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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Immunol. Author manuscript; available in PMC Jul 12, 2011.
Published in final edited form as:
PMCID: PMC3133676
NIHMSID: NIHMS298211

B Cell Subsets Contribute to Both Renal Injury and Renal Protection after Ischemia/Reperfusion

Abstract

Ischemia/reperfusion (I/R) triggers a robust inflammatory response within the kidney. Numerous components of the immune system contribute to the resultant renal injury including the complement system. We sought to identify whether natural antibodies bind to the post-ischemic kidney and contribute to complement activation after I/R. We depleted peritoneal B cells in mice by hypotonic shock. Depletion of the peritoneal B cells prevented the deposition of IgM within the glomeruli after renal I/R, and attenuated renal injury after I/R. We found that glomerular IgM activates the classical pathway of complement but does not cause substantial deposition of C3 within the kidney. Furthermore, mice deficient in classical pathway proteins were not protected from injury, indicating that glomerular IgM does not cause injury through activation of the classical pathway. We also subjected mice deficient in all mature B cells (μMT mice) to renal I/R and found that they sustained worse renal injury than wild-type controls. Serum IL-10 levels were lower in the μMT mice. Regarded together, these results indicate that natural antibody produced by peritoneal B cells binds within the glomerulus after renal I/R and contributes to functional renal injury. However, non-peritoneal B cells attenuate renal injury after I/R, possibly through the production of IL-10.

Introduction

I/R is an important cause of acute kidney injury (AKI). Work in animal models has demonstrated a robust inflammatory response within the renal tubulointerstitium after I/R (1). These studies have demonstrated that both cellular (B cells, T cells, neutrophils, and macrophages) and soluble (complement, cytokines, chemokines) components of the immune system contribute to tissue injury during reperfusion (2). Strategies that block various components of the immune response have proven protective in pre-clinical studies and hold promise as effective therapies for the prevention of AKI. The mechanisms by which the immune system is engaged after renal I/R are incompletely understood, however.

Several laboratories have demonstrated that the complement system is activated within the kidney after I/R, and complement activation is an important cause of tissue injury. For example, mice deficient in factor B [fB−/− mice; deficient in alternative pathway activity, (3)] and mice deficient in C3 [C3−/− mice; deficient in complement activation through any of the activation pathways, (4)] are protected from ischemic AKI. However, experiments using mice deficient in C4 (C4−/− mice; deficient in classical and mannose binding lectin activity) demonstrated that these mice were not protected from injury after renal I/R. Furthermore, C3 deposition was not observed in the kidneys of fB−/− mice after I/R. These studies suggest that intra-renal complement activation after renal I/R occurs through the alternative pathway following the disruption of normal inhibitory protein expression (5), and does not require an intact classical pathway.

Studies of ischemia in other organs, however, has demonstrated an important role for IgG and IgM in triggering complement activation and tissue injury. For example, natural antibody binds to neo-antigens expressed within the intestine after I/R and causes tissue inflammation by activation of the classical and lectin pathways of complement (68). One study did report that a soluble product of B cells contributes to renal injury too, although the renal injury did not appear to be mediated through the complement system (9). Work by another group using a similar model of renal I/R demonstrated that mice deficient in both T and B cells were not protected from injury (10). These discrepancies may be due to the various functions that B cells can serve. They act as positive mediators of inflammation through their production of immunoglobulin. They also support T cell activation by acting as antigen presenting cells and also through the production of cytokines such as IL-4 and IL-6 (11). On the other hand, some B cell subsets limit the immune response. Recent studies have demonstrated that IL-10 producing B cells regulate the adaptive immune response and attenuate tissue injury in diseases such as experimental autoimmune encephalitis and inflammatory bowel disease (11).

