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Division of Developmental and Clinical Immunology, Department of Microbiology, University of Alabama, Birmingham, AL 35294
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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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In rodents and humans two categories of mature, long-lived B cells, termed memory and naive, have been defined that either have or have not encountered the nominal Ag under study (8, 9, 10, 11). Phenotypic and immunohistological analyses subdivided mature splenic B cells into follicular (FO)3 comprising 80–90% and marginal zone (MZ) accounting for 5–10% of adult mouse splenic B cells (12, 13). Because of complex intrasplenic trafficking pathways that are not fully understood, there is not a perfect correlation between the phenotype and the topography of splenic B cell sub- sets. However, it is clear that the marginal zone of the spleen is enriched in IgMhighIgDlowCD21highCD23low B cells, while the follicles contain mostly IgMlowIgDhighCD21intCD23high B cells (12, 14, 15, 16).
It has been shown that memory B cells involved in T-dependent antigenic responses are long lived, have receptors with increased antigenic affinity resulting from somatic mutations, and sometimes switch isotypes (17, 18, 19, 20). At least some of the long-lived memory B cells generated in both T-dependent and T-independent responses reside in the splenic marginal zone for long periods of time after antigenic challenge (21, 22). Memory B cells, as described in humans, have a longer life span, a lower threshold of activation, sometimes a higher affinity for Ag, and an increased ability to present Ag to T cells (4).
Although the phenotype of different long-lived B cell subpopulations is well described, their functional capabilities in immune responses are less well understood (13, 23, 24). It has been shown previously that there are differences in the ability of mature B cell subsets, defined as IgMhighCD23lowCD21high and IgMlowCD23highCD21int,to respond in vitro to a variety of stimuli (12, 25, 26). Despite the fact that mature splenic B cells are constantly trafficking, and that the MZ of the spleen contains other cell types, including memory B cells, for the purpose of simplicity we refer to the two phenotypes as FO (IgMlowIgDhighCD21intCD23high) and MZ (IgMhighIgDlowCD21highCD23low) B cells (12).
MZ B cells are present in nude and thymectomized rats, suggesting that they do not represent postgerminal center T-dependent B cells, although this assumption needs further confirmation (27, 28). Human splenic MZ lymphomas overlap significantly in immunophenotype with the extranodal disease of mucosa-associated lymphoid tissue generically referred as the postfollicular MZ B cell (29). Taken together with recent results from lymphotoxin gene-targeted mice in which both mucosa-associated lymphoid tissue and the MZ of the spleen are lacking, these observations further suggest a link between mature long-lived B lymphocytes in these distinct anatomical sites (5, 30).
Here we report that compared with their nontransgenic littermates, certain Ig heavy chain transgenic (TG) mice have proportionally larger MZ compartments that may constitute up to 50% of splenic B cells, and that the development of this compartment is indeed independent of T cells. We have used these TG mice as a source of large quantities of purified MZ B cells to facilitate the analysis of their functional potential in early stages of in vitro activation. We show that MZ B cells respond better than FO B cells with respect to early in vitro activation events, resulting in up-regulation of T cell costimulatory molecules and a concomitant ability to function as APCs. We also show that MZ B cells proliferate much faster in response to very low doses of polyclonal mitogens and terminally differentiate into plasma cells within hours of activation. Collectively, these findings suggest that MZ B cells have unique signaling and subsequent differentiative potentials that might permit them to react faster than the majority of splenic B cells (FO) in the earliest stages of an in vivo immune response. These functional differences apply to both Ag presentation and the production of short-lived IgM Ab-forming cells that are involved in the early defense against blood-borne pathogens.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Eight- to twelve-week-old C57BL/6 and BALB/c mice were purchased from Charles River Laboratories (Raleigh, NC) or were bred in our mouse facility. All other mice were used at 8–16 wk of age; 81X-C57BL/6 (31), 81X-JHko (32), MD2 (gift from Dr. C. C. Goodnow, Australian National University, Canberra, Australia), MD4 and TCR T (TCR double knockout, C57BL6/J Tcrbtm1MomTcrdtm1Mom, purchased from The Jackson Laboratory, Bar Harbor, ME), and 81X-TCR T-C57BL/6 (generated by crossing 81X-C57BL/6 with TCR T-C57BL/6 and screening for the TCR double knockout by PCR) were bred and housed in a pathogen-free facility in accordance with institutional policies for animal care and usage.
