Hemorrhage decreases blood flow to the intestine and is followed by rapid intestinal damage and inflammation, which may initiate a systemic inflammatory response (1, 2). The intestinal inflammatory response involves multiple components of the innate immune system including neutrophils, complement, and cytokines (3-5). Hemorrhage-induced inflammation also includes activation of macrophages within the liver, spleen, and lungs (6-8). However, the specific complement activation pathway and the early intestinal effects on macrophage infiltration and activation in response to hemorrhage are not well defined.
Composed of more than 30 proteins in the blood, complement protects the body by removing pathogenic microorganisms. Initiated by multiple pathways, complement activation generates C3 opsonins, chemotactic peptides, and the cytolytic terminal membrane attack complex. Antibody or lectin deposition on surface membranes initiates the classic and lectin pathways, respectively. The spontaneously activated, alternative pathway amplifies the classical and lectin pathways. However, excessive complement activation increases susceptibility to infections (9), and the amplification induced by the alternative pathway increases pathology in multiple disease states (10, 11). Treatment with complement inhibitors, C5a receptor antagonist (3) or cobra venom factor (12), attenuated hemorrhage-induced intestinal damage without identifying the specific initiation pathway. It is likely that the alternative pathway either directly or by amplification contributes to hemorrhage-induced tissue damage. During excessive or inappropriate complement activation, natural inhibitors protect host cells and tissues. Factor H (fH) binds C3b, preventing or dissociating the alternative C3 convertase and the subsequent amplification loop. By inhibiting amplification of the classic and lectin pathways, fH is a logical therapeutic for excessive complement activation. In other disease models, complement receptor 2 (CR2)-targeted fH significantly enhanced the effectiveness of fH by targeting the inhibitor to sites of complement activation (13).
Hemorrhage induces neutrophil and macrophage infiltration into the hypoxic intestine (12, 14). Multiple studies indicated that a neutrophil response with oxidative burst is critical to hemorrhage-induced damage (14, 15). Hemorrhage also activates Kupffer cells in the liver and macrophages in the spleen (6, 7) to release cytokines. A recent study indicated that intestinal tissues release the macrophage inflammatory cytokines, TNF-α, IL-6, and NO in response to hemorrhage (12). In addition, inhibition of IL-6 (16) or TNF (17, 18) attenuated hemorrhage-induced inflammation. However, a role of macrophage-produced IL-12 in the intestinal response to hemorrhage is not well characterized. The proinflammatory IL-12p70 consists of two subunits, IL-12p40 and IL-12p35. IL-12p40 is also a subunit of IL-23, which stimulates TH17 cells. In addition, IL-12p40 homodimers directly antagonize both IL-12p70 and IL-23 (19). Therefore, hemorrhage-induced macrophage infiltration may control the inflammatory process through the production of specific IL-12 components.
We hypothesized that alternative complement activation interacts with macrophage-secreted IL-12, and all three innate immune components are critical for hemorrhage-induced inflammation. We show that administering the targeted complement inhibitor, CR2-fH, to mice after hemorrhage significantly decreases intestinal damage and inflammation by reducing macrophage infiltration and IL-12p40 production. Furthermore, macrophage depletion with clodronate, IL-12p40, or IL-12p35 depletion attenuated injury and inflammation. In contrast, IL-23p19 depletion did not attenuate injury. Thus, inhibition of the alternative complement activation pathway attenuates macrophage infiltration and IL-12p70 production during the hemorrhage-induced inflammatory response and provides multiple therapeutic targets within the innate immune response.
MATERIALS AND METHODS
C57Bl/6J male mice (6-8 weeks old) were bred and maintained in the Division of Biology at Kansas State University. IL-12p40−/− and IL-12p35−/− mice (N11F32) were obtained from Jackson Laboratories (Bar Harbor, Maine) and rested 1 week before use in experiments. All mice were housed in a 12-h light-dark, temperature-controlled room and allowed food and water ad libitum. All research was approved by the Institutional Animal Care and Use Committee and conducted in compliance with the Animal Welfare Act and other federal statutes and regulations concerning animals.
