Intestinal ischemia-reperfusion (IR) injury is initiated when natural IgM Abs recognize neo-epitopes that are revealed on ischemic cells. The target molecules and mechanisms whereby these neo-epitopes become accessible to recognition are not well understood. Proposing that isolated intestinal epithelial cells (IEC) may carry IR-related neo-epitopes, we used in vitro IEC binding assays to screen hybridomas created from B cells of unmanipulated wild-type C57BL/6 mice. We identified a novel IgM mAb (mAb B4) that reacted with the surface of IEC by flow cytometric analysis and was alone capable of causing complement activation, neutrophil recruitment and intestinal injury in otherwise IR-resistant Rag1−/− mice. mAb B4 was found to specifically recognize mouse annexin IV. Preinjection of recombinant annexin IV blocked IR injury in wild-type C57BL/6 mice, demonstrating the requirement for recognition of this protein to develop IR injury in the context of a complex natural Ab repertoire. Humans were also found to exhibit IgM natural Abs that recognize annexin IV. These data in toto identify annexin IV as a key ischemia-related target Ag that is recognized by natural Abs in a pathologic process required in vivo to develop intestinal IR injury.
Ischemia-reperfusion (IR)-induced3 injury is a pathologic process that occurs when the normal blood flow to an organ or tissue is interrupted for a period of time that is sufficiently long to result in marked hypoxia and ischemia, following which the recirculation of blood is restored. A central concept in this process is that the introduction of oxygenated blood during the reperfusion phase results in the development of more severe target organ injury than that caused by the ischemia per se (1, 2). The precise mechanism of injury during the reperfusion phase is a subject of intense investigation, because no specific therapy exists at the present time for the treatment of this pathologic process that underlies common clinical events such as myocardial infarction and stroke (3, 4, 5, 6). IR-induced injury is especially prominent in the intestine and is frequently followed by multiple organ dysfunction and infection as secondary complications (7, 8, 9, 10).
With regard to the pathogenesis of this condition, complement activation and neutrophil infiltration are two key events that are required for experimental intestinal IR injury induced by ligation and subsequent release of the mesenteric artery, as both neutrophil depletion (11, 12, 13) and complement blockade (14, 15, 16) protect mice from the development of local tissue damage. Initiation of complement activation by the classical and lectin pathways has been demonstrated after IR of the heart, intestine, and skeletal muscle (15, 17, 18, 19), although tissue injury also apparently requires the engagement of the alternative pathway amplification loop (20). Initial evidence that natural Abs are centrally involved in IR-induced injury came from seminal findings that Rag1−/− mice are resistant to the induction of IR injury to the intestine as well as other vascularized organs (19, 21, 22). The same mice, when reconstituted with IgM purified from natural Ab in pooled sera from wild-type (wt) mice, become fully susceptible to IR-induced injury (19, 23, 24). As a related finding, Cr2−/− mice that lack expression of the B lymphocyte complement receptor (CR)1 and CR2 are also resistant to IR-induced injury, despite exhibiting normal quantitative levels of polyclonal IgM. Infusion of natural IgM and IgG Abs from Cr2+/+ but not Cr2−/− mice, or transfer of peritoneal B cells from Cr2+/+ to Cr2−/− mice, restores intestinal IR injury (23, 25). These data have suggested that there are specific IR-related neo-epitopes against which Cr2−/− mice do not develop normal quantitative or qualitative levels of natural Abs.
Natural Abs are Igs that are produced in the absence of deliberate immunization, and they are a major component of the repertoire of B1 cells, which produce IgM and in some cases IgG Abs (26, 27). B1 cells in the adult mouse are found primarily in the peritoneum and pleura (28). Natural Abs are frequently found to be polyreactive at low affinity with multiple self Ags (29, 30), and they are considered as an important part of the innate immune system defense against infection. For example, natural Abs have been found to be protective against challenge with bacterial as well as viral pathogens, and to play an important role in the clearance of endotoxin (31, 32, 33). In addition, natural Abs play important roles in the recognition of apoptotic cells, oxidized low-density lipoprotein, and nuclear and cytoplasmic components released from damaged cells (34, 35, 36).
The possibility that Abs recognizing specific Ags, or subsets of Ags, could be identified to play essential roles in IR-induced injury was suggested by Fleming et al. (21) in experiments where IgG mAbs against negatively charged phospholipids and β-2-glycoprotein 1, as well as polyclonal antisera with high titers against the same Ags, were able to reconstitute mesenteric IR-induced intestinal and lung injury in Cr2−/− mice. Unlike Cr2−/− mice, though, reconstitution of IR tissue damage in injury-resistant Rag1−/− mice required the infusion of both anti-β-2-glycoprotein 1 and anti-phospholipid IgG mAbs, or polyclonal serum-derived Abs. Another Ab system important in IR injury has been shown to involve natural Abs recognizing an epitope on non-muscle myosin and glycogen phosphorylase (37, 38). In contrast to studies by Fleming et al., an IgM mAb (designated CM22) recognizing these two Ags was found to be capable alone of inducing mesenteric and skeletal muscle IR injury in Rag1−/− mice, and a peptide mimic of the Ag also blocked in vivo the IR injury of intestine and skeletal muscle in wt mice (22, 37, 38).
