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and IL-4 by B Cells: Positive and Negative Effects on CD40 Ligand-Induced Proliferation, Survival, and Division-Linked Isotype Switching to IgG1, IgE, and IgG2a1
*
Centenary Institute of Cancer Medicine and Cell Biology, Newtown, Australia;
Boehringer Ingelheim Pharmaceuticals Inc., Ridgefield, CT; and
Medical Foundation, University of Sydney, Sydney, Australia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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each have potent effects on B cell responses as
well as strong mutual antagonism. Here we have examined the
quantitative effects of these cytokines on CD40 ligand-induced B cell
proliferation, cell survival, and division-linked isotype switching.
Both IL-4 (strongly) and IFN- (weakly) enhanced the number of B
cells found in culture by reducing the average time cells take to enter
the first division cycle and by promoting B cell survival. When added
in combination, the net effect of IL-4 and IFN- on time to division
and survival was a response intermediate between that of the two
cytokines alone, indicating a partial antagonism of IL-4 by IFN-. By
modulating both time to division and cell survival, these small effects
of IFN- are amplified and give rise to large reductions in cell
number in the presence of IL-4. At higher concentrations, IFN- had
minor inhibitory effects on IL-4-induced isotype switching to IgG1 and
greater effects on IgE. A reciprocal relation was observed between the
ability to inhibit IgE at late cell divisions vs induction of IgG2a. In
contrast, IL-4 did not prevent switching to IgG2a induced by IFN-
alone. Therefore, antagonism between IFN- and IL-4 is observed at
multiple levels and over different concentration ranges, resulting in
complex net outcomes. The evolutionary significance of this complexity
is discussed. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,
has been shown to increase IgG2a levels in vivo (6) and to
promote IgG2a switching by B cells stimulated in vitro (7, 8). Thus, IFN- and IL-4 regulate selection of Ig isotypes
(9), which are responsible for different effector
functions. It has long been recognized that the Th1 cytokine IFN-
antagonizes IL-4 responses in a variety of systems (reviewed in Ref.
10), including IL-4-induced B cell switching to both IgG1
and IgE isotypes (9, 11, 12, 13, 14). Recently, isotype switching has been found to correspond to division numbers (15, 16). Thus, in cultures of B cells stimulated with CD40L and IL-4 (16, 17, 18), IgG1 expression commences after division three whereas IgE appears in culture only from division five onwards, revealing an additional variable in IL-4-induced B cell stimulation. Moreover, IL-4 has been reported to exert additional effects on regulation of the B cell response, whereby B cell survival and rate of proliferation are also enhanced (16, 19, 20).
To clarify the mechanism of antagonism between IFN- and IL-4, the
outcome of culturing CD40L-stimulated B cells with various combinations
of the two cytokines was examined by monitoring cell death,
proliferation, and isotype switching using methods based on fluorescent
cell division analysis and concurrent intracellular Ig measurement. Our
results demonstrate that the strong antagonistic effect of IFN- on
IL-4-induced B cell responses depends on a series of weak, but
consecutive, inhibitory events that influence cell proliferation and
survival as well as differentiation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Inbred CBA/H mice (8–12 wk old) of both sexes were used for all experiments. Mice were purchased from the Animal Resource Center (Perth, Australia) and maintained under specific pathogen-free conditions in the animal facilities of the Centenary Institute.
Reagents and Abs
Cell membranes expressing murine ligand for CD40 (CD40L) were
prepared as previously described (17) from the Sf9 insect
cell line infected by baculovirus vector containing a CD40L gene
construction. Recombinant mouse IL-4 was kindly provided by R.
Kastelein (DNAX Research Institute, Palo Alto, CA), and recombinant
mouse IFN- (sp. act. 5 U/ng) was purchased from Genzyme
(Cambridge, MA).
LOMG-1 biotin (anti-IgG1) and LOMG-2a biotin (anti-IgG2a) conjugates were purchased from Serotec (Oxford, U.K.). Unlabeled and biotinylated goat anti-mouse IgM, IgG1, and IgG2a Abs were purchased from Southern Biotechnology (Birmingham, AL). The IgE-specific mAbs 6HD4 and R1E4 (21) were purified by protein G (Pharmacia, Uppsala, Sweden) from cell culture supernatant. R1E4 and goat anti-mouse IgG1 Abs were conjugated to PE by using succinimidyl 4-(p-maleimidophenyl)butyrate (Pierce, Rockford, IL) according to the manufacturer’s instructions. R1E4 mAb was biotinylated using NHS-biotin (Sigma, St. Louis, MO). The anti-5-bromo-2-deoxyuridine (BrdU)-specific mAb (BR-3 biotin) and streptavidin-tricolor (SA-TC) were purchased from Caltag Laboratories (Burlingame, CA). SA-TC was used as the second step to detect biotinylated mAbs.