Given the growing role of therapies that target B cells, it is important to fully understand the pathologic and protective function of B cells in diseases such as AKI. Because tubulointerstitial complement activation occurs primarily through the alternative pathway, it seemed unlikely that immunoglobulin is an important activator of the complement system after renal I/R. We hypothesized, however, that other B cell functions, such as the production of IL-10, may modulate renal injury. To test this hypothesis we depleted mice of their peritoneal B cells through hypotonic shock and subjected them to renal I/R. The kidneys of these mice were evaluated to determine whether natural antibody produced by peritoneal B-1 cells contributes to complement activation and injury after renal I/R. We also subjected mice deficient in mature B cells or deficient in specific complement proteins to renal I/R in order to determine whether these immune factors are important in the development of renal injury after I/R.

Materials and Methods

Animals

Male mice aged 8–12 weeks were used for all experiments. C57Bl/6 mice were used for experiments in which peritoneal B cells were depleted and as control animals for other experiments. B-cell deficient μMT mice were obtained from Jackson Laboratories. Mice deficient in C4, C1q, and MBL A/C were generated as previously described (1214), and were each backcrossed >10 generations onto a C57Bl/6 background. Animal care before and during the experimental procedures was conducted in accordance with the policies of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All animal procedures were approved by the University of Colorado Denver – Institutional Animal Care and Use Committee.

IgM purification

Serum from C57Bl/6 mice was buffer exchanged with 20 mM NaPO4 and run over a Protein G column to remove IgG. PEG 4000 (Sigma) was then added to the flow-through at a final concentration of 7%, mixed by inversion, and incubated at room temperature for 3 hours. The mixture was then centrifuged at 2000 g for 30 minutes. The pellet was then resuspended in PBS, and the IgM containing fraction was further purified by size excusion chromatography using a Sephadex 26/60 Superdex 200 column (GE Amersham). The purity of the isolated IgM fraction was assessed by Coomassie staining and Western blot analysis.

Depletion of peritoneal B cells by hypotonic shock

Peritoneal B-cells were depleted as previously described (15). Mice received intraperitoneal injections of 0.5 mL of distilled water 12 days and 6 days prior to undergoing renal I/R. Control mice were injected with 0.5 mL of PBS. Depletion of peritoneal B-1 cells after injection with water was confirmed by FACS analysis. Peritoneal cells were obtained by lavage and stained with antibodies to B220 (SouthernBiotech), CD19 (BD Pharmingen) and CD5 (BD Pharmingen). Cells were then washed and resuspended in 500 μL of PBS, run on a FACSCalibur machine (BD Biosciences), and analyzed with CellQuest Pro software (BD Biosciences).

Renal I/R protocol

Mice were anesthetized with 300 μl of 2,2,2-Tribromoethanol (Sigma-Aldrich) injected intraperitoneally, and they were placed on a heating pad to maintain their body temperature during surgery. Laparotomies were performed and the renal pedicles were located and isolated by blunt dissection. The pedicles were clamped with surgical clips (Miltex Instrument Company, Inc.), and occlusion of blood flow was confirmed by visual inspection of the kidneys. The clamps were left in place for 24 minutes and then released. The kidneys were observed for approximately one minute to ensure blood re-flow, then fascia and skin were sutured with 4-0 silk (United States Surgical). Sham surgery was performed in an identical fashion, except that the renal pedicles were not clamped. The mice were volume resuscitated with 0.5 ml of normal saline injected subcutaneously and kept in an incubator at 29°C to maintain body temperature until the time of sacrifice. After 8, 24, 48, or 72 hours of reperfusion the mice were anesthetized, and blood was obtained by cardiac puncture. Laparotomy was performed and the kidneys were harvested.

Histology, TUNEL staining and Immunofluorescence microscopy

For light microscopy, sagittal sections were fixed, embedded in paraffin, and four μm sections were cut and stained with periodic acid Schiff. The sections were evaluated by a renal pathologist (DLj) in a blinded fashion. The cortex and outer stripe of the outer medulla were assessed for epithelial necrosis, loss of brush border, tubular dilatation and cast formation. At least ten fields (400×) were reviewed for each slide, and the percentage of tubules displaying these findings was determined. The kidney sections were scored as follows based on the percentage of affected tubules: 0, none; 1, <10%, 2, 11–25%, 3, 26–45%, 4, 46–75%, 5, >75%.

