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*
Division of Immunology, University of Cincinnati College of Medicine, Cincinnati, OH 45267;
Cincinnati Veterans Affairs Medical Center, Cincinnati, OH 45220;
Department of Microbiology, University of Alabama, Birmingham, AL 35294; and
§
Division of Pediatric Rheumatology, Children’s Hospital Medical Center, Cincinnati, OH 45229
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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3 days if IL-4
treatment is continued. The increase in splenic B cell number is IL-4
dose dependent, CD4+ T cell independent, Fc
RII/FcRIII
independent, and Stat6 independent. Decreases in the number of B cells
in the blood and the percentage of mature B cells in the bone marrow,
concomitant with the increase in splenic B cell number, suggest that
redistribution of circulating B cells to the spleen is partially
responsible for IL-4 induction of splenic B cell hyperplasia.
Considerable reduction in the effect of 5 days of IL-4 treatment on
splenic B cell number when B lymphopoiesis is blocked with
anti-IL-7 mAb suggests that generation of new B cells is also
involved in IL-4-induced splenic B cell hyperplasia.
5-Bromo-2'-deoxyuridine labeling experiments demonstrate that IL-4
modestly prolongs the life span of newly generated splenic B cells, and
experiments that measure B cell HSA (CD24) expression as an indicator
of B cell age suggest that IL-4 may also prolong the life span of
mature splenic B cells. Thus, IL-4 increases splenic B cell number
through two Stat6-independent effects: increased net migration of
circulating B cells to the spleen and increased B cell life span. Both
effects may promote Ab responses to a systemic Ag
challenge. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female BALB/c mice were purchased from the Small Animals
Division of the National Cancer Institute (Frederick, MD) and were used
at age 8–14 wk. Wild-type mice and mice deficient for the Stat6 gene
(both on a mixed C57BL/6 and 129 genetic background) were bred and
typed by PCR at the Cincinnati Veterans Affairs Medical Center from
mice heterozygous for the nonfunctional Stat6 gene (18)
that were provided by Dr. James Ihle (St. Jude’s Children’s Research
Hospital, Memphis, TN). BALB/c.IL-4R
-deficient mice
(19) were bred at the Cincinnati Veterans Affairs Medical
Center from mice given to us by Dr. Nancy Noben-Trauth (National
Institute of Allergy and Infectious Disease, National Institutes of
Health, Bethesda, MD).
IL-4
Murine rIL-4 was a gift from Dr. Robert Coffman (DNAX Research Institute, Palo Alto, CA).
Immunological reagents
The following Abs were produced and tested for specificity as
previously described: BVD4.1D11.2 (20) (a neutralizing rat
IgG2a anti-mouse IL-4 mAb), BVD6-24G2.3 (20) (a
nonneutralizing rat IgG2a anti-mouse IL-4 mAb), m25
(21) (a mouse IgG2b mAb that neutralizes both human and
mouse IL-7), GK1.5 (22) (a rat IgG2b mAb that kills
CD4+ T cells and blocks Th cell function), 24G2
(23) (a rat IgG2b mAb that binds to mouse FcRII and
blocks its ability to bind IgG), HB
6 (24) (a rat IgG2a
anti-mouse IgD mAb, also known as LO-MD-6), FF1-4D5
(25) (a mouse IgG2a mAb of the b allotype
specific for IgD of the a allotype that is not blocked by
HB6), DS-1 (26) (a mouse IgG1 mAb of the b
allotype that binds to mouse IgM of the a allotype), 6B2
(27) (a rat IgG2a mAb specific for mouse B220, the B cell
form of CD45), 1D3 (28) (a rat IgG2a mAb that binds to the
B cell marker CD19), B3B4 (29) (a rat IgG2a mAb that binds
to mouse Fc
RII), and MKD6 (30) (a mouse IgG2a
alloantibody specific for I-Ad). Some of these
Abs were labeled with FITC (31),
biotin-N-hydroxysuccinimide (32), or the
fluorochrome Cy5 (Research Organics, Cleveland, OH). M1/69
(33) (a rat IgG2b mAb that binds to heat-stable Ag (HSA;
CD24)) was purchased from PharMingen (San Diego, CA).