Cell culture
Small resting B cells were prepared as described previously (22) and cultured in B cell medium (BCM), which contained RPMI 1640 medium (Life Technologies, Grand Island, NY) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 10 mM HEPES (pH 7.4), 100 µg/ml streptomycin, 100 U/ml penicillin, 5 x 10-5 M 2-ME (all supplement ingredients were purchased from Sigma) and 10% heat-inactivated FCS (CSL, Victoria, Australia). Typically, 105–5 x 104 purified B cells were cultured in 1 ml or 2 x 104 cells in 0.2 ml cell culture medium at 37°C in a humidified atmosphere of 5% CO2.
CFSE labeling
Resting B cells were labeled with 5-(and -6)carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes, Eugene, OR) according to the original method described elsewhere (23). Briefly, CFSE was dissolved in DMSO at a concentration of 5 mM as a stock solution and kept at -20°C until further use. Before labeling, B cells were washed and resuspended at 107 cells/ml in PBS containing 0.1% BSA (PBS/BSA). The CFSE stock was diluted 1/10 in PBS/BSA, and 20 µl was added to 1 ml cell suspension (10 µM final concentration). Cells were incubated for 10 min at 37°C and then were quenched with cold PBS/BSA solution. After twice washing with PBS/BSA, the labeled cells were resuspended into BCM.
Intracellular staining and flow cytometry
The stimulated B cells were harvested at various times and
washed twice with PBS/BSA/0.1% NaN3 solution.
Cells (
5 x 105-106
per sample) were fixed with 0.25 ml 2% paraformaldehyde (BDH, Poole,
U.K.) for 10 min at room temperature. The fixed cells were
permeabilized overnight at room temperature by adding equal volume of
PBS and 0.5 ml 0.2% Tween 20 to yield a final concentration of 0.5%
paraformaldehyde with 0.1% Tween 20. After permeabilization, the cells
were washed twice with PBS/BSA/0.1% NaN3 and
then stained with primary mAbs for 45 min, washed twice, and incubated
with SA-TC for 30 min. The staining Abs and SA-TC were diluted in
PBS/BSA/0.1% NaN3 solution containing 1% normal
rat serum. All incubations were conducted on ice and after each step
cells were washed twice with cold PBS/BSA/0.1%
NaN3 containing 0.05% Tween 20. Analysis was
conducted on a FACScan flow cytometer (Becton Dickinson, San Jose, CA)
using CellQuest software (Becton Dickinson). CFSE peaks were
individually gated, and the proportion of Ig-positive cells within each
division round was calculated as described previously
(16).
Cell proliferation assay
B cell cultures were pulsed on day four with 50 µg/ml BrdU (Sigma) for 3 h before being harvested, fixed, and permeabilized as described above. DNA of fixed and permeabilized cells was digested by incubating for 30 min at 37°C in the presence of 10 µg/ml DNase I (Boehringer Mannheim, Mannheim, Germany), dissolved in Tris buffer (pH 7.4) with 1 mM CaCl2 and 1 mM MgCl2. Treated cells were stained with anti-BrdU-biotin mAb conjugate followed by SA-TC. Control cells were treated in the same way, but without a BrdU pulse, and used as a background control for flow cytometric analysis.
B cell proliferation assay by [3H]TdR incorporation was performed as described previously (24). B cell cultures were pulsed for 4 h with 1 µCi/ml [methyl-3H]TdR, sp. act. 50 Ci/mmol; ICN, Irvine, CA), and the scintillation counting was performed on a Betaplate counter (Pharmacia-LKB, Uppsala, Sweden). Results were expressed as a mean cpm and SE of triplicate cultures.
