The appropriate development of an inflammatory response is central for the ability of a host to deal with any infectious insult. However, excessive, misplaced, or uncontrolled inflammation may lead to acute or chronic diseases. The microbiota plays an important role in the control of inflammatory responsiveness. In this study, we investigated the role of lipoxin A4 and annexin-1 for the IL-10-dependent inflammatory hyporesponsiveness observed in germfree mice. Administration of a 15-epi-lipoxin A4 analog or an annexin-1-derived peptide to conventional mice prevented tissue injury, TNF-α production, and lethality after intestinal ischemia/reperfusion. This was associated with enhanced IL-10 production. Lipoxin A4 and annexin-1 failed to prevent reperfusion injury in IL-10-deficient mice. In germfree mice, there was enhanced expression of both lipoxin A4 and annexin-1. Blockade of lipoxin A4 synthesis with a 5-lipoxygenase inhibitor or Abs against annexin-1 partially prevented IL-10 production and this was accompanied by partial reversion of inflammatory hyporesponsiveness in germfree mice. Administration of BOC-1, an antagonist of ALX receptors (at which both lipoxin A4 and annexin-1 act), or simultaneous administration of 5-lipoxygenase inhibitor and anti-annexin-1 Abs, was associated with tissue injury, TNF-α production, and lethality similar to that found in conventional mice. Thus, our data demonstrate that inflammatory responsiveness is tightly controlled by the presence of the microbiota and that the innate capacity of germfree mice to produce IL-10 is secondary to their endogenous greater ability to produce lipoxin A4 and annexin-1.
The appropriate development of an inflammatory response is central for the ability of a host to deal with any infectious insult. Indeed, leukocyte recruitment and activation are essential for Ag processing and presentation, for lymphocyte priming and for the effector functions (such as Ab production and cell-mediated immunity) of any immune response (1). In the absence of inflammation, lethality is the usual outcome after an infectious challenge. In contrast, excessive, misplaced, or uncontrolled inflammation is commonly the cause of death after infection. Inflammation does not only occur in the presence of an infectious insult, but is also a common denominator of the tissue response to stressful stimuli of diverse nature (chemical, mechanical, infectious, etc.). In fact, the list of human diseases associated with inappropriate or uncontrolled inflammation in response to stimuli of known or unknown origin is growing and include ischemia and reperfusion injury, rheumatoid arthritis, asthma, chronic obstructive pulmonary disease, multiple sclerosis, and atherosclerosis (2, 3, 4). In the latter conditions, tissue inflammation is clearly deleterious and inhibition of the inflammatory response may be of therapeutic benefit.
In mice, reperfusion of the ischemic superior mesenteric artery is followed by severe local (intestine) and remote (lungs) tissue pathology, characterized by marked neutrophil influx, edema formation, hemorrhage, and tissue destruction (5). Not only is there tissue damage, but also marked systemic inflammation, as assessed by the elevation in the serum concentration of proinflammatory cytokines and chemokines (5). In contrast to these findings, germfree mice, which have no detectable bacteria (and indeed, no other known pathogen) in their gut, presented little evidence of local or systemic injury after intestinal ischemia and reperfusion (6). The inability of germfree mice to inflame in response to systemic LPS or reperfusion-induced injury was largely because of the innate capacity of these mice to produce IL-10 and, possibly, other anti-inflammatory molecules (6). Indeed, blockade of IL-10 production in germfree mice was accompanied by reversal of inflammatory hyporesponsiveness and significant inflammatory responses to intestinal reperfusion or LPS administration. Moreover, reposition of the microbiota was accompanied by loss of the ability to produce IL-10 and regained ability to inflame in response to diverse stimulation (6). Thus, the latter results suggested that the lack of microbiota was accompanied by a state of active IL-10-mediated inflammatory hyporesponsiveness.
