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*
Department of Immunology, University of Strathclyde, Glasgow, United Kingdom;
Institute for Animal Health, Pirbright, Surrey, United Kingdom; and
Department of Immunology, University of Glasgow, Glasgow, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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140
million people are infected with these three parasites, and a further
1000 million are at risk (1). Infection with filarial nematodes is usually lifelong, and individual adult worms generally live for more than 5 yr (2). A consensus of the results of studies to date is that such longevity may, at least in part, reflect the ability of the parasite to induce defects in the host immune system. These defects can occur in both T cell (3) and B cell (4) functions, and may extend from parasite-specific to more generalized responses (reviewed in Refs. 5 and 6). The molecular mechanisms underlying such defects remain to be elucidated, but of potential relevance is the finding that sera obtained from humans or animals infected with these parasites are often associated with a similar immunosuppressive property (7, 8). Such serum samples contain molecules released by the worms, excretory-secretory products (ES)4 (9, 10), leading to the proposal that these ES may play a role in down-regulating lymphocyte responses. Certainly, we (11) and others (12) have demonstrated that these molecules can inhibit lymphocyte proliferation in vitro. In addition, a recent study on humans infected with W. bancrofti has demonstrated an inverse relationship between levels of ES and levels of total Ab in the bloodstream (13).
Our own particular study (11) focused on the effect of ES-62, a
phosphorylcholine (PC)-containing glycoprotein that is a major ES of
the rodent filarial parasite Acanthocheilonema viteae (14),
on the proliferation of resting splenic murine B cells following
cross-linking of the Ag receptors (sIg). Having established that
inhibition of sIg-driven B cell proliferation was taking place (as a
consequence of the attached PC group), we investigated the effect of
the parasite product on some of the early signaling events underlying
sIg-mediated B cell activation (11, 15). Although the events leading to
B cell proliferation have not been fully elucidated, it is clear that
ligation of the B cell Ag receptor complex (BCR), which comprises a
clonatypic Ag-binding component, sIg, and its accessory immunoreceptor
tyrosine-based activation motif-containing transducing molecules,
Ig-
and Ig-ß, induces protein tyrosine kinase (PTK) activity (16, 17). Such tyrosine phosphorylation induces recruitment of Syk kinase
and the reorientation, enhanced binding, and activation of
immunoreceptor tyrosine-based activation motif-associated
src-family PTKs, Blk, Fyn, Lck, and Lyn, leading to BCR
association of a number of key signal transducers, implicated in
cellular activation and proliferation, such as phospholipase C
(PLC)-
, phosphoinositide-3-kinase (PI-3-K), and the components of
the Ras/MAP (mitogen-activating protein) kinase signaling cascades (16, 17). We have shown previously that ES-62-mediated inhibition of B cell
activation is not targeted against the early sIg-coupled
PLC--mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate
(PtdInsP2) but, rather, appears to down-regulate cellular
levels of protein kinase C (PKC) expression and activity (11, 15).
However, the unresponsiveness of B cells following treatment with ES-62
suggested that the parasite molecule may also modulate the activation
status of key sIg-coupled proliferative pathways such as the PI-3-K and
Ras/MAP kinase signaling cascades. We now report that ES-62 does indeed
modulate sIg coupling to Ras MAP kinase and PI-3-K, and we propose that
these findings provide a molecular mechanism to explain how ES-62 could
modulate B cell responsiveness during filarial infections.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Anti-phosphotyrosine (4G10) and agarose-conjugated anti-phosphotyrosine (4G10) were obtained from Upstate Biotechnology (Lake Placid, NY). Anti-Lyn (for immunoprecipitates) Abs were from Transduction Products (Lexington, KY), and anti-Blk, anti-Syk, and anti-Fyn Abs for immunoprecipitation were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). In addition, anti-mouse Ig HRP, anti-rabbit Ig HRP, and enhanced chemoluminescence reagents were obtained from Amersham International (Bucks, U.K.). The other Abs used were generated at the Institute of Animal Health (Pirbright, U.K.): Syk (rabbit anti-peptide 317–333, ESDRGPWANEREAQREA), Lyn (rabbit anti-C-terminal decapeptide, ATEGQYQQQP), Blk (rabbit anti-N-terminal (27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47) peptide, KAPPPLPPLVVFNHLAPPSPN), Fyn (rabbit anti-peptide 22–35, LNQSSGYRYGTDPT), Yes (rabbit anti-peptide 15–35, KYRPENTPEPVSTSVSHYGAE), and Erk2 (rabbit anti-peptide EETARFQPGYRS). In addition, a mAb to Erk2 (mouse anti-peptide EETARFQPGYRS) was generated in association with Dr. A. M. Campbell, Department of Biochemistry, University of Glasgow (Glasgow, U.K.). Protein A- and protein G-Sepharose were obtained from Sigma Chemical Co. (Poole, U.K.).
