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* Department of Cellular Injury, Walter Reed Army Institute of Research, Silver Spring, MD 20910;
Department of Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD 20814;
Beltsville Human Nutrition Research Center, U.S. Department of Agriculture, Agricultural Research Service, Beltsville Agricultural Research Center-East, Nutrient Functions and Requirements Laboratory, Beltsville, MD 20705;
Departments of Medicine and Immunology, University of Colorado Health Science Center, Denver, CO 80262; and
¶ Department of Pathology, University of Iowa College of Medicine, Iowa City, IA 52242
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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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Evidence that complement activation is involved in the effector phases of intestinal IR injury has been provided by studies which showed that inhibition of the complement pathway at the point of C3 or C5 activation can either prevent or substantially attenuate intestinal injury (2, 3, 4, 5, 6, 7). In support of these findings, inflammatory mediators generated during complement pathway activation, such as the anaphylatoxin C5a and the membrane attack complex (MAC), are known to be able to directly cause cellular activation and injury (8, 9, 10, 11, 12).
The exact mechanism of complement activation during intestinal IR remains unclear, and there is relatively little information regarding the specific complement activation pathways (classical, alternative, or lectin) involved in tissue injury. However, two observations have strongly implicated the classical pathway in this process. The first is that intestinal IR injury is markedly attenuated in RAG-1-/- mice, and this protective effect is lost after reconstitution of these Ig-deficient mice with purified IgM natural Ab to normal levels (6). The second is that mice with normal levels of natural Ab, but in which the gene encoding complement C4 is inactivated (C4-/-), are protected from injury (6). The importance of natural IgM Ab and the classical complement pathway in mediating IR injury of skeletal muscle has also been shown using a similar experimental strategy with C3, C4, and Ig-deficient mice (13).
From these and other findings, it has been proposed that natural IgM Abs bind to neoantigens revealed on the surface membrane of cells injured by hypoxia in the intestinal or skeletal muscle vasculature during the ischemic phase and subsequently activate complement by recruiting C1 and then cleaving C4 (6). This is followed by the generation of complement C3 and C5 activation fragments as well as the MAC, with ensuing increases in adhesion molecule expression and release of a cascade of inflammatory mediators, including leukotriene B4 (LTB4) and others (3, 7).
Natural Abs are produced primarily by B-1 B cells, which in the adult mouse are found primarily in the peritoneum, and are frequently found to be polyreactive at low affinity with multiple self Ags (14, 15, 16, 17). Natural Abs are an important part of the defense against infection and have been found to be protective against challenge with bacterial (18) as well as viral (19, 20) pathogens and to play an important role in the clearance of endotoxin (21).
Complement receptor (CR) type 2 (CR2/CD21) is an important membrane receptor that greatly enhances B cell receptor (BCR)-mediated activation by binding complement iC3b/C3d-bound Ags and engaging the CR2/CD19/CD81 signaling complex (22, 23, 24, 25, 26). This recognition mechanism lowers the threshold for activation of B cells (27). In mice, CR2 is encoded along with the larger CR1 by the Cr2 gene, which produces both proteins through alternative splicing of a common mRNA (28, 29). CR2-/- mice demonstrate a substantial defect in the generation of IgG-switched isotype responses (30, 31, 32) as well as impaired B cell memory following immunization with T-dependent Ags (33, 34).
CR2 and CR1 have been potentially linked to the regulation of B-1 B
cell-derived natural Abs by the observation that there is an 
50%
decrease in peritoneal B-1 B cell numbers in one strain of
Cr2-/- mice (30).
Cr2-/- mice made by our group did not
demonstrate decreased B-1 cells at either an early (32) or
later (see Results) backcross to C57BL/6, and, like the
other strain of receptor-deficient mice, has also consistently
demonstrated quantitatively normal levels of serum IgM. Nevertheless,
based on these reported B-1 B cell findings, our previous demonstration
that peritoneal B-1a and B-1b B cells express readily detectable levels
of CR2 and CR1 (35), and the well-established role of CR1
and CR2 in T-dependent B-2 B cell responses, we sought to determine
whether the complement system might be important not only in the
effector phases of tissue injury, as discussed above, but also in the
development of a pathogenic subset of natural Abs that recognize
neoantigens on ischemic tissue and then initiate intestinal IR
injury.
