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* Infectious Diseases Section, Department of Veterans Affairs Western New York Healthcare System and State University of New York at Buffalo, Buffalo, New York;
Genetics Institute, Cambridge, Massachusetts;
Department of Veterans Affairs, White River Junction, Vermont; and
Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire
Correspondence: Charles S. Berenson, Infectious Diseases Laboratory-151, Buffalo V.A. Medical Center, Buffalo, NY 14215. E-mail: berenson{at}acsu.buffalo.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: glycosphingolipids • surface accessibility • immune cells • thin-layer chromatography
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Alternative methods for B-lymphocyte isolation exist. Severson et al. utilized the ability of surface immunoglobulin (Ig)-positive (sIg+) cells to adhere to nonspecific proteins rather than to anti-Igs ("plastic panning") [13 ]. This offers a simpler and less expensive method that yields B-lymphocyte sIg phenotypes and purity comparable with those of B lymphocytes isolated by antibody panning.
Recent reports indicate that the surface accessibility of cellular gangliosides may be altered by phorbol ester-induced protein kinase C activation [14 , 15 ] and that B-lymphocyte ganglioside expression may be mediated by lipopolysaccharide (LPS). Thus, exposure to exogenous molecules, including anti-Igs and non-B-cell proteins, might directly affect ganglioside expression and surface accessibility. Both are used as selective modalities in antibody panning and in plastic panning for B-lymphocyte purification.
A third approach, purification by depletion of non-B-cell components ("subtraction"), offers the advantage of avoiding direct stimulation of cells by the method of selection because incubation with anti-Ig or with nonspecific proteins is not required. Therefore, we purified B lymphocytes of C3H/HeN and C3H/HeJ mice by plastic panning and by the subtraction method and compared their ganglioside patterns and ganglioside surface accessibilities with those of B lymphocytes purified by antibody panning. We further examined the effect of anti-Ig exposure on the ganglioside character of subtraction-purified B lymphocytes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Reagents
Culture media were obtained from Whittaker MA Bioproducts,
Walkersville, MD. All solvents were of high-performance liquid
chromatography grade (Baker Chemical Co., Phillipsburg, NJ).
B-lymphocyte purification
Mouse spleens were teased apart and filtered through sterile
nylon gauze. Cells were centrifuged at 200 g and 10°C for
10 min. Erythrocytes were lysed in 3–5 mL of a solution containing
0.15 M NH4Cl, 0.01 M KHCO3, and 0.1 mM
ethylenediaminetetraacetate for 60 s. Cells were rewashed and
suspended in medium for further culture [9
]. In separate
experiments, B lymphocytes were isolated from the population of total
splenocytes by one of the following methods.
Antibody-panning method
Some B lymphocytes were purified by the method of Wysocki and
Sato [10
]. Splenic cells [5 x 106/mL
in RPMI 1640 medium containing 20% fetal bovine serum (FBS)] were
incubated for 2 h in 100-mm-diameter plastic culture dishes at
37°C in an atmosphere containing 5% CO2 to permit
adherence of macrophages. Splenic cells depleted of macrophages were
then incubated at a density of 5 x 106/mL in
phosphate-buffered saline (PBS) containing 5% FBS for 70 min at 4°C
in polystyrene petri dishes coated with purified rabbit anti-mouse IgG.
Nonadherent cells were removed and discarded. Ig-positive cells were
recovered by gentle scraping. This method, as described, was used in
earlier studies of B-lymphocyte gangliosides [5
,
9
].
Plastic-panning method
Other B lymphocytes were purified by the method of Severson et
al. [13
]. Cells (12x 106–15 x
106/mL) were incubated in PBS containing 3% bovine serum
albumin in plastic tissue culture flasks (Corning, Corning, NY) at room
temperature. After 30 min, nonadherent cells were resuspended by gently
swirling the flasks. After a total incubation time of 1 h,
nonadherent cells were removed by pipetting and were discarded.