Annexin IV belongs to a family of proteins that are Ca2+- and phospholipid-binding proteins (39, 40). The structure of annexins consists of a conserved Ca2+ and membrane binding core of four annexin repeats (eight for annexin VI) and variable N-terminal regions (41). Annexins are soluble cytosolic proteins, but despite the lack of obvious signal sequences and the apparent inability to enter the classical secretory pathway, annexins have been identified in extracellular fluids or associated with the external cell surface through poorly understood binding sites (40, 42, 43, 44). Annexin IV is predominantly produced by epithelial cells and is also found at high levels in lung, intestine, pancreas, liver, and kidney. Depending on the cell type, annexin IV has been found either along the basolateral, basal, or apical domains of the plasma membrane, and in some cell types, it has been found to be present throughout the cytoplasm (45, 46, 47). With regard to its function, annexin IV has been shown to inhibit the epithelial calcium-activated chloride ion conductance (48), to play a role in the formation of pronephric tubules (49), and to regulate the passive membrane permeability to water and protons (50). Up-regulation of annexin IV has been found in renal cell carcinoma (51, 52). Finally, surface membrane expression of annexin IV has also been recognized as an early marker for apoptotic cell death (53, 54).
Herein we report the identification of a novel pathogenic IgM mAb that is capable of inducing intestinal IR injury in Rag1−/− mice. The mAb was found to specifically recognize mouse annexin IV. Importantly, we also found that normal mouse sera contain natural Abs to annexin IV epitopes and that treatment of wt C57BL/6 mice with recombinant annexin IV before the reperfusion phase prevents IR-induced intestinal injury. We propose that binding sites on annexin IV are essential neo-epitopes that are expressed on ischemic tissues and are targets for natural Ab binding during the reperfusion phase, with subsequent complement activation, neutrophil recruitment, and tissue injury.
Materials and Methods
Adult male Rag-1−/− mice were obtained from The Jackson Laboratory and maintained following shipment for at least a 7-day acclimation period in the Uniformed Services University of Colorado Denver animal facility. Adult male and female Cr2+/+ and Cr2−/− mice were maintained and bred as two sublines at University of Colorado Denver as described previously (23). Animal studies at sites were approved by the local institutional review board. Human studies were approved by the Colorado MultiInstitutional Review Board.
Abs and reagents used for analysis
Biotinylated and FITC-conjugated goat anti-mouse IgG (Fcγ specific) or anti-mouse IgM (μ-chain specific) streptavidin (SA)-FITC and SA-PE were obtained from Jackson ImmunoResearch Laboratories. Alkaline phosphatase-conjugated goat anti-mouse Ig Ab was obtained from Caltag Laboratories. Polyclonal rabbit anti-annexin IV Ab was obtained from ProteinTech Group, and anti-6× His mAb was obtained from Novagen. Synthetic peptides were obtained from Synthetic Biomolecules.
Development and purification of IgM mAbs
mAbs B4 and D5 were developed by the fusion of peritoneal, lymph node, and spleen cells from unmanipulated wt C57BL/6 mice with the Sp2/0-Ag14 myeloma cell line by the standard protocol to establish hybridomas. Successful fusions were screened by Western blot analysis using IEC lysates and by flow cytometric analysis of isolated IEC. To purify mAbs, Ab from the exhausted supernatants of cultured B4 and D5 hybridomas was affinity purified on a column of agarose beads with goat anti-human IgM (Sigma-Aldrich). Bound Ab was eluted with a buffer containing 0.1 M glycine (pH 2.3) and collected into a buffer containing 1.5 M Tris (pH 8.8). Eluted mAb was dialyzed against PBS (pH 7.4) for 48 h and concentrated using centrifugal filtration on Centricon Plus-20 (Millipore). Ab concentration was determined by measuring the A280 nm of the sample, and purity was confirmed by analysis on a 10% SDS-PAGE gel.
IEC isolation and flow cytometric analysis
Isolation of IEC was performed using previously described methods (55, 56). Briefly, after dissection of the mouse intestine into small pieces, the latter were incubated twice in HBSS with 1 mM DTT and 1 mM EDTA for 30 min at 37°C with shaking to detach IEC. By this method of isolation, the cell mixture consists of 93–95% IEC, with <2% intraepithelial lymphocyte contamination (55). Intraepithelial lymphocyte can be readily gated out in time of flow cytometric analysis by size characteristics. Detached free cells and intact crypts were centrifuged and resuspended in HBSS containing Ca2+ and Mg2+, or in 5% FBS DMEM culture media. Isolated IEC were washed in the staining buffer (2% FCS/0.01% NaN3/PBS), resuspended in the staining buffer containing hybridoma supernatant or pure mAb, and incubated for 30 min at room temperature. After incubation, cells were washed in the staining buffer three times and then incubated with the secondary goat anti-mouse IgM (μ-chain specific) Abs (Jackson ImmunoResearch Laboratories) for 30 min at room temperature. Following incubation, cells were washed as above described and then resuspended in the staining buffer. Flow cytometry was performed using a BD Biosciences FACSCalibur.