Preparation of IL-4-anti-IL-4 Ab complexes
IL-4-anti-IL-4 Ab complexes, which greatly extend the in vivo half-life of IL-4 (34), were prepared by mixing IL-4 with the neutralizing anti-IL-4 Ab BVD4-1D11.2 at a 2/1 molar (1/5, w/w) ratio. After 5 min at room temperature, complexes were diluted with 1% BALB/c serum in PBS to a concentration that would allow injection of a 0.2-ml volume. Complexes were always prepared freshly before use.
Preparation of lymphocytes from peripheral blood
Mice were tail-bled 1 min after i.v. injection of 100 IU of heparin sodium. Heparinized blood (0.5 ml) was pelleted by centrifugation, after which the cell pellet was resuspended in 2.5 ml of distilled water for 10 s to lyse erythrocytes. NaCl saline (0.28 ml of 1.5 M) was added, after which nucleated cells were pelleted, resuspended in HBSS supplemented with 10% newborn bovine serum and 0.2% sodium azide (HNA), counted with a Coulter counter (Hialeah, FL), and adjusted to a concentration of 20 x 106 cells/ml.
Preparation of Peyer’s patch lymphocytes
Peyer’s patches were carefully excised from the intestinal wall and dissociated using the neutral protease dispase (Roche, Indianapolis, IN) in Joklik-modified medium (Life Technologies, Gaithersburg, MD) to obtain single-cell preparations (35).
In vitro culture conditions
Spleen cells and nucleated peripheral blood cells were cultured in 1 ml of RPMI medium 1640 supplemented with 10% FBS, 10 ml/L of nonessential amino acids solution, 1% L-glutamine, 1 mM sodium pyruvate, 25 mM 2-ME, 10 mM HEPES, penicillin, streptomycin, fungazone, and gentamicin, with or without 1 ng of IL-4, for 24 h at 37°C in an atmosphere containing 5.5% CO2 in 24-well flat-bottom Costar culture dishes (Cambridge, MA) at a density of 4 x 106 cells/ml/well. At the end of the culture period, cells were washed once in HNA, counted with a Coulter counter, and resuspended in ice-cold HNA at a concentration of 20 x 106 cells/ml.
Immunofluorescence staining
Single-cell suspensions of spleen, peripheral lymph node
(axillary, supraclavicular, inguinal, and popliteal lymph nodes), or
bone marrow were depleted of erythrocytes by treatment with a buffered
ammonium chloride solution, resuspended in HNA, filtered through nylon
gauze, counted with a Coulter counter, and brought to a concentration
of 20 x 106 cells/ml. One hundred
microliters of cell suspensions from the above organs and blood were
stained for 30 min on ice with 1 µg each of an FITC-labeled Ab and a
biotin-labeled Ab. Cells were washed twice with HNA and then stained
for 30 min on ice with streptavidin-R-PE (Life Technologies). All
staining was performed in the presence of 10 µg/ml of unlabeled
anti-FcRII mAb (24G2) to block the binding of IgG staining
reagents to FcRII. After washing once more with HNA, cells were
washed once with HBSS/0.2% sodium azide, then fixed in PBS/2%
paraformaldehyde. Cells were analyzed with a FACScan (Becton Dickinson,
Mountain View, CA) and CellQuest software. Light scatter gates were set
to exclude cells that had died before fixation as well as nonlymphoid
cells, except that light scatter gates for analysis of bone marrow
cells were set to include all living nucleated cells. Spleen cells that
had been stained with a single fluorochrome-labeled Ab were used to
determine compensation for overlap between FITC and PE emission
spectra. Data were analyzed to determine the percentages of
specifically stained cells and the mean and/or median fluorescence
intensities of specifically stained cells. In some experiments cells
stained with FITC-, PE-, and Cy5-labeled reagents were analyzed by flow
cytometry with a FACScaliber flow cytometer (Becton Dickinson), and PE
fluorescence histograms of
Cy5+FITCdull and
Cy5+FITCbright cells were
prepared.