Preparation of cell lysates
ES-62 from A. viteae was prepared as described previously (11). Small dense resting B cells were prepared from the spleens of BALB/c mice by depleting T cells with anti-Thy-1 and complement, followed by Percoll density fractionation (11). B cells (5 x 106) were preincubated with either ES-62 (2 µg/ml in supplemented RPMI 1640 medium) or medium alone for 4 h, and then incubated with anti-Ig (50 µg/ml) or medium alone for the indicated time at 37°C. Following cell incubations, reactions were terminated by addition of lysis buffer (50 mM Tris (pH 7.4), 150 mM sodium chloride, 2% (v/v) Nonidet P-40, 0.25% (w/v) sodium deoxycholate, 1 mM EGTA, 10 mM sodium orthovanadate, 0.5 mM PMSF, chymostatin (10 µg/ml), leupeptin (10 µg/ml), antipain (10 µg/ml), and pepstatin A (10 µg/ml)), and the samples were incubated on ice for 20 min before microcentrifugation at 20,000 x g for 30 min at 4°C. The supernatants were transferred to fresh tubes and stored at -20°C until required. Fresh cell lysates used for immunoprecipitations were precleared with protein A- or protein G-Sepharose, as appropriate.
Ras assay
Freshly prepared lysates from B cells, prelabeled with inorganic phosphate ([32P]Pi; (0.5 mCi/ml; Amersham) in phosphate-free DMEM for 90 min, were treated with charcoal precoated with BSA. After mixing, charcoal was removed by centrifugation. Ras activity was immunoprecipitated by mAb Y13-259 preconjugated to protein A-Sepharose via anti-rat IgG antiserum (Serotech, Oxford, U.K.). Immune complexes were washed twice in lysis buffer and then twice in washing buffer (50 mM Tris-HCl, pH 7.5, 20 mM MgCl2, and 150 mM NaCl) before being resuspended in 20 µl of 20 mM Tris-HCl, pH 7.5, containing 20 mM EDTA, 2% (w/v) SDS, 0.5 mM GDP, and 0.5 mM GTP. Samples were heated at 65°C for 5 min and centrifuged to elute Ras-bound nucleotides. Supernatants were spotted onto polyethyleneimine-cellulose TLC plates and developed with 0.75 M KH2PO4, pH 3.4. Resolved GDP/GTP spots were scraped and counted by liquid scintillation counting (18).
In vitro PI-3-K assay
PI-3-K activity was immunoprecipitated from freshly prepared
cell lysates using 5 µl of anti-PI-3-K (UBI 05-195; Upstate
Biotechnology), followed by protein A-Sepharose. Immune complexes were
washed three times with PBS containing 1% Nonidet P-40; three times
with 100 mM Tris, pH 7.4, and 5 mM lithium chloride; and twice with TNE
(10 mM Tris, pH 7.4, 150 mM sodium chloride, 5 mM EDTA, and 1 mM
orthovanadate). Kinase buffer (50 µl TNE, 10 µl
PtdInsP2 (10 µg), and 10 µl 100 mM manganese
chloride) and 5 µl of ATP (0.88 mM ATP, 20 mM manganese chloride, and
30 µCi [-32P]ATP) were added to each sample, and
reactions were allowed to continue for 10 min with constant agitation.