Western blot analysis
IEC were lysed on ice for 20 min in a buffer containing 0.5% Triton X-100, 0.5% Chaps, 20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 10 μg/ml leupeptin, and protease inhibitor mixture (Roche Molecular Biochemicals). Lysates were cleared by centrifugation at 8000 × g for 5 min. After separation by 8% Tris-glycine SDS-PAGE, the proteins were transferred to a polyvinylidene difluoride membrane. The membrane was blocked overnight with 5% nonfat milk dissolved in PBS. The membrane was washed in PBS and then probed with a primary Ab for 1–2 h in 2% milk/PBS, washed, and then incubated with HRP-conjugated secondary Abs. A positive signal was visualized using the ECL system (PerkinElmer).
Intestinal IR injury
The intestinal IR injury model was performed as previously described (23) by the surgical procedure of opening the abdominal cavity of an anesthetized mouse and occluding the superior mesenteric artery for 30 min, followed by removal of the clamp and 2-h reperfusion of the tissue. Ketamine/xylazine was used for anesthesia.
Histology and immunohistochemistry
Immediately after euthanasia, segments of small intestine specimens were fixed in 10% buffered formalin, embedded in paraffin, cut transversely in 5-μm sections, and stained with Giemsa. Mucosal injury was graded on a six-tiered scale in a blinded manner, as described previously (23). Briefly, the average of villus damage in an ∼2-cm intestinal section (50–100 villi) is determined after grading each villus in the section on a 0–6 scale. A score of 0 is assigned to a normal villus; a score of 1 to villi with tip distortion; a score of 2 is assigned when in addition goblet cells and Gugenheims’ spaces are missing; a score of 3 is assigned to villi with patchy disruption of the epithelial cells; a score of 4 is assigned to villi with exposed but intact lamina propria with epithelial cell sloughing; when the lamina propria is exuding a score of 5 is assigned; and lastly, a score of 6 is assigned to villi that display hemorrhage or to villi that are denuded. Additional tissue sections were 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 blocking solution (DakoCytomation) for 30 min. After washing in PBS, the tissues were incubated with goat anti-mouse C3 (Cappel) or appropriate control IgG (Jackson ImmunoResearch Laboratories) Ab overnight at 4°C. The primary Ab was detected with a biotinylated donkey anti-goat Ig (Jackson ImmunoResearch Laboratories), then SA-HPR (Vector Laboratories), and developed with Vector Nova Red Substrate (Vector Laboratories). For immunohistochemistry, tissues were fixed at 4°C for 2 h in 4% paraformaldehyde in PBS. Nonspecific Ab binding sites were blocked by the incubation with a blocking solution PBS for 30 min. After washing in PBS, the tissues were incubated with isotype control Ab or FITC-conjugated anti-mouse C3, tetramethylrhodamine isothiocyanate-conjugated anti-mouse Ig, and 4′,6-diamidino-2-phenylindole (DAPI) to visualize the nucleus. After washing, the slides were mounted using ProLong Gold antifade reagent with DAPI and analyzed on an Olympus AX80 microscope.
The ex vivo generation of eicosanoids in small intestine tissue was determined using a previously described method (16, 23). Briefly, sections of minced fresh mid-jejunum were washed and resuspended in 37°C oxygenated Tyrode’s buffer (Sigma-Aldrich). After tissues were incubated for 20 min at 37°C, supernatants and tissue were collected and stored at −80°C until assayed. The concentrations of leukotriene B4 (LTB4), and PGE2 were determined using an enzyme immunoassay (Cayman Chemical). The tissue protein content was determined using the bicinchonic acid assay (Pierce) adapted for use with microtiter plates. LTB4 and PGE2 levels were expressed in pictograms per milligrams protein per 20 min.
Myeloperoxidase (MPO) 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 (16). Briefly, supernatants were incubated with equal volumes of 3,3′,5,5′-tetramethylbenzidine peroxidase substrate (Kirkegaard & Perry) for 45 min. The reaction was stopped by the addition of 0.18 M sulfuric acid, and the OD450 nm was determined. The concentration of total peroxidase was determined using HRP (Sigma-Aldrich) as a standard and plotted as picograms of MPO activity per milligram of tissue.
Purification of IEC protein reactive with mAb B4
To purify protein reactive with mAb B4 for identification, a three-step purification procedure based on the method described by Vossenaar et al. (57) was performed. In the first step, IEC lysates were resolved using preparative 8% SDS-PAGE. Two lanes from the gel were cut. The first lane was transferred to a membrane and probed with mAb B4, and the second lane was stained with Coomassie brilliant blue to localize precisely the position of the proteins. The rest of the gel was stained with a nonfixing gel stain GeBA SeeBand stain (Gentaur), and protein-containing areas were excised and stored at 4°C. In the next step, the gel strips were washed in a freshly prepared, warm (37°C) washing solution (2 mM Tris-HCl (pH 8.0), 8 M urea, and 1% Nonidet P-40). The washed gel strips were then loaded on an isoelectric focusing (IEF) gel (6 M urea, 1% Nonidet P-40, 15% (of total volume) acrylamide mix (39:1), 2% ampholytes (3, 4, 5, 6, 7, 8, 9, 10), 0.47 μg/ml ammonium persulfate, and 0.66 μl/ml tetramethylethylenediamine). IEF was performed on a gel (15 × 15 cm) overnight at 200 V. The upper and lower buffers were 0.09 M NaOH and 0.85% phosphoric acid, respectively. Immediately after migration, the gel was fixed in the 20% trichloroacetic acid for 20 min and then stained with Coomassie brilliant blue. Regions containing proteins in the IEF were excised and separated by 8% SDS-PAGE. Protein was localized by staining with GeBA SeeBand stain, and areas of interest were excised and used for identification of proteins using mass spectrometry (MS) (University of Chicago). A parallel SDS-PAGE was performed, and after transfer to polyvinylidene difluoride membrane, Western blot analysis was performed to confirm the location of the Ag in the gel.