Cell viability assay
B cell viability was assessed by propidium iodide (PI; Sigma) exclusion test. Triplicates of B cell cultures were pulsed with 1 µg/ml PI and analyzed on flow cytometry. PI-positive cell population (dead cells) was gated on forward scatter vs FL2 channel, and results were expressed as percentage of PI-positive cells (20). In some experiments, results were expressed as total live cell numbers in reference to a known number of CaliBRITE (Becton Dickinson) beads.
Determination of cell number per division
Peak fitting software (Pro fit: QuantumSoft, Zurich, Switzerland) was used to fit a series of gaussian curves to live cell-gated CFSE intensity histograms. The fitting algorithm calculated the mean position of each division peak from the intensity of undivided cells (D0) and the autofluorescence level of non-CFSE-stained cells (A) according to the equation: Di = [[D0 - A]/2i] + A, where Di is the mean CFSE intensity of cells in division i. Pro fit then calculated the area of consecutive gaussian curves that best accommodated the data, using the same variance for each curve. The proportion of live cells per division was then used to calculate the total cell number in each division per culture by reference to a known number of CaliBRITE beads (unlabeled; Becton Dickinson) run simultaneously with cells. Typically, 103 beads were added to each 1-ml cell culture before harvesting. Bead and live cell events were discriminated using side and forward scatter parameters. The cell numbers in culture were then calculated from the ratio of bead to live cell events and the known total bead number per culture (25).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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partially inhibits IL-4- and CD40L-induced B cell
proliferation
Previously, we have reported multiple effects of IL-4
upon CD40L-induced B cell proliferation, viability, and division-linked
isotype switching to IgG1 and IgE (16). To explore the
nature of antagonism between IL-4 and IFN-, it was necessary to
determine separately the effect of IFN- on each component of the B
cell response.
Initially the effect of IFN- on B cell proliferation was examined.
The addition of a saturating concentration of IL-4 (500 U/ml) to B
cells exposed to a moderate CD40L dose increased
[3H]TdR incorporation 10-fold (Fig. 1
A). IFN- reduced IL-4- and
CD40L-induced B cell proliferation 2.3-fold, with the plateau of
inhibition occurring at concentrations of IFN- higher than 0.2
ng/ml. In the absence of IL-4, IFN- enhanced CD40L-mediated B cell
proliferation, although to a much lesser extent than IL-4. Furthermore,
the nadir of IFN- inhibition of the IL-4 response remained
substantially above the level of proliferation induced by IFN- and
CD40L alone.
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on total cell numbers during culture was also
monitored. B cells were cultured for 4 days with CD40L and various
doses of IFN- in the presence or absence of an optimal
concentration of IL-4 (Fig. 1
B). IFN- reduced IL-4-
and CD40L-stimulated B cell numbers 2.3-fold while increasing the cell
yield in the absence of IL-4 by 3.2-fold. Taken together, these results
revealed that IFN- is both a weak enhancing stimulus for CD40L and a
partial antagonist of IL-4-mediated enhancement of B cell
proliferation.
IFN- reduces cell number without affecting cell division rate
Previously, we have used the CFSE-labeling method to determine the
number of B cells in each division cycle after stimulation with CD40L
and IL-4 (15, 16). These studies revealed that B cells
enter division rounds in a highly asynchronous manner and that IL-4
increases the number of cells found in the later divisions. To
determine the way in which IFN- interferes with IL-4-promoted
enhancement of B cell division, CFSE-labeled B cells were stimulated
with CD40L and various concentrations of IL-4 in the presence or
absence of IFN- (Fig. 2A).
Individual peaks of CFSE histograms were analyzed using peak-fitting
software to determine the optimal series of gaussian curves underlying
the CFSE profiles (illustrated in Fig. 2A, upper
left histogram). This analysis allowed the total number of cells
in each division in culture to be determined after reference to the
known number of beads (see Materials and Methods) added to
each culture before harvesting (Fig. 2B). Based on this mode of
analysis, it is clear that, on day four, B cells had divided up to 10
times in response to CD40L and various concentrations of IL-4 (500–20
U/ml), with most cells in culture having divided at least five times
(Fig. 2, A and B). B cells stimulated with CD40L
alone were also spread over a broad division range; however, there were
substantially fewer cells in each division, even though the maximum
division number was similar. Remarkably, the addition of IFN- to
cultures did not substantially alter the CFSE profile of IL-4- and
CD40L-stimulated B cells although a reduction of approximately one
division in the mean division number was evident. Despite the
similarity in CFSE profiles, the total number of live cells in each
division was markedly reduced when IFN- was included in cultures
containing IL-4 (Fig. 2B). Interestingly, IFN- alone
augmented CD40L-induced B cell proliferation and resulted in a 3-fold
increase in total cell number (Fig. 2A, lower
panel).
