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,
,¶,
,¶,¶,
Departments of
* Pediatrics,
Pathology, and
Surgery, Harvard Medical School, and
Center for Blood Research and
¶ Brigham and Women’s Hospital, Boston, MA 02115
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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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Natural Abs are difficult to define due to their characteristically low
affinity and polyreactivity. All B cell subsets are capable of
producing natural Abs; CD5+ B-1 cells have been
identified as a major source in humans (14) and mice
(15). CD5+ B-1 cells are readily
distinguishable from "conventional" B-2 cells by their surface
phenotype, their ability for self-renewal, their anatomic location
(peritoneum vs spleen) and their restricted usage of particular
VH genes (16). For example, over
90% of the natural Ig product against phosphatidylcholine
(PtC),4 a ubiquitous
self-Ag, is derived from CD5+ B-1 cells through
restricted VH/V
unmutated germline genes
(VH11-V9 or
VH12-V4)
(17).
Despite these unique features, little is certain regarding B-1 cell ontogeny, development, and function. Analyses of knockout mice reveals that defects in B cell receptor (BCR) signaling have a more pronounced affect on the survival of B-1 cells than on conventional B-2 cells. For example, a deficiency in BCR signaling protein CD19 (18, 19), Bruton’s tyrosine kinase (20), or vav (21, 22), or chronic treatment of mice with the Ab specific for CD19 (from birth), leads to a loss of B-1 cells (23). Moreover, segregation of PtC-specific B-1 cells into the B-1 subset occurs after BCR expression (24). These findings suggest that B-1 cells require intrinsic BCR signaling for maintenance in the repertoire. Alternatively, B-1 cells might require an encounter with cognate Ag in their initial positive selection, expansion, or survival (24, 25). Examination of knockout mice homozygous-deficient in complement receptors CD21/CD35 (Cr2def) revealed a partial deficiency in the CD5+ subpopulation of B-1 cells (26), although in another line of Cr2def mice, no reduction in B-1 cells was identified (27). Although both lines have a similar defect in binding activation products of complement C3 (26, 27, 28, 29, 30), the former appears to express a low level of the truncated form of the receptor (31).
CD21 and CD35 are expressed primarily on B lymphocytes and follicular dendritic cells (FDC) and are encoded at a single locus (Cr2) (32, 33). Both receptors bind complement ligands iC3b and C3d and form a coreceptor signaling complex on B cells along with CD19 and CD81 (34). Mice deficient in CD21/CD35 have impaired humoral responses to thymus-dependent (26, 27, 28, 29, 35, 36) and -independent Ags (30). This defect is largely due to the absence of coreceptor signaling, although receptor expression on FDCs is also important in humoral immunity (37). B cell encounter of C3-coated Ag complexes results in coligation of the BCR and CD21/CD19/CD81 coreceptor, the effect of which not only lowers the threshold for B cell activation (38), but also provides a survival signal to germinal center B cells (39).
The reduction in the frequency of CD5+ B-1 cells in Cr2def mice may stem from a general requirement for complement during the initial expansion or maintenance of the cell. Alternatively, complement and coreceptor signaling might be required only for certain low affinity Ags. Given this apparent reduction in CD5+ B-1 cells and the relative importance of CD21/CD35 in regulation of humoral responses, we examined serum IgM and CD5+ B-1 cells isolated from Cr2def mice for binding of PtC and injury in vivo using a model for IgM-dependent ischemia-reperfusion (I/R) injury (40, 41). I/R injury represents an inflammatory response following ischemia in diverse tissues such as the intestine, hind limb, or myocardium (42). Injury is largely dependent on complement, as treatment of mice with a soluble inhibitor of complement C3 blocks full injury (43). Mice deficient in C3 are also protected from full injury (40). Complement appears to be activated by the classical pathway, as mice deficient in C4 are protected to a degree similar to that of C3def mice (40). The finding that mice totally deficient in lymphocytes (recombination-activating gene (RAG)-1def) are also protected, and that reconstitution with pooled IgM restores injury, supports an important role for natural IgM (41). Although the initiating Ag/Ags expressed on ischemic tissues are unknown, the I/R model provides a functional assay to evaluate the repertoire of natural IgM in Cr2def mice.
