The major gangliosides of human peripheral blood monocytes/macrophages: absence of ganglio series structures

Herbert C. Yohe1,2, Paul K. Wallace3, Charles S. Berenson4, Song Ye5, Bruce B. Reinhold5 and Vernon N. Reinhold5

2Research Service, Veterans Administration Medical and Regional Office Center, 215 North Main Street, White River Junction, VT 05009, USA and Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, NH 03755, USA; 3Department of Microbiology, Dartmouth Medical School, Hanover, NH 03755, USA; 4Infectious Disease Section, Department of Veterans Affairs, Western New York Healthcare System, State University of New York at Buffalo, School of Medicine, Buffalo, NY 14215, USA; and 5Department of Chemistry, University of New Hampshire, Durham, NH 03824, USA.

Received on May 31, 2001; accepted on June 27, 2001.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Sialoglycosphingolipids (gangliosides) are membrane components of eukaryotic cells that modulate cell signal transduction events. Discrepancies exist in the published descriptions of the gangliosides present in the human peripheral monocyte/macrophage. Macrophages were isolated from healthy human volunteers by two different methods. Their ganglioside fractions were isolated and examined by 2D thin-layer mobility, enzymatic susceptibility, and mass spectral-collision induced dissociation-mass spectral analyses. Thin-layer ganglioside chromatographic patterns displayed four major doublets and were similar for monocytes/macrophages isolated by either apheresis/elutriation or density gradient centrifugation. All gangliosides were resistant to ß-galactosidase but sensitive to Clostridium perfringens sialidase, indicating the absence of terminal galactose residues and sialidase-resistant sialic acid moieties. Mass spectra indicated only three major sets of glycolipid components with mass heterogeneity in the ceramide portion of each set. In all the gangliosides, the ceramide moiety contained only C18 sphingosine with the heterogeneity produced by the presence of C16 or C24 fatty acid. One doublet was resistant to Newcastle disease virus sialidase, indicating the presence of an {alpha}(2-6)-linked sialic acid residue with the same mass as another doublet. All data was consistent with the following structures as the major gangliosides of human peripheral monocyte/macrophages: II3NeuAcLacCer (sialolactosyl ceramide, GM3), IV3- and IV6NeuAcnLcOse4Cer (sialoparagloboside, nLM1), and IV3NeuAcnLcOse6Cer (a sialohexosylceramide).

Key words: glycolipids/gangliosides/human/monocytes/tandem mass spectrometry


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Gangliosides, sialic acid–containing glycosphingolipids, are components of the cell plasma membrane, where they appear mainly in lipid rich areas of the plasmalemma termed rafts (Simons and Ikonen, 1997Go). These glycolipids are now emerging as important modulators of cell signal transduction (Yates and Rampersaud, 1998Go). Though early investigations centered on glycolipids of brain, work in murine models has shown that substantial glycolipid alterations can occur in immune cells on cell differentiation and in cells with genetic deficiencies not directly related to glycolipid biosynthesis (Schwarting and Gajewski, 1981Go; Yohe and Ryan, 1986Go; Yohe et al., 1991Go). Like brain, glycolipid patterns of immune cells are conserved within a species (Nakamura et al., 1988Go; Yohe et al., 1991Go), unlike glycolipid patterns from other peripheral tissues (Nakamura et al., 1988Go).

Macrophage gangliosides possess a unique array of structural and immunoregulatory attributes, distinct from gangliosides of other tissues. Furthermore, the ability of glycolipids to affect cell function appears dependent on not only the carbohydrate portion of these compounds but the ceramide as well (Kannagi et al., 1982Go). This is supported by several lines of evidence. Murine macrophage gangliosides inhibit T cell proliferation with far greater potency than do brain gangliosides and act at the level of the cell membrane, while the effect of neural gangliosides is primarily extracellular (Ryan et al., 1985Go; Berenson and Ryan, 1991Go). Furthermore, the immunologic effect of macrophage gangliosides is reversible, but brain ganglioside-mediated inhibition of T cell function is not (Berenson and Ryan, 1991Go). Murine macrophage gangliosides are also far more effective at down-regulating human monocytic CD4 expression than are brain gangliosides (Berenson et al., 1991Go).

Monoclonal antibodies raised specifically against human macrophage gangliosides activate human macrophages and functionally inhibit macrophage migration, much as antibody binding to GD3 initiates T cell activation (Norihisa et al., 1994Go; Ortaldo et al., 1996Go). Thus gangliosides of human macrophages and lymphocytes are likely receptors for activating ligands. In particular, minor endogenous human macrophage gangliosides bind nontypeable Haemophilus influenzae with high specificity, while gangliosides of brain origin do not (Fakih et al., 1997Go). Most notably, 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 disease (Berenson et al., 1998Go). This is accompanied by diminished responsiveness to ganglioside-stimulated activation, further emphasizing the clinical importance of macrophage gangliosides (Berenson et al., 1998Go). The conspicuous immunologic properties of macrophage gangliosides may be due to distinct molecular structures, and correct identification of such structures is critical to determining their function in immune cells. Unfortunately, the glycolipid structures of many peripheral cell systems are not well defined.