The reactions were stopped with 20 µl of 6 M HCl; 160 µl of
chloroform:methanol (1:1) was added to extract the lipid; and the
phases were then separated by centifugation. Lipids were resolved by
TLC on silica-60 plates treated with oxalate (19), and
PtdInsP3 spots were scraped and counted by liquid
scintillation.
Erk2 kinase assay
MAP kinase activity was immunoprecipitated from freshly prepared
cell lysates using the Erk2-specific mAb (1 µg), followed by protein
G-Sepharose (20 µl). Immune complexes were washed once in PBS,
followed by two additional washes in 0.5 M lithium chloride and 20 mM
Tris, pH 8, before being resuspended in MAP kinase buffer (40 mM HEPES,
pH 8, 2 mM DTT, 0.1 mM EGTA, 5 mM magnesium acetate, and 1 mM sodium
orthovanadate). Myelin basic protein (10 µg) and 15 µCi of
[-32P]ATP were added, and immune complexes were
incubated at room temperature for 30 min. Reactions were stopped by the
addition of 100 µl, 75 mM orthophosphoric acid. Samples were spotted
onto phosphocellulose paper (Whatman, Kent, U.K.), left to dry, then
extensively washed in 75 mM orthophosphoric acid. The papers were dried
and counted by liquid scintillation.
Measurement of tyrosine phosphorylation
B cell lysates were resolved by SDS-PAGE, and tyrosine phosphorylation was assessed by Western blotting using an anti-phosphotyrosine (4G10)/HRP anti-Ig enhanced chemoluminescence system. Even loading of lanes was checked by Ponceau Red staining.
In vitro PTK assays
The individual PTKs were immunoprecipitated from freshly
prepared cell lysates using the appropriate Abs (1 µg), followed by
protein G-Sepharose (20 µl), and washed once in PBS, pH 7.4, followed
by a further wash in 0.5 M lithium chloride and 20 mM Tris, pH 8.
Immune complexes were then washed once in kinase buffer (10 mM
manganese chloride, 50 mM Tris, pH 7.4, and 1 mM sodium orthovanadate)
and then finally resuspended in a further 40 µl of the kinase buffer.
Enolase (10 µg) was added along with 15 µCi of
[-32P]ATP, and the samples were incubated at room
temperature for 15 min. The reaction was stopped by the addition of 100
µl, 75 mM orthophosphoric acid. Samples were spotted onto
phosphocellulose paper, left to dry, and then extensively washed in 75
mM orthophosphoric acid. The papers were dried and counted by liquid
scintillation.
PLC assay: choline and choline phosphate generation
PLC was measured as described previously (20). B cells were labeled for 4 h with 2 µCi/ml [3H]choline chloride in DMEM containing 2% serum; for the final hour of the labeling period, the radiolabeled medium was replaced with fresh unlabeled DMEM to reduce the levels of free intracellular choline. The cells were then washed three times and resuspended (3 x 106 cells/200 µl) in DMEM containing 10 mM glucose, 20 mM HEPES, pH 7.4, and 1% (w/v) BSA. Following appropriate stimulation, reactions were terminated by addition of 1 ml ice-cold methanol and 310 µl CHCl3, and the samples were extracted overnight at 4°C. Phase separation was achieved by addition of CHCl3 (390 µl) and H20 (480 µl), followed by vortexing and centrifugation at 1200 x g for 5 min. Aliquots (0.8 ml) of the upper aqueous phase were made up to 5 ml with H2O and then loaded onto 1 ml Dowex-50 H+ column to resolve the water-soluble [3H]choline-labeled metabolites. Following calibration of the columns with standards, the glycerophosphocholine fraction is eluted by 8 ml H2O, the choline phosphate fraction is eluted within a further 15 ml of H20, and the choline fraction is then eluted with 7 ml of 1 M KCl. Aliquots of these fractions are counted by liquid scintillation counting.