Flow cytometric analysis and sorting
Fluoresceinated (FITC) anti-CD5, anti-CD23, and anti-CD69 as well as biotinylated anti-CD62L, anti-CD25, anti-MHC class I, anti-MHC class II, anti-CD95, anti-syndecan-1, anti-B7.1, and anti-B7.2 mAbs were purchased from PharMingen (San Diego, CA). The anti-CD21 (7G6) Ab-secreting hybridoma was a gift from Dr. Michael Holers (University of Colorado Health Science Center, Denver, CO) (33, 34). The purified Ab was conjugated to PE by Southern Biotechnology Associates (Birmingham, AL). Two-, three-, and four-color surface stainings were performed as previously described (31). Briefly, 5 x 105 cells depleted of RBC by lysis with an ammonium chloride-containing buffer were incubated with a mixture of fluorescein-, PE-, and biotin-conjugated Abs followed by SA-PECY5 (Southern Biotechnology Associates) or streptavidin-allophycocyanin (PharMingen). Cells were incubated for 15 min at each step and were washed with 2% FCS/PBS between steps. Cells were resuspended in either 1% paraformaldehyde or 2% FCS/PBS with 2 µg/ml of propidium iodide to exclude dead cells. Data from stained cell samples were acquired using FACSCalibur and CellQuest software (Becton Dickinson, Mountain View, CA) and were analyzed with WinList 2.01 (Verity Software House, La Jolla, CA) and WinMDI 2.0 (Trotter@scripps.edu) software programs.
FO and MZ cells were separated by cell sorting using anti-CD23-FITC and anti-CD21-PE Abs as previously described (12). Briefly, single-cell suspensions were made from five or six mouse spleens. T cells were removed from RBC-depleted spleen cells by treatment with anti-Thy1.2 and anti-CD4 Abs and rabbit complement (Cedarlane Laboratories, Hornby, Canada). Viable cells were recovered by centrifugation over a Lympholyte M gradient (Cedarlane Laboratories) at 900 x g and were stained with anti-CD23-FITC and anti-CD21-PE as described above. MZ and FO B cells were sorted based on their differential expression of CD21 and CD23 using a FACStar Plus (Becton Dickinson). When sorting T cells for the Ag presentation assays, BALB/c lymph nodes were used for the single-cell suspension and were stained with B220-FITC and CD43-PE. To control for a possible stimulating effect of CD21 ligation during the sorting process, some experiments were repeated using anti-CD38 (12, 35) with identical results.
5-Chloromethylfluorescein diacetate (CMFDA) labeling of B cells
Sorted FO and MZ B cells were loaded with CellTracker Green CMFDA (Molecular Probes, Eugene, OR) according to the manufacturer’s recommendations. In brief, sorted B cells were washed and resuspended at 107 cells/ml in serum-free RPMI 1640 with 1.25 µM CMFDA and were incubated for 30 min at 37°C. Cells were washed and incubated for another 30 min at 37°C in serum-free RPMI 1640, washed again, and plated in 96-well plates at 5 x 105/ml in 200 µl of medium containing either LPS (20 µg/ml) or anti-CD40 Ab (10 µg/ml).
Lymphocyte proliferation assays
Sorted B cells were cultured in triplicate at 1 x 105 cells/well in RPMI 1640 containing 10% FCS (HyClone Laboratories, Logan, UT) in 96-half-well flat-bottom plates (Costar, Cambridge, MA) in a total volume of 125 µl. LPS (20 µg/ml; Sigma, St. Louis, MO), polyclonal goat anti-mouse F(ab')2 anti-IgM (20 µg/ml; Southern Biotechnology Associates), polyclonal goat anti-rabbit F(ab')2 anti-IgM (20 µg/ml), anti-CD40 (FGK45, 10 µg/ml, a gift from Dr. Jan Anderson, Basel Institute Immunology, Basel, Switzerland), and IL-4 (100 U/ml) were added in different combinations to the plated cells. IL-4 was obtained from the supernatant of P3 x 63Ag8.653 cells transfected with the appropriate expression constructs. Cells were incubated for 48 h at 37°C in the presence of 10% CO2. Four hours before harvesting, 1 µCi of [3H]thymidine was added to each well. Cells were harvested on Filter MATs (Skatron Instruments, Sterling, VA), and [3H]thymidine incorporation was measured in a scintillation counter (Wallac, Gaithersburg, MD).
For the T cell alloreaction, sorted 81X TG C57BL/6 FO and MZ B cells were activated for 8 or 24 h with LPS (20 µg/ml), anti-CD40 Ab (10 µg/ml), or both in culture medium at 37°C, washed twice, irradiated at 2500 rad, and plated in 2-fold dilutions starting at 2.5 x 105/well. Sorted BALB/c T cells (2 x 105) were added to each well. The plate was pulsed 5 days later with [3H]thymidine and read on day 6.
Cell culture
Cells were sorted as described above and were plated in 0.2–1 ml at a total density of 5 x 105/ml in flat-well (24, 48, or 96 wells, depending on volume) tissue culture plates (Costar). Cells were cultured separately or in a combination with anti-CD40 (10 µg/ml), LPS (20 µg/ml), anti-IgM (20 µg/ml), and IL-4 (100 U/ml) for 1–4 days. At the end of the incubation, the cells were isolated, washed, and stained with the appropriate Abs to determine changes in the expression of cell surface Ags or were used for cytospin preparations.