Protein identification by MS
After separation the protein was digested in-gel with trypsin according to a modified protocol (University of Chicago). The aqueous peptide extract (10 μl) was analyzed using electrospray liquid chromatography MS (LC/MS/MS). An HPLC instrument (Agilent) was connected with an XCT ion trap MS (Agilent). Sample was loaded automatically at 10 μl/min. Chromatography buffer solutions (buffer A, 2.5% methanol, and 0.1% formic acid; buffer B, 99.9% acetonitrile and 0.1% formic acid) were used to make a 90-min gradient (8 min to 10% buffer B, 32 min from 10 to 45%, hold 5 min, 5 min from 45 to 90%, hold for 20 min, then 5 min to 0% B, hold for 15 min). A flow rate of 0.25 μl/min was used. The MASCOT program was used to search the mouse protein sequence database. Probability-based mouse score (−10*log P) was used for protein identification, where P is the probability that the observed match is a random event. Individual ion scores > 40 indicate identity or extensive homology (p < 0.05).
Annexin IV constructs
KpnI) to the start of annexin IV and XhoI restriction site to the end of annexin IV. The KpnI/XhoI fragment was amplified by PCR from genomic DNA using Pfu polymerase (Novagen). The KpnI/Xho
Expression and purification of recombinant annexin IV
The expression construct pETXa/LIC-A4 was transformed into E. coli Rosetta 2 (DE3) cells. Bacterial expression cultures were incubated at 37°C in Luria-Bertani medium containing ampicillin (50 μg/ml) until an A600 nm of 0.6 was reached. Recombinant protein expression was induced by an addition of isopropyl β-D-thiogalactoside (Sanland-Chem) to a final concentration of 0.3 mM. After 6 h of incubation at 32°C, bacteria were harvested by centrifugation at 10,000 × g for 10 min at 4°C. After harvesting the cells, they were resuspended in PBS with Complete, EDTA-Free Protease Inhibitor Cocktail Tablets (Roche Molecular Biochemicals). Bacteria were lysed by four freeze-thaw cycles. The lysate was then incubated with DNase and RNase for 30 min. Cell lysate was then centrifuged (10,000 × g for 40 min), and cell pellet was resuspended in 6 M urea for 30 min. Centrifugaton by 10,000 × g for 40 min was then used to remove undissolved debris. The precleared supernatant was diluted (2:1) by PBS, its pH adjusted to 7.6 with NaOH and then applied to a TALON resin column (BD Clontech) equilibrated with 4 M urea. Bound protein was refolded on the column using a discontinuous gradient from 4 to 0.25 M urea, starting with the equilibration buffer and finishing with a buffer containing 10 mM imidazole in PBS (pH 7.0). The refolded protein was eluted with a buffer containing a stepwise gradient of 19, 38, 75, and 150 mM imidazole. The presence of the protein and its purity was confirmed by SDS-PAGE and staining with Coomassie brilliant blue.
Annexin IV ELISA
Immulon 1B plates (Dynatech Laboratories) were coated with 5 μg/ml annexin IV-purified protein in PBS. Wells were blocked with 1% BSA in PBS. Serial dilution of serum samples were made in blocking buffer, and samples were applied to wells. After incubation and washing, bound Abs were detected using AP-conjugated anti-mouse IgG (Fcγ specific) or anti-mouse IgM (μ-chain specific) Abs, followed by p-nitrophenyl phosphate (Sigma-Aldrich) at 1 mg/ml. Plates were read at 405 nm.
Generation of mAbs
As a strategy to identify mAbs that would recognize neo-epitopes on ischemic tissues, we hypothesized that intact intestinal epithelial cells (IEC), when isolated as a single-cell suspension, might expose on the surface the same neo-epitopes that are targets on the ischemic cells for pathogenic IgM Abs in vivo during intestinal IR. Consistent with this, in pilot experiments, we found that whole serum from Cr2+/+ mice exhibited higher binding of Igs to IEC than Cr2−/− mice (data not shown). On the basis of this hypothesis, we used freshly isolated IEC to screen hybridomas obtained by fusion with the Sp2/0-Ag14 myeloma cell line of B cells from wt unmanipulated C57BL/6 mice derived from peritoneum, lymph nodes, and spleen. Wells were chosen for further subcloning based on the positive surface staining of IEC by flow cytometry or reactivity by Western blot analysis on tissue lysates. Cells were then serially recloned to obtain monoclonal cell lines stably producing a single mAb.
