pharmacia) on density gradient centrifugation. The washed PBMC (10 6 /ml) were cultured in U-form microtiter plates (Greiner, Nü rtingen, Germany) at 200 l in RPMI 1640 medium containing antibiotics, 10% heat-inactivated human serum, and the relevant glycolipid. After a 6-h incubation at 37°C (5% CO 2 ), the supernatants were collected by centrifugation at 1200 rpm for 5 min and investigated for cytokine activity. The supernatants of glycolipid-stimulated PBMC were analyzed by bioassay as to the cytokine activities of TNF-␣, IL-1, and IL-6 (19). The cytotoxicity of TNF-␣ was determined with the TNF-sensitive L929 fibrosarcoma cell line (20). The proliferative capacity of IL-1 was assayed with human dermal fibroblasts (21). The proliferation of IL-6-dependent murine B9.9 -3A4 hybridoma cells was applied to determine IL-6 activity.
Liquid Secondary-ion Mass Spectrometry (LSIMS)-LSIMS was carried out with a MAT 900 mass spectrometer (Finnigan MAT) equipped with a cesium gun, which was operated at an emission current of 2-3 A. Mass spectra were recorded at an acceleration potential of 5 kV with a resolution of approximately 3,000 and were acquired using a DEC 2100 data system. Spectra of native, peracetylated, or permethylated glycosphingolipids were recorded in the positive-ion mode using 3-nitrobenzyl alcohol (Aldrich) as matrix.
NMR Spectroscopy-The 1 H NMR spectra were recorded at 333 K on a Bruker DRX 600 spectrometer with deuterium-exchanged samples (0.9 mg each) for solutions in Me 2 SO-d 6 (99.96%; Aldrich) containing 2% (by vol) 2 H 2 O (99.96%; Aldrich) using the 1 H signal of dimethyl sulfoxide-d 5 (␦ H 2.49) as internal reference. All one-and two-dimensional NMR experiments like two-dimensional correlation spectroscopy (COSY) and two-and three-step related coherence transfer (RCT-1 and -2), were performed using standard Bruker software (XWINNMR, Version 1.3).
HF Treatment-Nz-glycosphingolipids (native or permethylated) were dried in a stream of nitrogen and incubated for 24 h at 4°C with 50 -200 l of HF (48%; Fluka, Neu-Ulm, Germany). Excess was removed in a stream of nitrogen at room temperature.
Endoglycoceramidase Cleavage-Nz-glycosphingolipids were resuspended in 100 l of 50 mM sodium acetate buffer, pH 5.0, containing 1 g/liter sodium taurodeoxycholate, and 0.5 milliunits of endoglycoceramidase (Sigma) were added. After incubation at 37°C for 24 h, another 0.5 milliunits of enzyme were added. The reaction was stopped after 48 h by adding 400 l of H 2 O and 400 l of water-saturated n-butanol for phase separation of the reaction products.
Exoglycosidase Treatment-Pyridylaminated oligosaccharides were cleaved after obligatory HF treatment with either ␣-D-galactosidase (EC 3. Methylation Analysis-Nz-glycosphingolipids (20 g) were permethylated both before or after HF treatment and hydrolyzed (25). Partially methylated alditol acetates obtained after sodium borohydride reduction and peracetylation were analyzed by capillary GLC/MS using the instrumentation and microtechniques described elsewhere (26).
Identification of Zwitterionic Substituents-Phosphocholine was released by HF treatment of Nz-glycosphingolipid components A and C. Liberated choline residues were derivatized with pentafluoropropionic acid anhydride (Supelco, Deisenhofen, Germany) and analyzed by LSIMS. Ethanolamine was identified as its 9-fluorenylmethoxycarbonyl-derivative by HPLC (28) after HF treatment of component C.
N-Methylation of Phosphoethanolamine-Nz-glycosphingolipid component C was treated with 200 l of 750 mM aqueous sodium carbonate containing 20 l of methyl iodide for 2 h at 50°C (29,30), and thereafter, desalted on a reverse-phase cartridge (31).