Immunofluorescence analysis of tissues sections and cytospins
Spleens embedded in OCT compound (Lab-Tek Products, Naperville,
IL) were flash-frozen in liquid nitrogen. Frozen sections were cut,
air-dried, fixed in ice-cold acetone, blocked with normal horse serum,
and stained with MOMA-1 (rat, IgG2a 
, a gift from Dr. Georg Kraal,
specific for mouse splenic metallophilic macrophages) developed with
goat anti-rat IgG-biotin and followed by a third layer of
strepavidin-AMCA (Vector Laboratories, Burlingame, CA). The sections
were washed and then stained with a mixture of FITC rat anti-mouse
CD5 (clone 53-7.3, 10 µg/ml; PharMingen) and rhodamine isothiocyanate
goat anti-mouse IgM (2 µg/ml; Southern Biotechnology Associates).
Cytospins were fixed in alcohol for 30 min at -20°C and stained with
rhodamine isothiocyanate goat anti-mouse IgM (2 µg/ml; Southern
Biotechnology Associates). Sections and cytospins were washed and
mounted in Fluormount G (Southern Biotechnology Associates) and viewed
with a Leica/Leitz DMRB fluorescence microscope equipped with
appropriate filter cubes (Chromatechnology, Battleboro, VT). Images
were acquired with a C5810 series digital color camera (Hamamatsu
Photonic System, Bridgewater, NJ). Images were processed with Adobe
Photo Shop and IP LAB Spectrum software (Signal Analytics Software,
Vienna, VA).
Ig secretion by mature B cell subsets
MZ and FO B cells were sorted based on their differential expression of CD21 and CD23 as described above and were plated in 96-well flat-bottom plates (Costar) at 1 x 105 cells in 200 µl with 20 µg/ml LPS. Supernatants were harvested at each of three time points (3, 7, or 10 days). Igs secreted into the supernatant were detected by ELISA. Briefly, 96-well plates were coated with purified goat anti-mouse IgM (2 µg/ml; Southern Biotechnology Associates) overnight at 4°C. The plates were washed and then blocked with 1% BSA in PBS for 1 h at room temperature. Supernatants were diluted and added to the plates along with titrated purified mouse IgM isotype control (Southern Biotechnology Associates) to establish a standard concentration curve. The plates were incubated for 4 h at 37°C and then washed, and goat anti-mouse IgM conjugated to alkaline phosphatase (1/500; Southern Biotechnology Associates) was added and incubated for 4 h. The ELISAs were developed by adding alkaline phosphatase substrate (1 mg/ml; Sigma). The ODs of wells were measured using a TiterTek Multiskan Plus (Labsystems, Helsinki, Finland) at 405 nm, and data were analyzed using ELISA LITE software (Meddata, New York, NY).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have used the differential expression of the CD23 and CD21
surface Ags by MZ and FO B cells to identify these mature subsets in
the mouse spleen. MZ B cells express high levels of CD21 and low levels
of CD23, while FO B cells express CD23 and moderate levels of CD21 (12, 13, 36). In adult C57BL/6 mice, MZ B cells comprise approximately 5%
of the lymphocytes (Fig. 1
). However, in
heavy chain VH81X TG (81X) mice (37), the MZ is almost 3 times higher
proportionally than that in littermate (LM) control mice (81X TG,
15.1 ± 1.3%; LM, 6 ± 0.9%; n = 5). This
increased percentage of MZ cells is relative and is due to an overall
decrease in the absolute numbers of FO B cells (3.4 ± 0.25 vs
10.1 ± 1.5 x 106/spleen, respectively, in TG
and LM; n = 5). In contrast, the absolute number of MZ
B cells is remarkably conserved between TG and LM (1.85 ± 0.1 and
1.9 ± 0.1 x 106/spleen; n = 5).
Topographic evidence of smaller follicles and larger MZ is observed in
spleen sections stained to display the MZ B cells separated from the FO
B cell population by MOMA-1-positive macrophages (Fig. 1). In another
heavy chain TG line, the anti-HEL heavy chain MD2 (38) MZ are also
proportionally increased (15.3 ± 1.1% in TG vs 7.5 ± 0.4%
in LM; n = 4), although the overall B cell compartment
is reduced due to a decrease in the absolute numbers of FO B cells
(2.36 ± 0.6 x 106/spleen in TG and 6.95 ±
0.5 x 106/spleen in LM; n = 4).
Again, the absolute numbers of splenic MZ B cells were similar in TG
and LM MD2 adult mice (1.5 ± 0.14 x 106/spleen
in TG and 1.33 ± 0.1 x 106/spleen in LM;
n = 4; Fig. 1). The establishment and maintenance of
this MZ B-cell population do not appear to depend on the presence of T
cells, because mice lacking the ß- and 
-chains of the TCR have
nearly equivalent numbers of MZ B cells compared with control mice (TCR
T in Fig. 1). Similar to the original 81X TG mice, in 81X mice crossed
to the TCRß, double knockout, the percentage of MZ B cells was
also about 3-fold higher than that in the LM controls (Fig. 1). In a
parallel experiment CD40L-deficient mice bred with 81X TG mice had a MZ
B-cell compartment similar in size to that in the 81X
CD40L+ controls (data not shown), again suggesting that the
establishment and maintenance of MZ B cells are independent of T cells
and also germinal centers. However, T cells, when present, might still
participate in the differentiation and overall function of MZ B cells.