TUNEL staining was performed using TACS-XL Blue Label Kit with Nuclear Fast Red counterstain (Trevigen) according the manufacturer’s instructions. To assess the prevalence of apoptotic cells, 25 glomeruli and 25 fields in the cortex and outer medulla were examined in a blinded fashion. The number of TUNEL positive bodies per field was counted, and the results for each kidney section were averaged.

For immunofluorescence microscopy, sagittal sections of the kidneys were snap frozen in OCT compound (Sakura Finetek). Four μm sections were cut with a cryostat and stored at −70° C. At the time of staining the slides were fixed with acetone and incubated with primary antibody. The antibodies used include FITC-conjugated anti-mouse C3 (ICN/Cappel), FITC conjugated antibodies to mouse IgG and IgM (Jackson ImmunoResearch Laboratories), and anti-mouse C4 (Hycult Biotech). Secondary antibodies were obtained from Jackson ImmunoResearch Laboratories. Sections were counterstained with hematoxylin (Vector Laboratories, Inc.). Staining was visualized using a Nikon T2000 inverted fluorescent microscope and slide book software (Intelligent Imaging Innovations). For all immunofluorescence at least three kidneys were examined per group, and greater than 10 high powered fields were examined per kidney. For quantitative analysis of glomerular IgM deposition, images of 14–30 glomeruli per section were obtained. The fluorescence intensity was determined using ImageJ software, and the mean value for each treatment group was determined. For the low-powered view of glomerular IgM (Figure 1E), the brightness and contrast have been adjusted to improve the visibility of the staining.

Figure 1
IgM is deposited in mouse glomeruli

Western blot analysis

Renal tissue was homogenized in RIPA lysis buffer containing 1% Triton X-100, 0.5% deoxycholic acid, 150 mM NaCl, 20 mM β-glycerophosphate, 20 mM Tris·HCl (pH 8.0), 5 mM EGTA, 3 mM MgCl2, 0.1% SDS, 1 mM DTT, 50 μM Na3VO4, and one tablet of complete, EDTA-free, protease inhibitor cocktail (Roche Applied Science). Homogenates were centrifuged at 14,000 rpm for 15 min. at 4° C and the supernatant was collected. Fifty μg of protein for each sample was resolved by electrophoresis with a 10% Bis-Tris polyacrylamide gel (Invitrogen) and transferred to a nitrocellulose membrane. The membrane was probed with an HRP-conjugated antibody to mouse IgM (SouthernBiotech) and was visualized using a chemiluminescence detection kit (Amersham Life Science).

Serum urea nitrogen measurements

Serum urea nitrogen (SUN) was measured using an Alfa Wasserman ACE Chemistry Analyzer.

Enzyme-linked immunosorbent assays

Serum IgM was measured by ELISA. Briefly, ELISA plates (Corning Incorporated) were coated with 100 ng of anti-mouse IgM (SouthernBiotech). After the plates were washed and blocked with 1% BSA (Sigma-Aldrich) for two hours, serum samples were diluted 1/50 and added to the wells. Standard curves were generated using serial dilutions of purified IgM (7.8125 ng/mL to 500 ng/mL). The plates were then washed, and IgM was detected with an HRP-conjugated anti-mouse IgM antibody (SouthernBiotech). The plates were then developed with ABTS (Sigma) and read on a Spectramax PLUS plate reader (Molecular Devices Corporation).

Serum IL-10 and CXCL1 were measured using ELISAs obtained from ELISATech according to the manufacturer’s protocol.

Statistical analysis

Data were graphed and analyzed using GraphPad Prism software. T tests were used for experiments involving two groups, and ANOVA with a Tukey-Kramer post-test was used for multiple group comparisons. A P value of < 0.05 was considered significant.