Ceramide Analysis-For fatty acid analysis, Nz-glycosphingolipids (1-10 nmol) were hydrolyzed according to Gaver and Sweeley (32). The resultant fatty acid methyl esters were analyzed by capillary GLC/MS using the instrumentation described previously (26). For the separation of fatty acid species, a fused silica capillary column (DB1, 0.25 mm internal diameter, 60 m; ICT, Bad Homburg, Germany) was used. The column temperature was increased from 80°C at 7°C/min to a final temperature of 320°C and held isothermally for 10 min. Spectra were recorded either after chemical ionization (CI-MS) with ammonia or electron-impact ionization (EI-MS) at an electron energy of 2.4033 ϫ 10 Ϫ17 J or 1.1215 ϫ 10 Ϫ17 J, respectively. For determination of the absolute configuration at C-2 of the contained hydroxy fatty acids, they were converted to the corresponding (R)-phenylethylamides and trifluoroacetylated as described previously (3). Sphingoid bases were analyzed after conversion to the corresponding fatty acids by periodate and periodate/permanganate oxidation as their methyl and picolinyl esters as described elsewhere (3).

Isolation of Zwitterionic Components A and C-Glycosphin-
golipids were separated into a neutral and acidic fraction by anion-exchange column chromatography. Two-dimensional HPTLC of the resultant neutral fraction indicated the presence of two groups of glycosphingolipids: N-neutral, Nz-neutral zwitterionic glycosphingolipids (see Fig. 1). For isolation of the two main zwitterionic components A and C, the neutral fraction was subfractionated into a neutral and neutral zwitterionic fraction by silica gel column chromatography. A further silica gel column chromatography yielded four fractions designed as components A, B1, B2, and C. The fractions B1 and B2 repre-sent nonhomogeneous, minor zwitterionic components and will not be discussed further in this publication.
Chemical and Immunochemical Characterization-The two zwitterionic components A and C were separated on HPTLC by chloroform/methanol/water 10:10:3 (by vol) as running solvent. Both components gave positive reactions on incubation with iodine vapor, spraying with orcinol/sulfuric acid, and molybdate reagent (organic phosphate groups). HPTLC-immunostaining with the phosphocholine-specific monoclonal antibody TEPC-15 is shown in Fig. 2. Due to the approximately 50-fold higher sensitivity of HPTLC-immunostaining, additional, minor species of components A and C resulting from heterogeneities in their lipid moieties were also visualized. The component C also reacted with ninhydrin, indicating the presence of a free amino group. HF treatment of the zwitterionic compounds yielded glycosphingolipids with migration properties on HPTLC similar to CPH, which showed no reaction with TEPC-15 or ninhydrin. Choline was identified after HF treatment of the components A and C by derivatization with pentafluoropropionic acid anhydride and analysis by LSIMS, yielding a molecular ion [M] ϩ at m/z 250. Ethanolamine was identified after HF treatment of compound C as its 9-fluorenylmethoxycarbonyl derivative by HPLC and co-chromatography with the standard (data not shown).
Zwitterionic Component A-and C-induced Monokine Production-Since we consider A. suum merely as a model for the human parasitic nematode Ascaris lumbricoides, all in vitro procedures were performed with human and not porcine PBMC. The zwitterionic components A and C and the component A-derived CPH were assayed as to their biological activity in inducing the inflammatory monokines TNF-␣, IL-1, and IL-6 because of the postulated similarities in physico-chemical properties and biological activity between glycosphingolipids and LPS. Components A and C, but not ceramide pentasaccharide, were shown to be biologically active in terms of a dose-dependant response in the release of TNF-␣, IL-1, and IL-6 (see Fig. 3). For IL-1 and IL-6, this dose dependence of cytokine release was evident up to and including 1000 ng/ml component A, with the apparent presumption that higher concentrations were inhibitory at the cellular level. Of the two zwitterionic glycolipids tested, component A was the more bioactive in inducing the monokines TNF-␣ and IL-1; component A and to a lesser extent component C were also capable of inducing low levels of IL-6 activity as demonstrated in three separate experiments (data FIG. 1. Two-dimensional HPTLC separation of N-and Nz-glycosphingolipids of A. suum. HPTLC separation was performed on silica gel 60 plates. The solvents used were chloroform/methanol/water 10:10:3 (by vol) for the first dimension and chloroform, methanol, 2% aqueous ammonia 10:10:3 (by vol) for the second dimension. Glycosphingolipids were detected by spraying the plate with orcinol/sulfuric acid. Components A and C are indicated by arrows.

FIG. 2. Chemical and immunochemical characterization of HPTLC-resolved, zwitterionic components A and C.