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could inhibit the synergistic
effect of IL-4 and CD40L on B cell activation without influencing the
rate of cell division. To confirm this finding, BrdU incorporation was
used to compare the rates of cell division under the various culture
conditions (Fig. 3). When the percentage
of BrdU-labeled cells in each cell division was determined, it was
found that cells from division two onwards were dividing at a similar
rate, as noted previously (16). Cells cultured with IL-4
incorporated BrdU at approximately twice the rate of those not exposed
to this cytokine. The addition of IFN- to cultures had little if any
significant effect on the rate of BrdU incorporation at any IL-4
concentration.
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reduces the number of cells entering division
According to the data shown in the previous section, stimulated B
cells divided at a similar rate in the presence or absence of IFN-.
Therefore, the one division reduction in average division number
induced by IFN- (Fig. 2) must have been due to a delay in the
average time of entry into the first division. This postulate was
tested by examining the number of cells in each division at an early
time point (Fig. 4). In the presence of
optimal concentrations of IL-4 and CD40L, more than 80% of live B
cells were found in the first two divisions on day two of culture, with
less than 10% reaching divisions three and four (Fig. 4). The addition
of IFN- delayed B cell entry into the first two divisions in a
concentration-dependent manner but could not completely inhibit
division entry, even at saturating doses, consistent with the data
presented in Fig. 2. Thus, IFN- delays the average time to first
division for B cells stimulated with CD40L and IL-4 by approximately
one division period.
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inhibits IL-4 enhanced cell viability
The delay in entry into the first cell division was not sufficient
to account for the 5-fold reduction in cell number at day 4 induced by
IFN- (Fig. 2). Since the rate of division was not altered, we
reasoned that IFN- must also affect the rate of B cell death in
culture. To test this hypothesis, CD40L- and IL-4 stimulated B cells
were cultured with various concentrations of IFN- for 2–4 days
(Fig. 5, A and B).
Cell viability was monitored by the exclusion of PI. As shown in Fig. 5A, PI-positive (dead) cells did not retain the
division-related CFSE intensity as well as live cells, suggesting some
decay of the CFSE label during cell death. Nevertheless, it was still
apparent from these profiles that the proportion of PI-positive vs live
cells was much greater in cultures with IFN-. In Fig. 5B,
the total proportion of PI-staining events is presented, indicating an
increase in dead cells from 25–30% to 75% on days 3 and 4. Thus, the
addition of various concentrations of IFN- clearly reduced the
viability of IL-4- and CD40L-stimulated B cells on days 3 and 4 of
culture.
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on the viability, purified B
cells were incubated for 48 h in the presence of optimal
concentrations of IFN- (20 ng/ml), IL-4 (1000 U/ml), or combinations
of both cytokines (Fig. 5C). The viability of small dense B
cells dropped rapidly in unstimulated cultures. Addition of IL-4
rescued B cells from spontaneous cell death, increasing the proportion
of live cells by 2-fold from 35% to 67%, as previously reported
(19, 20). In contrast, IFN- decreased the
IL-4-dependent increase in cell viability by 20%, while increasing the
viability of cells incubated without IL-4 by 7%. Titration of
IFN- dose indicated that higher IFN- concentrations did not
dramatically change the effect on cell viability (Fig. 5D).
These data demonstrate that IFN- can partially inhibit the ability
of IL-4 to promote B cell survival.
Effects of IFN- on IL-4-induced isotype switching
B cells differentiate into IgG1- and IgE-expressing cells in
response to IL-4 and CD40L stimulation. The effect of IFN- on
IL-4-stimulated Ig isotype switching was examined using intracellular
staining, which has been found to be more sensitive than surface
staining methods (data not shown). As shown in Fig. 6A, high concentrations of
IFN- reduced the total expression of IgG1 to 60%, and IgE to 15%
of the initial level. Over the same concentrations of IFN-,
IgG2a+ cells appeared reaching 9% of the total
cells in culture.