After the mice were anesthetized with ketamine (16 mg/kg) and xylazine (8 mg/kg), a drop of 0.5% proparacaine hydrochloride ophthalmic solution was applied to the appropriate eye. Mice subjected to hemorrhage sustained removal of 25% total blood volume via the retro-orbital sinus as described previously (3, 20). The blood volume removed was determined by weight (in grams) using the following equation: ∼25% = wt × 0.02 (3, 20), and completed within 5 min. Sham-treated mice were subjected to similar procedures with no blood removal. Body temperature of animals was maintained at 37°C using a water-circulating heating pad, and all procedures were performed with the animals breathing spontaneously. Two hours after hemorrhage, mice were killed, and sera and tissue were collected. Midjejunal sections (2 cm), approximately 10 cm distal to the gastroduodenal junction, were collected for histology and subsequent analyses. Additional mice underwent the same procedures as above with i.v. administration of CR2-fH (17.5uM) 5 min after bleeding. CR2-fH was produced as described previously (13). Some mice received neutralizing anti-IL-12p40 (R&D Systems, Minneapolis, Minn) or anti-IL-23p19 (R&D Systems) mAb (0.1-1 mg/kg) i.p. at 1 h before hemorrhage.
Histology and injury score
After fixation in 10% buffered formalin, paraffin-embedded tissue sections were cut transversely (8 μm) and hematoxylin-eosin stained for scoring mucosal injury. Using a six-tiered scale as described previously (21), the average damage score of midjejunum intestine (75-150 villi) was determined after grading each villus from 0 to 6 with the following categories: Normal villi were assigned a score of zero; villi with tip distortion were assigned a score 1; a score of 2 was assigned when Guggenheim spaces were present; villi with slight disruption of the epithelial cells were assigned a score of 3; a score of 4 was assigned to villi with exposed but intact lamina propria; a score of 5 was assigned when the lamina propria was exuding; last, villi that displayed hemorrhage or were denuded were assigned a score of 6.
Ex vivo secretions
Ex vivo intestinal supernatants were generated as described previously (3, 21) and used to determine the secretions released in a 20-min period. Briefly, a 2-cm midjejunal section was minced, washed, resuspended in 37°C oxygenated Tyrode buffer (Sigma-Aldrich, St. Louis, Mo) and incubated for 20 min at 37°C. After incubation, the supernatants and tissues were collected and stored at −80°C until assayed. After overnight digestion in 0.1 M NaOH at 37°C, protein content in the intestinal tissue was determined by BCA protein assay (Pierce, Rockford, Ill). Cytokine concentrations in the intestinal supernatants were determined with a Milliplex MAP kit (Millipore, Billerica, Mass), following the manufacturer's instructions, and analyzed using xPONENT 3.1 and Analyst (Millipore). Because liposomes interfered with the Milliplex assay, IL-12p40 was also determined by enzyme-linked immunosorbent assay (BioLegend, San Diego, Calif). Leukotriene B4 (LTB4) concentrations in the intestinal supernatants were measured using a commercially available enzyme immunoassay kit (Cayman Chemicals, Ann Arbor, Minn). All concentrations in the intestinal supernatants were normalized to the total intestinal protein content and reported as picograms per milligram of intestinal tissue. Sera C5a concentrations were determined by capture enzyme-linked immunosorbent assay (BD Biosciences, San Jose, Calif).
After sham or hemorrhage treatment, a 2-cm midjejunal section was frozen in O.C.T. freezing medium and stored at −80°C until used. Intestinal cryosections (8 μm) were fixed in cold acetone, and nonspecific binding was blocked using 10% donkey serum in phosphate-buffered saline (PBS). Tissues were stained for C3 deposition using a rat anti-mouse C3 antibody (Hycult Biotechnologies, The Netherlands) followed by an appropriate secondary antibody (Jackson Immunoresearch, West Grove, Pa) or for F4/80 using directly conjugated rat anti-mouse F4/80 (eBioscience, San Diego, Calif). Serial sections stained with isotype control antibodies were used as background. Slides were examined by a blinded observer by fluorescent microscopy using a Nikon 80i fluorescent microscope, and images acquired using a CoolSnapCf camera (Photometrics, Tucson, Ariz) and MetaVue Imaging software (Molecular Devices, Sunnyvale, Calif).