Staining for 5-bromo-2'-deoxyuridine (BrdU)-labeled cells
BrdU (Sigma, St. Louis, MO) was added at a concentration of 0.8 mg/ml to the drinking water. Water bottles were covered with aluminum foil, and water was changed daily. Spleen cell suspensions were stained for surface markers as described above, then washed in PBS and resuspended in 0.5 ml of ice-cold 0.15 M sodium chloride, to which 1.2 ml of ice-cold 95% ethanol was added dropwise as cells were being gently vortex mixed. Cells were incubated on ice for 30 min, pelleted by centrifugation at 1600 rpm for 15 min, and washed with PBS, after which they were incubated for 30 min at room temperature in 1 ml of PBS/1% paraformaldehyde/0.01% Tween 20. Cells were kept at 4°C overnight, after which they were pelleted by centrifugation at 1600 rpm for 15 min and resuspended in 1 ml of 0.15 M sodium chloride/0.0042 M magnesium chloride/50,000 Kuntz units of DNase I (Sigma). Cells were then incubated for 10 min at room temperature, washed with PBS, stained with 20 µl of FITC-anti-BrdU (Becton Dickinson) for 30 min at room temperature, washed twice in PBS, and analyzed for surface markers and BrdU by flow cytometry.
Determination of cell numbers
Cells were counted with a Coulter counter, using settings that excluded dead cells. Total cell counts were multiplied by the percentages of cells that stained specifically for a particular marker to determine the number of cells that expressed that marker.
Statistics
Hypotheses that IL-4 causes an increase in splenic B cell number
were tested with a one-tailed t test. All other experimental
results were tested with a two-tailed t test. The hypothesis
that IL-4 treatment causes a larger increase in splenic B cell number
than in splenic CD4+ T cell number was tested
with a two-tailed t test comparison of the ratios of splenic
B cell numbers in untreated vs IL-4-treated mice with the ratios of
splenic CD4+ T cell numbers in untreated vs
IL-4-treated mice (see Fig. 5
). Results were considered significant at
p < 0.05, to be highly significant at
p < 0.01, and to lack significance at
p > 0.05. The results of tests for significance are
provided in the figure legends.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A previous study showed that IL-4 treatment causes an increase in
the percentage of B cells in the spleen (34). We performed
additional studies to determine whether this reflects an increase in
the absolute number of splenic B cells or a decrease in splenic non-B
cell number. Treatment of BALB/c mice 5 and 2 days before sacrifice
with IL-4C that contained 1 µg of IL-4 caused both a 2-fold increase
in spleen cell number and a nearly 40% increase in the percentage of
B220+mIgM+ spleen cells
(Fig. 1). Thus, systemic IL-4 treatment
causes splenic B cell number to increase 2- to 3-fold and the number of
splenic non-B cells to increase to less of an extent.
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To determine the dose of IL-4 required to induce a maximal
increase in splenic B cell number, BALB/c mice were left untreated or
were injected i.v. 5 and 2 days before sacrifice with IL-4C that
contained 125-1000 ng of IL-4. Spleen cells were counted and stained
for B220 and Ia, and percentages of
B220+Ia+ cells and the
intensity of their Ia staining were determined by flow cytometry (Fig. 2). Both splenic B cell number and Ia
mean fluorescence intensity were increased at the lowest dose of IL-4
tested, but splenic B cell number peaked at a dose of 500 ng of IL-4,
while B cell Ia median fluorescence intensity continued to increase as
the dose of IL-4 was increased to 1000 ng.
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and
4A). Because mature splenic
marginal zone B cells stain more brightly for HSA than most other
mature splenic B cells, although not as brightly as immature B cells
(33, 36, 37), a preliminary experiment was performed to
determine the degree of HSA staining required to discriminate immature
from mature splenic B cells. This was done by comparing the HSA
staining profiles of splenic B cells from untreated mice and
anti-IL-7 mAb-treated mice (Fig. 3), because anti-IL-7 mAb
treatment prevents B lymphopoiesis and thus depletes most immature B
cells from the spleen (21, 33, 36).