There is growing evidence that, during an inflammatory response, there are active processes and mediators which prevent excessive inflammation, the so-called “mechanisms of anti-inflammation” (7), and that may induce resolution of inflammation (8). There has been much recent interest in a series of mediators of the inflammatory process which possess significant anti-inflammatory actions when given exogenously, including lipoxin A4 (LXA4)3 and annexin-1 (ANXA-1, previously referred to as lipocortin-1) (9, 10, 11, 12). The mechanisms by which these molecules modulate the inflammatory response are not clearly shown and a recent study suggested that control of SOCS-2 activation might be relevant for the action of LXA4 under certain conditions (13). It has been demonstrated that activation of the LXA4 receptor down-regulates polymorphonuclear (PMN) responses in vitro and promotes resolution of inflammation through up-regulation of NAB1, a transcriptional corepressor identified previously as a glucocorticoid-responsive gene (14). It is also possible that the effects of LXA4 and ANXA-1 may be mediated by the release of molecules with anti-inflammatory effect. For example, ANXA-1 may function via the release of IL-10 (15). Less is known about the possibility that “mediators of anti-inflammation” are capable of controlling the inflammatory process when released endogenously (16). In this regard, germfree mice could provide a powerful tool for a better understanding of the mechanisms and mediators involved in the control of inflammation. In the present study, using a mixture of immunological, biochemical, and pharmacological approaches, we evaluated the functional relevance of LXA4 and ANXA-1 release and activation of their shared ALX receptor for IL-10 production and the inflammatory hyporesponsiveness observed in germfree mice.
Materials and Methods
Germfree Swiss/NIH mice were derived from a germfree nucleus (Taconic Farms) and maintained in flexible plastic isolators (Standard Safety Equipment) using classical gnotobiology techniques (17). Conventional Swiss/NIH mice are derived from germfree matrices and considered conventional only after two generations in the conventional facility. All experimental procedures in germfree mice were conducted under aseptic conditions to avoid infection of animals. C57BL/6 or IL-10-deficient mice (8–10 wk), obtained from the Bioscience Unit of Instituto de Ciências Biológicas (Brazil), were housed under standard conditions and had free access to commercial chow and water. All animals were 8- to 10-wk-old males and females, and the experimental protocols used were approved by the animal ethics committee of Universidade Federal de Minas Gerais.
To evaluate the role of LXA4 and ANXA-1 in intestinal ischemia and reperfusion model several experimental protocols were performed. 1) To reproduce the action of LXA4 and ANXA-1, conventional mice were treated with their respective mimetics, the 15-epi-LXA4 analog ATL-1 (5 μg/mouse—a generous gift from Brigham and Women′s Hospital, Harvard Medical School, Boston, MA) (18) or the peptide Ac2–26 (10 mg/kg) (19), i.v. 10 min before reperfusion. 2) To prevent the action of LXA4 and ANXA-1, mice were treated with the ALX antagonist BOC-1 (2.0 mg/kg) (20), the 5-lipoxygenase inhibitor ZM230487 (5 mg/kg) (21) or the BLT1/2 antagonist CP-105696 (3 mg/kg) (22) i.v. 10 min before reperfusion, or with anti-ANXA antiserum (0.2 ml of hyperimmune serum/animal), or the Cys-LT antagonist Montelukast (5 mg/kg,) (23) s.c. 30 min before reperfusion. As nonimmune serum had no effect on the injury induced by reperfusion of the ischemic superior mesenteric artery (SMA) (data not shown), results in nonimmune serum- and vehicle-treated animals were pooled for presentation.
Ischemia and reperfusion
Mice were anesthetized with urethane (1400 mg/kg, i.p.) and laparotomy was performed. The SMA was isolated and ischemia was induced by totally occluding the SMA for 60 min. For measuring percentage of surviving mice, reperfusion was re-established, and mice were monitored for the indicated time periods. For the other parameters, reperfusion was allowed to occur for the indicated period of time before sacrifice. Sham-operated animals were used as controls.
Evaluation of changes in vascular permeability
The extravasation of Evans blue dye into the tissues was used as an index of increased vascular permeability, as previously described (24, 25). Briefly, Evans blue (20 mg/kg) was administered i.v. (1 ml/kg) via a tail vein 2 min before reperfusion of the ischemic artery. Thirty minutes after reperfusion, a segment of the duodenum (∼3 cm) or the flushed left lung were cut in small pieces and Evans blue extracted using 1 ml of formamide. The amount of Evans blue in the tissue (μg of Evans blue per 100 mg of tissue) was obtained by comparing the extracted absorbance with that of a standard Evans blue curve read at 620 nm in an ELISA plate reader.
The extent of neutrophil accumulation in the intestine and right lung tissues was measured by assaying myeloperoxidase activity, as previously described (25, 26). Briefly, a portion of duodenum and the flushed right lungs of animals that had undergone ischemia/reperfusion injury were removed and snap frozen in liquid nitrogen. Upon thawing and processing, the tissue was assayed for myeloperoxidase activity by measuring the change in OD at 450 nm using tetramethylbenzidine. Results were expressed as total number of neutrophils by comparing the OD of tissue supernatant with the OD of casein-elicited murine peritoneal neutrophils processed in the same way.