Phosphatidylcholine-specific phospholipase D (PLD) assay
PLD activity was measured by the definitive transphosphatidylation assay, as described previously (21). Briefly, cells prelabeled for 4 h with [3H]palmitate were then washed and resuspended (3 x 107/ml) in RPMI 1640 containing 20 mM HEPES, 0.1% BSA, and 0.3% butan-1-ol, and equilibrated at 37°C for 15 min. After the indicated time, incubations were terminated by the addition of 750 µl of chloroform:methanol (1:2, by vol), and the samples were left to extract on ice for approximately 10 min. Phase separation was achieved by the addition of 250 µl chloroform and 300 µl water, followed by vortexing and centrifugation at 270 x g for 5 min. Aliquots of the lower chloroform phase were dried in vacuo, redissolved in 25 µl of chloroform:methanol (19:1, by vol), and applied to Whatman LK5DF TLC plates. Plates were developed in the organic phase of a solvent comprising 2,2,4-trimethylpentane:ethyl acetate:acetic acid:water (5:11:2:10, by vol), and [3H]PtdBut, identified by its comigration with a cold PtdBut standard, was scraped off the plates and its radioactivity was determined by liquid scintillation counting.
Mass measurement of diacylglycerol (DAG) or ceramide:DAG kinase assay
DAG and ceramide levels were determined essentially as described
previously (22). Resting B cells were stimulated with the appropriate
ligand, and the reactions were quenched by two-phase separation, as
outlined above. Aliquots of the lower organic phase were dried in
vacuo, and the lipids were solubilized in a Triton
X-100/phosphatidylserine mixture. Briefly, phosphatidylserine (30 µl;
supplied as 25 mM stock from Lipid Products, Surrey, U.K.) was dried
under nitrogen and then probe sonicated in 2.5 ml of 10 mM Imidazole
buffer, pH 6.6, containing 0.6% (w/v) Triton X-100, until the solution
was optically clear. Aliquots (50 µl) were added to the lipid
samples, which were then sonicated in a bath for 30 min. Once
sonicated, 20 µl of 250 mM Imidazole buffer, pH 6.6, containing 250
mM NaCl, 62.5 mM MgCl2, 5 mM EGTA, and 10 µl of
freshly prepared 100 mM DTT, was added to the solubilized lipid.
Escherichia coli DAG kinase (Calbiochem, Nottingham, U.K.)
was added to a final concentration of 5 mU, and the reaction was
started by addition of 10 µl of 5 mM ATP containing 1 µCi of
[-32P]ATP made up in 100 mM Imidazole, pH 6.6; this
results in a final ATP concentration of 0.5 mM in a final reaction
volume of 100 µl. The tubes are incubated at 30°C for 30 min. The
reaction is stopped by addition of 1 ml of chloroform:methanol:HCl
(150:300:2). After 10 min, 300 µl of chloroform and 400 µl of
H2O were added. The tubes are vortexed and centrifuged at
270 x g for 5 min to promote phase splitting, and
washed once with 1 ml of a synthetic upper phase. The samples were then
dried in vacuo, solubilized in 40 µl of chloroform:methanol (19:1),
and 20 µl spotted onto silica TLC plate (Merck, Darmstadt, Germany;
5714, 5 x 20 cm 60F254). The plates were developed in
chloroform:methanol:acetic acid (38:9:4.5) to within 5 mm of the top of
the plate. Radiolabeled bands were located by autoradiography, and the
phosphatidic acid or ceramide bands (relative to standards) were
scraped and counted by liquid scintillation counting.
Presentation of results
All assays were performed in triplicate, and the results were presented as means ± SD. The data presented were representative of at least three independent experiments. In the PTK densitometry experiments, n = 3 refers to the number of experiments using independently prepared B cell lysates.