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The increased percentage of MZ B cells in Ig heavy chain TG mouse
models compared with normal mice permits the isolation of greater
numbers of these cells and facilitates subsequent functional analyses.
However, before using these mice as a source of MZ B cells, we first
determined whether their ability to respond differently from FO B cells
in vitro (12) was preserved. As shown in Fig. 2A, TG MZ B cells isolated
from mice that either have (81X) or do not have T cells (81X TCR T)
share functional characteristics similar to those reported previously
for MZ B cells from normal BALB/c mice (12) responding more vigorously
to CD40 (40,471 ± 4,325 cpm) and to LPS stimulation (228,959
± 2,540 cpm) compared with FO B cells (7,225 ± 640, 138,416
± 1,765 cpm, respectively). Similar to normal mice, TG FO B cells
(47,359 ± 5,140 cpm) proliferate better in response to BCR
cross-linking (Fig. 2A) than do MZ B cells (391 ± 130
cpm), which are very sensitive to BCR ligation and die by apoptosis
(12). Due to the fact that a similar increase in the percentage of MZ B
cells is observed when 81X TG is crossed onto a JH knockout background
(39) (data not shown), cells derived from either 81X-C57BL/6 or 81X-JH
T mice were used for functional experiments and gave similar results.
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B). The relative decrease in CMFDA levels also indicated
that MZ divide more than FO B cells within the first 48 h of
culture with anti-CD40 Abs (1.5-fold) or LPS (2- to 3-fold). By 4
days, the difference is even more distinct for anti-CD40
stimulation, while for LPS the MZ cultures are overgrown, but surviving
MZ and FO cells have gone through similar numbers of cycles. MZ B cells are not activated but can be quickly stimulated to up-regulate T cell costimulatory molecules
Since MZ B cells divide vigorously in response to stimuli, it was
of interest to determine whether these cells constitutively express
surface Ags that would indicate their readiness to collaborate with T
cells and engage in an immune response. MZ cells are not activated per
se, since they do not express increased levels of typical activation
markers, including CD69, CD25, MHC class I, MHC class II, and CD95,
compared with FO B cells (data not shown) (40, 41). However, these
cells do express surface Ags characteristic of a memory phenotype
indicative of a history of prior Ag encounter. In contrast to FO B
cells, MZ B cells express decreased levels of CD62L and IgD, but have
increased levels of CD44 (data not shown) (41). Moreover, the basal
level of the costimulatory molecules B7.1 and B7.2 on MZ B cells is
higher than that on FO B cells (Fig. 3A). These data further
support the hypothesis that MZ B cells have been previously primed, and
together with their ability to rapidly enter the cell cycle, suggest
that these B cells are potential candidates to provide T cell
costimulation.
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B). Stimulation with IL-4, LPS, or
anti-CD40 also increased the expression of B7.2 on both FO and MZ B
cells, with the MZ B cells always expressing higher levels. In
addition, B7.1 expression was amplified on MZ B cells to a greater
extent than on FO B cells when stimulated with either LPS or
anti-CD40 Abs. The expression of other activation Ags was also
analyzed after stimulation with either anti-CD40 or LPS, and
similar up-regulation of CD25 and CD69 was observed on both MZ and FO B
cells, although levels of CD25 on MZ B cells were slightly higher after
LPS stimulation (Fig. 3C).
We also determined the kinetics of B7.1 and B7.2 expression on
sort-purified FO and MZ B cells cultured with anti-CD40 (10
µg/ml), anti-IgM (20 µg/ml), or LPS (20 µg/ml) or alone. At
intervals, cells were isolated and stained to determine the relative
mean fluorescence intensity (MFI) of B7.1 or B7.2 (Fig. 4A). Anti-IgM, anti-CD40,
and LPS are all able to up-regulate B7.2 to comparable levels, although
anti-IgM is slightly more potent. As previously reported,
expression of B7.2 increases in the first 8 h after activation and
reaches peak levels at 12–24 h, declining by 48 h (42, 43). Both
MZ and FO populations exhibit similar kinetics of B7.2 expression, but
at all times, MZ cells respond more vigorously to stimulation with
anti-IgM Abs (MFI, 828 vs 329), anti-CD40 Abs (MFI, 503 vs
160), or LPS (MFI, 473 vs 169). In contrast to B7.2, in the first
48 h of stimulation, B7.1 is up-regulated quickly (peaking at
12 h) on MZ B cells by anti-CD40 and LPS and only marginally
on FO cells. Thus, MZ B cells quickly express more B7.1 and B7.2
molecules on their cell surface after acute stimulation.