Infiltrating macrophages were depleted using clodronate liposomes as described previously (22, 23). Briefly, mice were injected i.v. with 200 μL clodronate- or PBS-containing liposomes on days 0, 1, 3, and 5 with hemorrhage occurring on day 5 or 6. Liposomes were produced using phosphatidylcholine (Lipoid GmbH, Ludwigshafen, Germany) and cholesterol (Sigma, St Louis, Mo). Cl2MDP (clodronate) was a gift of Roche Diagnostics GmbH (Mannheim, Germany).
Data are presented as means ± SEM. Differences between treatment groups were considered significant if P ≤ 0.05 as determined by one-way ANOVA with a Newman-Keuls post hoc test.
CR2-fH attenuates hemorrhage-induced mucosal damage and inflammation
We verified the ability of CR2-fH to inhibit complement activation by measuring serum C5a in wild-type C57Bl/6 and CR2-fH-treated mice. As no significant difference was observed between shams from different treatment groups or mouse strains, these data are shown as pooled shams in all graphs. Similar to previous studies (3), serum from hemorrhaged C57Bl/6 mice contained significantly more C5a than sham-treated mice (Fig. 1A). In contrast, C5a was not significantly elevated in the serum of CR2-fH-treated mice with or without hemorrhage (Fig. 1A), indicating that CR2-fH attenuated hemorrhage-induced complement activation. Importantly, administration of CR2-fH significantly attenuated intestinal damage in response to hemorrhage (Table 1; Fig. 1, E and F). CR2-fH treatment also attenuated the intestinal inflammatory response. Intestinal LTB4 production was significantly elevated in hemorrhage-treated mice only in the absence of CR2-fH treatment (Fig. 1B). In addition, at 2 h after hemorrhage, C57Bl/6 mice expressed significantly elevated levels of intestinal IL-12p40 and TNF-α, and CR2-fH treatment reduced these cytokine secretions to the level of sham-treated mice (Fig. 1, C and D). These data indicate that in a fixed-volume mouse model of hemorrhage, inhibition of the alternative complement pathway attenuates the early LTB4 and cytokine response.
Macrophage depletion attenuates hemorrhage-induced intestinal damage and inflammation
As the above cytokines may be produced by activated macrophages or epithelial cells, we hypothesized that hemorrhage induces an infiltration of F4/80+ macrophages in intestinal sections. Immunohistochemistry showed few, if any, macrophages in the intestinal villi of sham-treated mice (Fig. 2, A and G). Intestinal villi from hemorrhage-treated mice contained significantly more F4/80+ cells (Fig. 2, B and G). Similar to its effect on cytokine levels, CR2-fH significantly reduced the number of F4/80+ macrophages infiltrating the villi in response to hemorrhage (Fig. 2, C and G).
To determine if macrophages are required in hemorrhage-induced intestinal injury, we subjected mice to hemorrhage after depletion of macrophages with Cl2MDP-containing liposomes. Immunohistochemistry confirmed macrophage depletion in Cl2MDP-treated mice (Fig. 2, D and G). As indicated in Table 1, macrophage-depleted mice did not sustain hemorrhage-induced intestinal injury when compared with mice treated with PBS-containing liposomes. Similar to untreated mice, in response to hemorrhagic shock (HS), mice treated with liposomes containing PBS produced significant quantities of LTB4 and IL-12p40 (3.2 ± 1.1 and 12 ± 1.11 pg/mg tissue, respectively). After treating with liposomes containing Cl2MDP, hemorrhage induced significantly lower concentrations of intestinal LTB4 and IL-12p40 (0.98 ± 0.13 and 4.6 ± 1.7 pg/mg tissue, respectively) compared with liposomes containing PBS. It was possible that macrophage depletion altered the mechanism of intestinal damage. Therefore, we examined C3 deposition in mice treated with liposomes containing PBS or Cl2MDP. Compared with sham-treated mice, intestinal sections from mice pretreated with PBS-containing liposomes displayed more C3 deposition (Fig. 3, A and B). In contrast, C3 deposition was not detected on the intestinal tissue of macrophage-depleted mice (Fig. 3C).
