The polyamines (putrescine, spermidine, and spermine) are polycationic compounds found in high concentrations in all eukaryotic cells (1). Their intracellular concentrations are dependent on the activity of ornithine decarboxylase (ODC), which is one of the rate-limiting enzymes in polyamine biosynthesis, and on the equilibrium between their uptake, excretion, and catabolism (2,3). Polyamines are of fundamental importance in biologic processes such as cell proliferation and differentiation (1,4,5).
During the postnatal development of the rat small intestine, the mucosa undergoes major modifications in its structural and functional properties. At the time of weaning, the large supranuclear vacuoles contained in the enterocytes of the immature ileum disappear (6), and some mucosal enzyme specific activities (SA) change, such as those of lactase, which decreases, and maltase and sucrase, which increase (7). During the same period, the mucosal ODC activity and the intestinal polyamine content increase, indicating the importance of these compounds in weaning (8).
Intestinal maturation, which appears at weaning, is dependent upon genetic, dietary, and hormonal factors (9). We have demonstrated that the ingestion of spermidine or spermine by suckling rats induces precocious maturation of the small intestine (10). The mechanism by which polyamines accelerate or induce intestinal maturation is not yet established. We previously reported that corticosterone is involved in the effects of spermine on intestinal functions (11,12) and that the cytokines interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α, appear in the plasma of suckling rats between 4 and 8 hours after the first spermine ingestion (13). Moreover, intraperitoneal injection of some of these cytokines induces modifications of disaccharidase SA (13), mainly an increase of maltase and sucrase SA. Cyclosporin A, an activator of T-cells and inhibitor of IL-2 production, reduces the spermine-induced increase of maltase and sucrase SA (14). In this report, we checked whether the inhibition of IL-1β and TNF-α biologic activity reduces the spermine-induced effect on the disaccharidase SA. We also analyzed the role of IL-2 in the spermine-induced maturation of the small intestine. Intraperitoneal injection of lipopolysaccharide (LPS) was used to mimic the effect of spermine ingestion on the immune system. Indeed, LPS injection rapidly induces the production of IL-1β and TNF-α in the plasma (15).
MATERIALS AND METHODS
Wistar rats, housed in an air-conditioned room at 23°C with a 12-hour light-dark cycle, were used throughout the study. They were fed with A03 10-mm pellets (Animalabo, Brussels, Belgium) and had water ad libitum. The litters were reduced to 12 pups per lactating mother with free access between mother and pups. The day of birth was designated as day 0. In our experiments there was no body weight difference between male or female pups. No distinction between sexes was made. Because it is well known that the experimental values vary from one litter to another, the comparisons of results were always made between animals from the same litter. All animal experiments were approved by the animal welfare committee of the University of Liege and of the Fonds de la Recherche Scientifique Médicale (FRSM).
Chemicals and Equipment
All chemicals were purchased from Sigma Chemical Co. (St Louis, MO, U.S.A.) or from Merck (Darmstad, Germany). Immune rabbit serum containing polyclonal antimurine TNF-α was from Genzyme diagnostics (Cambridge, MA, U.S.A.). Ten microliters of serum can neutralize 1,000 units of mouse TNF-α bioactivity in a standard L929 cell cytotoxicity assay. Mouse monoclonal anti-rat IL-1β (clone 38139.11, 500 μg/mL) was from R&D systems (Abingdon, United Kingdom). Neutralization Dose50 was determined to be 10 to 30 μ g/mL in the presence of 4 ng/mL of rat IL-1β using the murine T-helper cell line D10.G4.1. FR167653, an experimental molecule able to inhibit production of IL-1β and TNF-α in vivo (16), was a generous gift from the Fujisawa Co. (Osaka, Japan).
Spermine was dissolved in 50 μL of water (αQ system, Millipore) and administered orally (8 μmol/animal) once a day for 1 (11th postnatal day) or 3 days (11th, 12th, and 13th postnatal days). Control animals were treated in the same way but received only the vehicle. The dose used was almost the same as the amount of polyamines ingested daily by adult rats by the way of solid food (17).
Immune rabbit serum containing the polyclonal antimurine TNF-α, a neutralizing antibody, was injected intraperitoneally (75 μL), diluted with 25 μL of sterile saline (NaCl, 0.9%), or intracardiacally (25 μL), diluted with 25 μL of sterile saline. Control animals were injected in the same way with heat inactivated nonimmune rabbit serum. These injections were performed 2 hours before spermine administration.