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B). By contrast,
B7.1 expression was further up-regulated by the combined stimuli on MZ
cells. The dramatic increase in B7.1 on FO cells after LPS plus
anti-CD40 treatment is of interest, since both agents showed a
minimal effect when added alone. All these data predict a complex
picture in which the B cell subsets’ ability to provide T cell
costimuli will depend on the nature, timing, dose, and combination of
signals that the B cell received. Monoclonal and polyclonal MZ and FO B cells have similar functional characteristics
All previous experiments used a polyclonal population of B cells
from normal or Ig heavy chain TG mice. Since it was possible that the
results reported in the above experiments were influenced by
certain B cell clones in the mixture, we determined whether a
monoclonal population of B cells with either FO
(CD21intCD23high)or MZ
(CD21highCD23low) phenotype from the
anti-HEL TG mice MD4 had similar characteristics (44, 45). As shown
in Fig. 4C, B7.1 and B7.2 expression is higher on MZ than on
FO B cells 24 h after incubation with LPS. However, the
differences between MZ and FO B7 up-regulation were slightly smaller
than those observed with a polyclonal B cell population and faded by
72 h, indicating that individual clones might differ in their
ability to provide B7 cosignals (Figs. 3B and
4C). Also, similar to TG mice in which B cells contain only
a common heavy chain, MD4 MZ (14,754 ± 2,760 cpm) proliferate
better than FO B cells (386 ± 190 cpm) when stimulated with
anti-CD40 Abs, while FO (12,380 ± 870 cpm) out-proliferate MZ
B cells (1,622 ± 280 cpm) when treated with anti-IgM Abs. The
ability to proliferate in response to LPS still follows the same
pattern, with MZ cells cycling better than FO B cells, although the
differences between these two subsets are smaller than those seen with
B cells from normal or heavy chain TG mice. These data show that
independently generated B cell clones with identical receptors are
heterogeneous with respect to different stimuli (anti-IgM,
anti-CD40) depending on their FO or MZ phenotypes. This functional
heterogeneity indicates that unknown stochastic selective events
influence the fate of a B cell depending on whether it is present in a
certain microenvironment and not another at a particular time point in
its life span.
MZ B cells can function as potent APCs
Since MZ B cells express high basal levels of B7.1 and B7.2, which
are rapidly up-regulated within 8–24 h, we next analyzed the capacity
of FO and MZ B cells to present Ag and stimulate alloreactive T cells.
Purified FO and MZ B cells, when stimulated with either LPS or
anti-CD40 Abs for 8 h, increased their levels of B7.1, B7.2,
MHC class I, and MHC class II molecules (Fig. 4A and data
not shown). Stimulated or unstimulated B cells from 81X heavy chain TG
C57BL/6 mice were irradiated and mixed with sort purified BALB/c T
cells. As shown in Fig. 5A,
purified unstimulated B cells were unable to induce T cell
proliferation. In contrast, LPS- or anti-CD40-stimulated MZ, but
not FO, B cells induced vigorous T cell proliferation. T cells
stimulated with 2.2 x 105 LPS-activated MZ B cells
proliferated much more (132,486 ± 9,915 cpm) than T cells
incubated with LPS-activated FO B cells (31,068 ± 4,390 cpm).
Similarly, T cells stimulated with 1.1 x 105 MZ B
cells that had been treated with anti-CD40 Abs (55,024 ±
5,870 cpm) proliferated better than T cells stimulated with their FO
counterparts (3,849 ± 290 cpm). Thus, MZ, but not FO, B cells
when activated by LPS or anti-CD40 for a brief time can up-regulate
key costimulatory molecules on the cell surface that help to induce
allogenic T cell proliferation. To determine whether combinations of
stimuli and a longer activation time will enable FO to present Ags as
well as MZ cells, a similar experiment was performed using a mixture of
LPS and anti-CD40 to stimulate the sorted B lymphocyte populations
for 8 or 24 h. Although at 8 h the LPS-stimulated or LPS- and
anti-CD40-stimulated MZ cells presented alloantigen much better
than FO cells, by 24 h LPS- and anti-CD40-activated FO B cells
induced T cell proliferation comparable to that in similarly treated MZ
cells (Fig. 5B). This correlated with the equivalent level
of B7 expression achieved on MZ and FO cells after stimulation with LPS
and anti-CD40 in combination but not with each stimulus alone (Fig. 4B).
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Although we have clearly shown that MZ B cells express activation
molecules that would potentially enable them to participate in B-T cell
interactions, we also studied their involvement in T-independent
responses, which is another function potentially associated with these
B cells. Although MZ B cells overall are better LPS responders than FO
B cells, we next investigated this effect at limiting mitogen
concentrations. As already shown, MZ B cells proliferated about twice
as much as FO B cells (297,799 ± 7,240 vs 154,180 ± 4,650
cpm) when stimulated with 20 µg/ml LPS. Remarkably, a similar degree
of MZ B cell proliferation was seen even with a 10-fold lower LPS
concentration (297,799 ± 7,240 cpm at 20 µg/ml vs 292,379
± 34,010 cpm at 2 µg/ml LPS), while FO B cells showed very little
response at this dose (Fig. 6A). Even at 0.2 µg/ml LPS
stimulation, MZ B cells proliferated (28,053 ± 1,450 cpm), while
FO B cells were totally unresponsive. A kinetic study of LPS (20
µg/ml)-induced cell division showed a faster peak response for MZ
cells (48 h) that had decreased by 72 h compared with that for FO
cells (Fig. 6B).