Despite increasing evidence for their importance in cellular interactions, conflicting reports exist on the ganglioside structures present in human monocytes/macrophages. Investigations have demonstrated effects on macrophage function by Vibrio cholerae toxin, a ligand known to bind the sialidase-resistant monosialoganglioside, GMla (Corcoran et al., 1994Go; Krakauer, 1996Go) and previous investigations have claimed the presence of GMla in human peripheral blood monocytes. Progressive expression of GM1a on human mononuclear phagocytes was reported with advancing HIV infection (Auci et al., 1992Go; Fantini et al., 1998Go).

In contrast, Berenson et al. (1998)Go were able to detect neither any ganglioside peaks of human macrophages that comigrated with GM1a standards, nor any significant differences in relative expression of any ganglioside peaks on chromatograms between HIV-seronegative and -seropositive donors. Furthermore, an investigation of the glycolipids of human peripheral blood immune cells by Kiguchi et al. (1990)Go also did not report the presence of GMla or asialoGM1 structures in human monocytes. Kiguchi et al. (1990)Go indicated all human monocytic gangliosides were sialidase-sensitive, terminally sialylated, monosialo structures. This further contrasts with a report indicating the presence of the disialoganglioside, GD3, in human monocytes (Fantini et al., 1998Go). Nonetheless, all investigations agree that sialyllactosyl ceramide, GM3, is the major ganglioside component present. The immunologic functions of human monocyte/macrophage gangliosides are very likely modified by the sialidases released by many infectious bacteria. Such sialidase sensitivity would be dependent not only on the accessibility of these molecules but also on specific carbohydrate structures. However, the conflicting published information on ganglioside structure made it imperative to first examine definitively the core ganglioside structures of human monocytes and macrophages. The work presented here confirms the carbohydrate structures previously suggested by Kiguchi et al. (1990)Go. Our investigation further expands these findings by demonstrating that ceramide structures of murine immune cell gangliosides (Muthing et al., 1987Go; Yohe et al., 1991Go, 1997), which differ from those of mammalian brain glycolipids, are also present in human monocyte/macrophage glycolipids.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Characterization of isolated monocyte fractions
Isolated monocyte preparations were examined for purity prior to adherence by using a series of defined antibodies, selected to detect not only monocytes but also the presence of other cells. The results are given in Table I. Results indicate the minimum monocyte purity before adherence was at least 80%, with most preparations contained 5–10% T and B cells. Staining with a pan-leukocyte marker was consistently very high, indicating that contamination from nonlymphoid cells was very low. Adherent cells examined microscopically for esterase staining were 97–99% nonspecific esterase positive.


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Table I. Antibody analyses of the isolated monocyte preparations
 
HPTLC of the monocyte gangliosides
Two-dimensional thin-layer analyses of the monocyte gangliosides displayed minor differences between the samples from the two different isolation procedures employed (Figure 1). In all samples, the ganglioside pattern consisted of four major doublets with both visual (Figure 1) and distribution analyses (Table II) showing the majority of the sialic acid density residing in the fastest moving doublet. A slight reduction in the doublet designated 3,4 in density gradient-isolated monocytes was the only difference noted for the two methods of isolation. When apheresed cells were allowed to differentiate in culture, patterns were similar to the ganglioside patterns of freshly isolated monocytes.



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Fig. 1. Two-dimensional thin-layer chromatograms of gangliosides from human monocytes isolated by two different procedures. Monocytes were isolated by leukapheresis/elutriation (a) and by gradient centrifugation of whole blood (b). Gangliosides were isolated and chromatographed as described in Materials and methods. Chromatographic origin is in the lower right with solvents for the first and second dimensions developed as indicated by the numbered arrows. Gangliosides are visualized by the reaction of their sialic acid moieties with resorcinol.

 

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Table II. Relative percentages of human monocyte/macrophage gangliosides on thin layer chromatograms isolated by two different methods
 
Enzymatic and lectin analyses of monocyte gangliosides
Clostridium perfringens sialidase treatment. Experiments were performed to determine if sialic acid residues of any human macrophage gangliosides are susceptible to C. perfringens sialidase. Control samples demonstrated that the sialidase-susceptible standard GM3 treated with C. perfringens sialidase was no longer resorcinol-positive, whereas sialidase-resistant standards GM1a and GM2 were unaffected. Equimolar volumes of sialidase-treated and untreated macrophage ganglioside samples were run on thin-layer chromatograms (TLCs) and sprayed with resorcinol. Greater than 98% of the human monocyte ganglioside sialic acid was removed by sialidase treatment, compared with untreated controls. The presence of desialylated substrates was confirmed by exposure of the chromatograms to iodine vapors prior to resorcinol spraying. Incubation with buffer alone did not cause any degradation. Results indicate the absence of any C. perfringens sialidase-resistant residues.

ß-galactosidase treatment.
Human macrophage ganglioside susceptibility to ß-galactosidase was measured to determine if any possessed external galactose residues. Equimolar volumes of ß-galactosidase-treated and untreated samples were prepared on TLC and sprayed with resorcinol. To confirm activity of the enzyme lot and conditions used, control samples were simultaneously treated. A shift in the chromatographic mobilities of ganglioside standards which possess an external galactose was observed; GD1b converted to GD2 and GM1a converted to GM2. No change in the chromatographic mobility of standards GD1a or GD3, which both lack an external galactose, was seen. No changes in either the relative chromatographic mobility or content were seen in any ganglioside from human monocyte/macrophage preparations in either the buffer or ß-galactosidase-treated samples. The results indicate the absence of terminal galactose residues.