IL-12p35 RNA expression increases in response to hemorrhage
IL-12 family members, IL-12p70 and IL-23, play a role in other models of intestinal damage (24-27). Using real-time polymerase chain reaction, we evaluated IL-12p40, IL-12p35, and IL-23p19 mRNA to determine the critical family members. As indicated in Figure 4A, IL-12p40 mRNA was maintained within all treatment groups. In contrast, IL-12p35 was significantly elevated in response to HS in wild-type mice and in wild-type mice after treatment with PBS-containing liposomes (Fig. 4B). However, treatment with fH-CR2 or macrophage depletion eliminated the HS increase in IL-12p35 RNA (Fig. 4B). Although IL-23p19 RNA expression increased with HS, complement inhibition altered only IL-12p35 RNA expression, not production of IL-23p19 RNA expression (Fig. 4C). Clodronate treatment eliminated hemorrhage-induced IL-12p40, IL-12p35, and IL-23p19 RNA expression (Fig. 4). These data suggest that HS induces macrophages to produce IL-12p70.
IL-12p70 is critical to hemorrhage-induced damage and inflammation
To establish if IL-12p70 or IL-23 plays key roles in hemorrhage-induced intestinal injury and inflammation, we analyzed mucosal damage and complement activation in response to sham or hemorrhage treatment in IL-12p40−/− or IL-12p35−/− mice. Hemorrhage induced significantly less midjejunal damage in IL-12p40−/− or IL-12p35−/− mice compared with C57Bl/6 mice (Table 1). The integrity of the villi and intestinalepithelium was maintained in both strains of IL-12-deficient mice at 2 h after hemorrhage (data not shown). In response to hemorrhage, intestinal sections from IL-12p40−/− mice contained significantly fewer C3 deposits compared with wild-type mice (Fig. 3, D and E). Similar to sham-treated mice, no C3 deposits were observed on midjejunal tissues from hemorrhaged IL-12p35−/− mice. Similarly, the absence of IL-12p40 significantly attenuated hemorrhage-induced serum C5a compared with C57Bl/6 mice (138 ± 13 and 194 ± 25 ng/mL, respectively). However, the hemorrhage-induced C5a levels in IL-12p40−/− mice were significantly higher than sham-treated mice (76 ± 6 ng/mL). Thus, IL-12p40−/− and IL-12p35−/− mice are protected from intestinal damage with lower levels of complement activation than C57Bl/6 mice. Despite increased macrophage infiltration after hemorrhage (Fig 2, E and F), IL-12p40−/− and IL-12p35−/− mice produced significantly less intestinal LTB4 and TNF-α than wild-type mice (Fig. 5, A and B).
To confirm that IL-12p70, and not IL-23, is a critical intestinal cytokine produced in response to HS, we administered either IL-12p40- or IL-23p19-neutralizing antibodies or isotype control antibodies to additional wild-type mice. As indicated in Table 1 and Figure 5C, anti-mouse IL-12p40 mAb attenuated intestinal hemorrhage and inflammation similar to that of IL-12p40−/− or IL-12p35−/− mice. Mice receiving immunoglobulin control sustained midjejunal mucosal damage and inflammation similar to wild-type mice. In contrast, administration of even 100 times the neutralizing dose of anti-IL-23p19-specific antibodies did not attenuate intestinal damage, TNF, or LTB4 production (Table 1; Fig. 5C). Together, these data suggest that macrophage production of IL-12p70 is critical for intestinal damage and inflammation.