Measurement of hemoglobin concentrations
The determination of hemoglobin concentrations in tissues was used as an index of tissue hemorrhage. After washing and perfusing the intestines to remove excess blood in the intravascular space, a sample of ∼100 mg of duodenum was removed and homogenized in Drabkin’s color reagent according to instructions of the manufacturer (Analisa). The suspension was centrifuged for 15 min at 3000 × g and filtered using 0.2 μm filters. The resulting solution was read using an ELISA plate reader at 520 nm and compared against a standard curve of hemoglobin.
Measurement of mRNA expression by real-time RT-PCR
Total RNA was isolated from intestine using RNeasy mini kit (Qiagen). The RNA obtained was resuspended in diethylpyrocarbonate-treated water and stocked at −70°C until use. Real-time RT-PCR was performed on an ABI PRISM 7900 sequence detection system (Applied Biosystems) using SYBR Green PCR Master Mix (Applied Biosystems) after reverse transcription reaction of 2 μg of RNA using M-MLV reverse transcriptase (Promega). The relative level of gene expression was determined by the comparative threshold cycle method as described by the manufacturer, whereby data for each sample were normalized to hypoxanthine phosphoribosyltransferase and expressed as a fold change compared with naive animals. The following primer pairs were used: for hypoxanthine phosphoribosyltransferase, 5′-TTGGTTACAGGCCAGACTTTGTTG-3′ (forward) and 5′-GAGGGTAGGCTGGCCTATAGGCT-3′ (reverse); for il-10 5′-GCTCTTACTGACTGGCATGAG-3′ (forward) and 5′-CGCAGCT-CTAGGAGCATGTG-3′ (reverse).
One hundred milligrams of duodenum of sham-operated and reperfused animals were homogenized in 1 ml of cell lysis buffer (1% Nonidet P-40, 100 mM Tris-HCl (pH 8.0), 20% glycerol, 0.2 mM EDTA, 1 mM NaPO3, 1 mM DTT, 1 mM PMSF, 200 mM NaCl, leupeptin, and aprotinin). The samples were then centrifuged for 10 min at 3000 × g and the supernatant was collected, and total protein concentration was determined according to the instructions of Bio-Rad assay kit. To detect ANXA-1, protein extracts (30 μg) were loaded onto a 10% SDS-PAGE for electrophoresis together with the appropriate m.w. markers and transferred to ECL Hybond nitrocellulose membrane. Reversible protein staining of the membranes with 0.1% Ponceau S in 5% acetic acid was used to verify even protein transfer. Membranes were incubated for 1 h at room temperature in 5% nonfat dry milk in PBS with 0.1% Tween 20 (PBST). The membranes were washed three times for 5 min with PBST and incubated overnight with rabbit hyperimmune serum anti-ANXA-1 (1:100) in PBST with 5% BSA. After new washing, the membranes were incubated for 60 min at room temperature with peroxidase-conjugated goat anti-rabbit IgG (1:600), and immunoreactive proteins were detected using an ECL kit (Amersham Biosciences). Relative band intensity was quantified using NIH image software 1.63.
Measurement of lipoxin, cytokine/chemokine concentrations in serum, intestine, and lungs
The concentration of LXA4 was measured using commercially available Abs and according to the procedures supplied by the manufacturer (Neogen). The concentration of TNF-α, KC, and IL-10 in samples was measured in serum and tissue of animals using commercially available Abs and according to the procedures supplied by the manufacturer (R&D Systems). Serum was obtained from coagulated blood (15 min at 37°C, then 30 min at 4°C) and stored at −20°C until further analysis. Serum samples were analyzed at a 1/3 dilution in PBS. One hundred milligrams of duodenum or lung of sham-operated and reperfused animals were homogenized in 1 ml of PBS (0.4 M NaCl and 10 mM NaPO4) containing anti-proteases (0.1 mM PMSF, 0.1 mM benzethonium chloride, 10 mM EDTA and 20 KI aprotinin A) and 0.05% Tween 20. The samples were then centrifuged for 10 min at 3000 − g and the supernatant immediately used for ELISA at a 1/3 dilution in PBS.
