| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Cutting Edge |
Department of Medicine, University College London, London, United Kingdom
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Recent evidence has suggested that only a minority of newly formed B cells are selected for maturation through positive selection by Ag (7, 8, 9). Once the immature B cell has reached the spleen, it can develop into follicular (FO) or MZ B cells, and this decision may be governed by a number of factors including the strength of B cell receptor (BCR) signaling (10), and the drive to make a complete repertoire of natural Abs (6). Moreover, MZ and FO B cell numbers are also affected by other factors such as life span and retention and migration signals. B1 cell development also appears to be dependent on Ag selection (11). Whereas alterations in some molecules that affect B cell signaling result in parallel changes in B1 and MZ B cells, e.g., Aiolos (12), other examples demonstrate that these two B cell subsets are not always regulated in tandem as observed in CD22- mice in which B1 cells are increased but MZ B cells are reduced (13, 14).
Here we show that splenic MZ B cells, as well as B1 cells, are expanded in mice deficient in serum IgM, with a corresponding reduction in splenic FO B cells, and that replacement with polyclonal, polyreactive IgM, but not monoclonal or oligoclonal IgM, reverses the changes in these B cell subsets.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The generation of Sµ- mice has been described previously (2). Mice were backcrossed onto C57BL/6 mice for six generations and kept under specific pathogen-free conditions. Litter-matched controls were used for all experiments.
Generation of Vµ1s-transgenic mice
The Vµ1 construct (gift from Dr. M. Neuberger, Laboratory of
Molecular Biology, Cambridge, U.K.) encodes a VH
(a allotype) gene that binds to
anti-4-hydroxy-3-nitrophenenacetyl under control of a
VH promoter (15). It was
modified by restriction enzyme digestion so that the membrane exon of
the transgene was deleted but the secretory exon remained intact (Fig. 2
A). The resulting 11-kb construct Vµ1s was used for
pronuclear injection, and transgenic mice were generated by standard
techniques. The mice were tested for the presence of the transgene by
Southern blotting and PCR and were backcrossed six times onto C57BL/6
mice and subsequently onto Sµ- mice.
|
Flow cytometric analyses were performed using standard techniques, stained cells were analyzed on FACScan, and files were plotted using CellQuest software (BD Biosciences, San Jose, CA). The MZ:FO ratio was calculated using absolute numbers of B cells in the CD21highCD23low MZ compartment and the CD21intCD23high FO compartment.
Injections and immune responses
Sµ- and litter-matched controls were injected
i.p. on six occasions at regular intervals for 2 wk with 200 µg/mouse
of a purified monoclonal (either TEPC-183, unknown specificity (here
designated monoclonal A) or MOPC-104E, specific for 
-1,3-glucose
(here designated monoclonal B); Sigma-Aldrich, St. Louis, MO) or
polyclonal IgM Ab (Rockland, Gilbertsville, PA). To induce a
T-independent response, mice were immunized i.p. with 5 µg of
phosphorylcholine-Ficoll (PC-Ficoll; Bioresearch Technologies, Novato,
CA) in PBS. Mice were bled at regular intervals during the 2 wk after
the immunization.
ELISA
Ig and Ag ELISAs were performed as previously described (2) using anti-class or -allotype reagents (BD PharMingen, San Diego, CA). The myeloperoxidase ELISA was performed with a Captia myeloperoxidase ELISA kit (Trinity Biotech, Bray, Ireland).
Immunofluorescence analysis of tissue sections
Standard immunofluorescence staining procedures were used on frozen spleens sections stained with FITC-conjugated anti-mouse IgM and Texas Red conjugated anti-mouse IgD (BD PharMingen). Sections were viewed using a confocal microscope (Axiovert 100; Zeiss, Oberkochen, Germany).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Mature splenic B cells can be divided into MZ and FO B cells and
can be distinguished by surface markers using FACS. MZ B cells
(CD21highCD23low) are
expanded in 4-mo-old Sµ- mice, whereas FO B cells are
reduced (Fig. 1A). Whereas the
increase in MZ B cell numbers is 2.5-fold, the reduction in follicular
B cells is less (a 25% reduction) based on proportion of absolute cell
numbers for individual mice. Wild-type (WT) and Sµ- mice
have the same number of splenic B220+ B
lymphocytes (WT, 1.27 ± 0.26 x 107;
Sµ-, 1.12 ± 0.17 x
107). This contrasts with the cellular changes
observed after a T-independent response to PC-Ficoll where MZ B cell
numbers increase in both WT and Sµ- mice (compare
immunized with naive) whereas absolute FO B cell numbers are unchanged.