Examination of serum IgM and CD5+ B-1 cells isolated from Cr2def mice for binding of PtC and intestinal I/R injury in vivo revealed a similar frequency of PtC+ CD5+ B-1 cells in Cr2+ and Cr2def mice. By contrast, Cr2def mice were protected from reperfusion injury, despite normal levels of serum IgM. Full sensitivity to reperfusion injury was restored following reconstitution of the deficient mice with pooled IgM from wild-type (WT) mice or adoptive transfer of Cr2+ peritoneal B cells. Furthermore, in the I/R model, a direct comparison of RAG-2def mice reconstituted with IgM prepared from either WT or Cr2def mice revealed reduced pathogenic activity in the later.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The construction and generation of Cr2def mice was achieved through embryonic stem cell gene targeting as described (26). To verify deficiency, we tested tail DNA from the parents of all mice by Southern blot analysis. Male mice, 5–8 wk old, were used in all intestinal I/R experiments. WT control mice represent C57BL-6/129 Cr2+/+ littermates generated by crossing C57BL-6/129 Cr2 heterozygotes. Mice serving as donors for analysis of peritoneal B-1 cells were 4- to 5-wk-old female Cr2+ and Cr2def mice crossed onto a C57BL/6 background for 10 generations. Animals in this study were maintained in accordance with the guidelines of the Committee on Animals of Harvard Medical School (Boston, MA) and those prepared by the Committee on the Care and Use of Laboratory Animals of the Institute of Laboratory Resources, National Research Council (Department of Health, Education and Human Services, publication no. 85-23 (National Institute of Health), revised 1985).
Model of intestinal I/R injury
Surgically induced intestinal I/R has been described elsewhere (44). Briefly, mice anesthetized with pentobarbital i.p. (90 mg/kg) underwent laparotomy. Ligation of the jejunum and associated mesenteric vessels was achieved by application of a 10-cm loop tourniquet composed of 3-0 Prolene suture (Ethicon, Somerville, NJ) and 1-cm lengths of intramedic polyethylene tubing (BD Biosciences, Parsippany, NJ) used as a snugger. Ischemia was confirmed by lack of mesenteric pulsations and intestinal pallor. Incisions were closed and ischemia proceeded for 40 min. Each mouse received 1 µCi of 125I-labeled BSA i.v. 5 min before the reperfusion phase. Soluble complement receptor type 1-treated animals received 20 mg/kg protein simultaneously with radio-labeled BSA. After reopening the incisions and removing the tourniquets, mice were kept warm with heating pads for 3 h. All mice received 0.2 ml of saline i.v. 1 h after initiation of reperfusion. Sham-treated controls underwent a similar procedure without jejunal ligation.
Following euthanasia, blood was collected via cardiac puncture and jejunal loops were harvested, counted for radioactivity, and dried to constant weight for 3 days at 90°C. The intestinal permeability index (PI) was determined using the following ratio: (cpm/gram jejunal loop)/(cpm/gram blood). Mice were followed for 5 days after treatment for survival studies.
Reconstitution of Cr2def mice with purified WT IgM
The purification of normal mouse IgM from whole serum has been described previously (11). An immunoaffinity approach was used. Whole serum, obtained from either inhouse C57BL-6/129 mice or from commercial sources (EQUITECH-BIO, West Ingram, TX), was first precipitated by (NH4)2SO4 (50% saturation). The precipitate was dissolved in 1x PBS (10 mM Na2HPO4, 150 mM NaCl, pH 7.25) and dialyzed against 100 volumes of the same buffer. Recovered dialysate was then filtered through a 0.45-µm syringe filter (Acrodisc; Gelman Sciences, Ann Arbor, MI) and applied to a 2-ml protein G column (Gammabind Sepharose; Pharmacia Biotech, Uppsala, Sweden). After several passages over protein G, the flow-through was applied to a 4–8 ml anti-IgM agarose column (Sigma-Aldrich, St. Louis, MO). Eluates were obtained (0.1 M glycine-HCl, 0.15 M NaCl, pH 2.5) and neutralized. Peak fractions were pooled, dialyzed, and concentrated against 1x PBS using vacuum concentration (Schleicher & Schuell, Keene, NH). Simultaneous purification of murine IgG was achieved by elution of bound material from the protein G column in a similar fashion. Samples were assessed by 10% SDS-PAGE (45) for purity.