Results are shown as means ± SEM. Percent inhibition was calculated by subtracting the background values obtained in sham-operated animals. Differences were compared by using ANOVA followed by Student-Newman-Keuls post hoc analysis. Results with a p < 0.05 were considered significantly different.
Exogenous administration of LXA4 or ANXA-1 prevents reperfusion injury in conventional mice in an IL-10-dependent manner
Initial experiments evaluated the ability of LXA4 and ANXA-1 to protect mice from reperfusion injury. The treatment of mice with 15-epi-16-(para-fluoro)-phenoxy-LXA4-metil ester (ATL-1), a stable 15-epi-LXA4 analog (1 μg/mouse), or peptide Ac2–26 (10 mg/kg), which contains the active portion of ANXA-1, greatly inhibited the reperfusion-associated inflammatory response, as assessed by the decrease in vascular permeability, leukocyte influx, and hemorrhage in the intestine (Fig. 1, A–C). ATL-1 and peptide Ac2–26 were also capable of inhibiting the reperfusion-induced remote injury to the lungs (data not shown). Both compounds greatly suppressed the reperfusion-associated production of TNF-α in the intestine (Fig. 1,D), lungs (data not shown) and serum (Fig. 1,F). Furthermore, ATL-1 or Ac2–26 delayed and partially prevented lethality in conventional mice (Fig. 1, G and H).
Interestingly, the inhibition of tissue injury and lethality by the treatment with ATL-1 or Ac2–26 was accompanied by a significant increase in the levels of IL-10 in intestine (Fig. 1,E) and lungs (data not shown). To verify whether the IL-10 released was relevant for the action of ATL-1 and Ac2–26, we conducted experiments in IL-10 deficient mice (IL-10−/−). IL-10−/− animals submitted to reperfusion injury had a significant inflammatory response characterized by intense neutrophil influx, plasma extravasation, tissue hemorrhage and elevation of TNF-α that was not significantly different from wild type animals (compare Fig. 1 and Table I). However, neither ATL-1 nor Ac2–26 were able to prevent the inflammatory response in IL-10−/− mice subjected to intestinal reperfusion injury (see Table I). These results indicate that the protective effect of the exogenous administration of LXA4 and ANXA-1 mimetics is dependent on the production of IL-10.
|.||Sham .||Ischemia and Reperfusion Injury .||.||.|
|.||.||Vehicle .||ATL-1 .||Ac2–26 .|
|Plasma extravasation||0.9 ± 0.1||5.8 ± 0.2c||4.6 ± 0.5cd||4.0 ± 0.4cd|
|Neutrophil influx||1.0 ± 0.2||5.3 ± 0.9c||4.9 ± 0.6c||5.5 ± 0.4c|
|Hemorrhage||71 ± 9||355 ± 48c||295 ± 59c||262 ± 50c|
|TNF-α||NDb||163 ± 22c||151 ± 20c||148 ± 16c|
|.||Sham .||Ischemia and Reperfusion Injury .||.||.|
|.||.||Vehicle .||ATL-1 .||Ac2–26 .|
|Plasma extravasation||0.9 ± 0.1||5.8 ± 0.2c||4.6 ± 0.5cd||4.0 ± 0.4cd|
|Neutrophil influx||1.0 ± 0.2||5.3 ± 0.9c||4.9 ± 0.6c||5.5 ± 0.4c|
|Hemorrhage||71 ± 9||355 ± 48c||295 ± 59c||262 ± 50c|
|TNF-α||NDb||163 ± 22c||151 ± 20c||148 ± 16c|
IL-10-deficient mice were administered vehicle (PBS, 200 μl), a 15-epi-LXA4 analogue (ATL-1, 1 μg/mouse) or a peptide containing the active portion of annexin-1 (Ac2–26, 10 mg/kg) 10 min prior to reperfusion. After 40 min of reperfusion, the intestine was removed and evaluated for plasma extravasation (μg of Evans blue/100 mg of tissue), neutrophil influx (as assessed by measurement of myeloperoxidase activity), hemorrhage (μg of hemoglobin/100 mg of tissue), and TNF-α (pg of cytokine/100 mg of tissue). Results are the mean ± SEM of 6 animals in each group.
ND, Below the detection limit of the assay.
p < 0.01 when compared with sham-operated mice;
p < 0.01 when compared with vehicle-treated mice submitted to reperfusion injury.