Zwitterionic components A and C were separated on silica gel 60 HPTLC plates with chloroform/methanol/water 10:10:3 (by vol) as running solvent. Glycosphingolipids were detected chemically by spraying with orcinol/sulfuric acid (a) or ninhydrin (b) and immunochemically by staining with the phosphocholine-specific monoclonal antibody TEPC-15 (c). not shown). The apparent inconsistency in concentration levels measured was due to the inherent between-experiment variability of the bioassay system with different human donors.
Structural Analysis of Zwitterionic Components A and C-For structural analysis, the zwitterionic components A and C were subjected to MALDI-TOF-MS, LSIMS, methylation analysis, and exoglycosidase digestion. The results of MALDI-TOF-MS and LSIMS are summarized in Table I and methylation data in Table II Fig. 5), whereas pseudomolecular ions at m/z 2298, 2300 and 2379, 2381 and 2421, 2423, respectively, are most likely due to incomplete acetylation and/or ketene elimination.
To locate the monosaccharide linkage and phosphodiester substitution positions, methylation analysis of the two zwitterionic compounds A and C was performed with the permethylation procedures both before or after HF treatment (Table II). If the permethylation procedure was performed after HF treatment, the compounds A and C showed similar results with terminal galactose, 3-substituted mannose, 4-substituted glucose, 4-substituted N-acetylglucosamine, and 3-substituted Nacetylgalactosamine (Table II, columns A1 and C1). HF treat-FIG. 3. Zwitterionic component A-and C-induced monokine production by human PBMC. PBMC (10 6 /ml) were cultured for 6 h with varying concentrations of the glycosphingolipid components A, C, and CPH, and positive control LPS (1-10,000 ng/ml). After incubation, supernatants were collected, and their TNF-␣, IL-1, and IL-6 activities determined by bioassay. ment after the permethylation procedure revealed for compound A the presence of a 4,6-disubstituted N-acetylglucosamine (Table II, column A2) that indicated location of the phosphocholine substituent at the C-6 of N-acetylglucosamine. For compound C, a 4,6-disubstituted N-acetylglucosamine and a 3,6-disubstituted mannose were found along with 3-substituted mannose (Table II, column C2), the latter of which is formed due to the lability of the phosphoethanolamine substituent to the conditions of the permethylation procedure (33). If the phosphoethanolamine substituent was stabilized by methylation to choline before permethylation, the major mannose constituent was found to be 3,6-disubstituted mannose, indicating the localization of phosphoethanolamine at the C-6 of mannose (Table II, column C3).
The zwitterionic glycolipids were cleaved by endoglycoceramidase, and the liberated oligosaccharides were reductively pyridylaminated and isolated by amino-phase HPLC. For determination of the anomeric configurations of individual glycosidic bonds, pyridylaminated oligosaccharides, after HF-treatment, were sequentially incubated with ␣-galactosidase, ␤-N-acetylhexosaminidase, and ␤-mannosidase, resulting in the release of one galactosyl residue, two N-acetylhexosaminyl residues, and one mannosyl residue, as confirmed by amino-phase HPLC.
As a second method for structural confirmation, the ano-meric linkages of the sugar residues in components A and C were further elucidated by 1 H NMR spectroscopy as ␣-Gal V , ␤-GalNAc IV , ␤-GlcNAc III , ␤-Man II , and ␤-Glc I (Table III).
Chemical shift values and coupling constants ( 3 J 1,2 ) of the anomeric protons were found to be very similar, indicating identical linkages and composition in both glycosphingolipids. With the exception of the terminal ␣-Gal residue, all sugars were identified to express ␤-anomeric linkage. The anomeric linkage of the Man II could not be determined by a one-dimensional 1 H NMR experiment but was investigated following the connectivities of the spin system using two-dimensional correlation spectroscopy (COSY) and two-and three-step-related coherence transfer (RCT-1 and -2) (data not shown). All anomeric linkages determined by 1 H NMR were found to be identical as compared with results obtained by enzymatic degradation reactions (see above). In addition, glycosphingolipids A and C showed characteristic singlets (integral 9H) for the methyl protons of the choline residue [-ϩ N(CH 3 ) 3 ] (3.135 ppm, compound A; 3.156 ppm, compound C) originating from a phosphocholine residue being assigned by methylation analysis to position 6 of the GlcNAc III residue in both glycosphingolipids.