Before ischemic challenge, Cr2def mice were reconstituted with 0.35–0.4 mg of purified WT IgM via the retro-orbital plexus. Control Cr2def mice received an equal volume of HBSS (Sigma-Aldrich) i.v., serving as mock-reconstituted controls. A third group received 0.35–0.4 mg of WT IgG.
Reconstitution of Cr2def and RAG-1def mice with peritoneal B cells
Peritoneal B cells were obtained via peritoneal lavage of WT mice. Following euthanasia of 2- to 3-mo-old C57BL-6/129 mice, peritoneal lavage was performed using 5–10 ml ice-cold HBSS/1% BSA per mouse. The recovered cells were pooled, washed, and resuspended in lavage buffer before reconstitution. Trypan blue (Sigma-Aldrich) counterstained cells were counted via hemocytometer for viability. Approximately 1 x 106 cells were injected i.p. into 3- to 4-wk-old Cr2def male mice in a volume of 0.5 ml/mouse. Age-matched RAG-1def recipients (C57BL6/129 background) were used to determine the efficiency of reconstitution.
To verify successful reconstitution of CD21/CD35-deficient mice, peritoneal exudate cells (PEC) were harvested from reconstituted mice at 6–8 wk time points, erythrocyte-depleted via Ficoll-Hypaque separation (Ficoll PLUS; Pharmacia Biotech), washed with HBSS/10 mM EDTA, and incubated with biotin-conjugated B220 and 7E9, a monoclonal directed against CD35.
Immunohistological analysis of jejunal sections following I/R injury
Cryostat serial sections of intestinal tissue from saline-reconstituted, IgM-reconstituted, or peritoneal cell-reconstituted CD21/CD35-deficient mice were analyzed via immunoperoxidase labeling with goat anti-mouse IgM and C3 (5 µg/ml) (Organon Teknika, Durham, NC) and a previously described avidin-biotin protocol (46). WT, sham-treated WT, and Cr2def mice served as controls.
Flow cytometry analysis
PEC isolated from Cr2+ and Cr2def mice were obtained as previously described (26). Approximately 1 x 106 cells were washed twice in PBS containing 2% FCS and 0.02% sodium azide and subsequently incubated with a mixture containing either FITC-encapsulated anti-IgM, PE-conjugated anti-CD19 mAb (CD19-PE; BD PharMingen, San Diego, CA), and allophyocyanin-conjugated anti-CD5, or FITC-conjugated liposomes containing PtC- (a gift of Dr. L. A. Herzenberg, Stanford University, Palo Alto, CA), PE-conjugated CD19, and allophycocyanin-conjugated CD5 for 30 min on ice. After washing, cells were incubated with propidium iodide to identify dead cells. A fluorescence analysis was performed with aFACSCalibur (BD Biosciences) as described (26).
Statistics
PIs represent mean ± SD calculated using the Student t test. Comparison of survival following I/R over a 5-day period was performed by the Mantel-Cox rank test. Student’s t test was used to evaluate differences between the frequencies of peritoneal CD5+/CD19+ B-1 cells in Cr2def vs WT controls. Differences were considered statistically significant when p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although Cr2def mice bear normal levels of
serum IgM and serum complement proteins, we proposed that the
specificity of natural IgM might be altered given the general
impairment in B cell activation and the apparent reduction in
CD5+ B-1 cells. To test this hypothesis,
deficient mice were analyzed in an intestinal I/R model and compared
with mice deficient in complement proteins C3
(C3def) or C4 (C4def). In
this model, ischemia is induced by ligating the jejunum and mesenteric
vessels for 40 min with a tourniquet as described (36).
Before release of the tourniquet, mice were injected with radio-labeled
albumin. The extent of injury was evaluated after 3 h of
reperfusion based on increased permeability, i.e., the ratio of
radio-labeled protein in the blood vs dried tissues. Injury was also
assessed by histopathology. As expected, injury was reduced in
C3def and C4def mice
relative to WT controls (Fig. 1
a) (35, 36).