B220+ spleen cells that stained more brightly
than the channel used to demarcate HSAbright from
HSAdull decreased by >76% after 12 days
of anti-IL-7 mAb treatment, while this treatment caused no
appreciable change in the percentage of B220+
spleen cells that stained less brightly than this channel (Fig. 3).
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A). Numbers of mature, but not immature, splenic B cells
increased by an additional 80% during the next 2 days. Numbers of
mature splenic B cells decreased to day 1 levels by 6 days after IL-4C
injection and changed little during the subsequent 14 days. Additional
experiments demonstrated that splenic B cell number continued to
increase between 2 and 3 days after IL-4C injection and remained
elevated, but did not increase further, when mice were reinjected with
IL-4C on day 3 and sacrificed on day 5 (Fig. 4B) or
reinjected every 3 days for 14 days (data not shown).
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RII/III independent
Because IL-4 can stimulate CD4+ T cell
proliferation and helper activity (6) and can suppress B
cell FcRII expression and function (38), it was
possible that the IL-4-induced increase in splenic B cell number was
CD4+ T cell and/or FcRII dependent. To examine
these possibilities, the effect of treatment for 5 days with IL-4C was
studied in mice treated with anti-CD4 and anti-FcRII/III
mAbs. IL-4C induced similar increases in splenic B cell number in mice
treated with anti-CD4 and anti-FcRII/III mAbs and in mice
that did not receive these mAbs (Fig. 5
).
Treatment with IL-4C for 5 days also caused a doubling of the number of
splenic CD4+ T cells in mice that received
neither anti-CD4 nor anti-FcRII/III mAbs.
The IL-4C-induced increase in splenic B cell number is Stat6 independent
IL-4 stimulates cells through at least two molecular pathways, one
of which involves activation of Stat6 (18, 39). In
general, IL-4 effects on cell differentiation are Stat6 dependent,
while IL-4 effects of cell survival and proliferation are Stat6
independent (40). To determine the Stat6 dependence of
IL-4-induced migration of B cells to the spleen and increased B cell
survival, we examined the effects of treatment with IL-4C for 1 or 5
days on spleen cell number in wild-type and Stat6-deficient mice.
Although untreated Stat6-deficient mice had fewer splenic B cells than
untreated wild-type mice, IL-4C treatment caused similar
percent increases in splenic B cell number in both sets of mice; these
were larger after 5 days of IL-4C treatment than after 1
day of IL-4C treatment (Fig. 6).
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A). Secondly, we compared the
abilities of IL-4C (prepared with BVD4-1D11) to stimulate an increase
in splenic B cell number and B cell class II MHC expression in BALB/c
wild-type and IL-4R-deficient mice. IL-4C increased splenic B cell
number and Ia expression in wild-type, but not in IL-4R-deficient,
mice, demonstrating that these effects of IL-4C are IL-4R dependent
(Fig. 7B).
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IL-4-induced splenic B cell hyperplasia could result from the
redistribution of mature B cells, increased B lymphopoiesis, increased
migration of recently generated B cells from bone marrow to spleen,
increased splenic B cell survival, mature splenic B cell proliferation,
or a combination of these effects. To determine whether redistribution
of B cells might contribute to IL-4-induced splenic B cell hyperplasia,
we examined whether the increase in splenic B cell number is
accompanied by the loss of B cells from any other organ. BALB/c mice
were untreated or were treated with IL-4C that contained 1 µg of IL-4
1 day before sacrifice. IL-4C treatment caused spleen and mesenteric
lymph node number to increase by 60–100%, while blood B cell number
decreased by 35%, and most mature B cells disappeared from the bone
mar-row (Fig. 8 and data not shown). No
significant change was seen in the number of immature bone marrow B
cells or on the number of spleen or lymph node
CD4+ T cells, although a marginal decrease was
observed in the number of CD4+ T cells in
blood.