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Results |
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Treatment of B cells with ES-62 (2 µg/ml) in vitro did not
induce activation of either Ras or PI-3-K over a 4-h period (Fig. 1
and results not shown). However, this
exposure of B cells to ES-62 was found to modulate the Ras (Fig. 1A) and PI-3-K (Fig. 1, B and
C) activity normally associated with stimulation of
sIg. Moreover, PC was found to have similar effects to ES-62 on the
sIg-mediated activation of Ras and PI-3-K (Fig. 1C and
results not shown), results consistent with our previous study, which
showed that PC could mimic ES-62 in the induction of modulation of PKC
levels (11).
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B), albeit much less dramatically than at 30 min
(Fig. 1, B and C), an enhancement of the
early (5-min) sIg-Ras signal was observed (Fig. 1A).
These results showing that ES-62 differentially modulates sIg coupling
to the PI-3-K and Ras pathways suggest that PI-3-K is not a downstream
effector of sIg-mediated Ras activation in B cells, despite reports
that the p110 catalytic subunit of PI-3-K may be activated downstream
of Ras in other systems (23). ES-62 disrupts coupling of sIg to the MAP kinase cascade
sIg-mediated Ras activation couples the BCR to the MAP kinase
cascade via activation of Raf and MEK (16, 17, 24, 25). Since
phosphorylation and activation of MAP kinase is an important event in B
cell proliferation downstream of Ras activation, as demonstrated by the
use of dominant-negative mutants of Ras and Raf-1 (26), any
interference with MAP kinase activation could explain the inhibitory
effect of ES-62 on sIg-mediated proliferation. While pretreatment with
ES-62 was found to have little effect on the early peak (up to 5 min)
of sIg-coupled Erk2 activation, the sustained phase (5–30 min) was
completely abrogated (Fig. 2). Moreover,
pretreatment with ES-62 alone induced substantial Erk2 activity. Hence,
these results suggest that ES-62-mediated desensitization of subsequent
sIg-coupled MAP kinase activation may be due to abortive activation of
this signaling pathway by the parasite product.
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In an attempt to explain how ES-62 modulates the activity of such
key signaling molecules, we investigated its effect on the activation
of PTKs associated with the BCR (Fig. 3)
since tyrosine phosphorylation and/or association of signal transducers
with tyrosine-phosphorylated kinases or adaptor molecules initiate BCR
signaling. Activation of B cells via sIg induced considerable increases
in tyrosine phosphorylation of a variety of proteins that is sustained
for 20 to 30 min (Fig. 3). However, quite different kinetics of
phosphorylation was observed following stimulation of B cells via ES-62
or PC followed by sIg, indicating that the combination induces either
1) a desensitization of PTK activation or 2) a more rapid
dephosphorylation of target proteins (Fig. 3 and results not shown). In
vitro kinase studies of anti-phosphotyrosine immunoprecipitates
showed that preincubation of B cells with ES-62 or PC stimulated basal
PTK activity and blocked sIg-mediated PTK activation over 30 min by
some 70% (results not shown), suggesting that pretreatment with ES-62
induces a state of anergy in B cells that only allows an abortive
activation of sIg-coupled PTK-mediated events.
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A). While exposure to
ES-62 had no effect on the basal, or sIg-mediated, tyrosine
phosphorylation and hence, activation of Fyn and Yes, the parasite
product was found to stimulate tyrosine phosphorylation of Syk
(289 ± 107% of basal levels, n = 3), Lyn
(535 ± 279%, n = 3), and to a much lesser
extent, Blk (172 ± 63%, n = 3). Tyrosine
phosphorylation of Syk, Lyn, and Blk was also stimulated by culture
with PC (results not shown). Moreover, ES-62 substantially blocked
anti-Ig-mediated phosphorylation of Blk (53 ± 6% of control
anti-Ig response, n = 3), Syk (36 ± 3%,
n = 3), and Lyn (43 ± 15%, n =
3). More importantly, the subsequent desensitization to signaling via
sIg was absolute for Syk and Lyn, but not Blk, as anti-Ig failed to
tyrosine phosphorylate Syk (288 ± 122% of basal levels,
n = 3) and Lyn (416 ± 16%, n =
3), but not Blk (503 ± 116%, n = 3), to a
significantly higher level than that observed following culture with
ES-62 alone (Fig. 4A, legend).