Interestingly, mice deficient in Cr2 were also partially protected, as
the PI of the mutant mice was 
43% that of
Cr2+ controls. These findings correlate with a
significant increase in 5-day survival following treatment,
i.e., 80 and 20% for Cr2def and
Cr2+, respectively (Fig. 1b).
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, c and d). By
contrast, the mucosa of Cr2+ controls was
characterized by significant necrosis (Fig. 2, a and
b). Immunohistological analysis of tissues revealed abundant
deposits of C3 and IgM on the vascular endothelium of treated
Cr2+ mice. By contrast, both proteins were
significantly reduced on tissues from Cr2def mice
(Fig. 2, c and d). Thus, deficiency in complement
receptors CD21/CD35 was protective in the intestinal I/R model.
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To determine whether protection is due to a cellular or a serum
defect, Cr2def mice were reconstituted with 0.4
mg of pooled IgM i.v. before treatment. A similar concentration of IgM,
which is below physiologic levels in normal serum (1–2 mg), was
previously shown to restore injury in treated
RAG-1def mice (41). Reconstitution
of Cr2def animals resulted in a reversal of
protection equivalent to that of Cr2+ animals,
based on PIs of the two groups (Fig. 3) and histopathology of the
jejunal mucosa (Fig. 2
, e and f). Most
interestingly, reconstituted Cr2def mice exhibit
C3 and IgM deposits on the vascular endothelium of ischemic tissues
(Fig. 2, e and f). It is important to note that
IgM is the isotype of natural Ab required to produce injury, as IgG
reconstitution of deficient mice had no pathogenic effect (Fig. 3).
These analyses provide firm evidence that nonimmune
Cr2+ serum contains a natural IgM that is
specific against postischemic tissue, and is missing in
Cr2def mice despite their normal level of
IgM.
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To determine whether Cr2+ peritoneal B-1
cells are a source of pathogenic autoantibody in the intestinal I/R
model, peritoneal B cells from adult Cr2+/+ mice
were harvested and transferred i.p. into 3- to 4-wk-old MHC-gender- and
age-matched Cr2def (WT
PEC
Cr2def) or RAG-1def
(WT PECRAG-1def) mice. Because
RAG-1def mice are totally deficient in B
lymphocytes and serum Ig, they served as controls for ascertaining
transfer of peritoneal B cells and confirming that B-1, but not B-2,
cells make up the dominant population of B lymphocytes. Indeed, within
6 wk after engraftment of 1 x 106 cells,
the levels of serum IgM in RAG-1def recipients
were 50% that of normal (data not shown). To confirm that B-1 cells
were the major B cell subset, spleen, peritoneal, and mesenteric lymph
node cells were harvested from the WT
PECRAG-1def chimeric mice 6–8 wk following
transfer. Monocytic cells were treated with mAbs specific for IgM,
CD11b (B-1 cell marker), and CD23 (B-2 cell marker) and examined by
FACS. As expected, the majority of the surviving
IgM+ B cells expressed the B-1 cell phenotype,
i.e.,
IgM+/CD11b+/CD23-
and <2% of B cells stained positive for CD23; i.e.,
IgM+/CD11b+, 95.7 ±
1.9% SD and
IgM+/CD23+, 1.2 ±
0.9% SD for WT PECRAG-1def vs
IgM+/CD11b+, 49.6 ±
5.3% SD and IgM+/CD23+,
43.9 ± 5.5% SD for B6 (Fig. 4a). Forster et al.
(47) made a similar observation reporting that
Ly-1+ (CD5), but not
Ly-1-, peritoneal B cells were long-lived and
self-replenishing. Thus, Cr2def chimeric mice (WT
PECCr2def) were considered reconstituted
primarily with donor Cr2+ B-1 cells. The presence
of donor B-1 cells in chimeras was confirmed by two-color flow
cytometric analysis following treatment with mAbs specific for B220 and
CD21/CD35. By 6 wk posttransfer, >50% of the
CD5+ peritoneal B cells stained positive for
CD21/35 (Fig. 4b).