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60–70% decreases in blood B cell number and the percentage of
mature bone marrow B cells (Fig. 9);
(IL-4 had no significant effect on the numbers of cells recovered from
bone marrow, Peyer’s patches, or peripheral lymph nodes in this
experiment, but results were reported as percentages of B cells only
because of concerns about the completeness of bone marrow and Peyer’s
patch cell recovery and because of the considerable variability in
peripheral lymph node cell number within groups). Microscopic
examination of sections of small intestine that had been stained with
anti-B220 Ab showed no noticeable effect of IL-4 on B cell number
(3.0 ± .7 B cells/villus for untreated mice; 2.7 ± .9 for
IL-4C-treated mice). The increases in mesenteric lymph node B cell
number that were seen 1 day after the initiation of IL-4C treatment
were no longer observed on day 5. Taken together, these observations
suggest that IL-4 induces a redistribution of mature circulating B
cells to the spleen, but that this redistribution does not deplete B
cells from other peripheral lymphoid organs.
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The ability of 1-day treatment with IL-4C to increase splenic B
cell, but not splenic CD4+ T cell, number (Fig. 7) suggested that IL-4 induced migration of lymphocytes to the spleen
might be B cell specific. In contrast, treatment of mice with IL-4C for
5 days significantly increased splenic CD4+ T
cell number, although less than this treatment increased splenic B cell
number (Fig. 5). An additional, kinetic study was performed to evaluate
whether these different effects of IL-4C on B and T cells reflected
true kinetic differences or were due to experimental variability. As
was observed in our two previous studies that evaluated single time
points, treatment with IL-4C caused a significant increase in splenic B
cell number within 1 day as well as a larger increase that was seen
after 5 days of treatment (Fig. 10). In
contrast, IL-4C treatment did not significantly increase the number of
splenic CD4+ T cells until day 3, and, as
observed previously, the increase at that time point and on day 5 was
smaller than the increase in splenic B cell number. Thus, the effects
of IL-4C on splenic B cell and CD4+ T cell number
differ in both kinetics and magnitude.
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Because the number of B cells lost from the blood and bone marrow
was smaller than the number gained by spleen (Figs. 8 and 9), it seemed
possible that changes in B cell survival might also contribute to the
increase in splenic B cell number. Alternatively, large numbers of B
cells from an additional source that we could not evaluate, such as
lymph, may have migrated to the spleen. Experiments were performed to
evaluate the possibility that IL-4 contributes to splenic B cell number
by enhancing new B cell production or B cell survival. One experiment
examined the effect of 5 days of in vivo IL-4C treatment on B cell
expression of HSA, a surface marker that decreases in expression as B
cells age (36). If IL-4 primarily contributed to splenic B
cell hyperplasia by increasing B cell production and enhancing the
migration of newly generated (HSAbright) B cells
to the spleen, we would expect it to cause an increase in mean HSA
expression by splenic B cells. In fact, IL-4C treatment induced
significant decreases in mean HSA expression by both splenic and blood
B cells, but had no effect on mean HSA expression by lymph node B cells
(Fig. 11A). This effect is
consistent with IL-4 induction of accelerated splenic B cell maturation
and/or increased splenic B cell longevity. It cannot be explained
solely by migration of HSAdull blood B cells to
the spleen, because the mean HSA staining intensity of splenic B cells
from IL-4-treated mice is similar to or lower than that of blood B
cells from untreated mice (Fig. 11A). An alternate
possibility, that IL-4 directly down-regulates B cell HSA expression,
appears unlikely because in vitro IL-4 treatment, at a dose (1 ng/ml)
that substantially up-regulates B cell Ia expression, had no effect on
splenic B cell HSA expression (Fig. 11B).