For ceramide analysis, the two zwitterionic glycolipids were subjected to acid hydrolysis according to Gaver and Sweeley

TABLE II Methylation analysis of zwitterionic glycosphingolipids A and C
A. suum Nz-glycosphingolipids were treated according to the following sequence: with (C3) or without (A1, A2, C1, C2) PE-methylation, with (A1, C1) or without (A2, C2, C3) HF treatment before permethylation, with (A2, C2, C3) or without (A1, C1) HF treatment after permethylation. The partially methylated sugar derivatives obtained after reduction and peracetylation were analyzed by capillary GLC/MS. Results are expressed as peak ratios of the alditol acetates found based on 2,3,6-GlcOH ϭ 1.0. The low yields of terminal monosaccharides are due to their higher volatility, i.e. a higher level of methylation. Due to a lower sensitivity for N-acetylhexosamines, their presence or absence is indicated by ϩ/Ϫ.  linear (b, d, and f) and reflectron (a, c, e, and g) modes either before (a, b, e, and f) or after HF-treatment (c, d, and g) with 2,5-dihydroxybenzoic acid as matrix. Pseudomolecular ions are given in accurate mass values rounded to the nearest mass unit. a , Inset in a, after LiCl addition. (32). Fatty acids were extracted with n-hexane and analyzed by GLC/MS. In agreement with previous data on neutral A. suum glycosphingolipids (see Table III and Fig. 4 in Ref. 3), the results demonstrated the predominant presence of 2-hydroxytetracosanoic acid. The absolute configuration at C-2 was found to be (R) by GLC/MS analysis of the corresponding trifluoroacetylated (R)-phenylethylamide (data not shown). Sphingoid bases were analyzed after periodate and periodate/ permanganate oxidation as their methyl and picolinyl esters (data not shown). The results indicated the presence of C17 iso-branched sphingosine and sphinganine bases in agreement with Ref. 3.

DISCUSSION
A slowly emerging chemical characteristic of invertebrate glycoconjugates (glycolipids, glycoproteins) is their frequent substitution by electrically neutral but amphoteric moieties. The diversity of zwitterionic glycoconjugates among the various phyla of the Invertebrata would point to their biological importance, but as yet, unknown functional significance. A major post-translational modification of parasitic helminth antigens is apparently PC. This antigenic determinant has been detected in nematodes (5,8,34,35), in trematodes, including Schistosoma mansoni (9), and in the cestode Bothriocephalus scorpii (36). In fact, the frequency of serological cross-reactivity between cestodes, trematodes and, in particular, nematodes (37) may be accounted for by the broad distribution of PCbearing molecules. The (macro)molecular location of the PC moiety is in most cases unknown, but at least in the excretory/ secretory product (ES-62) of the adult filarial nematode, Acanthocheilonema viteae, it is attached to the protein backbone via an N-linked glycan (38).

and this publication).
Localization of zwitterionic substituents such as phosphocholine or phosphoethanolamine was performed by HF treatment, both before or after permethylation and subsequent hydrolysis, reduction, and peracetylation (in the range of 10 g of glycosphingolipid). The alkali instability of the phosphoethanolamine substituent, however, requires selective N-methylation before the permethylation procedure. MALDI-TOF-MS analysis of the zwitterionic glycolipids revealed a characteristic fragmentation in the reflectron mode, probably due to the loss of choline (M Ϫ 87) and ethanolamine (M-45), respectively, by metastable decay, which was not detectable in the linear mode. A similar fragmentation pattern has been observed in LSIMS after permethylation, whereas the peracetylated structures were found to be stable. This idiosyncratic fragmentation behavior may help to detect and identify zwitterionic substituents by mass spectrometry.
Structural elucidation of the two major, zwitterionic glycosphingolipids (components A and C) of the porcine, parasitic Numbers in parentheses mark pseudomolecular ions, probably resulting from incomplete acetylation and/or ketene elimination.

600-MHz 1 H NMR data of anomeric protons (H-1) for compounds A and C
Chemical shifts (␦) are given in parts/million in dimethyl sulfoxide-d 6 at 333 K; coupling constants ( 3 J 1,2 ) are given in hertz.  nematode A. suum has shown their common pentasaccharide core to belong to the arthro-carbohydrate series (as originally isolated from glycosphingolipids of the blowflies Calliphora vicina and Lucilia caesar). The amphoteric substituent PC is linked to C-6 of the third monosaccharide in the oligosaccharide chain, GlcNAc, of component A, and, uniquely, the amphoteric substituents PE and PC are simultaneously linked to C-6 of the second and the third monosaccharide in the oligosaccharide chain, Man and GlcNAc, respectively, in component C. Component C, therefore, represents the first member of the glycosphingolipids to carry two zwitterionic substituents. The carbohydrate and ceramide moieties of the two zwitterionic glycosphingolipids correspond to the recently elucidated arthropentaosyl ceramide of A. suum.