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Cr2def chimeric
mice in the reperfusion model revealed a dramatic increase in injury,
equivalent to WT and IgM-reconstituted knockout mice, and significantly
higher than untreated Cr2def mice (Fig. 4c). Restoration of injury was
confirmed by histological examination of the intestinal tissue of
treated chimeric mice. The pathology observed in treated WT
PECCr2def chimeras was similar to that of IgM
recipients and WT controls; murine jejunal mucosa was marked by
necrosis as well as the deposition of C3 and IgM (Fig. 2, g
and h). Presumably, IgM detected by immunoperoxidase
staining is derived from Cr2+ peritoneal
CD5+ B-1 cells, as ungrafted mice fail to stain
positive (Fig. 2d). We concluded that
Cr2+ donor B-1 cells are a major source of the
I/R pathogenic autoantibody that is missing or reduced in the
Cr2def animals. IgM isolated from Cr2def mice is not pathogenic
To directly assess the presence of pathogenic IgM in the sera of
Cr2def mice, RAG-1def mice
were reconstituted with pooled IgM and tested in the intestinal I/R
model. Ig-deficient RAG-1def mice were
reconstituted with 0.4 mg of purified IgM isolated from the sera of
Cr2def or Cr2+ mice. Injury
was evaluated by PI and histopathology as described above. The PI index
of RAG-1def mice administered WT IgM was
increased relative to saline controls, as previously reported
(36), while the PI of RAG-1def mice
receiving pooled IgM prepared from Cr2def mice
was similar to that of saline controls (Fig. 5b).
Examination of tissue sections from the region of ischemia revealed
extensive necrosis of the villous architecture in
RAG-1def mice reconstituted with IgM prepared
from WT, as reported (36). By contrast, negligible injury
was observed in mice reconstituted with saline or
Cr2def IgM (Fig. 5a). Thus, based on
two different assays, the IgM isolated from
Cr2def mice does not appear to induce I/R
injury.
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A common specificity of B-1 cells is PtC; 10–15% of mouse
peritoneal B-1 cells are specific for this common membrane Ag
(48, 49). To directly assess the frequency of peritoneal
CD5+ B-1 cells in Cr2+ and
Cr2def mice, PECs were isolated from 4- to
5-wk-old female mice and treated with mAbs in combination with
PtC-bearing liposomes encapsulated with FITC. As previously reported
(26), a significant decrease in the mean frequency of
CD5+ B-1 cells in Cr2def
mice was observed, i.e., 59 vs 41% in Cr2+ and
Cr2def, respectively (p
< 0.0005; Fig. 6a).
Three-color FACS analysis of the CD5+ B-1
population (CD5+/CD19+) for
PtC-liposome binding predictably revealed that the
CD5+ B-1 subpopulation bound PtC, whereas
negligible binding was observed in the CD5- B-1
and conventional B-2 populations (Ref. 49 ; Fig. 6b). Interestingly, comparison of the frequency of
PtC+ CD5+ B-1 cells between
the two groups of mice indicated a similar mean frequency of
PtC-binding B cells (Fig. 6b). Thus, despite an overall
reduction in CD5+ B-1 cells in the
Cr2def mice, the frequency of PtC-binding B cells
among the CD5+ population was similar, i.e., 21%
for both.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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a), survival (Fig. 1b), and histopathology
(Fig. 2). Reconstitution of Cr2def or
RAG-1def mice with purified WT IgM, but not IgG,
restored I/R injury (Figs. 2, e and f, and 3).
Therefore, WT serum contains an autoantibody (or autoantibodies) that
recognizes ischemic endothelium and initiates injury via classical
pathway complement. Interestingly, 0.35–0.4 mg of IgM, <40% of the
normal level of serum IgM, was sufficient to restore injury in
CD21/CD35-deficient animals. Therefore, the 40–45% reduction in
CD5+ B-1 cells observed in
Cr2def mice is an unlikely explanation for
reduced injury. Our results suggest that the natural Ab repertoire is
altered in the deficient mice.