Newcastle disease virus (NCDV) sialidase treatment.
Experiments were performed to independently verify the anomeric sialic acid linkage of human monocyte gangliosides by treatment with NCDV sialidase, which has relative specificity for removal of {alpha}2,3-linked sialic acids (Corfield et al., 1983Go; Berenson et al., 1995Go). Equimolar volumes of NCDV sialidase-treated and untreated samples were prepared, placed on TLCs, and sprayed with resorcinol. Sialic acid residues were lost from nearly all ganglioside peaks, except for the minor doublet of peaks designated 5 and 6 in Figure 2. The most abundant peak, peak 1 (GM3), which comprised approximately 50% of the total volume (Table II), also retained a faint amount of resorcinol positivity, likely due to its greater relative volume. The presence of resorcinol-negative glycolipid, in sialidase-treated samples, was confirmed in all TLCs by reversibly staining with iodine vapor prior to resorcinol spraying (Berenson et al., 1989Go). The results support previous data indicating a ganglioside sialylated by an {alpha}2,6 anomeric linkage (Kiguchi et al., 1990Go).



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Fig. 2. Schematic of human monocyte 2D thin-layer ganglioside chromatogram pattern. Pattern schematic corresponds to images in Figure 1 and to the sialic acid density distributions for cells from the two isolation methods given in Table II.

 

To confirm the sialidase activity, control samples were treated and demonstrated that externally {alpha}2,3 sialylated gangliosides, GM3 and GD3, treated with NCDV sialidase were no longer resorcinol-positive, and internally sialylated gangliosides, GM1a and GM2, remained resorcinol-positive.

Sambucus nigra lectin binding.
In a single experiment, an in situ thin-layer binding analysis was done using S. nigra lectin, which binds {alpha}(2-6)-linked sialic acid (Shibuya et al., 1987Go). S. nigra lectin linked to horseradish peroxidase was purchased from E Y Laboratories (Costa Mesa, CA). Thin-layer plates were prepared and analyzed as detailed previously for horseradish peroxidase–linked antibodies (Yohe et al., 1991Go). The doublet numbered 5,6 (Figure 2) displayed a weak but positive binding, also indicating this set contained an {alpha}(2-6)-linked sialic acid.

Radioisotope labeling and detection of macrophage glycolipids
Autoradiographic analyses of labeled monocyte gangliosides failed to reveal the presence of additional gangliosides following extended exposure of TLCs to film as described in Materials and methods. A sample autoradiogram following a 48-h exposure to Kodak BioMaxTM MS film is shown in Figure 3. Extension of the exposure to over 2 weeks revealed a few trace components migrating near the identified gangliosides, suggesting the components may be present as triplets rather than doublets (data not shown). One salient observation was that the components containing the {alpha}(2-6) linked sialic acid (designated 5, 6 in Figure 2) appeared to be synthesized at a different rate, as they were barely evident on the films exposed for less than 48 h (marked by central arrows in Figure 3).



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Fig. 3. Autoradiogram of human monocyte 2D thin-layer gangliosides following intracellular labeling. Autoradiograms were obtained as described in Materials and methods. Image shown was obtained from a film after a 48-h exposure to a chromatogram containing 400 DPM. Chromatographic origin is in the lower right with solvents for the first and second dimensions developed as indicated by the numbered arrows. Arrows with the chromatogram indicate the weakly labeled moieties corresponding with the gangliosides designated 5, 6 in the schematic.

 
Mass spectral analyses of monocyte gangliosides
Initial mass spectral analyses of the total methylated ganglioside fraction gave two intense peaks with parent ions of m/z 1372 and 1484; fragmentation analyses indicated the ganglioside GM3 with ceramides containing C16 and C24 fatty acids and a single sphingosine base of C18 (data not shown). Subsequent preparations of monocyte/macrophage gangliosides were prepared by altering the final elution of the ganglioside fraction from the Iatrobead columns (2 ml bed volume) by using 5 ml of chloroform:methanol (4:1, by vol.) followed by 15 ml of chloroform:methanol (1:2, by vol.). These solvent mixtures and volumes caused a large portion of the major peak, now identified as GM3, to elute in the first fraction, whereas the rest of the ganglioside fraction and a portion of the GM3 eluted in the second fraction. Methylation of this second ganglioside fraction followed by MS provided the ion pattern shown in Figure 4. Under the ionization conditions employed, the glycolipid acquires charge by adducting sodium cations. This results in a molecular ion peak at m/z = [M + n23]/n where M is the molecular mass of the permethylated glycolipid, n is the number of adducted sodium cations, and 23 is the mass of the sodium cation. Mass relationships suggest that the parent ion profile consists of three sets of ions containing two components each.



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Fig. 4. Parent ion mass spectrum derived from the isolated methylated monocyte ganglioside preparation. Conditions for preparation of the sample and the method for obtaining the mass spectrum are given in the text.