In the present study, we subjected mice to intestinal IR injury and found that Cr2-/- mice were protected from the induction of tissue injury. The defect was shown to lie in an altered repertoire of natural Abs by the demonstration that IgM and IgG purified from the serum of wild-type C57BL/6 mice, when injected before the ischemic phase, were able to reconstitute IR-induced injury in Cr2-/- mice. Of interest, transferred IgM and IgG each contributed to different aspects of tissue injury but together allowed the development of a complete injury phenotype. These findings demonstrate a previously unrecognized role for CRs in the development within the natural Ab repertoire of a tissue injury-inducing subset of Abs. We propose that these Abs have developed to serve a critical role in recognizing ischemic intestinal tissue and initiating processes that result in either repair or removal of the damaged regions.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Adult male and female Cr2-/- and control Cr2+/+ mice were produced following seven generations of backcrossing to the C57BL/6 strain by intercrossing a single pair of Cr2+/- mice to create Cr2-/- and Cr2+/+ progeny, which were then bred and maintained as two sublines.
Experimental protocol
Mice were prepared for surgery, after at least a 7-day acclimation period following shipment, in the Uniformed Services University for the Health Sciences animal facility. Anesthesia was induced with ketamine (16 mg/kg) and xylazine (8 mg/kg) administered by i.m. injection. All procedures were performed with the animals breathing spontaneously and body temperature maintained at 37°C using a water-circulating heating pad. Experiments were performed according to the principles set forth in the "Guide for the Care and Use of Laboratory Animals" (Institute of Animal Resources, National Research Council, Department of Health, Education, and Welfare publication no. 85-23).
Animals were subjected to IR as previously described (7).
Briefly, a midline laparotomy was performed before a 30-min
equilibration period. The superior mesenteric artery was then
identified and isolated, and a small nontraumatic vascular clamp (Roboz
Surgical Instruments, Rockville, MD) was applied for 30 min. After this
ischemic phase, the clamp was removed under direct visualization and
the intestine was allowed to reperfuse for 2 h. In some
experiments, 30 min before laparotomy animals were given 1 mg of
purified IgG or IgM, or both in combination, by i.v. injection. Some
mice received 1 mg IgA in an identical fashion while others received an
equal volume of normal saline. Sham animals were subjected to the same
surgical intervention, except they did not undergo superior mesenteric
artery occlusion. To control for the effects of Ab injection, Igs were
administered to sham-treated mice as well. The laparotomy incisions
were sutured and the animals were monitored during the reperfusion
period. Additional ketamine and xylazine was administered by i.m.
injection as needed (total given was 4 mg/kg ketamine and 2 mg/kg
xylazine) and immediately before euthanasia. After euthanasia, the
small intestine 10–20 cm distal to the gastroduodenal junction was
removed for histologic and immunohistochemical analysis as well as for
the measurement of inflammatory mediators as described below. There was
no significant difference in survival between treatment and control
groups.
Histology and immunohistochemistry
Immediately after euthanasia, segments of small intestine specimens were fixed in 10% buffered formalin, embedded in paraffin, sectioned transversely in 5-µm sections, and stained with Giemsa. Score of mucosal injury (SMI) was graded on a six-tiered scale as described previously (3, 36). In addition, the villus height of at least 10 villi from the same section was measured using an ocular micrometer.
Additional tissue was fixed for 2 h in cold 4% paraformaldehyde in PBS before transfer to PBS for paraffin embedding and preparation of transverse sections. Following removal of paraffin from sections, nonspecific Ab binding sites were blocked by treatment with a solution of 20% rat serum in PBS for 30 min. After washing in PBS, the tissues were incubated with goat anti-mouse C3, IgG, or IgM (ICN Pharmaceuticals, Costa Mesa, CA) Ab overnight at 4°C. The tissue was then incubated with a biotinylated rabbit anti-goat secondary Ab (Jackson ImmunoResearch Laboratories, West Grove, PA) followed by streptavidin conjugated to either fluorescein or 7-amino-4-methylcoumarin-3-acetic acid (Jackson ImmunoResearch Laboratories). The slides were then mounted with Fluoromount-G (Southern Biotechnology Associates, Birmingham, AL). A blinded observer examined the slides by fluorescent microscopy using a Leica DM RX/A fluorescent microscope (Leica Microsystems, Atlanta, GA) with SPOT diagnostic computer software (Diagnostic Instruments, Sterling Heights, MI).