Results

Glomerular IgM is seen in wild-type mice

We examined sham treated mice and mice subjected to renal I/R for tissue deposition of IgG and IgM. IgG was not seen in any of the kidneys, but IgM was seen in the mesangium of sham treated kidneys (Figure 1). The level of mesangial IgM increased after renal I/R (Figures 1E and 1F), suggesting that circulating IgM binds to the mesangium during reperfusion. IgM was not seen along the tubular basement membrane.

Depletion of peritoneal B cells protects mice from renal I/R

We depleted the peritoneal B cells in wild-type mice for two weeks by injecting distilled water into the peritoneum, and control mice were injected with an equal volume of PBS. Depletion of the B-1 population was confirmed by flow cytometry analysis of B220, CD5, and CD19 (Figure 2). This protocol has previously been reported to reduce peritoneal B-1 cells, thereby preventing tissue injury by pathogenic natural IgM antibody (15). Lysis of the peritoneal B-1 cells did not alter overall levels of circulating IgM (Figure 2C), but it did reduce levels of mesangial IgM after sham treatment and after renal I/R compared to control mice (Figures 2D–H). Depletion of peritoneal B-1 cells was also associated with a significantly attenuated increase in the SUN after 24 hours of reperfusion (the peak of injury; Figure 3A). SUN levels were not significantly different than control mice by 48 hours of reperfusion. Although the decrease in mesangial IgM was associated with protection of renal function, mice that underwent peritoneal B cell depletion still demonstrated tubular necrosis comparable to that seen in wild-type mice (Figures 3B–C).

Figure 2
Depletion of peritoneal B cells reduces glomerular IgM
Figure 3
Depletion of peritoneal B cells protects mice from renal ischemia/reperfusion

Mice deficient in the classical or MBL pathways of complement are not protected from renal I/R

IgM bound to antigen is a potent activator of the classical pathway of complement and also can activate the MBL pathway (7). To assess whether activation of these pathways by IgM contributes to renal dysfunction after I/R, we subjected C4−/−, C1q−/−, and MBL−/− mice to renal I/R. None of these strains showed functional protection from renal injury in our model (Figure 4). This suggests that injury caused by glomerular IgM is not mediated through activation of the classical or lectin pathways of complement.

Figure 4
Deficiency of the classical and mannose binding lectin complement pathways do not protect mice from injury after ischemia/reperfusion

The classical and alternative pathways of complement are activated in distinct compartments of the kidney after I/R

To further characterize the mechanisms of complement activation in the kidney after I/R we performed dual staining immunofluorescence for C3 and C4. C4 was present in the mesangium of sham treated mice (Figure 5A) and appeared to increase after I/R (Figure 5B), consistent with the mesangial deposition of IgM. The pattern of mesangial C4 was not notably different in fB−/− mice subjected to I/R (Figure 5C), as one would expect since these mice are only deficient in the alternative pathway. Mesangial C4 was not seen in C4−/− mice.

Figure 5
The classical and alternative pathways of complement are activated in distinct locations within the kidney after renal ischemia/reperfusion

C3 did not co-localize with mesangial C4 indicating that, although the classical pathway is engaged at this location, mesangial complement activation is well controlled by endogenous regulators (16). As has been previously described, C3 is deposited along the tubules of wild-type mice after I/R (Figure 5B) but was not seen in fB−/− mice. Tubular C3 was seen along the tubules of C4−/− mice (Figure 5D), indicating that tubulointerstitial complement activation requires the alternative pathway but does not require the classical or MBL pathways.