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C it can be seen that some MZ B
cells begin to express syndecan within 24 h after LPS treatment,
while FO B cells are still negative. By day 3 both MZ and FO B cells
have subpopulations expressing syndecan, but compared with FO, MZ has a
greater number of very bright syndecan cells. Similar to human
tonsillar memory B cells (47), sorted MZ and FO B cells treated with
anti-CD40 do not express syndecan-1 (data not shown). In addition
to syndecan expression, MZ B cells enlarge very rapidly to form blasts
within 24 h, and by day 3 mature to plasma cells containing high
levels of cytoplasmic Ig compared with FO B cells-derived blasts (Fig. 6D). Furthermore, MZ B cells from both 81X and MD4 TG mice
secrete significantly greater amounts of IgM in a shorter period of
time than FO B cells after in vitro LPS stimulation (Table I). These data show that MZ B cells
differentiate into mature plasma cells and secrete large quantities of
Abs in response to LPS stimulation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and 2) (28). They have the ability to perform effector
functions after brief periods of in vitro activation and either become
potent APCs for T cells through up-regulation and expression of B7
costimulatory molecules (
Figs. 3–5) or differentiate into plasma cells
in response to very low doses of polyclonal mitogens (Fig. 6). These
findings raise interesting questions about the role of these B cells
and their functionality in the course of in vivo immune responses.
It is well documented that besides long-lived B cells, the MZ of the
spleen contains several other cell types potentially involved in the
early stages of immune responses. MZ and metallophilic macrophages are
able to scavenge Ags from the blood, and dendritic cells transport them
to the T cell zones in the first few hours after antigenic exposure
(48, 49, 50, 51) (F. Martin and J. F. Kearney, unpublished observations).
MZ B cells also migrate from their normal sites very early after in
vivo immunization, with some of them traveling to the T cell zone and
subsequently to the B cell follicles (15). Our results predict that in
the first hour of an immune response, MZ B cells generate activated
effector B cells that can either present Ags to T cells or
differentiate into plasma cells for an early boost of IgM Ab
production. The rapid secretion of these Abs would function locally to
facilitate trapping of Ag and formation of immune complexes that
enhance Ag processing and presentation to T cells by APCs or to B cells
by follicular dendritic cells (52, 53, 54). Although similar effector
functions might also be performed at a slower pace by the bulk of
follicular B cells (Fig. 7), the majority
of the Ags, at least for those of a particulate nature, is probably
scavenged and processed in the marginal zone and reaches the B cell
follicles mostly on the surface of follicular dendritic cells.
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), and it will be interesting to test
this hypothesis in vivo in an Ag-specific immune response. MZ B cells share some similar functional properties with Ag-specific memory B cells that have been previously described in mice, rats, and humans (4, 21, 57). They are long lived, localize in the same compartment of the spleen, and have lower activation thresholds for specific Ag in the case of memory B cells (4) or in response to costimulation (CD40 or LPS) for MZ B cells. Human memory cells from tonsils are able to present Ags without any extra in vitro activation, while mouse MZ B cells need brief periods of CD40 or LPS activation (57). Although no true memory appears to be involved in T-independent responses, postimmune Ag-specific cells were characterized in rats (21, 58), and since these cells do not depend on germinal center formation, they exhibit even greater similarity to mouse MZ B cells.
The ability of B cells to up-regulate both B7.1 and B7.2 costimulatory
molecules has been associated with polyclonal mitogens (LPS), various
cytokines, anti-CD40, and, recently, anti-CD21 and
anti-CD19 cross-linking signals (59, 60, 61). In contrast, anti-BCR
signals, either through Ag or anti-IgM Abs, preferentially
up-regulate B7.2 (42). The differences in these events between MZ and
FO B cells is not simply one of kinetics, since FO cells never reach
the levels of B7 expressed by MZ B cells (Fig. 4). These data suggest
that there are intrinsic differences within these populations regarding
B7 expression following activation, and that the resulting activated MZ
and FO effector B cells differ in their ability to use these pathways
of costimulation. When these B cell subpopulations are stimulated with
a mixture of two different reagents (Fig. 4B, LPS plus CD40
or IL-4), for some combinations there is a dramatic increase in B7
molecules compared with the use of each stimulus alone. As a result,
the levels of B7.2 expression on FO B cells become comparable to those
on MZ B cells, which require only one of the stimuli alone to be
maximally stimulated. The attainment of similar levels of expression of
B7.2 by both populations is reflected by their equivalent ability to
present alloantigens. These findings predict that depending on the type
of in vivo Ag stimulation, MZ or both subsets will participate in Ag
presentation. These results also suggest that there is a qualitative
difference in the regulation of B7.2 and subsequently of the
costimulatory activity between FO and MZ B cell subsets. Recently,
constitutive expression of the B7.2 molecule has been shown to break
the inability of tolerant FO B cells to become activated (62). The
higher levels of B7 constitutive expression and its rapid up-regulation
on MZ B cells raise questions about mechanisms that control these B
cells to avoid autoimmunity and suggest that their repertoire selection
and activation are stringently controlled in a healthy organism.