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). IL-4C treatment induced 2- to
3-fold increases in HSAdull B cells at each time
point studied, but did not consistently affect the number of
HSAbright splenic B cells. Mice treated
simultaneously with IL-4C and BrdU (day 5 point) had similar
percentages of BrdU+ splenic B cells as mice that
received BrdU alone. This suggests that IL-4C did not stimulate B cells
to proliferate and is consistent with previous evidence that IL-4 is
not a B cell mitogen (6). Percentages of
HSAbright splenic B cells that were
BrdU+ were similar in untreated and IL-4-treated
mice on days 5 and 19 and declined from 40% on day 5 to 8% on
day 19. However, a significantly larger percentage of
HSAbright splenic B cells were
BrdU+ in the IL-4-treated than in untreated mice
on day 10 (5 days after the initiation of IL-4 treatment), although the
absolute number of splenic HSAbright B cells was
only slightly increased at this time. Both the absolute numbers of
BrdU+HSAdull splenic B
cells and percentages of HSAdull splenic B cells
that were BrdU+ peaked on day 10, at which time
they were substantially greater in IL-4-treated mice than in control
mice. By day 19, the percentage of HSAdull
splenic B cells that were BrdU+ had declined by
nearly 50% in the IL-4-treated mice and was only slightly greater than
that in mice that had not received IL-4. Taken together, these
observations suggest that IL-4 has relatively little effect on B cell
production or on the migration of immature B cells to the spleen, but
modestly prolongs their survival, and possibly promotes their
maturation, after they reach the spleen. These effects appear to be
sufficient for some of the newly produced B cells to acquire a mature
(HSAdull) phenotype.
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An IL-4-induced increase in spleen cell number that results from
the net migration of mature B cells from blood and bone marrow to the
spleen should not depend on the generation of new B cells. In contrast,
an IL-4-induced increase in splenic B cell number that results from
enhanced survival of newly produced B cells will depend on new B cell
production. In addition, increases in B cell number that result from
population shifts might occur relatively rapidly, while the
accumulation and maturation of newly produced B cells would occur over
a longer period of time. Based on these considerations, we examined the
dependence of the IL-4-induced increase in splenic B cell
number on B lymphopoiesis by determining whether anti-IL-7 mAb
inhibition of B lymphopoiesis would inhibit the IL-4-induced increase.
All mice were treated with anti-CD4 and anti-FcRII/III mAbs
in these experiments to block any effects that the injection of a large
quantity of IgG might have on Th cell activation or on B cell
FcR-dependent interactions.
Treatment of BALB/c mice for 1 day with IL-4C increased splenic B cell
number to at least the same extent in mice in which B lymphopoiesis had
been suppressed with anti-IL-7 mAb as in mice with normal B
lymphopoiesis (Fig. 13, left
panels). In contrast, the increase in splenic B cell number
induced by 5 days of IL-4C treatment was twice as large in mice with
normal B lymphopoiesis as in anti-IL-7 mAb-treated mice (Fig. 13, right panels). Thus, the initial IL-4-induced increase in
splenic B cell number is independent of B lymphopoiesis, while the
additional increase in splenic B cell number that results from further
treatment with IL-4C requires B lymphopoiesis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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dependent and CD4+ T cell, Stat-6, and
FcRII/III independent; and can be observed at doses of IL-4 low
enough to induce only small increases in B cell class II MHC expression
(Fig. 2A). Endogenously produced IL-4 may well induce
similar effects on splenic B cells; greater than 100-fold increases in
total body IL-4 secretion and large, IL-4-dependent increases in
splenic B cell class II MHC expression and splenic B cell number are
observed in mice infected with the nematode parasite
Nippostrongylus brasiliensis; the latter effects are blocked
when infected mice are treated with anti-IL-4 mAb (41)
(J. F. Urban and F. D. Finkelman, unpublished
observations). Five different mechanisms could account for IL-4-induced splenic B cell hyperplasia: increased B cell production, increased migration of recently produced B cells from bone marrow to spleen, increased peripheral B cell proliferation, increased B cell survival, and redistribution of mature B cells to the spleen from other organs. No evidence has been found for increased B cell production; no increase was observed in IL-4C-treated mice in the percentage or number of immature B cells in bone marrow or in the percentage of B cells in spleen that have an immature phenotype. Both of these changes, in contrast, are observed in mice treated with IL-7, an established stimulus of B lymphopoiesis (34). Furthermore, anti-IL-7 treatment, which blocks B lymphopoiesis (21), does not inhibit the initial (day 1) increase in B cell number in IL-4C-treated mice. These same observations are also incompatible with the possibility that IL-4-induced splenic B cell hyperplasia is caused by increased migration of recently produced B cells from bone marrow to spleen. In addition, our observations make it unlikely that IL-4-induced splenic B cell hyperplasia results from increased peripheral B cell proliferation; no increase in the percentage of BrdU+ splenic B cells was observed in mice that were treated with IL-4C while receiving BrdU.