One such product of this strategy, designated mAb B4, was obtained from a fusion with spleen cells and is an IgM κ isotype Ab. As shown using flow cytometric analysis, mAb B4 binds a surface epitope on IEC but not on freshly isolated splenocytes or thymocytes (Fig. 1⇓A). By Western blot analysis, mAb B4 recognizes a protein with a m.w. of 37 kDa in IEC lysates but not lysates from freshly isolated splenocytes or thymocytes (Fig. 1⇓B). When other tissue lysates were probed by Western blot with mAb B4, the epitope was found to be widely distributed, with the highest relative expression in lung and isolated IEC (Fig. 1⇓C). As an isotype control we used mAb D5 directed to an epitope on mouse cytokeratin 19 (our unpublished data). The results shown in Fig. 1⇓C demonstrate that when the whole spleen was taken to make lysates, a weak band corresponding to the B4 Ag is seen, and a band of the same size was seen in lysates from whole thymus (data not shown). These data suggest that the mAb B4 epitope is expressed in nonlymphoid stromal cells, perhaps of epithelial origin, in these tissues.
To determine whether mAb B4 is typical of polyreactive natural Abs or has a more restricted reactivity, microarray analysis was performed using a series of Ags typically found to be targets of this class of Abs (29, 30). The accession number for the data is GSE14862; the data can be accessed on the website www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc= GSE14862. Positive controls for the assay were mAb NC17-D8 that recognizes phosphatidyl choline (58, 59) and polyclonal mouse IgM. As shown in Fig. 1⇑D, mAb B4 demonstrates only minimal reactivity with a subset of Ags that is far below that demonstrated by mAb NC17-D8 or polyclonal IgM. mAb B4 also does not recognize negatively charged phospholipids when using ELISA analysis (see Fig. 5 below). Thus, mAb B4 does not appear to be typical of the polyreactive subset of natural Abs.
Monoclonal Ab B4 restores IR injury in Rag1−/− mice
To determine whether mAb B4 exhibited the desired characteristic of induction of intestinal IR injury, we used Rag1−/− mice that are normally protected from IR injury (19). Purified mAb B4 or control IgM mAb D5 was injected i.v. 60 min before the reperfusion phase, and the intestines from mice undergoing IR were examined for injury.
Analysis of intestinal injury revealed that the sham treated Rag1−/− mice as well as Rag1−/− mice that underwent the IR protocol did not show significant damage as compared with wt C57BL/6 mice that demonstrated an injury score of 2.82 ± 0.9 (Fig. 2⇓). In contrast, Rag1−/− mice injected with 25 μg of mAb B4 Ab demonstrated an injury score of 1.8 ± 0.42. In contrast to the effects of mAb B4, the isotype control mAb D5, generated during the same screening protocol, did not cause IR injury in Rag1−/− mice even at a dose of 100 μg per mouse (score 0.75 ± 0.09). In other experiments, treatment of mice with doses of mAb B4 ranging from 9 to 81 μg also led to significantly increased IR injury (data not shown), and thus, there is not a narrow dose-response interval for its biologic effect.
Monoclonal Ab B4 recognizes annexin IV
The finding that mAb B4 was able to mediate intestinal IR injury in Rag1−/− mice led us to perform further studies and identify the protein specifically recognized by this mAb on IEC. We used a multistep process described in Materials and Methods, which culminated in a 2D gel separation of the proteins according to their m.w. and charge, following which the protein reactive with mAb B4 was characterized by proteolysis and MS analysis (Fig. 3⇓). Of note, the IEF gel, wherein the separation of proteins occurs based on charge, revealed that the protein of interest formed a long smear, thus making it difficult to localize it in a single spot (Fig. 3⇓A). To overcome the problem, we cut two separate areas from the gel and analyzed the proteins in each of these areas, considering that the same protein would be found in each section. MS analysis of fragmented protein in the two areas identified the same protein recognized by mAb B4 as mouse annexin IV (Fig. 3⇓B). Consistent with this result, using commercial polyclonal Abs to annexin IV, we were able to determine that anti-annexin IV Abs recognize the same extended band on the IEF gel to which mAb B4 binds (data not shown).
Monoclonal Ab B4 recognizes recombinant annexin IV expressed in mammalian cells
To confirm that murine annexin IV is the protein recognized by mAb B4, we expressed recombinant annexin IV in mammalian and bacterial cell systems. When recombinant annexin IV was expressed in the human F-293 cell line using the pSecTag2/Hygro B expression vector, Western blot analysis showed that the recombinant annexin IV protein expressed was recognized by mAb B4 (Fig. 3⇑C). The presence of annexin IV on the Western blot was confirmed by probing the membrane with polyclonal anti-annexin IV and anti-6× His Abs (data not shown). We also performed flow cytometric analysis of transfected F-293 cells to show that mAb B4 recognizes cells expressing recombinant murine annexin IV (Fig. 3⇑D).
When transfected F-293-A4 cells were probed with mAb B4 using flow cytometric analysis, the protein was found to be localized on the surface of the cells (Fig. 3⇑D). These results are consistent with the known localization of annexin IV whereby the protein is found to be closely associated with the plasma membrane. Previous results have suggested that annexin proteins may bind to the plasma membrane through a Ca2+-dependent mechanism (41, 60). However, when the recombinant annexin IV-expressing F-293-A4 cells were incubated in a buffer containing 0.5 M EDTA, immunoreactive protein was not released from the cell surface (Fig. 3⇑E). These data correlate with the observation that IEC isolated in EDTA-containing buffer display the mAb B4 epitope on the surface of IEC as detected by flow cytometric analysis. In sum, several lines of evidence using mammalian recombinant protein expression methods confirm that mAb B4 specifically recognizes mouse annexin IV.