(3) Therefore, we have assumed that the biosynthetic pathway of the former involves zwitterionic substitution of the latter, either at the level of CPH or incomplete oligosaccharide cores. The 1 H-NMR data obtained for both glycosphingolipids A and C were found to be structurally closely related to that identified in a pentaglycosyl phosphoglycosphingolipid (Nz5a) isolated from the blowfly C. vicina Meigen (51). Comparing the glycosphingolipid Nz5a of the blowfly C. vicina Meigen and compound A described here, both belong to the arthro series, and only three structural differences were observed: (i) a terminal ␣-GalNAc V instead of ␣-Gal V , (ii) the terminal sugar (␣-GalNAc V ) being (134)-linked in Nz5a and ␣-Gal V being (133)-linked in compound A, and (iii) 2-aminoethyl phosphate instead of a phosphocholine substituent in position 6 of GlcNAc III . The biological properties of the glycosphingolipid Nz5a, however, were not investigated.
Glycosphingolipids have been shown to be immunomodulatory molecules that suppress cells of the immune system, both in vivo and in vitro. Thus, gangliosides inhibit the in vitro proliferative response of various classes of activated immune cells such as T-and B-lymphocytes, macrophages, and natural killer cells (52). However, the molecular mechanism(s) underlying the immunosuppressive activity of glycosphingolipids are incompletely understood but include the direct interaction of ganglioside micelles with IL-2 and IL-4 in the modulation of IL-2-/IL-4-dependent processes (53) and the interference of monocytes at the level of antigen presentation (54). The immunomodulation of T-lymphocyte activation in vivo and in vitro, observed in the case of Trypanosoma cruzi glycoinositol phospholipids, can be directly related to the ceramide moiety of the molecule (55). A functional resemblance of LPS to glycosphingolipids has been proposed and reinforced by the ability of the former to mimic the second messenger ceramide in TNF-␣-and IL-1-stimulated cells (56,57). Since there is no structural similarity between these two classes of lipid molecules, it may be assumed that this coincidence of biological activity is based on similar physico-chemical properties. The ability of A. suum zwitterionic components A and C to induce the human PBMC inflammatory cytokines of TNF-␣, IL-1, and IL-6 provides a further example for the parallelism between LPS and glycosphingolipid biological activity.
Interestingly, the zwitterionic glycosphingolipids of A. suum stimulated rather than suppressed human PBMC production of the cytokines TNF-␣ and IL-6 in a concentration range similar to that of LPS stimulation, whereas these molecules were at least a factor 100-fold less potent than LPS in the stimulation of IL-1. In general, component C was less active than component A. When compared with LPS with respect to the amount of cytokine induced, component A was as active in the stimulation of TNF-␣ but was decreasingly active in the sequence of IL-1 and IL-6. The expression of the inflammatory response cytokines TNF-␣, IL-1, and IL-6 is usually considered to be concomitant (58). Until concrete data are available, there are at least two plausible explanations for the detection of the anomalous, nonconcomitant levels of these cytokines induced by the A. suum-derived zwitterionic glycosphingolipids under study. First, kinetic studies of LPS-and zwitterionic glycosphingolipid-induced inflammatory cytokine expression demonstrated maximal activity in the temporal sequence of TNF-␣ and IL-1 at approximately the same time point and prior to that of IL-6 (18,19). The fixed-point determination of cytokine release in the assay used in this study at 8 h of incubation introduces an experimental artifact whereby TNF-␣ and IL-1 approach their maximal levels of activity, whereas IL-6 induction is suboptimal at this time point. Secondly, it is known that activation of monocytes by LPS is a receptor-mediated process that is transduced by the cell surface molecule CD 14 (59). The mechanism by which the A. suum-derived zwitterionic glycosphingolipids induce cytokine production is, however, unknown. It may be postulated that they act in a direct way by replacing intracellular lipid second messengers such as ceramide. Therefore, a different pattern of cytokines released by activated monocytes may be due to different mechanisms of activation.