IL-4 does appear, however, to contribute to the increase in splenic B
cell number by causing a redistribution of circulating B lymphocytes to
the spleen and by increasing the life span of recently generated B
cells. Evidence in favor of redistribution is provided by observations
that numbers of mature blood and bone marrow B cells decline at the
same time that the number of splenic B cells increases, and that the
initial (day 1) IL-4-induced increase in splenic B cell number does not
depend on the production of new B cells (it is not inhibited by
anti-IL-7 mAb treatment). Because the IL-4C-induced increase in
mature splenic B cell number is considerably greater than the loss in
mature blood or bone marrow B cells (Figs. 8 and 9), and IL-4C
treatment does not cause a net loss in B cells from lymph nodes,
Peyer’s patch, or intestinal lamina propria, it is necessary to
hypothesize that IL-4 induces B cells from an additional source to
redistribute to the spleen. The most likely source, which we have not
been able to directly evaluate, is lymph; thoracic duct drainage of an
adult mouse may yield 2 x 107 B cells in
24 h (42). Thus, we propose that IL-4 has little
effect on the net flux of parenchymal B cells from lymph node, Peyer’s
patch, or lamina propria but induces actively circulating bone marrow,
blood, and lymph B cells to redistribute to the spleen. Because most B
lymphocytes rapidly circulate to the spleen under normal conditions
(43), this net migratory effect most likely results from
decreased exit of B cells from the spleen, perhaps as a result of
increased adhesion molecule expression or loss of a molecule require
for exit from the spleen, rather than from increased entry of B cells
into the spleen.
Our experiments also support the possibility that IL-4C treatment
increases splenic B cell number by increasing the survival of newly
generated B cells: 1) IL-4C treatment causes a decrease in splenic B
cell expression of HSA, consistent with an increase in average B cell
age (36); 2) BrdU labeling studies indicate that IL-4
extends the normally short life span of recently generated splenic B
cells (44) (the percentage of BrdU-labeled splenic B cells
is increased 10 days after the start of a 5-day pulse with BrdU); and
3) the additional increase in splenic B cell number when mice are
treated for >1 day with IL-4C, unlike the initial (day 1) increase, is
IL-7 dependent and thus probably dependent on new B cell production. It
is unlikely that this IL-7 dependence reflects IL-7 stimulation of T
cell help, because our anti-IL-7 experiments were performed in mice
treated with anti-CD4 mAb and because anti-CD4 mAb treatment
did not affect IL-4 induction of splenic B cell hyperplasia. The IL-7
dependence of the increase in splenic B cell number that occurs from
1–3 days after the initiation of IL-4C treatment taken together with
the failure of splenic B cell number to increase further when IL-4C
treatment is continued for >3 days suggests that IL-4 increases the
survival of newly generated B cells by 2 days. Small, but
persistent, increases in the number of splenic B cells that have a
mature phenotype after IL-4C treatment is discontinued (Fig. 4A), however, suggest that IL-4 may also contribute to
increased splenic B cell number to a slight extent by accelerating B
cell maturation, inducing some immature splenic B cells to survive long
enough to mature into long-lived B cells, and/or enhancing the survival
of mature B cells.
Thus, our observations provide evidence that IL-4 increases splenic B cell number by increasing the net migration of circulating B cells to the spleen and by increasing splenic B cell survival. These effects of IL-4 may contribute to both the large, IL-4-dependent Ab responses that are made to nematode parasites (45) and to the humoral autoimmunity that is induced in mice by chronic IL-4 overproduction (46).
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Footnotes |
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2 Current address: Department of Pediatrics, Yokohama City University School of Medicine, 3-9 Fuku-ura, Yokohama 236-0004, Japan.