Enhanced production of LXA4 and ANXA-1 by germfree mice
Germfree mice submitted to ischemia and reperfusion or given other inflammatory stimuli do not present an inflammatory response (6). In contrast, germfree mice produce high levels of IL-10 that actively prevents TNF-α production and inflammation. Here, we performed real-time-PCR to assess enhancement of IL-10 expression after reperfusion injury. Our data clearly demonstrate that IL-10 mRNA expression in germfree mice was significantly greater than that found in conventional mice (Fig. 2 A).
As LXA4 and ANXA-1 may drive IL-10 production in vivo, we evaluated whether there was greater production of LXA4 or ANXA-1 in germfree when compared with conventional mice. Our results demonstrated that there were no differences in basal LXA4 production in intestines of germfree and conventional mice. After reperfusion, there was a time-dependent increase of LXA4 production that peaked at 40 min after reperfusion (Fig. 2,B). Levels of LXA4 in germfree mice were ∼3-fold higher than those found in conventional mice (Fig. 2 B).
The expression of ANXA-1 was evaluated by Western blot. There was greater basal expression of ANXA-1 in germfree mice when compared with conventional mice. After reperfusion, the expression of ANXA-1 was enhanced in both germfree and conventional mice, but the enhancement appeared to be greater in the former (Fig. 2 C). Altogether, the latter results suggest that germfree mice have an innate ability to express basally or produce greater quantities of endogenous anti-inflammatory mediators when submitted to an inflammatory stimulus. The next series of experiments were designed to investigate whether the production of the latter mediators was relevant for the inflammatory hyporesponsiveness of germfree mice.
Inhibition of LXA4 partially reverses the inflammatory hyporesponsiveness of germfree mice
To inhibit lipoxin action, we used two different strategies, an inhibitor of 5-lipoxygenase (ZM230487), a central enzyme in lipoxin biosynthesis, and BOC-1, an antagonist of the ALX receptor (27). Postischemic treatment of germfree mice with ZM230487 (5.0 mg/kg) or BOC-1 (2.0 mg/kg) was accompanied by a significant increase in reperfusion-induced tissue injury, as assessed by intestinal (Fig. 3, A–C) and pulmonary (data not shown) vascular permeability, neutrophil influx, and hemorrhage. Germfree mice had undetectable concentrations of TNF-α in intestine and serum after ischemia/reperfusion injury. In germfree mice treated with ZM230487 or BOC-1, there was a marked increase in the intestinal (Fig. 3,D) and serum (Fig. 3,F) levels of TNF-α. The production of TNF-α in the lungs of mice which received ZM230487 or BOC-1 was also elevated (data not shown). Treatment with ZM230487 or BOC-1 was also accompanied by significant reperfusion-induced lethality, which contrasts to the lack of lethality observed in vehicle-treated germfree mice (Fig. 3, G and H). Vehicle-treated germfree produced large quantities of IL-10 when submitted to reperfusion, an effect greatly reversed by treatment with ZM230487 or BOC-1 both in the intestine (Fig. 3 E) and lungs (data not shown).
The production of lipoxins and leukotrienes (LT), including LTB4 and cysteinyl-LTs, relies on a biosynthetic pathway dependent on the activation of 5-lipoxygenase. As such, treatment with the 5-lipoxygenase inhibitor ZM230487 would prevent the production of both lipoxins and of LTB4 and cysteinyl-LTs (28). To exclude the participation of leukotrienes in our system, we used the specific BLT1/2 and cysteinyl-LT receptor antagonists CP-105696 (3 mg/kg) and Montelukast (5 mg/kg), respectively. At the doses used, both compounds selectively prevent the action of the ligand on the relevant receptor (29, 30). Treatment with either drug failed to alter the inflammatory hyporesponsiveness of germfree mice, as shown by the lack of effect of the compounds on reperfusion-induced lethality (Fig. 3 G). The latter results suggest that LXA4 is the 5-lipoxygenase product relevant for the inflammatory hyporesponsiveness of germfree mice. Treatment with CP-105696 or Montelukast had no significant effects in the lethality rates of conventional mice submitted to ischemia and reperfusion injury (data not shown).
It is relevant to note that BOC-1 was more effective than ZM230487 in reversing the inflammatory responsiveness of germfree mice, as shown by all the parameters assessed in Fig. 3. As BOC-1 is an antagonist of the ALX receptor, these data indicate that a ligand other than LXA4 may also mediate IL-10 production and inflammatory hyporesponsiveness in germfree mice.