Mice lacking mature B cells sustain more severe injury than wild-type controls

We also subjected μMT mice to renal I/R. Contrary to the mice with depletion only of the peritoneal B cells, we found that the μMT mice sustained more severe renal dysfunction than wild-type mice as assessed by SUN levels after 24 hours of reperfusion (the peak of injury; Figure 6A). Although the mean SUN value for the μMT mice remained higher than that for wild-type mice out to 72 hours of reperfusion, the difference was no longer significant after 24 hours. Significant necrosis was seen in the tubulointerstium of both μMT and wild-type mice (Figure 6B–D). TUNEL staining was used to assess the prevalence of apoptotic cells in the glomeruli and tubulointerstitium (Figures 6E), and a greater number of apoptotic cells were seen in the tubulointerstitium of μMT mice than in wild-type controls. Few apoptotic cells were seen in the glomeruli of either strain, and differences between the strains were not significant. After 8 hours of reperfusion serum levels of IL-10 were undetectable in μMT mice. The chemokine CXCL1 has also been implicated in the development of renal injury after I/R (Figure 7). Serum CXCL1 levels were not significantly different between the two groups, but showed a trend towards being higher in the wild-type mice.

Figure 6
B cell deficient mice develop more severe injury than wild-type controls after renal ischemia/reperfusion
Figure 7
Serum levels of IL-10 and CXCL1 after ischemia/reperfusion

Reconstitution of mice with IgM does not restore protection from renal injury after I/R

IgM was purified from the serum of wild-type mice by PEG precipitation. μMT mice were injected with IgM or with vehicle and were subjected to renal I/R. The reconstituted μMT mice had levels of IgM comparable to wild-type mice (Figure 8A). However, reconstitution of circulating IgM did not restore the mesangial IgM deposits (Figure 8B). Renal injury was no different than that seen in control μMT mice (Figure 8C). These results indicate that the purification procedure we used did not restore IgM with specificity for mesangial antigens, and suggest that the effect of IgM on renal function may be mediated by specific clones of IgM. It is unclear why the purified IgM failed to restore the mesangial deposits. Possible explanations are that the mesangial-specific IgM circulates at very low concentrations since most of it is bound up in tissue, or that the IgM purification process that we used depletes the mesangial specific clone(s).

Figure 8
Reconstitution of μ MT mice with purified IgM does not protect the mice from renal injury after ischemia/reperfusion

Discussion

These studies demonstrate that B cells play a complex role in the development of injury after renal I/R. Natural antibody IgM was present within the mesangium of mice at baseline, and increased during reperfusion. Depletion of peritoneal B-1 cells by hypotonic shock reduced glomerular IgM. This treatment only slightly attenuated the degree of renal dysfunction after I/R, and reduction of glomerular IgM did not prevent tubular injury. Reconstitution of B cell deficient mice with purified IgM failed to restore mesangial IgM, highlighting that the interaction of IgM with mesangial structures is a specific finding and not due to passive trapping of circulating IgM.

Our results also demonstrate that the complement system is activated in the glomeruli and the tubulointerstitium by distinct mechanisms after I/R. The classical pathway was engaged by IgM deposits in the mesangium leading to C4 deposition, but this activation was well controlled and did not proceed to the level of C3 cleavage. Although the deposition of IgM in the mesangium contributed to renal dysfunction during reperfusion, this effect was not mediated by the complement system. In contrast, the classical and MBL pathways were not required for complement activation in the renal tubulointerstitium after I/R, and complement activation at this location required only an intact alternative pathway. Tubulointerstitial alterative pathway complement activation proceeded through the cleavage of C3. Previous work has also shown that tubulointerstitial complement activation generates C5b-9 and contributes to renal injury (3, 4).

In spite of the fact that natural IgM contributes to renal injury after I/R, mice wholly deficient in mature B cells developed more severe injury than wild-type control mice. These mice had lower levels of serum IL-10 than wild-type mice during reperfusion. IL-10 is an anti-inflammatory cytokine that has previously been demonstrated to play an important role in protecting mice from ischemic AKI (17). Several recent studies have demonstrated that a population of IL-10 producing B cells helps to regulate the adaptive immune response in various diseases (11), and our results suggest that IL-10 producing B cells may also control the inflammatory response in this acute injury model. In contrast to the glomerular location of the pathogenic IgM, the protective effect of B cells manifested as reduced tissue injury in the tubulointerstitium (Figure 6).