Antimurine IL-1β (monoclonal antibody) was injected intraperitoneally (40 μg), diluted in 200 μL of sterile saline. Control animals were injected with the same sterile saline. These injections were performed 30 minutes before spermine administration.
FR167653 was injected intracardiacally (0.3 mg) in 50 μL of sterile saline. Control animals were injected in the same way with the vehicle. These injections were performed 15 minutes before spermine administration.
The dose of anticytokines was based on information available in the literature (16,18). Concerning anti-TNF-α, we used 75 μL of antiserum. In the literature, 50 μL antiserum was injected intraperitoneally before lethal (400 μg) LPS challenge. This treatment significantly protected animals from death. We reduced the dose for intracardiac injection because of direct administration in the blood flow. Concerning anti-IL-1β treatment, we used 40 μg antibody to inhibit a IL-1β plasma concentration of about 6 ng/mL (13). This dose is close to the ID50. Concerning FR167653, we used one bolus injection of 15 μg/g body weight. The literature reported a continuous injection of 0.3 μg/g/15 minutes in animal injected with LPS (1 μg/g body weight). This injection began 15 minutes before the LPS challenge and continued for 4 hours. So the total amount of FR167653 injected was 5 μg/g body weight. This treatment reduced TNF-α and IL-1β concentration to the level observed in untreated animals. Schedules of anticytokine therapy were based of the delay of production of cytokines after spermine administration. TNF-α appeared in the plasma 2 hours and IL-1β 4 hours after the ingestion of spermine. This timing is similar to those observed after LPS injection. We used the same treatment schedule as for LPS inhibition.
Murine IL-2 was injected intraperitoneally (100 μL, 20 μg/kg body weight), once or three times a day for 3 days (11th, 12th, and 13th postnatal days) in sterile NaCl solution (0.9%). Control animals were treated in the same way but received only the vehicle.
LPS from E. coli 026:B6 (10 μg) was injected intra-peritoneally in 100 μL of sterile saline to 11-day-old rats. Control rats received only the vehicle. This treatment was repeated on days 12 and 13.
In the first and second set of experiments, the litter was divided into three groups of four rats and the groups designated as CONT, SPM, and SPM-anti-TNF-α. The SPM group received spermine and an intraperitoneal (first experiment) or intracardiac (second experiment) injection of nonimmune rabbit serum. The SPM-anti-TNF-α rats received spermine and an intraperitoneal or intracardiac injection of polyclonal antimurine TNF-α. Rats in the CONT group received only the vehicles. Animals were killed on the 14th postnatal day by cervical dislocation; tissues were collected immediately after the sacrifice.
In the third set of experiments, the litter was divided into three groups of four rats and the groups designated as CONT, SPM, and SPM-anti-IL-1. The SPM group received spermine and an intraperitoneal injection of sterile saline. The SPM-anti-IL-1 rats received spermine and an intraperitoneal injection of monoclonal antimurine IL-1β. Rats in the CONT group received only the vehicles. Animals were killed on the 14th post-natal day by cervical dislocation; tissues were collected immediately after the sacrifice.
In the fourth set of experiments, the litter was divided into four groups of three rats and the groups designated as CONT, SPM, FR167653, and SPM-FR167653. The SPM group received spermine and an intracardiac injection of sterile saline. FR167653 rats received spermine-vehicle per os and an intra-cardiac injection of FR167653. SPM-FR167653 rats received spermine and an intracardiac injection of FR167653. Rats in the CONT group received only the vehicles. Animals were killed on the 14th postnatal day by cervical dislocation; tissues were collected immediately after the sacrifice.
In the fifth set of experiments, a litter was divided into two groups of four rats; groups were designated as CONT and LPS. The LPS group was injected intraperitoneally with LPS. The CONT group received only the vehicle. Animals were killed on the 14th postnatal day by cervical dislocation; tissues were collected immediately after the sacrifice.
In the sixth set of experiments, a litter was divided into two groups of four rats. Groups were designated as CONT and IL-2. The IL-2 group was injected with IL-2, and the CONT group received only the vehicle. Animals were killed on the 14th postnatal day by cervical dislocation; tissues were collected immediately after the sacrifice.
In the seventh set of experiments, a litter was divided into two groups of four rats. Groups were designated as CONT and SPM. The SPM group received spermine per os, and the CONT group received only the vehicle. Animals were killed on the 11th postnatal day by cervical dislocation; tissues were collected immediately after the sacrifice.