An earlier report identified a significant reduction in peritoneal
CD5+ B-1 cells in Cr2def
mice on a mixed C57BL-6/129 background (26), although in a
similar study using the same line of mice, no reduction in this subset
of peritoneal B-1 cells was reported (31). In the current
study, analysis of Cr2def mice bred onto a B6
background for 10 generations also revealed a significant reduction
(30–40%) in peritoneal CD5+ B-1 cells, i.e.,
59 vs 41% mean frequencies for Cr2+ and
Cr2def, respectively. Given the reduction in
CD5+ B-1 cells in the
Cr2def mice, it seemed likely that B-1 cells were
a possible source of pathogenic natural IgM. Adoptive transfer of
1 x 106 peritoneal B cells from unimmunized
Cr2+ mice to Cr2def mice
fully restored normal levels of injury (Figs. 2, g and
h, and 4c). Chimerism was confirmed by expression
of CD21/CD35 on the majority of CD5+ B-1 cells
harvested at 6–8 wk posttransfer (Fig. 4b). Analysis of
lymphoid tissues harvested from WT PECRAG-1def
chimeras 6–8 wk following transfer revealed that
CD11b+ B-1 cells were the major subset of
surviving B cells and that <2% were B-2 (Fig. 4a). Because
full injury is restored in the WT PECCr2def
chimeric mice, we concluded that B-1 cells are a primary source of I/R
pathogenic Ab, although we cannot rule out a role for conventional B-2
cells in normal mice. Studies by Forster and Rajewsky
(47), through transfer of CB.20 peritoneal cells to the
allotype-specific congenic mouse strain BALB/c, revealed that the
percent of donor-derived natural IgM in recipient mice posttransfer was
30% at week 10 and 50% at week 20. Similarly, reconstitution of
RAG-1def mice with 1 x
106 peritoneal B cells led to serum IgM
concentrations 50% those of normal by 6 wk posttransfer (R. R.
Reid and M. C. Carroll, unpublished observation). Considering the
kinetics of natural Ab production by donor cells, and the dramatic
reversal of protection seen in Cr2def chimeric
mice, it can be surmised that only minimal concentrations of specific
natural Ab are required for the induction of injury.
The finding that there was no difference in the frequency of PtC-binding CD5+ B-1 cells isolated from Cr2+ and Cr2def mice suggests that complement is not involved in the development or maintenance of this population. Although considered primarily a common self-Ag, PtC or cross-reacting Ags are also common among bacteria. Mice deficient in IgM are highly susceptible to acute septic peritonitis, but reconstitution with a PtC-specific hybridoma is partially protective (9).
Reperfusion injury represents an acute inflammatory response following ischemia that can affect both local and remote tissues (42). In at least four of the tissues examined, i.e., intestinal (41, 50, 51, 52, 53), hind limb (40, 44), myocardium (54), and CNS (55), injury is complement-dependent. Complement appears to be activated by the classical pathway and injury appears to be IgM-dependent in at least the hind limb (40) and intestinal models (41, 56). However, other mediators, such as mannan-binding protein, can also activate early complement components C1-C3 and could be involved in pathology following formation of the initial cellular injury by natural Ab (57). Classical pathway activation of C3 could trigger the alternative pathway and might be important in amplification of the cascade (51). It will be important to examine injury in mice bearing a defect in alternative pathway components, such as factor D-deficient mice (58).
The finding that Cr2def mice are protected, but that reconstitution with normal IgM restores injury, supports the model that pathogenic IgM is specific and not the result of binding of polyspecific natural Ab. Identification of a specific clone or clones of B cells that secrete pathogenic IgM will be important in the development of a possible therapy to block injury and to identify tissue Ags involved in injury. Whether the specificity of pathogenic IgM is similar for all tissues or varies for each tissue type, i.e., intestine, myocardium, or hind limb, cannot be determined from these studies. Identification of the specific ischemia Ag or specific B cells will hopefully resolve this important question.
In a recent study, Holers and colleagues (56) reported
that their line of Cr2def mice was protected from
injury in the intestinal I/R model based on necrosis of intestinal
mucosa, infiltration of neutrophils, and release of inflammatory
mediators such as leukotriene 
4 and
peroxidase. In their study, restoration of full injury required both
IgM and IgG. They also found that based on pathology and C3 deposition,
reconstitution with normal IgM alone restored injury, in concordance
with observations in our study. However, normal IgG was required for
neutrophil influx and release of mediators such as peroxidase and
leukotriene 4. The latter parameters were not
examined in our study. Thus, both lines of Cr2def
mice appear to have an altered repertoire of natural Ab.