Adherent cells were removed by flushing with RPMI 1640 containing 0.3%
bovine serum albumin.
Subtraction method
Still other B lymphocytes were purified by affinity
chromatography to eliminate non-B-cell splenic components
[16
] (Cytovax Biotechnologies, Edmonton, Alberta,
Canada). Cells (0.75 x108–1.0 x108)
were suspended in 1.5 mL of Hanks’ balanced salt solution containing
2% FBS and incubated in sterile vials with monoclonal antibodies (rat
anti-mouse CD4, rat anti-mouse CD8a, and rat anti-mouse CD11b/CD18) at
0°C for 30 min. The suspension was centrifuged (200 g, 10
min), and the supernatant was discarded. Cells were resuspended in
medium and washed one additional time to remove unbound antibody. Cells
(5 x107/mL) were loaded onto columns of activated
agarose beads. After two 2-mL rinses of each tube with medium, the B
cells were eluted with an additional 15–25 mL of Hanks’ balanced salt
solution–2% FBS. Purified B cells were collected in the column
eluates and combined.
In selected experiments, subtraction-purified B lymphocytes were incubated on polystyrene plates coated with rabbit anti-mouse (anti-mu) IgG (70 min at 4°C) and recovered as described earlier. For all experiments, cell viability was assessed by trypan blue exclusion. Cells were stained with fluorescein isothiocyanate-conjugated µ-chain-specific goat anti-mouse IgG (ICN Pharmaceuticals, Aurora, OH) and assessed for B-lymphocyte purity by direct immunofluorescence microscopy.
Purification of gangliosides
Total B-lymphocyte lipids were extracted as previously detailed
[17
]. B-lymphocyte pellets (3 x 108
cells) were solubilized in 20 mL of chloroform-methanol [1:1 (v/v)]
in glass tubes. Insoluble debris was removed by filtration through a
sintered glass funnel overlaid with a glass fiber mat. Gangliosides
were isolated from the total lipid extract by anion-exchange
chromatography followed by base treatment and desalting. Finally,
purified gangliosides were eluted by column chromatography (Iatrobeads
6RS-8060; Iatron Laboratories, Tokyo, Japan) and evaluated for purity
as previously detailed [18
]. Gangliosides were
quantitated by the method of Svennerholm [19
].
Thin-layer chromatography
B-lymphocyte gangliosides were separated by two-dimensional
thin-layer chromatography (TLC) on high-performance TLC plates (Silica
Gel 60; E. Merck, Darmstadt, Germany), as previously described
[15
, 16
]. The first solvent was
chloroform–methanol-0.25% aqueous KCl [50:45:10 (v/v/v)]. After
being dried in vacuo over P2O5 for 90 min, the
plate was rotated 90° counterclockwise and developed in a second
solvent [chloroform- methanol- and 2.5 M NH4 in 0.25%
aqueous KCl (50:40:10 v/v/v)]. Gangliosides on chromatograms were
visualized by spraying with resorcinol reagent. Optimal chromatograms
were obtained using 4–6 µg of ganglioside sialic acid per TLC plate.
Radioactive labeling of surface gangliosides
As previously described [9
], purified B
lymphocytes of both murine strains were labeled by an adaptation of the
procedure of Gamberg et al. [20
]. A suspension of viable
cells (5 x107/mL) was incubated (3 h at 37°C) with
galactose oxidase (type V; 5 IU/mL; Sigma Chemical Co., St. Louis, MO).
After being washed with PBS, cells (108/mL) were gently
agitated (30 min at room temperature) in sodium
[3H]borohydride (1 mCi/mL; specific activity, 15–20
Ci/mmol; Research Products International, Mount Prospect, IL, and
American Radiolabel Chemicals, St. Louis, MO). After several additional
rinses, total 3H incorporation of an aliquot was measured.
Cells were pelleted for extraction of gangliosides.