A previously published study reported that recombination-activating gene-1 deficient mice (which lack mature B cells and mature T cells) are not protected from renal I/R (10). In contrast, a report by Burne-Taney et al. found that a soluble factor produced by B cells contributes to renal injury after I/R, and that this B cell mediated injury is not mediated through complement activation (9). It is noteworthy that we used identical mice and a similar protocol as that used in this latter study, but the B cell deficient mice were protected in the study by Burne-Taney et al. (9). However, the pathogenic factor B cell factor was found to be present in serum, and transfer of splenic and lymph node B cells into the μMT mice was not pathogenic. These results are consistent with the divergent B cell effects that we have described – production of a pathogenic soluble factor but cell-mediated protection of the kidneys. Full understanding of the role of B cells in renal I/R will require further experiments to dissect the different roles of the different B cell subsets.

Our results indicate that, as with the intestine, natural antibody binds to antigens expressed in the post-ischemic kidney. In the case of renal I/R, however, the IgM mediated injury does not appear to be mediated through activation of the classical complement pathway. The decline in renal function associated with mesangial IgM deposition may be due to direct effects of IgM on the mesangial structures. A previously published study has also shown that MBL contributes to tubular complement activation after renal I/R (18). It is possible that MBL contributes to initiating complement activation, and the alternative pathway then amplifies the activation. It is also possible that the longer ischemic time used in that study (45 minutes) generated more endogenous ligand for MBL, and the MBL pathway therefore played a greater role in that model.

The above studies do not conclusively demonstrate that protection of the kidney by B cells is directly mediated by production of IL-10. Such a hypothesis is consistent, however, with the protective role of IL-10 in ischemic AKI and with the recent work describing a population of IL-10 producing regulatory B cells (11). Future studies that specifically deplete or restore the CD1dhiCD5+ regulatory B cell population may help to confirm a direct role of regulatory B cells in protecting the kidney after I/R. It is also worth noting that the contribution of IgM to renal injury in our model was modest. This is not surprising given renal ischemia primarily causes injury of tubules in the outer medulla, not glomeruli. Nevertheless, the effect of natural IgM on the increase in SUN after renal I/R appeared to be mediated by a specific clone of IgM with specificity for mesangial structures. This interaction may also have pathologic effects on renal function in other models of injury, particularly those that primarily cause severe mesangial injury.

B cell depleting antibodies have emerged as a promising therapy for several renal diseases, including lupus nephritis, membranous disease and focal segmental glomerulosclerosis. It has become clear, however, that B cells have a complex role in modifying disease. Our results have several implications. First, they demonstrate that B cells can dampen renal injury after I/R, and that therapies that non-specifically target B cells may exacerbate AKI. Second, our results demonstrate that natural antibody IgM may bind to the injured kidney and reduce renal function. Therefore, the optimal approach to treating renal diseases may require the development of agents that can target the discrete B cell populations, or agents that interfere with binding of immunoglobulin to specific renal antigens. Such an approach may also improve the treatment of non-renal autoimmune diseases in which B cells may play a similarly complex role.

In summary, our results demonstrate that B cells can have both protective and pathogenic functions during the development of AKI. The pathogenic role appears to be mediated by natural antibody IgM which binds to the mesangium after I/R. Depletion of peritoneal B-1 cells reduced mesangial IgM and protected mice from injury after I/R. B cells also play a role in protecting the kidneys after I/R, probably by production of the anti-inflammatory cytokine IL-10.

Acknowledgments

The authors would like to acknowledge Marina Botto for generously providing the C1q−/− mice.

This work was supported in part by National Institutes of Health Grants DK076690 and DK77661 (JMT), AI311052 (VMH), AI077081 (TK) and AI07450 (TK). This work was also supported by a Norman S. Coplon Extramural Grant from Satellite Healthcare (JMT).

Abbreviations used in this paper

IR
ischemia/reperfusion
AKI
acute kidney injury
SUN
serum urea nitrogen

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