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, 182 (9), 5363-73

Pathogenic Natural Antibodies Recognizing Annexin IV Are Required to Develop Intestinal Ischemia-Reperfusion Injury

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Pathogenic Natural Antibodies Recognizing Annexin IV Are Required to Develop Intestinal Ischemia-Reperfusion Injury

Liudmila Kulik et al. J Immunol.

Abstract

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.

Figures

Figure 1
Figure 1
Characterization of mAb B4 epitope expression. A, Representative flow cytometric histograms show binding of mAb B4 to a single cell suspension of IEC (top), splenocytes (middle) and thymocytes (bottom). Cells were incubated with mAb B4, and bound Ab was detected by using anti-mouse IgM (μ-chain specific) for IEC and thymocytes. In the case of splenocytes, mAb B4 labeled with biotin was used. B and C, Monoclonal Ab B4 epitope expression was determined by Western blot analysis of isolated from thymus, spleen or intestine cell in (B) or lysates were prepared from whole organs in (C). Data are representative of two independent experiments. D, Binding of mAb B4 to proteins typically found as targets of polyreactive natural Abs was studied by micro-array analysis. The positive control for mAb B4 binding are C1q and anti-mouse IgM antibody, positive controls for the micro-array are mAb NC-17D8 and polyclonal IgM, and the negative control is designated blank.
Figure 2
Figure 2
Restoration of IR injury in Rag1−/− mice by mAb B4. Sixty min prior to the induction of ischemia by mesenteric artery occlusion, 25 μg of purified mAb B4, or 100 μg of control IgM mAb D5, was injected i.v. Following completion of the IR protocol, mice were sacrificed and Giemsa-stained intestinal sections from each treatment group were scored for mucosal injury (0–6) as described in Materials and Methods. Monoclonal Ab B4 injected into Rag1−/− mice induced substantial injury in the mice undergoing IR (mAb B4 IR) in contrast to sham operated mice (Rag1−/− sham) or Rag1−/− mice undergoing IR (Rag1−/− IR). MAb D5 treated Rag1−/− mice (mAb D5 IR) did not demonstrate substantial injury. As a positive control for the level of intestinal injury, wildtype C57Bl/6 mice undergoing IR were included (wt IR). All measurements were obtained at x200 magnification. The figure is a representative of two independent experiments. Each bar is the average ± SEM with 3–6 animals per group. Statistical analysis was performed by Student’s t test.
Figure 3
Figure 3
Identification of the antigen recognized by mAb B4. A, Lysates prepared from isolated IEC were separated using a sequential three gel separation protocol as described in Materials and Methods. The presence of the B4 antigen was confirmed by Western blot analysis (right). Two gel samples (gray color boxes) were analyzed by MS. B, The MASCOT search results for the protein isolated from first sample was identified as mouse annexin IV based on the highest score number of 785; 78 matched peptides covering 53% of the annexin IV sequence were identified (underlying bold letters). C, Lysates of untransformed F-293 cells (lane 1) and F-293 cells transformed with the pSecTag2/Hygro B expression vector carrying annexin IV insert (F-293-A4) (lane 3) together with culture supernatants from transformed cells (lane 2) were probed by Western blot analysis with mAb B4. D, Flow cytometric analysis of F-293 cells expressing recombinant annexin IV. F-293-A4 cells were probed in flow cytometry with mAb B4. E, Supernatant (lane1) and lysate (lane 2) from F-293-A4 cells expressing recombinant annexin IV that had been incubated in PBS alone, or supernatant (lane 3) and lysate (lane 4) from the same cells incubated with 0.5 M EDTA, were probed by Western blot analysis with mAb B4. Recombinant annexin IV was not released from F-293-A4 cells in the absence of free Ca2+.
Figure 4
Figure 4