In all cases, after the animals were beheaded, blood was collected from the trunk on edetic acid, and the small intestine was removed and divided into two pieces of equal length. The proximal and distal pieces were respectively named jejunum and ileum. Both were washed in cold NaCl (9 g/L) and homogenized in 4.15 mL/g of cold water with an Ika Ultra-Turrax T8 (Staufen, Germany) equipped with an S8N-8G dispersing element. Homogenates were kept at −70°C until analysis.
Protein Content Analysis
The protein content of the homogenates was estimated by the Bradford method (19), using bovine serum-albumin as protein standard.
Disaccharidase Activity Analysis
Sucrase (EC 22.214.171.124), maltase (EC 126.96.36.199) and lactase (EC 188.8.131.52) activities were assayed according to Dahlqvist (20). Enzyme activities were expressed as micromoles substrate hydrolyzed per minute and per gram of intestinal protein (specific activity: SA).
Intestinal polyamine concentrations were measured by reverse phase, high-performance liquid chromatography according to Bontemps et al. (21) and Brown et al. (22).
IL-2 Concentration Analysis
Plasma IL-2 concentration was assayed by enzyme-linked immunosorbent assay following manufacturer recommendations (OptEIA from Pharmingen).
The results are reported as means with the standard deviation (SD). Statistical analysis was performed using Student t test or one-way analysis of variance (ANOVA) for comparison between groups. Mann-Whitney U or Kruskall-Wallis test was used when heteroscedasticity was suspected.
Inhibition of Spermine Effects by Cytokine Inhibition
We analyzed intestinal spermine-induced maturation after the use of several strategies to inhibit the spermine-effect. The disaccharidase SA, markers of intestinal maturation, were analyzed after spermine administration and after a TNF-α neutralizing antibody treatment (Table 1). Spermine administration led to a decrease of the lactase SA and to an increase of the maltase and sucrase SA. Anti-TNF-α treatment (intraperitoneal or intracardiac), before spermine ingestion, prevented neither the lactase SA decrease nor the maltase and sucrase SA increase induced by the spermine ingestion. These observations were similar in the jejunum and in the ileum.
The same parameters were analyzed after spermine ingestion and IL-1β neutralizing antibody treatment (Table 2). Spermine administration induced a decrease of the lactase SA and an increase of the maltase and sucrase SA. Anti-IL-1β treatment (intraperitoneal), before spermine ingestion, did not prevent the lactase SA decrease or the maltase and sucrase SA increase induced by the spermine ingestion. These results were similar in the jejunum and in the ileum.
Disaccharidase activities were analyzed after spermine and FR167653 treatment. As previously described, spermine induced a modification in disaccharidase-specific activities. FR167653 treatment before spermine ingestion did not prevent the spermine-induced lactase SA decrease and maltase SA increase. The same treatment significantly reduced the spermine-induced increase of sucrase SA (Fig. 1).
Mimicry of Spermine Effects by LPS
The effects of LPS on disaccharidase SA are shown in Table 3. In the jejunum, LPS injection (10 μg) induced a significant increase of the lactase, maltase, and sucrase SA. The ileum was not significantly affected by these experimental conditions, except for maltase SA.
Role of IL-2 in Spermine-induced Effects
Lactase SA, measured in the jejunum of animals treated once a day with IL-2, is significantly lower than in control rats (Fig. 2). However, in the ileum no significant difference was observed between the two groups. Maltase SA estimated in the jejunum of IL-2 treated rats (once a day for 3 days) is significantly lower than that in control animals (Table 4). No significant difference was observed in the ileum of these animals. Sucrase SA was low in both parts of the small intestine and in both experimental groups (Table 4). There was no difference between control and treated groups.