 
The six major ion peaks of the parent fraction were selectively transmitted into the collision cell followed by mass analyses of the collision products of mass spectral–collision-induced dissociation–mass spectral analysis (MS-CID-MS). Fragmentation analyses of the parent ion m/z 922.2 and 978.2 indicated that these ions were doubly charged components of monosialylated tetraosylceramides with the heterogeneity residing only in the alkane (ceramide) portion of the molecules. The resulting spectra are shown in Figure 5 with the derived structures in Figure 6. Fragmentation analyses gave a common carbohydrate structure with their ceramides having common C18-sphingosine bases, but differing fatty acids of C16 (m/z 922.2) and C24 (m/z 978.2). Carbohydrate fragmentation indicated terminal sialylation, consistent with its sialidase sensitivity. The assignment of the structures as sialoneolactosylceramides (sialoparaglobosides) was also based on the absence of any N-acetylgalactosamine when the monosaccharide composition of a single total monocyte ganglioside preparation was examined by gas-liquid chromatography as done previously for murine monocytes (Yohe et al., 1991Go; Griffin and Yohe, unpublished observations). Fragmentation analyses of the parent ions m/z 1147 and 1202 also indicated terminally sialylated structures, consistent with the sialidase analyses, but with a hexosyl carbohydrate backbone containing the same ceramide heterogeneity. An example of the fragmentation of the m/z 1147 ion is given in Figure 7. The last set of doublets of m/z 1372 and 1484 was again identified as singly charged ions from GM3 having the same ceramide pattern as seen for the other ganglioside components.



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Fig. 5. Mass spectra derived from fragmentation of parent ions m/z 922.2 (a) and m/z 978.2 (b). Fragmentation analyses with derived structures are given in Figure 5. Fragmentation also shows the parent ions are doubly charged ions. The spectra show a large number of common fragments for the two parent ions with heterogeneity only in the ceramide containing fractions.

 


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Fig. 6. Fragmentation analyses of ions m/z 922.2 (a) and m/z 978.2 (b). Fragmentation analyses indicate the only difference between the two structures resides in the ceramide moiety as indicated in the diagrams and specifically in the fatty acid entity.

 


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Fig. 7. Fragmentation mass spectrum (a) and analysis (b) of the parent ion m/z 1147.2. Fragmentation showed many mass units in common with m/z 922.2. Analysis shows the compound to be a terminally sialylated monosialostructure with an extended saccharide backbone and the same ceramide moiety as m/z 922.2.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Samples from human monocytes/macrophages isolated by two different methods show 2D thin-layer human monocyte/macrophage ganglioside patterns to consist of four doublets, with the fastest migrating doublet as the major component. Consistent with all prior investigations, this major component was determined to be the sialylated lactosylceramide molecule, GM3. Published discrepancies exist between investigators (Kiguchi et al., 1990Go; Auci et al., 1992Go; Fantini et al., 1998Go) as to the identity of the other ganglioside components of the human monocyte/macrophage. In a detailed report of the glycolipids of peripheral blood lymphocytes, Kiguchi et al. (1990)Go described the gangliosides of monocytes as terminally sialylated monosialo species consisting of GM3, sialoparagloboside, and a monosialohexosyl structure. In our investigations, combined enzymatic data show only sialidase susceptible species with no susceptible galactose residues present. The mass spectra show that all structures analyzed were terminally monosialylated. All data is consistent with the absence of GM1a as a component of the human monocyte/macrophage. Our structural data, including the mass spectral collision analyses of gangliosides, are in complete agreement with that described by Kiguchi et al. (1990)Go.

Previous investigations on murine B-cells indicated that the method of isolation had a profound impact on the glycolipid profile (Berenson et al., 2001Go). However, the method of isolation or culture of human macrophage/monocytes had little effect on their sialylated glycolipid patterns or distribution. Therefore, it seems unlikely that the discrepancies in the literature are due to cell isolation methods. Auci et al. (1992)Go described an increase in the surface expression of the GM1a component of human monocytes with AIDS. This report is also in direct contrast with that of Berenson et al. (1998)Go who found a decrease in surface accessibility but no decrease in ganglioside content in macrophages of adults with AIDS. An examination of the report reveals that Auci et al. (1992)Go used an anti-asialoGM1a antibody as the tool for detection. Although the cross-reactivity of the antibody used is not described, they may have detected an HIV-induced alteration in neutral glycolipid component(s) content or accessibility of the neutral sphingolipid constituents of the monocyte membrane. Berenson et al. (1998)Go did not examine the neutral glycolipid fraction for content or accessibility. Regardless, based on the antibody used, the data of Auci et al. (1992)Go cannot definitively show GM1a to be a component of the human monocyte/macrophage membrane. A detailed report by Fantini et al. (1998)Go also describes alterations in glycolipid metabolism in the human monocyte on HIV infection. These investigators described a slow moving component as the disialolactosylceramide structure, GD3. However, our mass spectra examination found no evidence for a disialo moiety. The data for the presence of GD3 as reported by Fantini et al. (1998)Go is mainly based on thin-layer mobility and the failure to detect a neutral tetraosyl structure following sialidase treatment of the ganglioside fraction. These investigators may have had the same problem that we observed in our early attempts to obtain mass spectra: the large percentage of GM3 overwhelmed the ability to obtain structural information on the other components. The analyses are further compounded by similar thin-layer chromatographic mobilities of some of the monocyte monosialo gangliosides and of disialo entities with a lactosyl backbone.