In older mice (12 mo) the MZ:FO B cell ratio is increased in WT mice
and the difference in MZ/FO B cell ratio between Sµ- and
WT mice is less striking at this age (Fig. 1, A and
B). Spleen sections taken from naive 4-mo-old
Sµ- mice stained with anti-IgM/anti-IgD
demonstrated a larger
IgMbrightIgDdull MZ area on
the periphery of the follicle compared with WT controls, further
demonstrating the expansion in MZ B cells in Sµ- mice
(Fig. 1C). There is also an increase in the number of
splenic CD21lowCD23low B
cells which predominantly consist of newly formed B lymphocytes but
would also contain splenic B1 cells. Although the numbers of splenic B1
cells are small compared with B2 cells, there is an increase in B1
cells (CD5+) in Sµ- mice (4.5
± 0.7% within the splenic B lymphocyte gate) compared with WT mice
(2.26 ± 0.3%), consistent with previous observations
(1).
|
To determine whether polyclonal IgM was critical in
governing the B cell compartments, two approaches were used. The first
was to generate a transgenic mouse line (Vµ1s, IgH a
allotype) in which B lymphocytes secreted a µ H chain, but the
construct was engineered so that no transgene expression was detectable
on the B cell surface (Fig. 2
A). These Vµ1s-transgenic
mice were backcrossed onto C57BL/6 mice and subsequently onto
Sµ- mice to test whether MZ expansion would be reversed.
Consistent with the removal of the CµM exons, B lymphocyte membrane
expression of the transgene was undetectable by FACS (Fig. 2B), but IgM (derived only from the transgene) was present
in the serum of Sµ-/Vµ1s mice (Fig. 2C).
These mice would therefore have a secreted oligoclonal repertoire
generated by a number of L chains pairing with the
VH chain. The second method of replacing
IgM was to administer a polyclonal preparation of IgM or two IgM
monoclonals i.p. every 3 days for 2 wk. The serum concentration of
these preparations during the 2-wk period were identical (Fig. 2C).
The main conclusion from these series of IgM replacement
experiments is that only the polyclonal IgM preparation returned the
increased splenic MZ:FO B cell ratio found in Sµ- mice
back to that observed in control mice as shown by FACS and tissue
staining (Fig. 2, D–F). A much smaller reduction in the
MZ:FO B cell ratio occurred after administration of monoclonal A. The
increased IgM staining on the periphery of the follicle seen in
Sµ- mice was restored to control levels by the
administration of polyclonal IgM but not by the other monoclonals or in
Vµ1/Sµ- mice (Fig. 2F).
Effects of IgM reconstitution on B1 cells
Consistent with previous observations, peritoneal B1a
cells were increased in Sµ- mice (1, 2).
This expansion in B1a cells was reversed by polyclonal IgM and to a
lesser extent by monoclonal A but not in those mice expressing the
Vµ1s transgene or the mice that had received the monoclonal IgM B
preparation (Fig. 3).
|
To determine whether the reactivity of the different IgM
preparations was associated with their ability to influence MZ and B1
cells, binding to four different Ags was assayed. Analysis of the
binding of these IgM preparations to the Ags tested confirmed the
multireactivity of polyclonal IgM which bound all four Ags strongly and
monoclonal A bound two Ags strongly, whereas monoclonal B bound only
one Ag (Fig. 4). Analysis using HEP-2
cells for antinuclear Ab staining demonstrated that the polyclonal
preparation and monoclonal A but not monoclonal B were positive
(data not shown). Thus, the Ag reactivity of the IgM preparations
correlated with their ability to reverse the changes in B cell subsets
found in Sµ- mice.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
It has been proposed that the balance between splenic MZ and FO B cells is regulated by B cell signaling and that increasing clonal B cell signals favors the formation of FO over MZ B cells (10). The pentameric structure of IgM, in contrast to IgG, can potentially yield a high avidity for Ag despite a low Ag-specific affinity. Moreover, one molecule of IgM can activate complement, whereas two IgG molecules are required; this may lead to enhanced complement coating of selected self Ags when complexed to IgM, resulting in more efficient engagement of coreceptor signals (16, 17). Consequently, BCR signaling could be reduced and MZ B cells preferentially formed in the absence of IgM, despite normal levels of serum IgG. A similar process may apply to B1 cells, which are also sensitive to Ag drive (11), because polyclonal IgM also reversed the expansion of this B cell subset. The MZ:FO B cell ratio increases in mice as they age (this work and Ref. 18), which may be explained by defective signaling through the BCR which is also associated with aging (19), because MZ B cells are preferentially formed when BCR signals are reduced (10). Thus, the less striking difference between older Sµ- and control mice could be attributed to the already dampened BCR signaling associated with age.