Autoradiography
For autoradiographic analysis, lipids were extracted from
0.4–1.0 x 108 B lymphocytes. The total lipid extract
contained 35–60 x 104 cpm/108 cells. The
purified 3H-labeled ganglioside fraction contained
0.5–5.0 x 104 cpm/108 cells for B
lymphocytes purified by any method. TLC plates of
3H-labeled gangliosides were sprayed with
En3Hance spray (New England Nuclear Corp., Westwood, MA)
and were visualized via autoradiography by exposure to hypersensitized
XAR-5 film (Eastman Kodak Co., Rochester, NY) for 2–3 weeks at
-70°C. X-ray film was hypersensitized with 7% hydrogen–93%
nitrogen at 48°;C for 16 h [21
].
Densitometric analysis
Densitometric analyses of gangliosides on thin-layer
chromatograms and autoradiograms were done with a two-dimensional
analytical scanning system (Molecular Dynamics, Sunnyvale, CA) as
previously described [9
]. Because of slight differences
in quantity of total ganglioside placed on each chromatogram, data from
TLC pattern analyses and autoradiograms were compared by determining
relative percentages of individual homologous spots. Because of the
number of mice required for each experiment (
50 mice of each strain,
100 per experiment), it was not logistically feasible to determine
significance for minor spots. The minimum quantity of lipid-bound
sialic acid needed for B-lymphocyte ganglioside analysis was 4 µg.
Therefore, significant differences in gangliosides containing <6% of
the total sialic acid content (i.e., <0.2 µg of sialic acid per
chromatogram) were not assigned.
Statistics
All statistical comparisons, including those of homologous
peaks, were performed using Student’s t-test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Evaluation of sIg+ cells by direct immunofluorescence microscopy indicated that B-lymphocyte purity was 83–93% by the plastic-panning method and 82–94% by the subtraction method, both values being comparable with the 94–98% purity achieved for antibody-panned B lymphocytes (P >0.2). Cell viability was consistently 92–96%, as determined by trypan blue exclusion, for all three methods.
Ganglioside sialic acid content of murine B lymphocytes
The lipid-bound sialic acid contents of B lymphocytes of the two
strains did not differ significantly. Plastic-panned C3H/HeN
B-lymphocyte gangliosides contained 1.9 ± 0.8 µg of sialic
acid/108 cells, whereas C3H/HeJ B lymphocyte gangliosides
contained 1.7 ± 0.6 µg of sialic acid/108 cells.
Subtraction-purified C3H/HeN B lymphocytes had 1.5 ± 0.2 µg of
sialic acid/108 cells, but C3H/HeJ B lymphocytes contained
1.8 ± 0.3 µg of sialic acid/108 cells. Results are
means ± SE derived from three separate experiments.
Expression of murine B-lymphocyte gangliosides by TLC is method
dependent
Two-dimensional TLC was used to determine the distribution of the
total ganglioside content of B lymphocytes. We have previously
demonstrated almost identical patterns for antibody-panned B-cell
gangliosides of both strains [9
]. Figures 1
and 2
show a comparison of ganglioside patterns of C3H/HeN
and HeJ murine B lymphocytes isolated by plastic panning and
subtraction, respectively. As with antibody-panned B cells, HeN and HeJ
B cells isolated by plastic panning (Fig. 1)
and by subtraction (Fig. 2)
show near identity between strains.
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In all instances, the differences noted were in relative quantities of individual peaks; no entirely new or absent gangliosides were noted. Results given are means ± SE of data from three separate experiments. Similarly, gangliosides of C3H/HeJ B lymphocytes obtained by antibody panning differed from those obtained by plastic panning, indicating that method-related differences in ganglioside distribution were not strain specific.