Kiguchi et al. (1990)Go indicated the presence of a ganglioside in human monocytes that was susceptible to Clostridium perfringens sialidase but not to sialidase derived from NCDV and concluded that this component contained an {alpha}(2-6) linked residue. The residue yielded a paragloboside neutral glycolipid and was thus identified as {alpha} (2-6) linked sialoparagloboside (Kiguchi et al., 1990Go). We also found a single ganglioside pair (5, 6 in Figure 2) that was resistant to NCDV sialidase. In addition, we noted the same ganglioside pair weakly bound an {alpha}(2-6) linked-sialic acid-binding lectin. The results based on all the data indicate the presence of {alpha}(2-6)-sialoparagloboside and explain the observation of four major ganglioside pairs on thin-layer chromatography (Figure 1), but only three pairs seen in the mass spectra parent ion profile (Figure 4).

Select ceramide moieties of macrophage gangliosides may also comprise important conserved immunoregulatory components. The diverse effect of specific ceramide structure on glycolipid activity is well established (Kannagi et al., 1982Go, 1983; Ladisch et al., 1994Go). The ceramide structures of human monocytes/macrophages seen in this work were originally observed in murine macrophages. Brain gangliosides have predominantly C18 as the ceramide fatty acid, with the major heterogeneity in the sphingosine as C18 and C20 entities (Ando and Yu, 1984Go). The ceramide component of gangliosides of macrophages, from either murine and now human sources, is structurally distinct from ceramide of gangliosides of brain origin in having a single C18-sphingosine entity with heterogeneity in the fatty acid portion as either C16 or C24 structures.

The structural distinctions of murine and human macrophage gangliosides may well explain many of the unique immunoregulatory properties that distinguish them from brain gangliosides. These properties include far more effective down-regulation of human macrophage CD4 expression and more potent, reversible down-regulation of lectin-induced T cell proliferation, compared with brain gangliosides (Berenson and Ryan, 1991Go; Berenson et al., 1991Go).

The minor gangliosides of human macrophages, designated as 3 and 4 in this investigation, bind nontypeable H. influenzae with high specificity and high affinity, while gangliosides of brain origin do not (Fakih et al., 1997Go). The selective affinity of gangliosides of human macrophages for activating ligands has only recently been explored and is likely to be dependent on specific core carbohydrate structures of ganglioside receptors. Binding of nontypeable H. influenzae to gangliosides is dependent on the presence of sialic acid, indicating the importance of the core carbohydrate structure to this interaction (Fakih et al., 1997Go). The findings of this current study now permit us to identify this putative receptor as {alpha}(2-3) sialoparagloboside.

Effects of the GM1a-binding ligand, cholera toxin, on human monocyte/macrophages have been described. Cholera toxin has been shown to induce production of cytokines in human monocytes via protein kinase c activity (Krakauer, 1996Go). This toxin has also been shown to alter other human monocyte activities via adenylyl cyclase (Corcoran et al., 1994Go). Our data as given here suggest the toxin could be binding to a membrane component other than GM1a. Pessina et al. (1989)Go demonstrated a profound effect of cholera toxin on the murine myelomonocytic cell line WEHI-3D, which we found also contained no GM1a (Yohe et al., 1992Go). In WEHI cells, an extended GM1b structure containing a galactose-N-acetylgalactosamine terminus was the probable target for the toxin. Cholera toxin has also been shown to bind fucosyl-GM1 (Masserini et al., 1992Go) and, to a reduced extent, the disialo ganglioside GD1b (Cumar et al., 1982Go). However, even with extended exposure of film to radiolabeled samples, no such alternate glycolipid target appeared in the human monocyte ganglioside fraction. Attempts to analyze fluorescein isothiocyanate (FITC)-tagged cholera toxin b subunit binding by fluorescence-assisted cell sorting (FACS) or by fluorescent microscopy indicated low nonspecific binding or were completely negative (Bergeron et al. unpublished data). Conceivably, a low level of surface protein with the requisite glycan structure could bind this toxin. An alternate possibility is a small, but still effective, level of contaminating endotoxins in the cholera toxin preparations as has been recently noted for lipoteichoic acid preparations (Gao et al., 2001Go). This possibility is now under investigation.

It has long been known that many infectious bacteria release sialidase (Corfield et al., 1981Go). Such exogenous bacterial enzymes may serve as important virulence factors by modifying accessible sialic acid residues of host cell sialoglycoconjugates. The data presented here, and that of Kiguchi et al. (1990)Go, indicate all major sialylated glycolipids in the human monocyte are sialidase-susceptible and, along with sialylated proteins, may be targets for exogenous bacterial sialidases. The enzymatic alteration of a sialoconjugate in a cell is dependent not only on its core carbohydrate structure and the specific sialidase, but also on its accessibility. In situ accessibility of these compounds and the effect of sialidases on monocyte function are now under investigation.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Isolation of human blood monocytes
Human blood monocytes were isolated by two different procedures.