Inhibition of ANXA-1 partially reverses the inflammatory hyporesponsiveness of germfree mice
There is greater expression of ANXA-1 in germfree mice and ANXA-1 may share the ALX receptor with LXA4 (30). The next series of experiments evaluated whether ANXA-1 was relevant for inflammatory hyporesponsiveness of germfree mice. To this end, the action of ANXA-1 was prevented by the administration of anti-ANXA-1 antiserum. Similarly to the treatment with ZM230487, treatment of germfree mice with anti-ANXA-1 antiserum was accompanied by an increase in reperfusion-induced increase in Evans blue extravasation, neutrophil recruitment and hemoglobin content in the intestine (Fig. 4, A–C) and lungs (data not shown). Anti-ANXA-1 treatment induced a significant enhancement in TNF-α concentration and a decrease in IL-10 concentration in intestine (Fig. 4, D–E) and lungs (data not shown) of germfree mice. The inhibition of reperfusion-induced IL-10 production and enhancement of TNF-α and tissue inflammation in anti-ANXA-1-treated germfree mice was accompanied by a significant increase in lethality (Fig. 4 G).
Simultaneous inhibition of LXA4 and ANXA-1 prevents IL-10 production and reverses the inflammatory hyporesponsiveness of germfree mice
It is interesting to note that treatment with BOC-1 was significantly more effective than treatment with either ZM230487 or with anti-ANXA-1 antiserum (compare Figs. 3 and 4). There is evidence showing that both LXA4 and ANXA-1 may share the same receptor, ALX, and that effects mediated by this receptor can be inhibited by BOC-1 (27, 30). To examine whether a combined action of LXA4 and ANXA-1 would be sufficient to fully activate the ALX receptor and mediate the inflammatory responsiveness of germfree mice, we administered ZM230487 and anti-ANXA-1 Ab concomitantly to germfree mice submitted to intestinal ischemia and reperfusion.
The concurrent treatment completely reversed the inflammatory hyporesponsiveness of germfree mice (Fig. 5), as demonstrated by the reperfusion-induced increase of vascular permeability, neutrophil influx, hemorrhage, and cytokines production in the intestine. It is particularly interesting to note that the innate capacity of germfree animals to produce great quantities of IL-10 was abolished by concomitant administration of ZM230487 and anti-ANXA-1 antiserum (Fig. 5,F). Indeed, all the parameters assessed, including systemic levels of TNF-α and survival rates, returned to levels similar to those observed in conventional mice (Fig. 5). Similar results were observed in the lungs of reperfused germfree mice treated with ZM230487 and anti-ANXA-1 antiserum (data not shown).
There are two main conclusions that may be reached from the results presented above: (i) the ability of the exogenous administration of LXA4 or ANXA-1 to prevent the local, remote and systemic injury induced by reperfusion of the ischemic small intestine in mice is IL-10-dependent; (ii) the production of LXA4 and ANXA-1 is enhanced in germfree mice, and both mediators appear to cooperatively activate the shared ALX receptor to induce IL-10 production and mediate the inflammatory hyporesponsiveness of germfree mice.
Lipoxins (LX), such as LXA4, constitute the first recognized class of anti-inflammatory lipid-based autacoids which may function as endogenous “stop signals” that down-regulate or counteract the formation and actions of proinflammatory mediators (31, 32) and promote resolution (33, 34). ANXA-1 is another mediator of anti-inflammation that was identified originally as the responsible for several of the anti-inflammatory actions of glucocorticoids (35, 36, 37). Both LXA4 and ANXA-1 or compounds which mimic their actions have anti-inflammatory effects in several models of acute and chronic inflammation, and in models of inflammation-mediated tissue injury (30, 38, 39, 40). A few studies have demonstrated the inhibitory effects of LXA4 or its analogues in the context of reperfusion injury (41, 42, 43). Similarly, the ANXA-1-derived peptide Ac2–26 has been shown to protect against intestinal (44) or myocardial (45) reperfusion injury. Thus, the findings demonstrating a protective anti-inflammatory effect of ATL-1 and Ac2–26 in our model of intestinal ischemia and reperfusion injury are consistent with these latter studies.