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B). Moreover, there is some evidence, although
caution should be used due to the low levels of PTK activities
detected, that ES-62 exerts some subtle and complex effects on sIg
coupling to PTK activation. For example, while pretreatment with ES-62
appears to enhance coupling to Lyn (5 min), Syk (10 min), Blk (5 min),
and Fyn (10 min) at some time points during the earlier stages of
anti-Ig stimulation, it seems to marginally reduce sIg coupling to
Lyn, Syk, Blk, but not Fyn at the 30-min time point (Fig. 4B). However, as ES-62 does not substantially inhibit
sIg responses at any point over this 30-min time period, these results
suggest overall that Syk, Lyn, and Blk are unlikely to be major targets
in the ES-62-mediated desensitization of sIg-mediated tyrosine
phosphorylation and coupling to the PI-3-K and the Ras MAP kinase
cascades. ES-62 does not stimulate Erk2 or desensitize B cells by inducing phosphatidylcholine-PLC (PtdCho-PLC), PtdCho-PLD, or sphingomyelinase (SMase) activation
It was initially rather surprising that ES-62 stimulated Erk2 in the absence of any Ras activation. There is, however, increasing evidence for Ras-independent pathways of MAP kinase activation (27, 28) mediated, for example, via PtdCho-PLC (27, 28, 29)-, PKC (30, 31)-, or ceramide-dependent protein kinase routes (32). We decided to investigate whether ES-62 could use one or more of these lipid-derived second messenger signals to stimulate Erk2 activity and desensitize subsequent sIg coupling to the Ras MAP kinase cascade.
We therefore investigated whether ES-62 stimulates PtdCho-PLC to
generate DAG and choline phosphate (phosphorylcholine) in B cells; in
addition, we also determined whether ES-62 stimulates PtdCho-PLD as the
resultant product, phosphatidic acid can be further metabolized to DAG
and/or modulate PKC activity (33). However, we found that ES-62 does
not stimulate either PtdCho-PLC or -PLD activity, over a 4-h period, as
judged by measurement of release of [3H]choline
phosphate or [3H]choline from
[3H]choline-labeled B cells (Fig. 5, A and B).
Moreover, ES-62 and PC do not stimulate PtdCho-PLD activity over the
same period, as determined by the definitive
transphosphatidylation-PtdBut accumulation assay using
[3H]palmitate-labeled B cells (Fig. 5C and
results not shown). Furthermore, the lack of stimulation of these
pathways is further supported by experiments showing that mass levels
of DAG generation are not altered following culture of B cells with
ES-62 over this 4-h period (Fig. 5D). Finally, ES-62
did not alter the mass levels of ceramide generation in B cells over a
similar 4-h period, and hence did not activate Erk2 by
ceramide-dependent kinase-mediated phosphorylation of Raf
(32).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) and coupling to key sIg-coupled
proliferative pathways such as PI-3-K (Fig. 1) and the Ras MAP kinase
(Figs. 1 and 2) cascade. We therefore propose that pre-exposure to
molecules such as ES-62 provides a mechanism to explain possible
defects in B cell responsiveness (4) during filarial infections. Thus,
while ES-62 pretreatment has little inhibitory effect on, and may
indeed enhance, the initial coupling of sIg to Ras (Fig. 1A) and MAP kinase activation, it profoundly inhibits
the later stages of sIg coupling to sustained activation of Ras (Fig. 1A), PI-3-K (Fig. 1, B and
C), and MAP kinase activation (Fig. 2). One model
that is consistent with the antiproliferative effects of ES-62 is that
pre-exposure to the parasite molecule may induce an abortive,
nonproductive activation of certain signaling elements, such as MAP
kinase, thereby desensitizing B cells to subsequent sustained
proliferative signaling via the BCR.