Ig ELISA
Sera collected from 14 Cr2+/+ and 10
CR2-/- age-matched mice were added in
serial dilution to ELISA plates that had been coated overnight with
goat anti-mouse 
(Southern Biotechnology Associates) or rat
anti-mouse IgM (Southern Biotechnology Associates) and then blocked
with 1% BSA/PBS. Plates were washed with PBS/0.1% Tween 20 and test
serum samples or purified mouse Abs (IgG and IgM; Southern
Biotechnology Associates) for standard curves were added and incubated
overnight at 4°C. Following washing, plates were incubated with
alkaline phosphatase-conjugated goat anti-mouse IgG or goat
anti-mouse IgM (Caltag Laboratories, Burlingame, CA), respectively.
Plates were washed, substrate was added, and then the
OD405 was measured. The concentration of IgM and
IgG was calculated using the standard curve.
Eicosanoid determination
The ex vivo generation of eicosanoids by small intestine tissue was determined as previously described (37). Briefly, fresh mid-jejunum sections were minced, washed, and resuspended in 37°C oxygenated Tyrode’s buffer (Sigma-Aldrich, St. Louis, MO). After incubating for 20 min at 37°C, supernatants were collected and stored at -80°C until assayed. The concentration of LTB4 was determined using an enzyme immunoassay (Cayman Chemical, Ann Arbor, MI). The tissue protein content was determined using the bicinchoninic acid assay (Pierce, Rockford, IL) adapted for use with microtiter plates. LTB4 levels were expressed per milligram of protein per 20 min.
Peroxidase activity
Supernatants generated for the eicosanoid assays were also used to determine peroxidase activity by measuring oxidation of 3,3',5,5'-tetramethylbenzidine as described previously (38). Briefly, supernatants were incubated with equal volumes of 3,3',5,5'-tetramethylbenzidine peroxidase substrate (Kirkegaard & Perry, Gaithersburg, MD) for 45 min. The reaction was stopped by the addition of 0.18 M sulfuric acid, and the OD450 was determined. The concentration of total peroxidase was determined using HRP (Sigma-Aldrich) as a standard and plotted as picograms of myeloperoxidase activity per milligram of tissue.
Ig preparation and purification
A total of 100 ml of serum from C57BL/6 mice was purchased from Charles River Breeding Laboratories (Wilmington, MA). For IgM purification, the serum was first fractionated by the addition of ammonium sulfate (50%). The precipitate was dialyzed into PBS and then passed through a Superdex HL-200 gel filtration column. The IgM containing fractions were pooled, concentrated, and dialyzed into sterile saline. The IgG fraction was obtained by passing the dialyzed supernatant of the 50% ammonium sulfate precipitate through a HiTrap DEAE column equilibrated in 50 mM Tris-HCL buffer (pH 8.6). A continuous gradient to 50 mM Tris-HCL (pH 8.6) and 1 M NaCl was used to elute the IgG material. Positive fractions were pooled, concentrated, and dialyzed into sterile saline. Purity of each preparation was assured by SDS-PAGE and isotype-specific Western blot analysis with either HRP-conjugated polyclonal anti-mouse IgG or anti-IgM. Purified IgA was purchased from Sigma-Aldrich. For some experiments, blood from Cr2-/- mice was collected by cardiac puncture, following which serum was obtained and pooled for injection into additional Cr2-/- mice that underwent intestinal IR injury.