There are several possible mechanisms to explain the ability of LXA4 and ANXA-1 to prevent inflammation in general and reperfusion injury in particular. For example, LXA4 has been shown to inhibit neutrophil chemotaxis (46, 47), neutrophil adhesion to and transmigration across endothelial cells and across monolayers of human intestinal epithelial cells (48, 49), and neutrophil-mediated increases in vascular permeability (50). Furthermore, a previous study demonstrated that an overexpression of the human LXA4 receptor in vivo could have a downstream effect on signal generation and reduce the number of PMN infiltrating into tissues (16). ANXA-1 has been shown to promote the detachment of neutrophils adhered to the inflamed endothelium, hence reducing the number of cells that migrate into the subendothelial space (51). In addition, the latter mediators have also been shown to prevent the production of TNF-α in various models of inflammation (52, 53). As neutrophils and TNF-α cooperate to mediate reperfusion-induced injury and lethality, the above-mentioned effects could account for the protective effects of LXA4 and ANXA-1 in mice. An interesting finding was the marked elevation of the production of IL-10 when either the ATL-1 or Ac2–26 was administered to reperfused mice. This is consistent with an in vitro study demonstrating the capacity of ANXA-1 in stimulating a macrophage cell line to produce IL-10 (15). More importantly, neither ATL-1 nor Ac2–26 prevented inflammatory injury or lethality in IL-10-deficient mice, suggesting that the enhanced production of IL-10 was relevant to their action. It is clear that IL-10 modulates proinflammatory cytokine production and tissue injury following ischemia and reperfusion injury (54, 55, 56, 57). For example, exogenous administration of IL-10 reduced the systemic inflammatory response during intestinal reperfusion injury, an effect associated with inhibition of TNF-α production and neutrophil accumulation (6, 55, 58). Thus, the results above suggest that the inhibitory effects of the administration of LXA4 or ANXA-1 mimetics during reperfusion injury are secondary to their ability to enhance IL-10 production and to modulate TNF-α production and neutrophil influx in an IL-10-dependent manner. This is to the best of our knowledge the first demonstration that LXA4 and ANXA-1 play a relevant role in inducing IL-10 production in vivo and modulating inflammation in an IL-10-dependent manner.
It has been recently reported that SOCS-2 is a critical intracellular regulator of the immunoregulatory and anti-inflammatory actions of lipoxins in mice infected with the intracellular parasite Toxoplasma gondii (13). In the latter study, lipoxins were not found to induce IL-10 production and the actions of IL-10 in the system differed from those of lipoxins (13). Moreover, in a model of renal ischemia reperfusion injury in vivo, mice treated with the same analog used in this work displayed increased mRNA levels for SOCS-1 and SOCS-2 (42). Whether SOCS-2 is expressed and mediates the induction of IL-10 and inhibitory actions of ATL-1 in mice undergoing ischemia and reperfusion injury is not known. Similarly, it is not know whether ANXA-1 is capable of inducing SOCS-2 or mediating its anti-inflammatory effects in vivo via this transcription factor. Other possible transcription factor that may participate in ALXR signaling is NAB1, a transcriptional corepressor identified previously as a glucocorticoid-responsive gene (29, 31, 59, 60). In this context, Qiu et al. (14) demonstrated that activation of the LXA4 receptor increased NAB1 expression, down-regulated PMN responses in vitro and promoted resolution of inflammation.