In humans, chronic activation is considered one of the potential steps involved in the induction of lymphomas; in particular for MZ lymphomas, molecular studies support the concept that they are derived from a postfollicular, postgerminal center or a memory B cell (29). In NFS.V+ mice, used as a model of lymphomagenesis, >40% of the spontaneous developing lymphomas derive from the MZ and, surprisingly, express low levels of CD5 and B220, a characteristic of the B1 subset of mouse B lymphocytes (63). In normal mice, splenic B1 cells are infrequent (13) and have not been assayed functionally, but the similarities between peritoneal B1 cells and MZ B cells (lack of proliferation to anti-IgM, larger size, ability to rapidly secrete Abs) suggest some kind of link, if not in their developmental origins at least in function. However, the selection and developmental expansion of B1 cells, in addition to being dependent on costimulation, are probably also linked to the specificity of their receptors (64, 65, 66, 67). Since B1 cells are depleted in several Ig TG mouse models that have normal or increased MZ populations (MD4, 81X), these B cells might not have receptors with permissive specificities that select them into the B1 compartment (16, 31).
Recently, several groups have reported that the level of expression of CD1 on MZ B cells is high and comparable to that found on dendritic cells, but there are no data yet on the functional significance of this finding (68, 69). NK T cells, which respond to Ags presented by CD1 molecules, are thought to be early participants in an immune response. The evidence we have presented indicates that MZ B cells show phenotypic and functional characteristics that would indicate that they are also early responders to antigenic challenge. We propose that certain nonprotein components of bacteria cell walls (Gram-negative derived mitogens such as LPS, newly described mitogens from Gram-positive bacteria (70), and glycolipids presented by CD1 (71)) would be targeted, in the first stage of an immune response, to this special subset of MZ B cells and prepare them for the subsequent phases of plasma cell generation and B-T cell interaction.
Based on the strategic localization of MZ B cells in the spleen and the findings described in this paper, we propose that MZ B cells constitute the first cohort of B cells that respond and participate in an immune response, in particular in conditions of limited T cell help, low concentration of TI mitogens, or low numbers of blood-borne particulate Ags reaching the spleen.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. John F. Kearney, University of Alabama, 378 Wallace Tumor Institute, 1824 6th Avenue South, Birmingham, AL 35294-3300. E-mail address:
3 Abbreviations used in this paper: FO, follicular B cell; MZ, marginal zone B cell; TG, transgenic; CMFDA, 5-chloromethylfluorescein diacetate; LM, littermate; BCR, B cell receptor for Ag; MFI, mean fluorescence intensity.
Received for publication December 14, 1998. Accepted for publication March 24, 1999.
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double-deficient mice. Int. Immunol. 8:23.
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A. Suzuki, T. Kaisho, M. Ohishi, M. Tsukio-Yamaguchi, T. Tsubata, P. A. Koni, T. Sasaki, T. W. Mak, and T. Nakano Critical Roles of Pten in B Cell Homeostasis and Immunoglobulin Class Switch Recombination J. Exp. Med., March 3, 2003; 197(5): 657 - 667. [Abstract] [Full Text] [PDF]
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H. Amano, E. Amano, T. Moll, D. Marinkovic, N. Ibnou-Zekri, E. Martinez-Soria, I. Semac, T. Wirth, L. Nitschke, and S. Izui The Yaa Mutation Promoting Murine Lupus Causes Defective Development of Marginal Zone B Cells J. Immunol., March 1, 2003; 170(5): 2293 - 2301. [Abstract] [Full Text] [PDF]
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H. Dalwadi, B. Wei, M. Schrage, T. T. Su, D. J. Rawlings, and J. Braun B Cell Developmental Requirement for the G{alpha}i2 Gene J. Immunol., February 15, 2003; 170(4): 1707 - 1715. [Abstract] [Full Text] [PDF]
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S. G. Tangye, D. T. Avery, E. K. Deenick, and P. D. Hodgkin Intrinsic Differences in the Proliferation of Naive and Memory Human B Cells as a Mechanism for Enhanced Secondary Immune Responses J. Immunol., January 15, 2003; 170(2): 686 - 694. [Abstract] [Full Text] [PDF]
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D. C. Otero, A. N. Anzelon, and R. C. Rickert CD19 Function in Early and Late B Cell Development: I. Maintenance of Follicular and Marginal Zone B Cells Requires CD19-Dependent Survival Signals J. Immunol., January 1, 2003; 170(1): 73 - 83. [Abstract] [Full Text] [PDF]
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T. Samardzic, D. Marinkovic, P. J. Nielsen, L. Nitschke, and T. Wirth BOB.1/OBF.1 Deficiency Affects Marginal-Zone B-Cell Compartment Mol. Cell. Biol., December 1, 2002; 22(23): 8320 - 8331. [Abstract] [Full Text] [PDF]