Statistical analysis
Data are presented as mean ± SEM and were compared by one-way analysis of variance with post hoc analysis using Newman-Keuls test (Instat; GraphPad, Philadelphia, PA). The difference between groups was considered significant when p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We tested the hypothesis that the tissue damaging natural Ab
repertoire is altered in Cr2-/- mice by
subjecting these mice and their Cr2+/+
controls to mesenteric IR. We first showed that the intestines of
sham-treated Cr2+/+ mice were
macroscopically and microscopically normal (Fig. 1
A), whereas those that had
undergone mesenteric ischemia for 30 min followed by 2 h of
reperfusion were swollen, edematous, and dusky, indicative of
hemorrhage and severe injury (Fig. 1B). In addition, many
villi were denuded with exposed lamina propria and hemorrhage. Tissue
injury was also quantitatively scored on a scale of 0–6 as previously
described to derive a SMI (3, 36). The SMI for
Cr2+/+ mice following IR was 3.3 ±
0.3, significantly elevated when compared with sham-treated
Cr2+/+ mice (Fig. 2A). In addition, the villus
height was significantly lower in mice with IR injury compared with
sham-treated Cr2+/+ mice (Fig. 2B), indicative of loss by sloughing of ischemic portions of
the intestinal tissue.
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and 2).
In contrast to Cr2+/+ mice, though, IR did
not induce significant injury in Cr2-/-
mice (Fig. 1D), with an injury score of 0.6 ± 0.2 and
average villus height of 33.4 ± 3.1 µm (Fig. 2). These results
demonstrate that the presence of CR2 and CR1 is necessary for the
induction of intestinal IR injury. Cr2-/- mice have a qualitative but not quantitative difference in serum IgM and IgG
Because CR2 and CR1 are involved in B cell responses to foreign Ags, it was considered possible that these mice might manifest substantially decreased serum levels of IgM and IgG Abs. If this were the case, intestinal IR injury might be prevented in Cr2-/- mice simply because of a lower level of serum natural Abs that was then insufficient to initiate injury. However, as was found in the initial analysis of this strain of mice at the F1 stage of breeding (32), both IgM and total IgG levels were not significantly different when comparing Cr2+/+ to Cr2-/- mice (data not shown). These results indicate that the lack of intestinal IR injury in Cr2-/- mice is not due to a markedly lower level of serum IgM and IgG but rather to apparent differences in the composition of the natural Ab repertoire.
As part of this analysis, we have also re-examined Cr2-/- mice to determine the numbers of B-1 B cells in the spleen and peritoneum following backcrossing seven generations to the C57BL/6 strain. As in our first analysis of these mice (32), we were again unable to find substantial differences in the numbers of B-1a and B-1b B cells in the peritoneum and B-1a cells in the spleen of Cr2+/+ and Cr2-/- mice, and the numbers of splenic B-2 B cells were also identical (data not shown). Therefore, although these data, as well as those presented below, indicate that there is a relative defect in B cell function, there are no apparent differences in the numbers of B-1 or B-2 B cells that develop in this strain of Cr2-/- mice.
In situ analysis of IgM, IgG, and complement C3 deposition
Previous studies have found that IgM and C3 are deposited in
ischemic tissues (2, 3, 4, 5, 6, 7). The deposition of these proteins
as well as IgG in the intestines of mice subjected to IR was determined
by immunohistochemistry. As expected, there was no significant
deposition of IgM, IgG, or C3 in the intestines of sham-operated mice
(Fig. 3). Similar to that seen by others,
we also found that IgM, IgG, and C3 were deposited on the ischemic
tissue of Cr2+/+ mice after IR injury.
However, consistent with the lack of injury, the intestines of
Cr2-/- mice subjected to IR injury did
not appear to have significant deposition of IgM, IgG, or C3.
Importantly, we have shown that sera from
Cr2+/+ and
Cr2-/- mice have equivalent ability to be
activated by a heterologous IgG-opsonized target (data not shown),
demonstrating that alterations in the endogenous complement system do
not underlie the phenotypic differences. These results suggest that
even though Cr2-/- mice have normal
levels of serum IgM and total IgG there is a qualitative difference in
the Ab produced in these mice manifested by the lack of reactivity with
neoepitopes on ischemic tissue.