We have previously demonstrated that germfree mice are hyporresponsive to a range of inflammatory stimuli (6). Indeed, the production of TNF-α and recruitment of neutrophils is greatly suppressed in germfree mice undergoing ischemia and reperfusion injury. In contrast, there is an enhanced production of IL-10. The IL-10 produced is very important for the inflammatory hyporesponsiveness of germfree because inhibition of this cytokine is accompanied by an increase of TNF-α production, neutrophil recruitment and reperfusion-induced tissue injury and lethality (6). As our initial experiments showed that both LXA4 and ANXA-1 enhanced IL-10 production when given exogenously and exerted their anti-inflammatory actions in an IL-10-dependent manner, it was relevant to evaluate the participation of these mediators in germfree mice. It is of note that levels of both LXA4 and ANXA-1 were elevated in germfree mice. Interestingly, elevated levels of these mediators could be detected before the enhancement of IL-10 mRNA levels in germfree mice, supporting that LXA4 and ANXA-1 act via IL-10 synthesis. LXs are lipoxygenase-derived arachidonate metabolites whose production may be inhibited by 5-lipoxygenase inhibitors and whose actions can be prevented by antagonists at ALX receptors. In agreement with a role for LXA4 in mediating the hyporesponsiveness of germfree mice, animals treated with 5-lipoxygenase inhibitors or the receptor antagonist had reduced IL-10 production and, consequently, significant inflammatory injury and lethality. Overall, the effects of the receptor antagonist were of much greater intensity than those of the 5-lipoxygenase inhibitor suggesting that another molecule acting on the same receptor could be mediating the enhanced production of IL-10 and inflammatory hyporesponsiveness. It is also important to note that blockage of BLT1/2 or cysteinyl leukotriene receptors had no effect on the inflammatory hyporesponsiveness of germfree mice. This is an important observation as inhibition of 5-lipoxygenase would be accompanied by inhibition of lipoxins and leukotrienes. Furthermore, in previous studies of our group, we have demonstrated that there is augmented LTB4 production after ischemia and reperfusion in conventional animals (25). Blockade of BLT1/2 resulted in reduced injury after reperfusion of the ischemic SMA in these animals (22, 25). These results suggest a proinflammatory role played by leukotrienes in the present model. However, BLT1/2 blockade did not change inflammatory hyporesponsiveness of germfree mice. This suggests that the results obtained with ZM230487 treatment are due mainly to inhibition of lipoxins action. There are evidences that acute inflammation resolution involve temporal regulation of lipid mediator generation, with early coordinate appearance of leukotrienes and prostaglandins followed by lipoxin production in the next phase of the inflammatory response (61). In our system (germfree mice), it is possible that absence of microbiota conferred an “innate switch” in lipid profile production.
Previous studies have now shown that LXA4 and ANXA-1 may share the ALX receptor (27, 62, 63), also known as FPRL-1. It was, thus, possible that ANXA-1 was the other agonist acting on the ALX receptor to mediate IL-10 production and inflammatory hyporesponsiveness. Treatment with the anti-ANXA-1 antiserum partially prevented IL-10 production and partially reversed the inflammatory hyporesponsiveness. When animals were given a combination of the antiserum and the 5-lipoxygenase inhibitor, IL-10 production was completely ablated. Inhibition of IL-10 production was accompanied to a level of reperfusion-induced neutrophil recruitment, TNF-α production and lethality similar to that found in conventional mice or germfree mice given the ALX antagonist. Altogether, these results are consistent with the hypothesis that an elevated production of LXA4 and ANXA-1 and cooperative action on ALX receptors mediate the enhanced production of IL-10 and IL-10-dependent inflammatory hyporesponsiveness of germfree mice. The present study did not investigate in any detail the cell types responsible for the production of IL-10. However, we previously demonstrated that macrophages derived from germfree mice and stimulated with LPS responded by producing IL-10, whereas those from conventional mice produced TNF-α (6). Overall the latter results show that the difference in macrophage function between conventional and germfree mice may be fundamental and suggest that studies of macrophage function may provide clues to answer why germfree mice preferentially produce IL-10. More recently, mast cells have been suggested as potential source of IL-10 in models of immune and innate inflammation in mouse skin (64). We have not evaluated expression of IL-10 by mast cells in our experiments but the function of these cells could also contribute to the hyporresponsive phenotype observed in germfree mice.
Most studies evaluating the relevance of LXA4 and ANXA-1 in the control and resolution in the inflammatory process have evaluated the effect of the exogenous addition of these molecules (9, 10, 11, 12). These studies have suggested that mediators of anti-inflammation and resolution may be useful in the treatment of acute and chronic inflammatory diseases. The present findings indicate that enhancement of IL-10 production may be an important action of LXA4 and ANXA-1 in vivo. Moreover, they indicate that the endogenous production of these substances mediates inflammatory hyporesponsiveness and is tightly controlled by the presence of the microbiota (See Fig. 6). This may be relevant in newborns who have not yet been colonized and in whom excessive inflammation may be detrimental. If this tenet is true it is possible we learn from germfree animals how to enhance LXA4 and ANXA-1 production. These studies may lead to finding of relevant new therapies for both acute and chronic inflammatory conditions.
The authors have no financial conflict of interest.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This work was supported by Fundação de Amparo à Pesquisa do Estado de Minas Gerais and Conselho Nacional de Desenvolvimento Cientifico e Tecnológico.
Abbreviations used in this paper: LXA4, lipoxin A4; ANXA-1, annexin-1; SMA, superior mesenteric artery; PMN, polymorphonuclear; LT, leukotriene.