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A was division sliced as described
(16) to determine the relation between switching and cell
division number for each culture condition. As previously observed,
IL-4 induced a progressive division-related increase in
IgG1+ cells, which reached 50% in the later
cell divisions (Fig. 6B). The addition of increasing
concentrations of IFN- had the effect of retarding IgG1 expression,
with the highest dose of IFN- resulting in a delay of approximately
two divisions. Thus, IFN- had the same effect as lowering slightly
the IL-4 concentration but did not prevent division-linked IgG1
switching (16).
Fig. 6B also illustrates the results of division slicing for
IgE and IgG2a. IgE+ cells appeared after division
five and increased in frequency with each division. IFN- had little
effect on IgE switching at concentrations that had maximal antagonistic
effects on IL-4 enhancement of proliferation and viability (0.08–1
ng/ml). However, higher concentrations yielded a progressive decline in
IgE+ cells per division illustrating a greater
sensitivity of this isotype to antagonism by IFN- than IgG1. In
parallel with the inhibition of IgE expression, IFN- promoted B cell
switching to IgG2a (Fig. 6B). IgG2a+
cells were seen to arise after division five, indicating that this
isotype is similar to IgE with respect to division-related expression.
Furthermore, the IFN- dose response range for inhibiting IgE and
promoting IgG2a were similar (Fig. 6B). Finally, Fig. 6C shows that division-linked switching to IgG2a induced by
CD40L and IFN- is not inhibited by the presence of IL-4.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is well described to have antagonistic
effects on IL-4-mediated activities (10) including isotype
switching to IgG1 and IgE (8, 9). Given the
well-documented ability of IL-4 to enhance the B cell response at
numerous levels including proliferation and viability (4, 26) in addition to effects on switching (15, 16),
it was of interest to determine in more detail how the antagonism
between IFN- and IL-4 was operating. The CFSE-labeling method
enabled us to explore this question in a novel manner, separating the
interdependent variables differentiation, rate of proliferation, and
survival.
Our analysis explored the direct effect of IFN- on division-linked
isotype switching. When B cells are cultured in the presence of CD40L
and IL-4, they switch isotype with a predictable probability related to
division number (15, 16). Such division-linked isotype
switching occurs independently of division rate or CD40L dose and
serves as a useful reference for defining the effects of cytokines on
cell differentiation (15, 16). Without this reference, it
is difficult to distinguish effects on cell proliferation from those of
differentiation so that treatments influencing the former can
indirectly appear to be altering the latter.
Analysis of the combined effect of IL-4 and IFN- on isotype
switching yielded a surprising result: IFN- induced only a small
delay in the divisions at which switching to IgG1 occurs. However, this
delay did not exceed two division cycles even at very high IFN-
concentrations. The effect on IgE switching was more marked, reducing
switching by 80% at high concentrations. These results illustrate that
IFN- is a partial antagonist of IL-4-switching functions. In other
words, IFN--induced inhibition follows a dose response that does not
reach 100% reduction, but plateaus at an intermediate level. This
level for IgG1 is approximately equivalent to reducing the IL-4
concentrations to 10 U/ml (16). Despite the partial
effects, these results are consistent with previous observations that
IFN- mediates reduction of IL-4-induced isotype switching by
reducing expression of sterile transcripts (11, 13).
IFN- promoted CD40L-stimulated B cells to switch to IgG2a after the
fifth division, following a division-linked pattern similar to
IL-4-induced IgE expression but different to that observed for IgG1,
which begins at earlier divisions. The IFN--induced switch to IgG2a
was unaffected by the presence of IL-4, when assessed as a function of
division number.
In addition to promoting isotype switching, IL-4 is a strong enhancer
of CD40L-induced B cell proliferation (16), as well as a
survival factor for B cells in culture (20). Our
examination of the effects of IFN- on these activities revealed the
following. First, IFN- inhibited IL-4-mediated enhancement of
CD40L-stimulated B cell proliferation, although, even at the highest
saturating concentrations, IFN- could not completely abrogate the
stimulatory effect of IL-4. Second, the addition of IFN-
reduced the ability of IL-4 to promote B cell viability, but once again
the effect was partial.