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Prevention of injury in Cr2-/- mice
could in principle be due to the lack of receptors on a cell population
that are key to inducing local injury, rather than a lack of a
particular subset of natural Abs. For example, CR1 and CR2 are
expressed on activated murine polymorphonuclear cells
(39). To determine whether this was the case, we asked
whether infusion of Cr2-/- mice, before
the ischemic phase, with IgM and IgG purified from the serum of
wild-type C57BL/6 mice would result in intestinal IR injury. As a
control, some mice were also pretreated with an identical amount of
purified IgA. Additional controls included sham-treated animals treated
with IgM, IgG, IgA, or IgM and IgG. As shown in Figs. 1 and 2,
pretreating Cr2-/- mice with purified IgM
alone induced edema and damage to the epithelial cells in the tips of
villi, resulting in an injury score of 2.3 ± 0.05. Pretreatment
of Cr2-/- mice with purified IgG alone
did not generate IR-induced mucosal injury. However, when
Cr2-/- mice were treated with both IgM
and IgG, intestinal IR injury was significant, with pronounced
destruction of villi and a SMI of 3.8 ± 1. The damage was not the
result of transfer of Abs alone, as sham-operated
Cr2+/+ or
Cr2-/- mice that received IgM, IgG, or
both IgM and IgG did not develop significant injury (data not shown).
In addition, pretreatment with IgA did not result in the induction of
intestinal IR injury (Fig. 2), suggesting that the injury-inducing Ig
is found only in IgM and IgG isotypes. Finally, to determine whether
Cr2-/- mice had only slightly lower
levels of injury-inducing Abs, we pretreated
Cr2-/- mice before the initiation of the
ischemic phase with 400 µl of pooled serum derived from other
Cr2-/- mice containing 250 µg of IgM
and 450 µg of IgG. There was no significant injury in these animals,
as indicated by an injury score of 0.2 ± 0.1.
Cooperative effects of transferred IgM and IgG Ab in the induction of intestinal IR injury in Cr2-/- mice
To confirm that restoration of the mucosal injury in
Cr2-/- mice treated with IgM and/or IgG
activated complement locally in the intestine, intestinal tissues from
Cr2-/- mice, which were treated with IgM
or IgG, were analyzed for C3 deposition. Fig. 4 demonstrates that C3 was deposited only
in the intestines of mice subjected to IR and treated with IgM.
Therefore, IgM is the most potent complement-fixing isotype in this
model.
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, when compared with
sham-operated animals, Cr2+/+ mice
subjected to IR demonstrated substantially elevated
LTB4 generation. Consistent with the lack of
injury in Cr2-/- mice, there was no
increase in LTB4 production following IR.
Interestingly, in contrast to the lack of significant injury
measured histologically, reconstitution of
Cr2-/- mice with wild-type IgG
significantly increased LTB4 production. In
contrast, reconstitution with wild-type IgM did not result in increased
LTB4 generation. Reconstitution with both IgG and
IgM also resulted in a significant increase of
LTB4 generation. These data suggest that the IgG
natural Abs are critical for IR injury-induced
LTB4 production and provide one explanation for
their additive effects with wild-type IgM.
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demonstrates that, as expected, PMN
infiltration and peroxidase activity were increased substantially in
tissues of Cr2+/+ mice subjected to IR when
compared with sham-operated mice. In contrast, tissue from
Cr2-/- mice subjected to IR did not
demonstrate an increase in either PMN numbers or peroxidase activity.
This is again consistent with the lack of injury induced in this
setting. In Cr2-/- mice reconstituted
with IgG, there was a substantial increase in both PMN infiltration and
peroxidase activity. Although reconstitution of
Cr2-/- mice with IgM also demonstrated a
minor increase, the pronounced increase with IgG alone provides a
further potential explanation for its additive effects in the induction
of local injury.
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B). In contrast, IR did not
induce ICAM expression in tissues from
Cr2-/- mice (Fig. 7C). Of
interest, Cr2-/- mice reconstituted with
IgM did express ICAM on the vascular endothelial cells after IR (Fig. 7E), while those treated with IgG did not (Fig. 7D). As expected, Cr2-/- mice
reconstituted with both IgG and IgM strongly expressed vascular
endothelial cell ICAM-1 (Fig. 7F).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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There are several major findings that support these conclusions. First, Cr2-/- mice do not develop intestinal IR injury despite having levels of serum IgM and total IgG that are indistinguishable from Cr2+/+ mice. Second, the lack of intestinal IR injury in Cr2-/- mice is associated with a lack of local complement activation, generation of LTB4, and recruitment of polymorphonuclear cells as measured by tissue peroxidase levels. Third, transfer of purified IgM and IgG obtained from wild-type C57BL/6 mice together into Cr2-/- mice before the ischemic phase restores the development of intestinal IR injury in these mice. Transfer of serum from Cr2-/- mice does not reconstitute injury. Finally, pretreatment of Cr2-/- mice with wild-type IgM and IgG individually results in a pattern of isotype-specific local activation of complement as well as the generation of LTB4 and the recruitment of polymorphonuclear cells.