In the jejunum of IL-2 treated rats (3 times a day for 3 days), the protein content expressed as milligrams per gram organ fresh weight was significantly higher (82.622 ± 2.254 v 91.132 ± 3.87; N = 4; P = 0.03) than in the control group. On the other hand, in the ileum, this content was significantly lower (80.880 ± 4.627 v 73.056 ± 1.16; N = 4; P < 0.05) in IL-2 treated rats than in control group. Lactase SA (Fig. 3), estimated in IL-2 treated rats, was significantly lower than in control rats. This observation was noticed in both parts of the intestine. In the jejunum, maltase SA did not vary significantly with IL-2 treatment (Table 4). However, in the ileum, the treatment with IL-2 significantly increased the SA of this enzyme. Sucrase SA was low in both parts of the small intestine and in both experimental groups (Table 4). There was a significant increase of this activity in the IL-2–treated group compared with control group. The intestinal content of putrescine, expressed as nano-moles per gram organ fresh weight or as nanomoles per milligram protein, increased significantly in the jejunum of IL-2–treated rats in comparison with controls (Table 5). In the ileum, the increase did not reach a significant level. In the jejunum, the spermidine content, expressed as nanomoles per gram organ fresh weight or as nano-moles per milligram protein, increased significantly in IL-2–treated rats (Table 5). In the ileum, a significant difference was found only when results were expressed as nanomoles per milligram protein. In both parts of the small intestine, spermine content, expressed as nano-moles per gram organ fresh weight, did not vary between animals from the control and IL-2–treated groups (Table 5). In the jejunum, spermine content, expressed as nano-moles per milligram protein, did not vary between the two groups. However, in the ileum, treatment of rats with IL-2 significantly increased the spermine content expressed as nanomoles per milligram protein (Table 5).
The concentration of IL-2 in plasma was analyzed at the 11th postnatal day in control and spermine-treated rats (Fig. 4). Six hours after the spermine treatment, rats showed an IL-2 plasma concentration significantly higher than that of control rats.
In previous studies, we demonstrated that the oral administration of spermine induces precocious intestinal maturation in suckling rats (10). This observation has been confirmed by other investigators (23–26). Food is the major source of polyamines for humans (27). Spermine content can be as high as 300 nmol/g food. Ingested polyamines appear quickly in the lumen, from whence they are taken up by the gut. Polyamines are initially bound to the apical membrane of enterocytes and subsequently transported across this lipid bilayer via specific carriers. In the case of the transport of polyamines across the basolateral membrane of enterocytes, the mechanism seems to be carrier-mediated (28).
Since then, much work has been done on the mechanism of spermine-induced maturation of the small intestine. Kaouass et al. (29) showed that spermine must be in contact with the intestinal mucosa to induce the maturation of the small intestine. Intravenous and intraperitoneal administrations of this polyamine were ineffective. The same authors (11,12) suggested an indirect action of spermine on the intestinal mucosa by way of glucocorticoids. After bilateral adrenalectomy, spermine did not increase the maltase or sucrase SA. Kaouass et al. (13) proposed that the immune system plays a relay role between the intestinal mucosa in contact with spermine and the adrenal glands. For these authors, secretion of TNF-α, IL-6, and especially IL-1β, explains the effects of spermine on the maltase and sucrase SA. To support this hypothesis, it was shown that spermine induces the maturation of the immune system in the small intestine (30) as well as in the spleen (31,32).
Our results show that spermine-induced maturation is prevented neither by the injection of TNF-α neutralizing antibody nor by the injection of IL-1β neutralizing antibody. These results do not agree with the efficacy of anticytokine therapies shown by other investigators (33). However, we must point out that these neutralizing therapies seem to be useless in some animal models of sepsis (34) and in clinical trials (35). Thus, our results do not allow rejection of the hypothesis of a role of these cytokines in the spermine-induced maturation. In agreement with this point, we report here two different observations: spermine-induced maturation of the small intestine is partially reduced by FR167653, an inhibitor of both IL-1β and TNF-α synthesis, and LPS injection leads to an increase of disaccharidase activities. LPS induces an increase of proinflammatory cytokines (IL-1β, TNF-α) in plasma shortly after its injection (15), and the injection of these cytokines leads to an increase of disaccharidase activities (13) similar to the LPS injection reported here. In addition, the data obtained with FR167653 and LPS confirm that these cytokines are involved in spermine-induced maturation of the small intestine. However, the simultaneous inhibition of IL-1β and TNF-α seems required to inhibit maturation. This result suggests a compensation of the remaining factor when only one is neutralized.