An intriguing feature of these results is that stimulation of B cells
with ES-62 alone does not induce activation of Ras or PI-3-K despite
the fact that ES-62, like anti-Ig, can induce activation of Lyn,
Syk, and Blk (Fig. 4). These data therefore suggest either that
activation of these key BCR-associated PTKs and their tyrosine
phosphorylation and/or recruitment of downstream-transducing molecules
are not sufficient for Ras or PI-3-K activation, or alternatively, that
ES-62 recruits additional, as yet undefined, signals that act to
negatively modulate Ras and PI-3-K activity. Moreover, our finding that
ES-62 does not substantially inhibit sIg coupling to these PTKs is
consistent with the above hypotheses and suggests that ES-62 does not
mediate its uncoupling of the BCR from the PI-3-K or Ras MAP kinase
cascades by targeting activation of Syk, Lyn, or Blk. Of course the
finding that ES-62 alone can selectively stimulate Blk, Syk, and Lyn
and MAP kinase activation, while it does not appear to modulate either
Ras or PI-3-K activity, also suggests that MAP kinase can be activated
in B cells via alternative PTK-dependent pathways. This proposal is
consistent with the increasing evidence for Ras-independent pathways of
MAP kinase activation involving lipid second messengers and PKC
(27).
We have not found any evidence to support the proposal that ES-62 (Fig. 5) or sIg stimulates MAP kinase activity via PtdCho-PLC, -PLD, or
SMase-dependent pathways, but PKC--mediated activation of MAP kinase
via Raf and MEKK pathways (30, 31) could provide a rationale for our
earlier finding that while ES-62 initially up-regulates PKC-
expression, prolonged pretreatment with ES-62 acts to reduce this PKC
activity (11, 15). Indeed, it is tempting to speculate that ES-62 may
exert its suppressive effects on B cell proliferation by inhibiting sIg
activation of this key signaling cascade and downstream regulators of
gene transcription via Ras, PTK-, and PKC-mediated pathways. Recent
findings that are consistent with this hypothesis of convergent
signaling are that the product of PI-3-K, PtdInsP3,
reportedly activates PKC
and the novel PKCs, 
, 
, and 
in
vitro (34, 35). Since PKC and are expressed in B cells (15, 36),
ES-62-mediated abrogation of sIg-coupled PI-3-K activity could
contribute to the suppression of PKC activity, which results from
exposure to the parasite product (11, 15). Moreover, given that
activation of PKC leads to phosphorylation and inactivation of
I
B, and hence activation of the transcription factor, nuclear
factor-B (37, 38), and that PI-3-K has also been implicated in
activation of S6 kinases (39, 40), ES-62-mediated disruption of PTK,
PKC-, and Ras-mediated activation of MAP kinase and such downstream
pathways would be likely to have profound inhibitory effects on B cell
activation.
How does ES-62 selectively, but nonproductively, stimulate the tyrosine phosphorylation and hence, activation of certain key signal transducers involved in B cell activation with the result that subsequent activation via sIg is apparently desensitized? The parasite product, like many filarial ES, contains covalently bound PC (41). PC is a structure recognized as possessing immunomodulatory properties (42), and indeed PC-containing molecules of B. malayi have previously been shown to inhibit the response of human T cells to mitogens (43). In addition, and as stated earlier, our previous studies have shown that the effect of ES-62 on anti-Ig-mediated proliferation and PKC levels can be largely mimicked by PC (11). Moreover, experiments designed to investigate whether PC could also mimic the effects of ES-62 on tyrosine phosphorylation, and Ras, PI-3-K, and PtdCho-PLD activation in this study established that its behavior was virtually identical to that observed with ES-62.