The observation that IFN- is only a partial antagonist of the
isolated actions of IL-4 on B cell behavior was surprising. However, by
examining models of cell growth, it is apparent that even partial
antagonism of consecutive events associated with growth can result in
marked reductions. B cells in culture do not proliferate according to
classic exponential growth equations (27). Most notably
the time of entry into the first division cycle is variable, and the
CD40L dose and presence of IL-4 alters the average time cells take to
reach their first division (16). Once cells have entered
the first division, however, they divide at an approximately constant
rate, although this rate is not fixed and can be increased in the
presence of IL-4. Evidence for this conclusion comes from the BrdU
experiments shown in Fig. 3, which revealed uniform incorporation
across all division cycles irrespective of whether IL-4 was present in
culture, even though the overall rate of incorporation was increased
approximately 2-fold. Furthermore, B cells die spontaneously in
culture, and, again, the probability of death is reduced by the
presence of IL-4. These experimental features can be used to show with
a mathematical model how multiple weak effects can together result in a
profound (10-fold) net inhibition of cell number. This model is built
on the following experimental observations: 1) the strength of B cell
stimulation by CD40L determines the time taken by the B cell population
to enter into the first division; 2) once initiated into division, B
cell cycle time is relatively constant through successive divisions,
and 3) B cells exhibit a fixed probability of dying on passaging
through each cell division. Given these assumptions, the cell number at
any time after initiation of culture can be approximated using the
following deterministic equation (adapted from Ref. 27):
 | (1) |
IFN- was also shown to enhance B cell stimulation induced by CD40L
and to promote cell viability, although the effects were significantly
less than those seen in the IL-4-induced response. This result is
consistent with a previous report demonstrating a mitogenic action of
IFN- on CD40L-stimulated murine B cells (28).
Interestingly, the BrdU incorporation studies indicate that
IFN-, in contrast to IL-4, does not appear to enhance the
division rate. Rather, the small reduction in the time to first
division and in the rate of cell death act in synergy to effect a small
increase in B cell number.
When the two antagonistic cytokines were combined in culture, IFN-
partially inhibited the ability of IL-4 to shorten the time to first
division and to promote B cell viability, but did not prevent IL-4 from
enhancing the rate of B cell division. Since IFN- has weak effects
on the time to first division and B cell viability in the absence of
IL-4, these observations could be interpreted as showing that the
weaker positive effects of IFN- were dominant over those of IL-4.
However, this is only partly correct, since, even at very high
concentrations, the response induced by IFN- in the presence of IL-4
did not decline to the same level as that achieved with IFN- alone.
By using the equation defined above, it is possible to show that the
net effect of delaying the time to first division and reducing
viability is a marked amplification of the weak antagonistic behavior
of IFN-. For example, in the IL-4-enhanced cultures mentioned above,
an increase in the time to first division of 20% and a reduction
in the survival rate of dividing cells of 20% leads to a predicted
amplification of the net difference in cell number of 4-fold after 3
days of culture and 9-fold at 4 days, which are consistent with the
actual reduction in cell number observed (Fig. 2).
The effect of IFN- on B cell proliferation and survival exhibited a
markedly greater sensitivity to cytokine concentration than either the
inhibition of IgG1 and IgE or the promotion of switching to IgG2a. This
difference was 100-fold. This disparity complicates the prediction
of the final net outcome of the dual effects of two cytokines on the
number of switched cells. The ability to separate these effects and
study them independently should lead to a more complete description and
understanding of how B cells will behave in vivo when confronted with
multiple signals.
In conclusion, IL-4 and IFN- both induce profound alterations in the
kinetics of B cell activation, expansion, and cellular differentiation.