In a recent study (14), we suggested that IL-2 might also be a mediator of the effects of spermine on disaccharidase SA. Treatment of suckling rats with cyclosporin A, an IL-2 transcription inhibitor, drastically reduces the increase of maltase and sucrase SA observed after spermine ingestion. Further evidence supports a role for IL-2 in intestinal maturation: (a) during weaning, the alterations of growth, structure and intestinal functions seem to be mediated by T cells (36); (b) neutralization of the IL-2 receptor reduces intestinal growth (37); (c) during weaning, the IL-2 synthesis increases from almost undetectable to significant secretion in the spleen of the rat (38). Our observations indicate that intraperitoneal injection of IL-2 induces a modification in lactase, maltase, and sucrase SA that is qualitatively comparable to that observed during weaning or after spermine treatment. Our results suggest that IL-2 is mainly involved in the decrease of lactase SA after spermine ingestion because the effect of IL-2 injection is primarily on this SA. This proposal is important because at present few substances can decrease lactase SA, and the mechanisms controlling the decrease of activity of this enzyme at the time of weaning are largely unknown. The substances now recognized to induce a decrease of lactase SA are bombesin (39) and thyroxin (40,41). The effect of thyroxin is contested (42).
To more precisely state whether the effects of spermine on lactase SA are mediated by IL-2, it was necessary to find a measurement of this interleukin in plasma of rats treated with spermine. Treatment with spermine induced an increase of the plasma concentration of IL-2. This increase, although weak, was statistically significant 6 hours after spermine administration. This low level of the increase may be explained by the short half-life of this protein (4 minutes in sera) (43), as well as by its binding to soluble receptors (44) and its sensitivity to proteases (45). The increase occurs at the same time as an even smaller increase of the IL-2 plasma concentration in control animals. This small basal increase seen in controls may be a result of stress at the time of spermine or vehicle administration or to a circadian rhythm. A stress effect on IL-2 secretion is not well supported by experimental data. (46). In humans, the IL-2 plasma concentration increases progressively in the morning to a maximum concentration at noon (47). We also observed a peak concentration in IL-2 four hours after the beginning of the treatment, at 1:00 PM.
Our results obtained after IL-2 injection were not in agreement with one of our previous hypotheses. In a previous work (14), we showed that the effect of spermine on lactase SA was not reduced by cyclosporin A, whereas the effects of spermine on maltase and sucrase SA were strongly altered by cyclosporin A. We suggested that spermine acts on sucrase and maltase SA but not on lactase SA via an IL-2 mediated process. Our current results showed an increase of ileal maltase and sucrase SA after IL-2 administration. Nevertheless, these suckling rats increases were much smaller compared to the marked effect of cyclosporin A. IL-2 by intraperitoneal injection strongly reduced lactase SA, a result not in agreement with our previous hypothesis (14). This difference could be explained by the mechanism of cyclosporin A action which blocks the activation of T cells and consequently induces an inhibition of the transcription of some lymphokines, notably IL-2 (48,49). This inhibition of transcription is caused by an interference between cyclosporin A, complexed with its targets cyclophilin, and calcineurin, a serine/threonine phosphatase depending on calmodulin. The inhibition of calcineurin phosphatase activity by the complex cyclophilin-cyclosporin A prevents the activation of nuclear factors (NF-AT, AP-3, NF-κB) involved in the regulation of the transcription of the IL-2 coding gene (50). These nuclear factors also mediate the transcription of many other genes, the products of which could be involved in the mechanism of spermine action. Therefore, in the observations we made previously (14), we could not exclude a nonspecific mechanism of cyclosporin A on enterocytes or a spread effect of the transcription inhibition of other cytokines.
Intraperitoneal injection of IL-2 (3 times a day for 3 days) modified in an important manner the intestinal metabolism of polyamines, inducing an increase in the content (nmol/mg protein and nmol/g fresh weight) of putrescine and spermidine, mainly in the jejunum. The spermine content (nmol/mg protein) was increased only in the ileum. These results were similar to those observed 3 days after spermine ingestion, especially for spermidine and spermine content (51).
We propose that spermine, in contact with the intestinal mucosa, induces the secretion of some soluble factors of an immune origin. Each of these factors would be at the origin of one or a few biochemical modifications of the intestinal mucosa, e.g., IL-2 would act specifically on lactase SA perhaps via bombesin or thyroxin. The experimental data we collected here allow a better understanding of some observations reported in the literature suggesting that the expression of genes coding for intestinal disaccharidases are independent.
Studies indicate that spermine inhibits synthesis of proinflammatory cytokines (52). Other analyses show that spermine exerts its effects in the gut via a mechanism involving proinflammatory cytokines (IL-1β, TNF-α). These molecules are also involved in some intestinal conditions such as Crohn disease. Moreover, this condition is characterized by a decrease of ODC activity in mucosa (53). Thus, the question of the utility of polyamines in the treatment of intestinal bowel diseases can be raised.
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