Surface accessibility of gangliosides of C3H/HeN versus C3H/HeJ B
lymphocytes is method dependent
Surface labeling of gangliosides was performed to determine
whether the striking differences in membrane architecture between
C3H/HeN and HeJ B lymphocytes isolated by antibody panning
[10
] are also present in B lymphocytes isolated by
plastic panning and by subtraction-purification method. Autoradiograms
of surface-labeled gangliosides of plastic-panned or
subtraction-purified B lymphocytes of C3H/HeN and C3H/HeJ mice are
shown in Figures 3
and 4
, respectively.
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As with antibody panning, 12 identical 3H-labeled spots were seen in plastic-panned B lymphocytes of both strains, and densitometric analysis confirmed that no differences in relative intensities existed (Table 2 ). However, in distinct contrast to antibody-panned B lymphocytes, the patterns of surface-accessible gangliosides from plastic-panned C3H/HeN and C3H/HeJ B lymphocytes were identical. Results were reproducible in three separate experiments (Fig. 3) .
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our present investigation expands on these findings and demonstrates the following:
(1) Dramatic changes in relative intensity were seen in expression of five ganglioside peaks of C3H/HeN and HeJ B lymphocytes, contingent on the method of B-cell purification (Fig. 1 and 2) . In all instances, no interstrain differences in total ganglioside content existed between HeN and HeJ B lymphocytes.
(2) The previously reported differences in ganglioside surface accessibility between HeN and HeJ B cells were limited to B cells isolated by antibody panning [5 , 9 ]. No interstrain differences in ganglioside surface accessibility were found in B cells isolated by plastic panning or by subtraction.
The contrast in overall ganglioside contents of B lymphocytes obtained by different purification protocols was not due to discrepant cell purity or viability, since both were comparable in all B-lymphocyte preparations. The presence of small numbers of contaminating T lymphocytes in any of the preparations also cannot adequately explain this difference, since murine T lymphocytes have a far smaller ganglioside content (0.65 µg of sialic acid/108 cells) than do B lymphocytes and major murine T lymphocyte gangliosides are structurally distinct from gangliosides of B lymphocytes [27 , 28 ].
Our findings, as well as those of others, raised concern that direct stimulation of B cells might alter ganglioside content or surface accessibility. For example, the surface accessibility of neuronal gangliosides is altered on the basis of differential susceptibility to sialidase in response to protein kinase C [14 , 15 ]. A similar scenario might exist for ganglioside surface accessibility of B lymphocytes purified by antibody panning or plastic panning, in which the methodology used might potentially provoke an alteration in relative ganglioside surface expression in B lymphocytes from either strain. Most of the previously cited B-cell ganglioside studies utilized B cells purified by direct selection rather than by depletion of non-B-cell components. Therefore, it was critical that we also study B lymphocytes purified by subtraction of non-B-cell components. Adherence of subtraction-purified B cells to plastic and subsequent exposure to anti-Ig failed to induce detectable ganglioside changes. Thus, our findings do not implicate direct stimulation of antibody-panned HeN and HeJ B cells as the cause of altered ganglioside surface accessibility in either strain. Rather, the relative differences in ganglioside expression of B lymphocytes isolated by the different methods favors the hypothesis that the different protocols isolate B-lymphocyte populations exhibiting differences in phenotypic expression of surface glycolipids.
The potential for lability of expression of immune cell gangliosides, including gangliosides of B cells, has been the focus of several investigations. Although early studies suggested that exposure to LPS might not induce changes in B-cell ganglioside expression [5 ], Portner et al. found distinct structural changes in gangliosides of murine B lymphocytes that resulted in greater heterogeneity in response to bacterial endotoxin and that differed in intensity with different endotoxin preparations [29 ]. Most notably, endotoxin-induced ganglioside changes featured a relative shift in predominant sialic acid from N-glycolylneuraminic acid to N-acetylneuraminic acid and increased relative expression of the disialoganglioside fraction. The increased structural heterogeneity of stimulated murine B lymphocytes parallels ganglioside changes seen in murine peritoneal macrophages in response to LPS [26 ]. Shifts between major glycosphingolipid series were also observed with B-cell differentiation due to activation of glycosyltransferases [30 ]. Human B-lymphocyte disialogangliosides also contain the CDw60 epitope [31 ], previously identified as a specific marker of functional subpopulations of both CD4+ and CD8+ T-lymphocyte subsets [32 , 33 ]. Moreover, splenic B-lymphocyte monosialogangliosides provide key carbohydrate epitopes for preferentially reactive serum autoantibodies in animal models of lupus, indicating their potential significance as immune targets [34 ].