Several additional issues relevant to this mouse strain were also explored in these studies. The numbers of B-1 (B-1a and B-1b) as well as B-2 B cells were found to be essentially identical in Cr2+/+ and Cr2-/- mice following seven generations of backcrossing to C57BL/6, which is the same result as that originally reported for the F1 analysis of this CR2/CR1-deficient strain (32). In addition, the lack of expression of either CR2 or CR1 in this strain was verified by flow cytometry, and a functional deficiency was shown by the inability to flux Cai2+ in response to co-cross-linking of the B cell receptor with CR2/CR1 (Ref. 40 and data not shown). Finally, Cr2-/- mice at this backcross also continue to manifest the original phenotype that consists of a severe defect in the switched IgG response to T-dependent Ags (41).
In our studies we have not yet determined whether the lack of generation of a pathogenic subset of natural Abs is due to a defect in B-1 or B-2 B cells, as both contribute to the serum pool of IgM and IgG (15, 16, 19). In support of the concept that B-1 B cells generate this subset is the extensive previous analysis of these cells demonstrating that they produce a large repertoire of low-affinity self-reactive Abs that result from positive selection during B-1 cell development (14, 42, 43, 44). If CR2/CR1 coreceptor function on B-1 B cells is required for the efficient recognition of a critical self Ag that is either identical to, or antigenically related to, the neoantigen revealed on ischemic tissue, this could, in principle, explain our findings relative to the lack of development of this subset of Abs. A previous review of the role of CR2/CR1 in B-1 cell selection has also suggested this possibility (45, 46).
It is important to point out that our results do not exclude a role for B-2 B cells in this phenotype. B-2 B cells generate a portion of the IgM natural Ab repertoire as well as the great majority of the natural IgG repertoire, the latter in likely response to environmental Ag exposure (44). In particular, marginal zone B cells are responsible for the immediate IgM response to particulate Ags and, like B-1 cells, are responsible for a substantial portion of the IgG3 response (15, 47, 48). It is apparent from our studies that both IgM and IgG must be given to Cr2-/- mice to generate a full injury phenotype. It is also intriguing that marginal zone B cells in Cr2-/- mice, while present in apparently normal numbers (Ref. 49 and data not shown), do not appear to trap blood-borne, complement activating, T-independent Ags appropriately (50). Therefore, it is possible that B-2 B cells recognize a foreign environmental Ag and generate a response that cross-reacts with the neoantigen(s) revealed on ischemic intestinal tissue. The lack of efficient T-dependent follicular and marginal zone B-2 B cell responses in Cr2-/- mice would be consistent with this finding. Of course, it is also possible that both B cell subsets are necessary in this complex phenotype. Also not explored yet are two additional possibilities. One is the potential role of CR2 acting as a target for the alternative pathway of complement (51), and the other the role of intestinal mast cell CR1 and CR2 (52) in potentiating injury. Although the IgM and IgG transfer experiments rule out a primary role for either of these mechanisms in this phenotype, each may play an enhancing role.
Two additional points regarding the mechanism of tissue injury deserve further comment. First, LTB4 is generated by arachidonic acid metabolism via the 5-lipoxygenase pathway in the intestinal mucosa in response to reperfusion and complement activation (52, 53, 54, 55). The specific cells that mediate the generation of LTB4 are likely to be phagocytic cells (either resident macrophages or polymorphonuclear cells) that are present in the intestinal muscle and mucosa (56). Second, our studies themselves do not rule out a role for FcR engagement and cell activation, especially because it is clear that IgG Abs are required for the full intestinal injury phenotype. However, studies by ourselves and others have shown that complement blockade alone completely ameliorates intestinal IR injury. Therefore, the major effect of IgG deposition in this setting may be polymorphonuclear cell activation, consistent with our findings herein that pretreatment of Cr2-/- mice with purified wild-type IgG results in local infiltration by these cells. In contrast, the role of polymorphonuclear cells in intestinal IR injury is unclear, as previous studies have provided evidence both for and against an important role of these cells (57, 58). Therefore, although the recruitment of polymorphonuclear cells is IgG dependent, we do not know whether this is a critical cell type for generating tissue injury in the setting of intestinal IR injury.