In one series of experiments, peripheral mononuclear-enriched cell preparations were obtained by leukapheresis using a Cobe Spectra Apheresis cell separator (Lakewood, CO) and citrate-acid dextrose as anticoagulant. For each procedure, 3.1–5.3 L of whole blood was processed at a flow rate of 4.9 to 7.0 ml/min to collect a total of 68 ml of product. The leukapheresis preparation was counted and checked for sterility.

Monocytes were further purified from the leukapheresis preparation by countercurrent flow elutriation using a Beckman J2-MI centrifuge equipped with a JE-6B elutriation rotor, standard (4 ml) elutriation chamber (Beckman Instruments, Palo Alto, CA) and Masterflex L/S variable flow peristaltic pump (Cole-Palmer, Niles, IL). Elutriation was performed under sterile conditions using disposable, single-use tubing and collection bags (Baxter Fenwal, Deerfield, IL) designed for blood collection and processing. Before each procedure the elutriator chamber, chamber assembly, rotating seal and transfer tube were cleaned and rinsed with sterile distilled water for injection. The assembled rotor with attached inflow and outflow tubing was then sterilized by running 6% hydrogen peroxide through the system for 30 min and then rinsed with sterile elutriation buffer (physiological saline, 0.9%; D-glucose, 1 mg/ml; sodium heparin, 10 U/ml; human serum albumin, 0.5%; American Red Cross, Washington DC). During the elutriation procedure a centrifuge temperature of 22°C and a fixed rotor speed of 600 x g was used with a gradually increasing counterflow rate. Cells from the leukapheresis preparation were pumped into the chamber at 12 ml/min. Platelets, red blood cells, and most lymphocytes were eluted under these conditions, but the monocytes were retained in the chamber.

After loading was completed, elutriation buffer was pumped through at an initial flow rate of 12 ml/min. After a total of 300 ml had passed through, the counterflow rate was sequentially increased to 15, 18, and 25 ml/min and 150 ml of elutant was collected at each speed. Each fraction was centrifuged at 250 x g, resuspended in AIM V (Life Technologies, Rockville, MD), analyzed by Coulter Counter with channelyzer for white blood cell differential based on cell size, and immunophenotyped by flow cytometry using an anti-CD14 PE/anti-CD45 FITC cocktail (Becton Dickinson, San Jose, CA). The fractions containing greater than 85% monocytes (typically the fractions collected at 18 and 25 ml/min flow rates) were pooled for additional analysis and culture. Monocyte purity was verified by FACS analyses using a selected battery of fluorescent antibodies. Pooled monocyte fractions were allowed to adhere overnight to sterile glass 100 mm petri dishes in RPMI 1640 supplemented with 5% heat-inactivated fetal calf serum at 5% CO2, 95% humidity and 37°C. Non-adherent cells were then removed with serial rinses of warm phosphate-buffered saline and the cells extracted for glycolipids as detailed below. With two separate preparations, freshly elutriated monocytes were resuspended in hydrophobic culture bags at a density of 2.5–3 x 106 cells/ml in monocyte culture medium (Iscove’s Modified Dulbecco’s Medium [provided by IDM]) or AIM V, supplemented with 2.5% autologous serum; 2 mM fresh L-glutamine; 25 mM HEPES (BioWhittaker); 5 x 10–5 M 2-mercaptoethanol (Sigma, St. Louis, MO) containing 10 ng/ml GM-CSF, generously supplied by Immunex (Seattle, WA) and 1 x 10–8 M 1,25-dihydroxyvitamin D3 (Abbott Pharmaceuticals, Abbott Park, IL).

Cells, in a volume of 100–200 ml per bag, were cultured for 5–7 days at 37°C in a 5% CO2 humidified incubator. During the final 18 h of culture, the cells were further activated by the addition of 750 IU/ml of recombinant human interferon gamma (IFN{gamma}; Genetech, San Francisco, CA) and then centrifuged to obtain a cell pellet at 400 x g for 10 min. The pellet was then resuspended in a 0.32 M isotonic nonionic pentaerythritol solution, repelleted, and finally extracted to obtain lipids as described below.

In a second series of experiments, human mononuclear phagocytes were purified from buffy coat suspensions obtained from HIV-seronegative volunteers from the Red Cross of Western New York as previously described in detail by Berenson et al. (1998)Go. All donors were also seronegative for hepatitis B and C. Briefly, buffy coat suspensions were prepared from whole blood centrifuged at 3800 rpm for 4 min at 4°C. Mononuclear cells were further purified by Ficoll-Hypaque density centrifugation and seeded onto 100-mm glass petri dishes (5 x 106 cells/ml) in RPMI 1640 supplemented with 10% heat-inactivated human AB serum. After incubation at 5% CO2, 95% humidity, 37°C for 7 days, nonadherent cells were removed with serial rinses of warm phosphate-buffered saline. Remaining monocyte-derived macrophages were incubated in RPMI 1640 with 10% fetal calf serum until extracted for lipids as detailed below.