Monoclonal Ab B4 does not cross-react with previously published antigens recognized by pathogenic natural Abs in intestinal IR injury. A, Monoclonal Ab B4 was tested in an anti-phospholipid Ab ELISA for the binding to cardiolipin (CL), phosphorylcholine-BSA (PC-BSA), phosphatidic acid (PA), phosphatidylserine (PS), and phosphatidylglycerol (PG). A broadly reactive IgM anti-phospholipid mAb (our unpublished data) was used as a positive control. Bound Abs were detected by AP-conjugated goat anti-mouse IgM. Data are shown as OD405nm. B, Bar graph shows the lack of reactivity of mAb B4 in ELISA with synthetic peptides reported to be targets for the pathogenic CM22 IgM mAb. Synthetic peptides coated to an ELISA plate were incubated with either mAb B4 or control mAb D5. Bound antibodies were detected by AP-conjugated goat anti-mouse IgM Ab. Pooled sera from C57BL/6 mice at a dilution of 1/50 were used as a positive control. Data are shown as OD405nm and are representative of two independent experiments.
Figure 5
Figure 5
Association of neutrophil infiltration and eicosanoid generation with IR in Rag1−/− mice treated with mAb B4. Intestinal tissue from sham operated wildtype C57Bl/6 mice (black bar), wildtype mice undergoing IR (horizontally stripped bar), or sham operated Rag1−/− mice (unfilled bar), and Rag1−/− mice pre-injected with mAb B4 and undergoing IR (gray bar) were collected. The concentrations of MPO (left), LTB4 (middle) and PGE2 (right) were determined ex vivo using enzyme immunoassays. The data are presented as picogram per milligram of tissue for LTB4 and PGE2, and as MPO activity. Each bar is the average ± SEM with four to six animals per group. Statistical significance was determined using an unpaired 2-tailed Student’s t test. ND: data not obtained.
Figure 6
Figure 6
Monoclonal Ab B4, when injected into Rag1−/− mice and inducing IR injury, clusters in the microvillus beneath the enterocyte layer. Three color immunofluorescence staining for C3 (green), mouse Ig (red), and DAPI (blue) was performed. Clusters of C3 deposition co-localized with mouse Ig (arrow head) and C3 deposition alone (arrow) were seen in intestine from Rag1−/− mice reconstituted by mAb B4 before IR (upper picture). Slides made from the same intestine were probed with anti-mouse Ig and isotype control antibody for C3 antibody (lower picture). Note absence of the yellow color clusters therein.
Figure 7
Figure 7
A, Intestinal IR injury is ameliorated in wildtype C57Bl/6 mice receiving recombinant annexin IV prior to the ischemic phase. Reduction of the of IR induced injury to the level of sham operated animals (B6 sham) was observed in wild type mice when they were injected with 50 μg/mouse of annexin IV 5 min prior to the reperfusion phase (A4 IR). Injury in mice pre-injected with control CPK19 protein (CP K19 IR) was comparable with the injury in C57Bl/6 mice undergoing IR (B6 IR) (a). Small intestine tissue samples were processed as in Fig. 6 for myeloperoxidase activity (b) and the eicosanoids LTB4 (c) and PGE2 (d). Each bar is the average ± SEM with 3–6 - animals per group. Statistical significance was determined using one way ANOVA. B, Small intestine of annexin IV pre-injected mice (far right), similarly to sham operated wild type mice (far left) does not show C3 deposition in IR induced injury, in contrast to wild type mice (second from left) and mice pre-injected with the CPK19 control protein undergoing IR (second from right).
Figure 8
Figure 8
Presence of natural antibodies to annexin IV. A, Cr2+/+ in contrast to Cr2−/− mice demonstrate higher levels of IgM natural Ab to annexin IV. Serum samples from three cohorts of mice, Cr2−/− (n=9), Cr2+/+ mice (n=14) and negative control Rag1−/− (n=5) mice were evaluated for the presence of natural IgM (left) and IgG (right) Abs to bacterial annexin IV. The figures are representative of two independent experiments. Statistical significance was determined using a Wilcoxson test. B, 9 serum samples from healthy humans and three from agammaglobulinemic patients were tested in the anti-annexin IV ELISA. Substantial levels of anti-annexin IV IgM antibody IgM is present in the sera of normal humans.

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