An additional question one must consider is why CRs would be necessary for the generation of this subset of IgM and IgG natural Abs. We believe that this is consistent with the major evolutionary role of complement, and of natural Ab itself, which is self protection for the organism. Certainly in a much less medically advanced, presurgical era the human species was entirely dependent for survival upon endogenous innate and adaptive immune responses. Tissue injuries, often of a severe nature, would have been a common event, and for survival it would be necessary to rapidly identify injured tissue and then isolate and remove it before overwhelming infection sets in. The evolution of a system by which natural Ab would recognize neoantigens revealed on ischemic tissue and activate complement, another ancient member of innate immunity (59), is an elegant solution to this problem. In addition, given the known requirement for the CR2/CD19/CD81 coreceptor complex for efficient cellular activation (24, 25), one would anticipate that B cells would use these same coreceptor functions to generate this subset of "protective" natural Abs. Therefore, the finding that Cr2-/- mice lack this particular subset would not be unexpected.
These findings also suggest the possibility that complement is activated on the Ag that selects this B cell reactivity, resulting in the generation of covalently bound C3 or C4 ligands for CR2/CR1. In this regard, if the relevant Ags for generating intestinal neoantigen reactivity are natural Ags derived from apoptotic cells, and the B cell subset that generates them is the B-1 subset, this would be consistent with the findings that apoptotic cell membranes bind complement C1 and activate complement directly (60). Thus, CR2/CR1 may be involved in positive selection of this subset of B-1 cells. In contrast, if the relevant Ags are foreign in origin, perhaps infectious organisms, and the B-2 B cell subset is recruited, then it is already well established that CRs are important coreceptors for their function (24, 25). Recently Nielsen et al. (53) found that blocking the CR2 binding site with Ab decreased both C3 and MAC deposition by inhibiting the alternative complement pathway. Therefore, another possibility is that the Abs are forming immune complexes and activating the alternative complement pathway via CR2.
These studies also suggest an interesting dichotomy related to the function of CR2/CR1 in B-2 vs B-1 B cells. As noted above, our data suggest that CR2/CR1 may function in positive selection of B-1 cells to complement-bound Ags. In contrast, several other studies have suggested that CR2/CR1 may function in negative selection of B-2 B cells. That hypothesis is supported by studies demonstrating that B6/lpr Cr2-/- mice manifest accelerated autoimmunity (61) and that Cr2 itself is a New Zealand White-derived lupus susceptibility gene in the NZM2410 murine model of systemic lupus erythematosus (62, 63). In those particular studies, the protein encoded by the Sle1c allele was found to contain an additional glycosylation site that interferes with receptor binding of its C3d ligand and subsequent signal transduction (63). Thus, while the role of CR2/CR1 on peripheral lymphoid follicular and marginal zone B-2 B cells is to enhance responses to foreign Ags (26), during early B-2 cell development CR2/CR1 may be required to impart a negative signal to immature or transitional B cells that are self reactive. In sum, the biologic outcome of ligating CR2/CR1 appears to greatly depend on the lineage and stage of development of the particular B cell.
Future studies will be focused on determining whether B-1, B-2, or both B cell subsets are required for the generation of these Abs. In addition, while the nature of the neoantigen(s) that are recognized by natural Abs is still obscure, the availability of Cr2-/- mice and the demonstration of their defect in the generation of this subset of Abs should be helpful in identifying the relevant Ag-Ab reactivity that initiates injury. Identification of relevant target Ags is very important for the development of therapeutic strategies that seek to interrupt the development of tissue injury by interfering with the binding of natural Ab to ischemic tissues before the generation of inflammatory mediators.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. George C. Tsokos, Walter Reed Army Institute of Research, Room 1A32, Building 503, Robert Grant Avenue, Silver Spring, MD 20910-7500. E-mail address: gtsokos{at}usuhs.mil
3 Abbreviations used in this paper: IR, ischemia-reperfusion; CR, complement receptor; LTB4, leukotriene B4; SMI, score of mucosal injury; TMB, 3,3',5,5'-tetramethylbenzidine; MAC, membrane attack complex.