Monoclonal Ab B4 does not recognize Ags previously shown to be targets for pathogenic Abs in intestinal IR injury
Two classes of Ags have previously been suggested as candidates for IR-related neo-epitopes and as targets for pathogenic natural Abs. Although mAb B4 was found to recognize annexin IV, we wanted to specifically determine whether it would also cross-react with these other Ags. First, since it had been shown previously that Cr2−/− and Rag1−/− mice reconstituted with Abs recognizing negatively charged phospholipids demonstrated IR injury (21), we used an anti-phospholipid Ab ELISA to determine whether mAb B4 would recognize this class of Ags. We were unable to detect any binding of mAb B4 to the same phospholipids recognized in previous studies (Fig. 4⇓A). Second, as reported by the Carroll group, the CM22 IgM mAb (37, 38) that is capable of inducing IR injury in Rag1−/− mice recognizes a synthetic peptide (designated N2) derived from one of its Ags, nonmuscle myosin, or a mimicking peptide (designated P8) that was identified from a phage display library. When the same synthetic peptides were probed in ELISA for binding by mAb B4, no detectable signal was apparent, in contrast to pooled sera from wt C57BL/6 mice that demonstrated robust binding to both peptides (Fig. 4⇓B). Thus, mAb B4 does not appear to cross-react with previously described Ags or epitopes important in this process.
IR injury restored by mAb B4 in Rag1−/− mice is accompanied by neutrophil infiltration and the local generation of proinflammatory eicosanoids
To confirm that the intestinal IR injury in Rag1−/− mice reconstituted with mAb B4 demonstrated similar characteristics to wt mice, we evaluated complement C3 deposition (see Fig. 6⇓) as well as the infiltration of neutrophils and generation of eicosanoids (Fig. 5⇓), all of which have been found in wt mice undergoing IR injury (16). To measure the level of neutrophil invasion into injured tissue, we performed biochemical analyses of MPO activity (Fig. 5⇓). Rag1−/− mice reconstituted with mAb B4 demonstrated elevated MPO activity indistinguishable from wt mice undergoing IR injury. Previous studies have shown that the neutrophil chemoattractant LTB4 is rapidly produced in response to intestinal IR injury (14, 16), and consistent with the increases in neutrophil invasion, the level of LTB4 in Rag1−/− mice reconstituted with mAb B4 was greatly increased as compared with LTB4 generation in sham-operated animals (Fig. 5⇓). Finally, the proinflammatory eicosanoid PGE2 also was elevated in samples from mice injected with mAb B4, but with marginal significance. In toto, mAb B4 appears to induce a proinflammatory response in Rag1−/− mice that is similar in nature to those in wt mice.
In situ analysis of mAb B4 localization and complement C3 deposition
It has been previously shown that complement C3 is deposited during intestinal IR injury (19, 23). The deposition of C3 in relation to mAb B4 in the intestines of mAb B4 treated Rag1−/− mice subjected to IR was determined by multicolor immunofluorescence microscopy (Fig. 6⇓). As shown, when Rag1−/− mice were reconstituted with mAb B4 and IR injury was induced, mAb B4 was detected in the intestinal microvillus under the enterocyte layer, possibly being attached to the basal membrane of these cells, and colocalizing with C3.
Recombinant annexin IV blocks intestinal IR injury in wt mice
Although our data using mAb B4 in Rag1−/− mice revealed that this Ab was sufficient to mediate intestinal IR injury, it was uncertain as to whether this reactivity was necessary in context of the entire natural Ab repertoire in wt mice to develop injury. To address this question, we used recombinant annexin IV as an “inhibitor” of the annexin IV reactive natural Ab repertoire. We used annexin IV made in bacteria because of the low yields and inability to purify large amounts of recombinant protein from F-293 cell membranes. Bacterial annexin IV was produced as described in Materials and Methods and was >95% pure by SDS-PAGE (data not shown). As a control for the experiments, we also produced and purified to a similar level a soluble fragment of cytokeratin 19, to which the control mAb D5 reacted (data not shown).
Following i.v. injection of wt mice 5 min before intestinal reperfusion phase with 50 μg/mouse of purified recombinant annexin IV or cytokeratin 19 as a relevant control, it was observed that the injection of annexin IV significantly reduced intestinal injury to the level of the sham-operated animals (Fig. 7⇓Aa). Reduction of IR injury in the intestine of mice injected with annexin IV was confirmed using additional approaches. First, the tissue MPO content (Fig. 7⇓Ab), which correlates with neutrophil activation and invasion, was also specifically reduced when compared with controls. Absence of inflammation in intestine of mice injected by annexin IV before reperfusion phase was also confirmed by the finding of specifically reduced levels of LTB4 and PGE2 (Fig. 7⇓A, c and d). Finally, immunofluorescence analysis revealed a greatly diminished C3 deposition in annexin IV-treated mice as compared with controls (Fig. 7⇓B). These data demonstrate that natural Ab reactivity with annexin IV is required for the full elaboration of intestinal IR injury.