As noted above, the surface architectural differences between HeN and
HeJ B cells appear to be limited to B-cell subpopulations isolated by
antibody panning. Endotoxin-hyporesponsive C3H/HeJ mice have served as
models for studying the effects of bacterial endotoxin. Although recent
studies have identified a structural defect in TLR4 as a likely cause
of susceptibility to LPS in HeJ mice [6
,
7
], earlier studies also discovered subtle alterations of
plasma membranes that were associated with the C3H/HeJ defect
[35
]. These findings were supported by restoration of
endotoxin responsiveness to C3H/HeJ B lymphocytes by fusion with
liposomes containing membrane fragments of endotoxin-responsive B
lymphocytes [36
]. Partial proteolysis of C3H/HeJ B
lymphocytes with trypsin also restored sensitivity to endotoxin
[12
]. While these data initially suggested that the
mechanism for endotoxin responsiveness might be intact but obscured
because of an alteration in the C3H/HeJ B-lymphocyte membrane
architecture, subsequent investigations indicated that trypsin itself
might serve as a B-lymphocyte mitogen, affecting a select B-lymphocyte
population [37
]. These observations support the idea of
heterogeneity among B lymphocytes and further indicate that different
protocols for isolation of B lymphocytes may not be comparable. The
data presented here indicate that this is the case. Ligand induction of
TLR-mediated activation triggers downstream kinases and transcriptional
factors that result in activation of nuclear factor 
B
[8
]. Although the precise means by which TLR4 signaling
affects B-cell ganglioside synthesis remains speculative, it is likely
that the mutation of TLR4 in HeJ mice alters numerous downstream events
dependent on cell activation, including shifts in ganglioside patterns
and surface accessibility. We have further reported altered ganglioside
expression and surface accessibility in activation of HeN and HeJ
macrophages [26
, 38
].
In summary, our studies indicate distinct protocol-dependent differences in glycolipid phenotypes of murine B lymphocytes and specifically reveal altered surface accessibility of gangliosides of B lymphocytes purified by antibody panning. Because endogenous gangliosides have critical immunomodulatory roles, including those in B lymphocytes explored herein, we speculate that the structural distinctions of B lymphocytes isolated by different methods are likely accompanied by differences in function. It is conceivable that differential ganglioside expression of each subpopulation is indicative of rearrangement of glycolipid-enriched membrane microdomains governing intracellular signaling. The latter has been demonstrated in a subfraction of murine B lymphocytes that express phosphatidylserine, which colocalizes with GM1 in lipid rafts in response to anti-IgM [39 ]. Cross-linking of class II major histocompatibility complex molecules in THP-1 cells is also associated with aggregation with glycolipid-enriched domains, which trigger intracellular kinase activity [40 ]. The distinctions in intracellular signaling may indicate that endotoxin sensitivity of B lymphocytes resides in given subpopulations that are isolated by specific protocols. This topic is the focus of ongoing studies.
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ACKNOWLEDGEMENTS |
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Received October 23, 2000; revised January 4, 2001; accepted January 18, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This article has been cited by other articles:
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  C. S. Berenson, M. A. Gallery, J. M. Smigiera, and R. H. Rasp The role of ceramide of human macrophage gangliosides in activation of human macrophages J. Leukoc. Biol., September 1, 2002; 72(3): 492 - 502. [Abstract] [Full Text] [PDF]
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