Isolation of glycolipids
The total human monocytic glycolipid fraction was isolated, separated by ion-exchange chromatography into neutral and ganglioside fractions and the fractions purified using reverse phase and spherical silica gel chromatographies as previously reported (Yohe et al., 1991Go; Macala and Yohe, 1995Go). Total lipid extracts are prepared by overnight extraction of the cell pellet using 10 ml chloroform:methanol (1:1, by vol)/108 cells. Cells adhered to glass petri dishes were extracted as previously detailed (Macala and Yohe, 1995Go). To remove membrane fragments, all resulting lipid extracts were filtered through sintered glass funnels (15 ml, medium porosity) overlaid with a glass fiber mat. The filtered residue was rinsed with 3 x 5 ml of chloroform:methanol (1:1, by vol). The combined extracts and rinses were taken to dryness by rotary evaporation. Using a slight modification of our earlier procedure (Yohe et al., 1991Go), the total lipid extract was redissolved, with sonication, in 20 ml of chloroform:methanol:water (30:60:8, by vol) and applied to a 4-ml bed volume column of DEAE-Sephadex A-25, acetate form (Pharmacia, Piscataway, NJ). The neutral lipid fraction was eluted with an additional 30 ml of the same solvent. The acidic lipid fraction, containing the gangliosides, was then eluted with 35 ml of chloroform:methanol:aqueous 0.8 M sodium acetate (30:60:8, by vol). The acidic lipid fraction was taken to dryness by rotary evaporation, redissolved in 5 ml of 0.1 N aqueous NaOH, and heated at 37°C for 90 min in a water bath to saponify any acidic phospholipids. The sample was chilled in an ice-water bath and the sample pH reduced to 5–6 by adding 0.1 N HCl. The sample was then diluted to 20 ml with chilled water and immediately desalted on a 2 ml bed volume reversed phase silica gel column (SepPak, Waters Associates, Waltham, MA). Each sample was applied to the column three times by passing the first and second eluates back through the same column. Following the third application of sample, flask rinses of 5 ml of 0.1 N NaCl and 2 x 5 ml of water were applied to the column. Remaining salts were eluted with 30 ml of water. Lipids were then eluted with 7 ml of methanol followed by 35 ml of chloroform:methanol (1:2 by vol). The desalting column was regenerated by rinsing with 7 ml of methanol followed by 40–50 ml of water.

The desalted lipid sample was taken to dryness by rotary evaporation, frozen, and then lyophilized to remove all traces of water. The lyophilized sample was dissolved, with sonication, in 1.5 ml of chloroform:methanol (1:1, by vol) followed by 3.5 ml of chloroform, making the final proportion of chloroform:methanol, 85:15. The sample and two 2.5 ml flask rinses were loaded sequentially onto a 2-ml Iatrobead 6RS-8060 column (Iatron Laboratories, Tokyo). Low polarity contaminants were eluted with an additional 10 ml of chloroform:methanol (85:15, by vol). The total ganglioside fraction was eluted with 20 ml of chloroform:methanol (1:2, by vol). After removal of the solvent by rotary evaporation, the purified ganglioside sample was transferred to a 12 x 75 conical screw cap tube with 4 x 1.5 ml rinses of chloroform:methanol (1:2, by vol), and dried under nitrogen. Samples were stored at –20°C until analyzed.

HPTLC of the ganglioside fractions
The macrophage ganglioside patterns were examined by 2D high-performance thin-layer chromatography (HPTLC) (Yohe et al., 1988Go, 1991). Briefly, the ganglioside sample (2–5 µg sialic acid) was spotted 15 mm in and up from the lower left corner. A human brain ganglioside standard (0.4 µg sialic) was spotted 5 mm in and 15 mm up from the lower right corner. The plate was chromatographed for 45 min in chloroform:methanol:0.25% aqueous KCl (50:45:10, by vol) and dried with forced air for 5 min and then over P2O5 for 90 min in a vacuum desiccator. The dried plate was rotated 90° counterclockwise, and a second brain ganglioside standard was spotted 5 mm in and 15 mm up from the new lower left corner of the plate. The plate was chromatographed in the second dimension in chloroform:methanol:2.5 N aqueous NH4OH containing 0.25% KCl (50:40:10, by vol) for 30 min. The plates were dried with forced air until the NH3 odor was gone. Ganglioside sialic acid was visualized by resorcinol-hydrochloric acid spray (Svennerholm, 1957Go) and the distribution determined by densitometry (Yohe et al., 1988Go; Berenson et al., 1998Go).

Enzymatic degradation of human macrophage gangliosides
C. perfringens sialidase treatment.
Human macrophage gangliosides containing 5–10 µg sialic acid were incubated with C. perfringens sialidase (2 U/ml) (Sigma) in 0.5 ml of 50 mM sodium citrate-phosphate buffer, pH 5.5, at 37°C, for 2 h, as previously described (Yohe et al., 1991Go). 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 columns and tested for hydrolytic products on TLCs. TLCs were run in chloroform:methanol:0.25% KCl (50:45:10, by vol), sprayed with resorcinol, and heated (92–94°C). Resorcinol-positive intensity was quantitated by scanning densitometry and desialylation was determined by loss of resorcinol-positivity, compared with untreated samples. The percent desialylation was expressed as: [volume of (resorcinol-positive) sialidase-treated sample ÷ volume of (resorcinol-positive) untreated sample] x 100. To verify efficacy of enzymatic activity, positive and negative control gangliosides were treated with sialidase, including gangliosides with external sialic residues (GM3) and gangliosides lacking external sialic acid residues (GM1a, GM2), respectively (Matreya, Pleasant Gap, PA).