Natural Abs to annexin IV are diminished in Cr2−/− mice
Because of the protection from the development of intestinal IR injury in Cr2−/− mice, we sought to determine whether differential reactivity to this particular Ag might underlie the difference in IR susceptibility. To address this question, we used an annexin IV ELISA to directly compare the level of anti-annexin IV Ab in serum samples from Cr2+/+ and Cr2−/− mice. As a negative control, sera from Rag1−/− mice were used (Fig. 8⇓A). Although we were not able to see complete abrogation of anti-annexin IV Ab in Cr2−/− mice, it is likely that the diminished natural Ab reactivity found is one reason that Cr2−/− mice are protected from IR injury.
Human annexin IV is a similar protein to mouse annexin IV and exhibits a 92% protein identity. We reasoned that it was relevant to determine whether similar natural Ab reactivity to annexin IV is present in humans. Indeed, when compared with sera from patients with agammaglobulinemia, there is IgM reactivity to annexin IV demonstrable in human serum (Fig. 8⇑B). These data suggest that a similar process of neo-epitope recognition could be present in humans with IR injury.
At the initiation of these studies, we hypothesized that neo-epitopes necessary for the development of IR injury could be identified by creating mAbs reactive with IEC, a major cellular target of reperfusion injury. Using this approach, we successfully identified a pathogenic IgM mAb, designated B4, that could alone mediate the induction of IR injury in Rag1−/− mice. In this experimental setting mAb B4 could lead to the fixation of complement C3 as well as the infiltration of neutrophils and elaboration of proinflammatory leukotrienes. Using several approaches, annexin IV was demonstrated to be the specific target Ag of mAb B4, and no evidence of cross-reactivity of mAb B4 with two previously suggested natural Ab targets, phospholipids and nonmuscle myosin, was found. Most importantly, the essential role for pathogenic natural Abs reactivity with annexin IV was confirmed by the specific inhibition of IR injury in wt mice using systemically administered recombinant annexin IV. Pretreatment of mice with annexin IV blocked tissue injury as measured by histologic criteria as well as greatly diminished the deposition of C3 and the elaboration of additional proinflammatory mediators in the intestine. Sera from Cr2−/− mice, which are relatively protected from intestinal IR injury, showed diminished reactivity with annexin IV. Finally, sera from normal human subjects also demonstrated IgM Ab reactivity with annexin IV, suggesting that a similar recognition process may play a key role in IR injury in man. In sum, annexin IV and natural Ab reactivity to this membrane-associated protein play an essential role in this medically important pathologic process.
One major question is how access to annexin IV by natural Abs is regulated. It is likely, based on what is known regarding the normal distribution of annexin IV in vivo, that IEC have an intracellular or internal membrane bilayer-associated pool of protein that transfers to the extracellular membrane upon initial cellular injury. Whether endothelial cells also elaborate this protein during IR injury is not known but such a situation might also contribute to the initial recognition of annexin IV by natural Abs. There was no evidence of reactivity of mAb B4 with endothelial surfaces as shown in Fig. 6⇑; however, recognition of endothelial cells may be below the level of detection of that immunofluorescence technique. Natural Abs then likely bind to several epitopes on annexin IV, activate complement and through this mechanism initiate the inflammatory cascade.
Annexin IV is a relatively widely distributed protein and a member of a family of largely membrane-associated proteins. Several annexin family members are expressed on the cell surface, for example, annexin II, which serves as an angiostatin receptor (61). Annexin VI and annexin II are found in the extracellular space and bind fetuin-A 43. The precise mechanisms by which annexins are transported to the surface of the cells are not certain. The N-terminal domain of annexins is responsible for binding to other proteins (62, 63, 64), and it is thus possible that the extracellular annexin IV we have detected is attached to the IEC membrane by being bound to another intrinsic membrane protein. Annexin IV has also been found in lipid rafts (65). The mechanism of raft inclusion is unknown; however, on the basis of this analogy to other lipid raft-associated proteins, this may be through attachment to a GPI anchor or through acylation with palmitate or myristate (66). A new ligand for annexin IV in the pancreas, GPI-anchored sialoprotein GP-2, was described recently (67). Our finding that annexin IV is detected on the IEC surface even if the purification protocol for these cells contains EDTA, as well as the finding that recombinant annexin IV, despite of presence of a leader peptide, was not released from F-293 cells but rather remained attached to the surface in a Ca2+-independent manner, strongly suggests that it is not using the well-described Ca2+-dependent phospholipid binding mechanism (40). The possibility of such Ca2+-independent binding by annexins was recently shown by others while demonstrating that annexin IV could bind phosphatidylserine- and phosphatidic acid-containing liposomes at low pH in a Ca2+-independent manner (68) and that hypoxia sufficient to induce an intracellular pH change can promote certain annexins to translocate to the surface of the plasma membrane (69).
The histological analyses clearly showed that when mAb B4 was injected into Rag1−/− mice, it was bound beneath the basal membrane of enterocytes in the villi. mAb B4 colocalizes with C3 deposition in intestine of mice undergoing IR injury, strongly suggesting that the binding of the mAb to the Ag results in complement activation. It is likely that annexin IV translocates to the cell surface when cells undergo hypoxia, early apoptosis, and perhaps necrosis. This translocation could also be facilitated by posttranslational modifications of annexin IV (53).