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S. Julien, P. Soulas, J.-C. Garaud, T. Martin, and J.-L. Pasquali B Cell Positive Selection by Soluble Self-Antigen J. Immunol., October 15, 2002; 169(8): 4198 - 4204. [Abstract] [Full Text] [PDF]
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Z.-Q. Wu, Y. Shen, A. Q. Khan, C.-L. Chu, R. Riese, H. A. Chapman, O. Kanagawa, and C. M. Snapper The Mechanism Underlying T Cell Help for Induction of an Antigen-Specific In Vivo Humoral Immune Response to Intact Streptococcus pneumoniae Is Dependent on the Type of Antigen J. Immunol., June 1, 2002; 168(11): 5551 - 5557. [Abstract] [Full Text] [PDF]
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W.-J. Won and J. F. Kearney CD9 Is a Unique Marker for Marginal Zone B Cells, B1 Cells, and Plasma Cells in Mice J. Immunol., June 1, 2002; 168(11): 5605 - 5611. [Abstract] [Full Text] [PDF]
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S. Itoh, M. Itoh, K. Nishida, S. Yamasaki, Y. Yoshida, M. Narimatsu, S. J. Park, M. Hibi, K. Ishihara, and T. Hirano Adapter Molecule Grb2-Associated Binder 1 Is Specifically Expressed in Marginal Zone B Cells and Negatively Regulates Thymus-Independent Antigen-2 Responses J. Immunol., May 15, 2002; 168(10): 5110 - 5116. [Abstract] [Full Text] [PDF]
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 H Schuster, T Martin, L Marcellin, J-C Garaud, J-L Pasquali, and A S Korganow Expansion of marginal zone B cells is not sufficient for the development of renal disease in NZBxNZW F1 mice Lupus, May 1, 2002; 11(5): 277 - 286. [Abstract] [PDF]
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T. L. Carvalho, T. Mota-Santos, A. Cumano, J. Demengeot, and P. Vieira Arrested B Lymphopoiesis and Persistence of Activated B Cells in Adult Interleukin 7-/- Mice J. Exp. Med., October 15, 2001; 194(8): 1141 - 1150. [Abstract] [Full Text] [PDF]
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Z. Hao and K. Rajewsky Homeostasis of Peripheral B Cells in the Absence of B Cell Influx from the Bone Marrow J. Exp. Med., October 15, 2001; 194(8): 1151 - 1164. [Abstract] [Full Text] [PDF]
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X. Li, F. Martin, A. M. Oliver, J. F. Kearney, and R. H. Carter Antigen Receptor Proximal Signaling in Splenic B-2 Cell Subsets J. Immunol., March 1, 2001; 166(5): 3122 - 3129. [Abstract] [Full Text] [PDF]
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S. R. Dillon, A. Constantinescu, and M. S. Schlissel Annexin V Binds to Positively Selected B Cells J. Immunol., January 1, 2001; 166(1): 58 - 71. [Abstract] [Full Text] [PDF]
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P. M. Dammers, A. Visser, E. R. Popa, P. Nieuwenhuis, and F. G. M. Kroese Most Marginal Zone B Cells in Rat Express Germline Encoded Ig VH Genes and Are Ligand Selected J. Immunol., December 1, 2000; 165(11): 6156 - 6169. [Abstract] [Full Text] [PDF]
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A. Cariappa, H.-C. Liou, B. H. Horwitz, and S. Pillai Nuclear Factor {kappa}b Is Required for the Development of Marginal Zone B Lymphocytes J. Exp. Med., October 16, 2000; 192(8): 1175 - 1182. [Abstract] [Full Text] [PDF]
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P. Sanchez, A.-M. Crain-Denoyelle, P. Daras, M.-C. Gendron, and C. Kanellopoulos-Langevin The level of expression of {micro} heavy chain modifies the composition of peripheral B cell subpopulations Int. Immunol., October 1, 2000; 12(10): 1459 - 1466. [Abstract] [Full Text] [PDF]
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H. Ogata, I-h. Su, K. Miyake, Y. Nagai, S. Akashi, I. Mecklenbrauker, K. Rajewsky, M. Kimoto, and A. Tarakhovsky The Toll-like Receptor Protein Rp105 Regulates Lipopolysaccharide Signaling in B Cells J. Exp. Med., July 3, 2000; 192(1): 23 - 30. [Abstract] [Full Text] [PDF]
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,
control FO; 
, control MZ; 
, anti-CD40 FO; 
, anti-CD40
MZ; 
, LPS FO; 
, LPS MZ; 
, anti-IgM-FO; •,
anti-IgM-MZ. Results represent one of three similar experiments.
B, Combinations of mitogens are even more potent in
up-regulating B7 molecules. Sorted FO and MZ B cells from 81X TG mice
were stimulated with LPS (thin line), anti-CD40, or IL-4 (normal
line) or with a combination of either LPS plus anti-CD40 or LPS
plus IL-4 (dotted line), and their B7 expression was assayed at 24
h by flow cytometry. Results represent one of three similar
experiments. C, Homogeneous populations of MZ and FO B
cells with the same specificity (anti-HEL) have different
functional properties. Sorted FO and MZ B cells from MD4 anti-HEL
TG mice were cultured with LPS (20 µg/ml) and stained for B7 at 24
and 72 h (left) or were assayed for proliferation
with anti-CD40 (10 µg/ml) and anti-IgM (20 µg/ml;
middle) or LPS (20 µg/ml; right).
Results represent one of three similar experiments.