NCDV sialidase treatment.
To further investigate the anomeric sialic acid linkage of minor monocyte gangliosides, human monocyte gangliosides were incubated with sialidase from NCDV (Glyko, Novato, CA), which is specific for removal of {alpha}2,3-linked sialic acid (Corfield et al., 1983Go). Human monocyte gangliosides containing 5–10 µg sialic acid were incubated with 0.2 U of NCDV sialidase in 100 µl of 50 mM sodium acetate buffer containing 2 µg/µl sodium cholate (Sigma), pH 5.5, at 37°C, for 18 h as previously described (Berenson et al., 1995Go). A duplicate sample of equal quantity of gangliosides was incubated in buffer alone. Reactions were terminated by placement of samples on ice in 5 ml of 0.1 M NaCl. After adjustment of pH to 5.0, samples were desalted on SepPak columns, and re-eluted over Iatrobead columns, as described earlier. The entire content of each sample was loaded onto a TLC plate, which was run in two dimensions (chloroform:methanol:0.25% KCl, 50:45:10, by vol, and chloroform:methanol:2.5N NH3 containing 0.25% KCl, 50:40:10, by vol), also as described earlier. TLC plates were sprayed with resorcinol and heated (92–94°C). Resorcinol-positive intensity was quantitated by scanning densitometry, and desialylation was determined by loss of resorcinol-positivity, compared with untreated samples. The presence of resorcinol-negative spots on NCDV sialidase–treated samples was confirmed on TLCs by reversible staining with iodine vapor prior to resorcinol spraying (Berenson et al., 1989Go).

Conditions for effective desialylation of {alpha}2,3-linked gangliosides were established, using known ganglioside (GM1a, GM2, GM3, GD3) standards (Matreya). Under the established conditions, gangliosides with external {alpha}2,3-linked sialic acids (GM3, GD3) were successfully desialylated, while those with internal {alpha}2,3-linked sialic acids (GM1a, GM2) were not.

ß-galactosidase treatment.
Human macrophage gangliosides containing 5–10 µg sialic acid were incubated with bovine testes ß-galactosidase (0.3 U/ml) (Sigma) at 37°C in 100 µl of 50 mM citrate phosphate buffer (pH 4.3), containing Triton X-100 (0.5 µg/ml), for 18 h, as previously described (Yohe et al., 1991Go). A duplicate sample of an equal quantity of gangliosides was incubated in buffer alone. Reactions were terminated with 0.1 M NaOH, and neutralized to pH 4–5 with 0.1 M HCl. As with sialidase treatment, solutions were desalted on SepPak columns and tested for hydrolysis on TLCs, run in chloroform:methanol:0.25% KCl (50:45:10, by vol) and sprayed with resorcinol. Enzymatic degradation was determined by a shift in chromatographic mobility of bands compared with untreated samples and with controls of known structures. To verify efficacy of enzymatic activity, positive and negative control gangliosides were treated with ß-galactosidase and included gangliosides with external galactose residues (GM1a, GD1b) and gangliosides lacking external galactose residues (GD3, GD1a), respectively (Matreya).

Radioisotope labeling and detection of macrophage glycolipids
Monocyte/macrophages were isolated by apheresis/elutriation and allowed to adhere as described above. The isolated cells were exposed to 14C-galactose (ARC, St. Louis, MO) (5µCi/20 x 106 cells) for 48–96 h and then extracted for glycolipids as detailed above. The isolated ganglioside fractions containing 400–1000 DPM were separated by 2D thin-layer chromatography as above and the resulting chromatograms exposed to Kodak BioMaxTM MS film enhanced with a BioMax TranScreenTM LE intensifying screen (Eastman Kodak, Rochester, NY). Films were exposed from 24 h to 4 weeks at –70°C and developed in a Kodak X-omatTM processor using the standard conditions. Extensive testing with 14C-labeled brain gangliosides indicated discrete spots containing as little as 30 DPM could be detected with a 24 h exposure and less than 5 DPM at 4 weeks (Degregorio et al., unpublished data).

Mass spectrometric analyses of macrophage gangliosides
The total ganglioside fraction was permethylated and subjected to electrospray ionization mass spectrometry performed on a triple quadrapole mass spectrometer as described previously for murine glycolipids (Reinhold et al., 1994Go; Yohe et al., 1997Go).


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The authors wish to acknowledge the assistance of Mary E. Griffin, Tamar J. Kitzmiller, and Robin H. Rasp in different phases of this investigation. This work was supported by Veterans Affairs Merit Reviews (H.C.Y. and C.S.B.) and supported in part by grants from The National Institute for Heath, AI-34478 (P.K.W.) and GM05445 (S.Y., B.B.R., and V.N.R.)


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate; HPTLC, high-performance thin-layer chromatography; MS-CID-MS, mass spectral–collision-induced dissociation–mass spectral analysis; NCDV, Newcastle disease virus; TLC, thin-layer chromatograms.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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