The expansion in MZ B cell numbers in Sµ- mice may be explained by a number of mechanisms apart from B cell signaling. Mice bearing Ig chain transgenes with a variety of Ag specificities have an increased MZ B cell compartment. Because these mice have a restricted Ab repertoire, it has been proposed that the drive to make a complete repertoire increases MZ B cell numbers (6). Clearly, in Sµ-mice, the secreted IgM repertoire is absent whereas a full membrane IgM repertoire is present. If this proposed drive arises from the serum Ab repertoire then one would predict, as is observed here, that in Sµ- mice MZ B cell numbers would be increased and that polyclonal but not monoclonal IgM would reverse the changes. The transgenic mouse Vµ1s crossed onto the Sµ- mouse also has a restricted secreted IgM repertoire with a normal membrane BCR repertoire, supporting the notion that it is the secreted repertoire that is important in governing B1 and MZ B cell numbers. This line of reasoning, however, presumes that the serum IgG repertoire does not constitute a full Ab repertoire. A loss of diversity in the Ab repertoire is also observed in aging (associated with increased production of autoantibodies) and may link the increased MZ:FO B cell ratio seen in the absence of IgM and as animals age (18, 20).
The fact that the administration of monoclonal and polyclonal IgM have different effects despite achieving the same serum concentration suggests that the increase in MZ B cells is not due to a feedback mechanism that simply senses the IgM concentration. However, polyclonal IgM may have an advantage even in this situation if a putative FcµR binds preferentially to the altered confirmation arising out of IgM complexed with Ag. An FcµR has been described, although not with these properties (21). However, a recent report has demonstrated that serum IgM is a ligand for CD19 (22), and B1 and MZ B cells are affected by alterations in CD19 expression (8, 23).
One apparent paradox of these observations is the short time (2 wk) required to reverse the MZ:FO ratio coupled with the evidence that MZ and FO B cells have a relatively long life span (24). This long life span, however, is somewhat dependent on the numbers of immature B cells entering the mature B cell pool. The finding that newly formed B cells increase in Sµ- mice may suggest either that these cells are being retained in this pool longer or that there are more immature B cells exiting from the marrow. It is known, and confirmed here, that MZ B cell numbers change during a 2-wk period after encountering T-independent Ags (6). Thus, changes in these B cell subsets could occur through a variety of mechanisms including alterations in cell proliferation or apoptosis, or by interchange between the MZ and FO B cell pools, although how IgM influences these processes remains to be investigated. It is tempting to speculate that IgM, through regulation of these B cell subsets, may have a role in suppressing the autoimmunity that occurs in mice strains where the MZ and B1 B cells are expanded.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Michael Ehrenstein, Department of Medicine, University College London, Arthur Stanley House, 40-50 Tottenham Street, London, W1T 4NJ U.K. E-mail address: m.ehrenstein{at}ucl.ac.uk
3 Abbreviations used in this paper: Sµ-, serum IgM deficient; MZ, marginal zone; FO, follicular; BCR, B cell receptor; PC, phosphorylcholine; WT, wild type.
Received for publication September 12, 2002. Accepted for publication October 24, 2002.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  S. Yu, P. K. Maiti, M. Dyson, R. Jain, and H. Braley-Mullen B cell-deficient NOD.H-2h4 mice have CD4+CD25+ T regulatory cells that inhibit the development of spontaneous autoimmune thyroiditis J. Exp. Med., February 21, 2006; 203(2): 349 - 358. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Kanayama, M. Cascalho, and H. Ohmori Analysis of Marginal Zone B Cell Development in the Mouse with Limited B Cell Diversity: Role of the Antigen Receptor Signals in the Recruitment of B Cells to the Marginal Zone J. Immunol., February 1, 2005; 174(3): 1438 - 1445. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. D. de Stahl, J. Dahlstrom, M. C. Carroll, and B. Heyman A Role for Complement in Feedback Enhancement of Antibody Responses by IgG3 J. Exp. Med., May 5, 2003; 197(9): 1183 - 1190. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
, IgMa allotype.
D, Representative density plots to define proportion of
MZ and FO cells using CD21 and CD23 as markers after administration of
monoclonal A, monoclonal B, and polyclonal IgM (poly) and in
Sµ-/Vµ1s mice. The density plots shown are gated on
B220+ B cells. E, Histogram showing splenic
MZ:FO B cell ratio calculated using absolute cell numbers in individual
cell fractions as shown in D. MZ and FO were defined
from FACS analysis as illustrated in Fig. 1