Although hypothesized to be the motor of systemic inflammation and subsequent mortality, intestinal damage and inflammation occur rapidly (within 2 h), and repair occurs equally quickly (within 3-4 h) in the presence or absence of resuscitation (2, 12). Previous studies concluded that the hemorrhage-induced response requires complement activation without identifying the specific activation pathway (3, 20, 28, 29). The present study indicates that inhibition of the alternative complement pathway with CR2-fH attenuates midjejunal mucosal damage and complement activation in a fixed-volume mouse model of hemorrhage. Complement inhibition also decreased the proinflammatory intestinal secretions TNF-α, IL-12p40, and LTB4. Furthermore, pretreatment with Cl2MDP-containing liposomes significantly reduced cytokine secretion and attenuated damage and inflammation, suggesting that hemorrhage induces macrophage secretion of TNF and IL-12. Finally, IL-12p40−/− and IL-12p35−/− sustained limited to no complement activation or hemorrhage-induced intestinal damage despite an intestinal macrophage infiltration. Together, these data suggest that the hemorrhage-induced inflammatory response requires complement activation, macrophage infiltration, and IL-12 production.
Previous studies showed that systemic blockade of central complement components, C3, C5, or C5a, attenuated hemorrhage-induced intestinal damage (3, 30, 31). However, each of these systemic inhibitors increases the risk of sepsis by blocking multiple complement activation pathways. CR2-fH binds tissue-bound C3 activation products and provides local but not systemic complement inhibition (13, 32). In addition, when administered early after hemorrhage, CR2-fH effectively prevented hemorrhage-induced tissue damage and inflammation. These results are similar to the ability of CR2-fH to attenuate tissue damage in models of collagen-induced arthritis, complement-dependent macular degeneration, and direct intestinal ischemia/reperfusion (13, 32). Treatment with CR2-fH also significantly decreased TNF-α mRNA in arthritic joints (32), suggesting a decrease in macrophage cytokine production. We extended the anti-inflammatory properties of CR2-fH to include significantly reducing macrophage infiltration and associated cytokines in response to hemorrhage. Although the specific mechanism is not known, the alternative complement activation pathway is critical for hemorrhage-induced intestinal damage and inflammation.
Within the intestinal mucosa, blood monocytes infiltrate into inflamed tissues to replenish mucosal macrophages (33). In response to hemorrhage, macrophage infiltration increased significantly within 2 h after hemorrhage. These data correlate with intestinal production of macrophage chemotactic factors, which are produced in response to hemorrhage (20). When macrophages were depleted with liposomes containing Cl2MDP, intestinal damage and IL-12 and LTB4 production were significantly reduced. Similar to these results in the intestine, depletion of Kupffer cells by gadolinium chloride significantly reduced hemorrhage-induced liver damage (6). Thus, hemorrhage seems to induce an early excessive innate response by the macrophages.
Macrophage cytokine production mediates the recruitment of innate immune cells and coordinates activation of adaptive immune responses. Multiple studies indicate that TNF-α and IL-6 increase in the serum within 2 h after hemorrhage (6, 7, 34). However IL-12 is more complex. IL-12p70 is involved in the differentiation of helper T cells to the proinflammatory TH1 subset, with macrophages and dendritic cells as the primary sources of this cytokine. IL-12p40 homodimers seem to have regulatory effects, and, as a subunit of IL-23, IL-12p40 may induce IL-17 (35). With limited complement activation, hemorrhage of IL-12p40−/− or IL-12p35−/− mice resulted in significantly less tissue damage and inflammation. In addition, hemorrhage induced significant damage in mice treated with IL-23p19-neutralizing antibodies. Thus, it seems that IL-12p70 is the critical family member in hemorrhage-induced intestinal damage.
In response to hemorrhage, the alternative complement activation pathway induces a macrophage infiltrate into the intestine and IL-12 secretion. These data indicate that all three components of the innate immune response are required for hemorrhage-mediated midjejunal damage. Thus, therapeutics targeting local alternative complement activation not only prevent complement-mediated damage, but also attenuate macrophage activation and cytokine production.
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Inflammation; mouse; intestine; cytokines; complement inhibitor