3 Address correspondence and reprint requests to Dr. Fred D. Finkelman, Department of Internal Medicine, Research Service 151, Cincinnati Veterans Affairs Medical Center, 3200 Vine Street, Cincinnati, OH 45220.
4 Abbreviations used in this paper: m, cell membrane; BrdU, 5-bromo-2'-deoxyuridine; HSA, heat-stable Ag.
Received for publication August 31, 1999. Accepted for publication March 22, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RII expression by CD4+ T helper subsets. J. Immunol. 143:2133.[Abstract]
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  S. Sehra, H. A. Bruns, A.-N. N. Ahyi, E. T. Nguyen, N. W. Schmidt, E. G. Michels, G.-U. von Bulow, and M. H. Kaplan IL-4 Is a Critical Determinant in the Generation of Allergic Inflammation Initiated by a Constitutively Active Stat6 J. Immunol., March 1, 2008; 180(5): 3551 - 3559. [Abstract] [Full Text] [PDF]
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A. Itakura, M. Szczepanik, R. A. Campos, V. Paliwal, M. Majewska, H. Matsuda, K. Takatsu, and P. W. Askenase An Hour after Immunization Peritoneal B-1 Cells Are Activated to Migrate to Lymphoid Organs Where within 1 Day They Produce IgM Antibodies That Initiate Elicitation of Contact Sensitivity J. Immunol., December 1, 2005; 175(11): 7170 - 7178. [Abstract] [Full Text] [PDF]
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A. Choudhury, M. A. Maldonado, P. L. Cohen, and R. A. Eisenberg The Role of Host CD4 T Cells in the Pathogenesis of the Chronic Graft-versus-Host Model of Systemic Lupus Erythematosus J. Immunol., June 15, 2005; 174(12): 7600 - 7609. [Abstract] [Full Text] [PDF]
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V. Taneja, N. Taneja, M. Behrens, M. M. Griffiths, H. S. Luthra, and C. S. David Requirement for CD28 May Not Be Absolute for Collagen-Induced Arthritis: Study with HLA-DQ8 Transgenic Mice J. Immunol., January 15, 2005; 174(2): 1118 - 1125. [Abstract] [Full Text] [PDF]
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R. R. Singh, V. Saxena, S. Zang, L. Li, F. D. Finkelman, D. P. Witte, and C. O. Jacob Differential Contribution of IL-4 and STAT6 vs STAT4 to the Development of Lupus Nephritis J. Immunol., May 1, 2003; 170(9): 4818 - 4825. [Abstract] [Full Text] [PDF]
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H. A. Bruns, U. Schindler, and M. H. Kaplan Expression of a Constitutively Active Stat6 In Vivo Alters Lymphocyte Homeostasis with Distinct Effects in T and B Cells J. Immunol., April 1, 2003; 170(7): 3478 - 3487. [Abstract] [Full Text] [PDF]
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 K. Saijo, I. Mecklenbrauker, A. Santana, M. Leitger, C. Schmedt, and A. Tarakhovsky Protein Kinase C {beta} Controls Nuclear Factor {kappa}B Activation in B Cells Through Selective Regulation of the I{kappa}B Kinase {alpha} J. Exp. Med., June 17, 2002; 195(12): 1647 - 1652. [Abstract] [Full Text] [PDF]
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 A. Q. Khan, Y. Shen, Z.-Q. Wu, T. A. Wynn, and C. M. Snapper Endogenous Pro- and Anti-Inflammatory Cytokines Differentially Regulate an In Vivo Humoral Response to Streptococcus pneumoniae Infect. Immun., February 1, 2002; 70(2): 749 - 761. [Abstract] [Full Text] [PDF]
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E. F. Wagner, N. Hanna, L. D. Fast, N. Kouttab, P. R. Shank, A. Vazquez, and S. Sharma Novel Diversity in IL-4-Mediated Responses in Resting Human Naive B Cells Versus Germinal Center/Memory B Cells J. Immunol., November 15, 2000; 165(10): 5573 - 5579. [Abstract] [Full Text] [PDF]
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