Furthermore, this control is achieved in a subtle manner, acting to
enhance or inhibit consecutive steps that strongly multiply the overall
number of responding B cells and the differentiation state of those
cells. Clearly IL-4 will behave as a powerful amplifier of T and B cell
collaboration. Since this cytokine appears to be important in immunity
to helminthic parasites (29, 30), it is interesting to
speculate why such amplification may have evolved. One possibility is
that parasites, which typically are slow growing, present little Ag
during the early stages of infestation, and, therefore, without
enhancement would not evoke a strong immune response. In contrast,
IFN- is normally produced upon challenge with replicating viruses
(31, 32). Under these conditions, Ag is less likely to be
limiting, perhaps explaining why this cytokine has evolved a much
weaker positive effect on the B cell response. Furthermore, the ability
of IFN- to act as a dampener of IL-4-promoted B cell proliferation
and survival might suggest that IL-4-mediated amplification may be
disadvantageous during a viral response. It seems possible that
activation of excessive numbers of B cell precursors of low affinity,
as will occur in the presence of IL-4, may inhibit the efficient and
rapid operation of affinity-based Ab selection and weaken the
effectiveness of the developing antiviral Ab response. In contrast,
with a slower growing pathogen, such as a parasite, which is not
presenting an acute threat, the entry of a larger pool of starting V
gene sequences into the hypermutating B cell response may eventually
lead to a higher quality Ab response.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Philip D. Hodgkin, Centenary Institute of Cancer Medicine and Cell Biology, Locked Bag No. 6, Newtown NSW 2042, Australia. E-mail address:
3 Abbreviations used in this paper: CD40L, ligand for CD40; CFSE, 5-(and -6)carboxyfluorescein diacetate succinimidyl ester; SA-TC, streptavidin-tricolor; BCM, B cell medium; PI, propidium iodide; BrdU, 5-bromo-2-deoxyuridine.
Received for publication March 17, 1999. Accepted for publication July 30, 1999.
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 N. Gao, P. Jennings, and D. Yuan Requirements for the natural killer cell-mediated induction of IgG1 and IgG2a expression in B lymphocytes Int. Immunol., May 1, 2008; 20(5): 645 - 657. [Abstract] [Full Text] [PDF]
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 E. D. Hawkins, M. L. Turner, M. R. Dowling, C. van Gend, and P. D. Hodgkin A model of immune regulation as a consequence of randomized lymphocyte division and death times PNAS, March 20, 2007; 104(12): 5032 - 5037. [Abstract] [Full Text] [PDF]
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A. Ghochikyan, M. Mkrtichyan, D. Loukinov, G. Mamikonyan, S. D. Pack, N. Movsesyan, T. E. Ichim, D. H. Cribbs, V. V. Lobanenkov, and M. G. Agadjanyan Elicitation of T Cell Responses to Histologically Unrelated Tumors by Immunization with the Novel Cancer-Testis Antigen, Brother of the Regulator of Imprinted Sites J. Immunol., January 1, 2007; 178(1): 566 - 573. [Abstract] [Full Text] [PDF]
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 J. P. Jayasekera, C. G. Vinuesa, G. Karupiah, and N. J. C. King Enhanced antiviral antibody secretion and attenuated immunopathology during influenza virus infection in nitric oxide synthase-2-deficient mice. J. Gen. Virol., November 1, 2006; 87(Pt 11): 3361 - 3371. [Abstract] [Full Text] [PDF]
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 O. Koch, D. P. Kwiatkowski, and I. A. Udalova Context-specific functional effects of IFNGR1 promoter polymorphism Hum. Mol. Genet., May 1, 2006; 15(9): 1475 - 1481. [Abstract] [Full Text] [PDF]
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 G. Hajishengallis, S. Arce, C.M. Gockel, T.D. Connell, and M.W. Russell Immunomodulation with Enterotoxins for the Generation of Secretory Immunity or Tolerance: Applications for Oral Infections Journal of Dental Research, December 1, 2005; 84(12): 1104 - 1116. [Abstract] [Full Text] [PDF]
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P. P. Ho, P. Fontoura, M. Platten, R. A. Sobel, J. J. DeVoss, L. Y. Lee, B. A. Kidd, B. H. Tomooka, J. Capers, A. Agrawal, et al. A Suppressive Oligodeoxynucleotide Enhances the Efficacy of Myelin Cocktail/IL-4-Tolerizing DNA Vaccination and Treats Autoimmune Disease J. Immunol., November 1, 2005; 175(9): 6226 - 6234. [Abstract] [Full Text] [PDF]