The combined results from the analysis of two B-1 cell assays support the hypothesis that the complement system is important in shaping the repertoire of IgM natural Ab to certain, but not all, Ags. Complement and its receptors CD21/CD35, could be involved at several distinct stages in B-1 cell development. B-1 cells are thought to derive from a positive selection event at the immature B cell stage in the fetal liver and early neonatal period (24, 25). Because they are self-renewing, contact with cognate Ag may be important for B-1 cell expansion and/or maintenance within the repertoire. CD21/CD35 receptors are expressed primarily on B cells (late transitional and mature stages) and FDCs (44, 59). Because repopulation of Cr2def mice with Cr2+ B-1 cells restores I/R pathogenic IgM, and these cells are maintained in the Cr2def chimeras, it seems unlikely that CD21/CD35 expression on stromal (FDC) and myeloid cells is required for the maintenance of B-1 cells. Therefore, it is more probable that the role of complement is to enhance early positive selection or expansion of B-1 cells during neonatal development.
In summary, the repertoire of natural Ab in Cr2def mice is limited by at least one specificity, i.e., I/R pathogenic Ab. Adoptive transfer of peritoneal B cells or passive transfer of pooled IgM can restore the deficiency. By contrast, no reduction in the frequency of CD5+ PtC-binding CD5+ B-1 cells is observed in the deficient mice. We propose that the complement system is critical in shaping the repertoire of IgM natural Ab to certain, but not all, Ags.
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Acknowledgments |
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Footnotes |
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2 R.R.R. and S.W. contributed equally to the manuscript.
3 Address correspondence and reprint requests to Dr. Michael C. Carroll, Center for Blood Research, 200 Longwood Avenue, Boston, MA 02115. E-mail address: carroll{at}cbr.med.harvard.edu
4 Abbreviations used in this paper: PtC, phosphatidylcholine; BCR, B cell receptor; FDC, follicular dendritic cell; I/R, ischemia/reperfusion; RAG, recombination-activating gene; WT, wild type; PI, permeability index; PEC, peritoneal exudate cells.
Received for publication March 29, 2002. Accepted for publication September 10, 2002.
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 M. Zhang, E. M. Alicot, I. Chiu, J. Li, N. Verna, T. Vorup-Jensen, B. Kessler, M. Shimaoka, R. Chan, D. Friend, et al. Identification of the target self-antigens in reperfusion injury J. Exp. Med., January 23, 2006; 203(1): 141 - 152. [Abstract] [Full Text] [PDF]
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M. L. Hart, K. A. Ceonzo, L. A. Shaffer, K. Takahashi, R. P. Rother, W. R. Reenstra, J. A. Buras, and G. L. Stahl Gastrointestinal Ischemia-Reperfusion Injury Is Lectin Complement Pathway Dependent without Involving C1q J. Immunol., May 15, 2005; 174(10): 6373 - 6380. [Abstract] [Full Text] [PDF]
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S. D. Fleming, R. P. Egan, C. Chai, G. Girardi, V. M. Holers, J. Salmon, M. Monestier, and G. C. Tsokos Anti-Phospholipid Antibodies Restore Mesenteric Ischemia/Reperfusion-Induced Injury in Complement Receptor 2/Complement Receptor 1-Deficient Mice J. Immunol., December 1, 2004; 173(11): 7055 - 7061. [Abstract] [Full Text] [PDF]
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M. Zhang, W. G. Austen Jr., I. Chiu, E. M. Alicot, R. Hung, M. Ma, N. Verna, M. Xu, H. B. Hechtman, F. D. Moore Jr., et al. Identification of a specific self-reactive IgM antibody that initiates intestinal ischemia/reperfusion injury PNAS, March 16, 2004; 101(11): 3886 - 3891. [Abstract] [Full Text] [PDF]
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M. S. Diamond, E. M. Sitati, L. D. Friend, S. Higgs, B. Shrestha, and M. Engle A Critical Role for Induced IgM in the Protection against West Nile Virus Infection J. Exp. Med., December 15, 2003; 198(12): 1853 - 1862. [Abstract] [Full Text] [PDF]
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M. J. Burne-Taney, D. B. Ascon, F. Daniels, L. Racusen, W. Baldwin, and H. Rabb B Cell Deficiency Confers Protection from Renal Ischemia Reperfusion Injury J. Immunol., September 15, 2003; 171(6): 3210 - 3215. [Abstract] [Full Text] [PDF]
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