One recent finding that could help explain these observations is that
PC can act as a second messenger; thus, generation of PC was shown to
be an essential event in platelet-derived growth factor-induced DNA
synthesis in fibroblasts (44). Generation of PC was dependent upon the
sequential action of PLD and choline kinase (44, 45), and if
platelet-derived growth factor-induced activation of choline kinase is
blocked, an abrogation of cell proliferation associated with a failure
to activate Raf-1 and MAP kinase is observed (45). This is a finding
consistent with our reported Ras-independent effects of ES-62 on the
MAP kinase cascade in B cells. Thus, since we have shown that ES-62
does not itself induce the generation of cellular PC (Fig. 5), it is
conceivable that the PC component of ES-62 could result in a partial
activation of B cells that renders them desensitized to subsequent
activation via the Ag receptor. Such a course of events would, however,
require the internalization of ES-62, but it is possible that this
could occur via the receptor for platelet-activating factor (PAF),
which is expressed on B cells (46). Certainly, Streptococcus
pneumoniae, which express PC on their surface, have been shown to
enter human cells by this mechanism (47). Alternatively, PC-ES could
simply desensitize the cells to subsequent challenge by Ag by
abortively signaling via such PAF receptors (48, 49). This latter
proposal may be particularly pertinent, as not only does signaling via
the PAF receptor augment certain B cell activation responses, such as
Ig secretion, but also, there is no heterologous desensitization of
IP3-sensitive calcium mobilization between the BCR and PAF
receptors (46), a situation reminiscent of the PLC-independent
suppression of B cell activation by ES-62. Moreover, stimulation with
high concentrations of ES-62 (>20 µg/ml) is weakly mitogenic for B
cells, in vitro (11), and if comparable localized levels of PC-ES exist
in humans, this finding may go some way to explaining why filariasis,
rather than being associated exclusively with B cell
hyporesponsiveness, actually in some cases appears to induce polyclonal
Ab production (6).
The important challenge now will be to establish whether elucidation of the exact molecular mechanism underlying induction of the anergic state by PC-ES can lead to the development of therapeutic intervention, which will induce affected B lymphocytes to recover reactivity. To this end, although we are primarily concerned with elucidating this mechanism, its selective action indicates that this parasite product, as shown in this study, can also be used as a tool for dissecting pivotal B cell proliferative signals, per se.
In summary, we have shown that a filarial nematode PC-ES renders B cells anergic to stimulation via the BCR by selectively uncoupling sIg from key proliferative pathways such as the PI-3-K and Ras MAP kinase cascades. Although anti-parasite Ab responses can generally be detected in filarial infections, a number of studies suggest that in some patients at least, these may be impaired (4, 13, 50). We propose that the results we have obtained in this study provide a molecular mechanism that could contribute to impaired B cell responses during filarial infection. Moreover, our approach, by elucidating the nature of B cell defects at the molecular level, may ultimately lead to the design of strategies to overcome such immune dysfunction.
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Footnotes |
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2 Current address: Division of Infection and Immunity, IBLS, University of Glasgow, Glasgow G12 8QQ.
3 Address correspondence and reprint requests to Dr. William Harnett, Department of Immunology, University of Strathclyde, The Todd Centre, 31 Taylor Street, Glasgow G4 0NR, United Kingdom.
4 Abbreviations used in this paper: ES, excretory-secretory; BCR, B cell antigen receptor complex; DAG, diacylglycerol; HRP, horseradish peroxidase; MAP, mitogen-activating protein; PAF, platelet-activating factor; PC, phosphorylcholine; PI-3-K, phosphoinositide-3-kinase; PKC, protein kinase C; PLC, phospholipase C; PLD, phospholipase D; PtdBut, phosphatidylbutanol; PtdCho, phosphatidylcholine; PtdInsP, phosphatidylinositol phosphate; PTK, protein tyrosine kinase; sIg, surface immunoglobulin; SMase, sphingomyelinase.
Received for publication February 11, 1997. Accepted for publication November 24, 1997.
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. EMBO J. 14:6157.[Medline]
isozyme of protein kinase C by phosphatidylinositol 3,4,5-trisphosphate. J. Biol. Chem. 268:13. subspecies blocks NF-B activation. Mol. Cell. Biol. 13:4770.PKC induces phosphorylation and inactivation of IB-. EMBO J. 13:2842.[Medline]
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