There is also a possibility that the chronic injury of epithelial cells may increase autoantibody production to annexin IV, as it has been demonstrated that patients with chronic alcoholism exhibit elevated levels of IgG autoantibodies to annexin IV (70, 71). This effect may increase the level of self-reactivity and promote the greater development of IR injury in the setting of similar levels of initial ischemia.
Natural Abs to IR related neo-epitopes are a component of the class of self-protein-directed Abs circulating in the body, the majority of which are products of B-1 B cells. The development of B-1 B cells secreting self-reactive Abs is believed to be Ag dependent (72). In this regard, it is notable that Cr2−/− mice, despite exhibiting normal quantitative levels of IgG and IgM isotype Abs (24), clearly show differences in natural Ab reactivity to annexin IV. It is likely that the absence of mouse CR2 is the major cause of that difference (23, 25, 73). In addition, while it has been shown that there is a decrease in autoantibody production in experimental immunization-induced models of self-reactivity in the absence of CR2, for example, collagen-induced arthritis (74), this is the first report to our knowledge of a marked decrease in natural Ab reactivity to specific self-Ags in Cr2−/− mice in the absence of directed immunization.
Of note, CD5+ B-1 cells do express mouse CR2; however, they do not amplify Ca2+ influx in response to BCR/C3dg coengagement (75) and also demonstrate a lower level of CR2 expression (76, 77). These data have been interpreted to indicate that, since these cells are already preactivated by Ag binding to the BCR, the cells are anergic due to chronic, low-grade, chronic stimulation in vivo. The lower levels of mouse CR2 may be due to receptor shedding or internalization after being engaged with its ligand (76, 77). If natural Ab reactivity to annexin IV is derived primarily from B-1 cells, and CR2 is required on B-1 cells for this process, this suggests that in vivo responses through CR2 are substantially different than those found in vitro using coligation as an analytic tool. Of course, since coligation of BCR with CR2 lowers the activation signal in B-2 cells several thousand-fold (78), it is also possible that B-2 cells play the major role in driving natural Ab self-reactivity to annexin IV. Finally, since the absence of mouse CR2 not only changes the B-2 cell activation threshold to immune complexes but also greatly alters follicular dendritic cell/immune complex networks (79) and diminishes Ab responses to T-independent Ags (73), additional mechanisms may play important roles in the lack of development of natural Abs to annexin IV in the absence of this receptor.
Finally, it is worth considering how natural Ab reactivity to annexin IV relates to nonmuscle myosin and phosopholipids, both of which have been previously suggested as targets for natural Abs in IR-induced injury and, at least with regard to nonmuscle myosin, as necessary for the development of tissue injury in vivo. There are several reasons that might explain these findings. One possibility is that serial recognition of several epitopes that are displayed in a stereotypic fashion during the development of cell apoptosis/necrosis is required to initiate complement-dependent injury. In that setting, interruption of reactivity of natural Abs to any one of the Ags could ameliorate the process. A second possibility is that epitopes are expressed at the same time but on unique cell populations, for example, nonmuscle myosin on endothelial cells and annexin IV on IECs, and recognition of each cell type is necessary for full injury development. A third possibility is that all of these proteins come together in protein/phospholipid complexes that are exposed during IR, and binding of natural Abs to several of the epitopes is necessary to activate complement and induce injury. What argues against these hypotheses is that individual IgM mAbs can transfer injury in Rag1−/− mice. However, it is possible that a high dose of individual injected IgM mAbs can overcome restriction points that would be present in wt mice with lower levels of natural Abs. Certainly going forward, it will be necessary to better understand the physicochemical and quantitative relationships between the various neo-epitopes that are important in injury as well as the natural Abs that recognize them.
In summary, we propose that annexin IV is a major IR related neo-epitope that is recognized by pathogenic natural Abs. Further explorations of the mechanisms of natural Ab induced injury, a determination of how and where annexin IV epitopes are displayed, and the development of a better understanding of how this component of the natural Ab repertoire is positively selected by the presence of CR2 are particularly relevant areas for future investigation.
The authors have no financial conflict of interest.
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↵1 This work was funded by U.S. Army Medical Research and Materiel Command (MRMC) Award W81XWH-06-1-0520 and MRMC Award W81XWH-07-1-0286; the Alliance for Lupus Research; and National Institutes of Health Grants R01 AI31105, A161691, AI46637, DK41873, and DK55357.
↵2 Address correspondence and reprint requests to Dr. V. Michael Holers, Departments of Medicine and Immunology, University of Colorado Denver School of Medicine, Mail Stop B115, P.O. Box 6511, Aurora, CO 80045. E-mail address:
↵3 Abbreviations used in this paper: IR, ischemia-reperfusion; CR, complement receptor; DAPI, 4′,6-diamidino-2-phenylindole; IEC, intestinal epithelial cell; IEF, isoelectric focusing; LIC, ligation-independent cloning; LTB4, leukotriene B4; MPO, myeloperoxidase; MS, mass spectrometry; SA, streptavidin; wt, wild type.
- Received November 26, 2008.
- Accepted January 17, 2009.
- Copyright © 2009 by The American Association of Immunologists, Inc.