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J. S. Rush, M. Liu, V. H. Odegard, S. Unniraman, and D. G. Schatz Expression of activation-induced cytidine deaminase is regulated by cell division, providing a mechanistic basis for division-linked class switch recombination PNAS, September 13, 2005; 102(37): 13242 - 13247. [Abstract] [Full Text] [PDF]
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 N. D. Huntington, Y. Xu, S. L. Nutt, and D. M. Tarlinton A requirement for CD45 distinguishes Ly49D-mediated cytokine and chemokine production from killing in primary natural killer cells J. Exp. Med., May 2, 2005; 201(9): 1421 - 1433. [Abstract] [Full Text] [PDF]
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 S. Al-Darmaki, K. Knightshead, Y. Ishihara, A. Best, H. A. Schenkein, J. G. Tew, and S. E. Barbour Delineation of the Role of Platelet-Activating Factor in the Immunoglobulin G2 Antibody Response Clin. Vaccine Immunol., July 1, 2004; 11(4): 720 - 728. [Abstract] [Full Text]
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M. D. Woolard, L. M. Hodge, H. P. Jones, T. R. Schoeb, and J. W. Simecka The Upper and Lower Respiratory Tracts Differ in Their Requirement of IFN-{gamma} and IL-4 in Controlling Respiratory Mycoplasma Infection and Disease J. Immunol., June 1, 2004; 172(11): 6875 - 6883. [Abstract] [Full Text] [PDF]
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 M. Rodriguez, L. J. Zoecklein, C. L. Howe, K. D. Pavelko, J. D. Gamez, S. Nakane, and L. M. Papke Gamma Interferon Is Critical for Neuronal Viral Clearance and Protection in a Susceptible Mouse Strain following Early Intracranial Theiler's Murine Encephalomyelitis Virus Infection J. Virol., November 15, 2003; 77(22): 12252 - 12265. [Abstract] [Full Text] [PDF]
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Y. Zheng and M. Monestier Inhibitory Signal Override Increases Susceptibility to Mercury-Induced Autoimmunity J. Immunol., August 1, 2003; 171(3): 1596 - 1601. [Abstract] [Full Text] [PDF]
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T. G. Phan, M. Amesbury, S. Gardam, J. Crosbie, J. Hasbold, P. D. Hodgkin, A. Basten, and R. Brink B Cell Receptor-independent Stimuli Trigger Immunoglobulin (Ig) Class Switch Recombination and Production of IgG Autoantibodies by Anergic Self-Reactive B Cells J. Exp. Med., April 7, 2003; 197(7): 845 - 860. [Abstract] [Full Text] [PDF]
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S. G. Tangye, D. T. Avery, and P. D. Hodgkin A Division-Linked Mechanism for the Rapid Generation of Ig-Secreting Cells from Human Memory B Cells J. Immunol., January 1, 2003; 170(1): 261 - 269. [Abstract] [Full Text] [PDF]
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S. G. Tangye, A. Ferguson, D. T. Avery, C. S. Ma, and P. D. Hodgkin Isotype Switching by Human B Cells Is Division-Associated and Regulated by Cytokines J. Immunol., October 15, 2002; 169(8): 4298 - 4306. [Abstract] [Full Text] [PDF]
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T. K. Varma, C. Y. Lin, T. E. Toliver-Kinsky, and E. R. Sherwood Endotoxin-Induced Gamma Interferon Production: Contributing Cell Types and Key Regulatory Factors Clin. Vaccine Immunol., May 1, 2002; 9(3): 530 - 543. [Abstract] [Full Text] [PDF]
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J. S. Rush, J. Hasbold, and P. D. Hodgkin Cross-Linking Surface Ig Delays CD40 Ligand- and IL-4-Induced B Cell Ig Class Switching and Reveals Evidence for Independent Regulation of B Cell Proliferation and Differentiation J. Immunol., March 15, 2002; 168(6): 2676 - 2682. [Abstract] [Full Text] [PDF]
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D. Rabah, S. Grant, C. Ma, and D. H. Conrad Bryostatin-1 Specifically Inhibits In Vitro IgE Synthesis J. Immunol., November 1, 2001; 167(9): 4910 - 4918. [Abstract] [Full Text] [PDF]
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N. Gao, T. Dang, and D. Yuan IFN-{gamma}-Dependent and -Independent Initiation of Switch Recombination by NK Cells J. Immunol., August 15, 2001; 167(4): 2011 - 2018. [Abstract] [Full Text] [PDF]
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 E. L. Munson, B. K. Du Chateau, D. A. Jobe, S. D. Lovrich, S. M. Callister, and R. F. Schell Production of Borreliacidal Antibody to Outer Surface Protein A In Vitro and Modulation by Interleukin-4 Infect. Immun., October 1, 2000; 68(10): 5496 - 5501. [Abstract] [Full Text] [PDF]
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) or absence
(
) of IL-4 (500 U/ml) and various doses of IFN-