Received for publication February 28, 2002. Accepted for publication June 12, 2002.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 I. Shimizu, T. Kawahara, F. Haspot, P. D. Bardwell, M. C. Carroll, and M. Sykes B-cell extrinsic CR1/CR2 promotes natural antibody production and tolerance induction of anti-{alpha}GAL-producing B-1 cells Blood, February 15, 2007; 109(4): 1773 - 1781. [Abstract] [Full Text] [PDF]
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J. M. Thurman, A. M. Lenderink, P. A. Royer, K. E. Coleman, J. Zhou, J. D. Lambris, R. A. Nemenoff, R. J. Quigg, and V. M. Holers C3a Is Required for the Production of CXC Chemokines by Tubular Epithelial Cells after Renal Ishemia/Reperfusion J. Immunol., February 1, 2007; 178(3): 1819 - 1828. [Abstract] [Full Text] [PDF]
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M. Zhang, K. Takahashi, E. M. Alicot, T. Vorup-Jensen, B. Kessler, S. Thiel, J. C. Jensenius, R. A. B. Ezekowitz, F. D. Moore, and M. C. Carroll Activation of the Lectin Pathway by Natural IgM in a Model of Ischemia/Reperfusion Injury J. Immunol., October 1, 2006; 177(7): 4727 - 4734. [Abstract] [Full Text] [PDF]
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R. Asokan, J. Hua, K. A. Young, H. J. Gould, J. P. Hannan, D. M. Kraus, G. Szakonyi, G. J. Grundy, X. S. Chen, M. K. Crow, et al. Characterization of Human Complement Receptor Type 2 (CR2/CD21) as a Receptor for IFN-{alpha}: A Potential Role in Systemic Lupus Erythematosus J. Immunol., July 1, 2006; 177(1): 383 - 394. [Abstract] [Full Text] [PDF]
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F. D. G. McGrath, M. C. Brouwer, G. J. Arlaud, M. R. Daha, C. E. Hack, and A. Roos Evidence That Complement Protein C1q Interacts with C-Reactive Protein through Its Globular Head Region. J. Immunol., March 1, 2006; 176(5): 2950 - 2957. [Abstract] [Full Text] [PDF]
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J. M. Thurman and V. M. Holers The Central Role of the Alternative Complement Pathway in Human Disease J. Immunol., February 1, 2006; 176(3): 1305 - 1310. [Abstract] [Full Text] [PDF]
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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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T. Lyubchenko, J. Dal Porto, J. C. Cambier, and V. M. Holers Coligation of the B Cell Receptor with Complement Receptor Type 2 (CR2/CD21) Using Its Natural Ligand C3dg: Activation without Engagement of an Inhibitory Signaling Pathway J. Immunol., March 15, 2005; 174(6): 3264 - 3272. [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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S. D. Fleming, M. Monestier, and G. C. Tsokos Accelerated Ischemia/Reperfusion-Induced Injury in Autoimmunity-Prone Mice J. Immunol., September 15, 2004; 173(6): 4230 - 4235. [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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K. Yamada, T. Miwa, J. Liu, M. Nangaku, and W.-C. Song Critical Protection from Renal Ischemia Reperfusion Injury by CD55 and CD59 J. Immunol., March 15, 2004; 172(6): 3869 - 3875. [Abstract] [Full Text] [PDF]
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R. R. Reid, S. Woodcock, A. Shimabukuro-Vornhagen, W. G. Austen Jr., L. Kobzik, M. Zhang, H. B. Hechtman, F. D. Moore Jr., and M. C. Carroll Functional Activity of Natural Antibody is Altered in Cr2-Deficient Mice J. Immunol., November 15, 2002; 169(10): 5433 - 5440. [Abstract] [Full Text] [PDF]
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indicates significant
difference from IR group (p < 0.05).