The gut has been called the "engine" of multiorgan failure (1), and proinflammatory mediators carried in mesenteric lymph are believed to have an important role (2). Clark and Coopersmith (3) have recently made the argument that the multiorgan dysfunction syndrome likely involves interactions ("cross talk") between "the intestinal epithelium, the gut-associated immune system, and the intestine's own endogenous bacteria." An important component of the gut-associated immune system is the secretion of antimicrobial peptides and proteins (AMPs) by the Paneth cells and other cells in the intestine. The AMPs play a pivotal role in our innate defense against bacterial pathogens through both their direct bactericidal action (4) and their immunomodulatory activity (5). A specific role for the Paneth cell in the response to hemorrhage is suggested by the study of Condon et al. (6), which showed that the messenger RNA for α-defensin 5 (HD5) increases 10-fold in the Paneth cells of the ileum during hemorrhage. The defensins are known to stimulate the production of IL-8 in enterocytes (7), but a more distant immunomodulatory role might be predicted if the AMPs were present in mesenteric lymph. Other mediators in lymph could potentially influence distant organ response to hemorrhage by influencing the production of NO.
Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of NOS. Asymmetric dimethylarginine has been shown to cause increased oxidative stress in cultured endothelial cells (8) and cytokine-stimulated lung epithelial cells (9). A role in the pathogenesis of multiorgan failure is suggested by the findings that an increased plasma level of ADMA is an independent risk factor for death from multiorgan failure (10) and sepsis (11). Nijveldt et al. (12) have studied the net flux of ADMA in the gut (i.e., stomach, pancreas, spleen, small intestine, and colon) and concluded that the normal gut is a source of ADMA. It is possible that these studies may have underestimated the production of ADMA in the gut because it was not measured in mesenteric lymph.
In the present studies, we determined if ADMA and α-defensin 4 increased in the mesenteric lymph during hemorrhage and resuscitation. Because α-defensin 4 is normally expressed in the Paneth cells, we also examined the lymph for other secretory products of the Paneth cells, namely secretory phospholipase A2 (sPLA2) and Reg 2 protein. The results indicate that ADMA and α-defensin 4 may contribute to the increase in inflammatory activity of mesenteric lymph during hemorrhage, but they are unlikely to be the mediators responsible for the further increase in the inflammatory activity seen in postresuscitation lymph.
MATERIALS AND METHODS
α-Defensin 4 and CD15 antibodies were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, Calif). Antibodies direct to Reg 2 (R&D Systems Inc) and sPLA2 (Abcam Inc, Cambridge, Mass) were obtained. The ADMD direct enzyme-linked immunosorbent assay (ELISA) kit was bought from ALPCO Diagnostics (Salem, NH).
Animal handling and treatments were conducted in compliance with the Animal Welfare Act and other Federal statutes and regulations related to animals and experiments involving animals, and adhered to principles stated in the Guide to the Care and Use of Laboratory Animals, National Research Council. The facilities are fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International.
Male Sprague-Dawley rats were commercially obtained and quarantined for 10 days in a temperature- and light-controlled environment. On the day of the experiment, the animals were anesthetized with ketamine (60 mg/kg, intraperitoneally) (NLS Animal Health, Owings Mills, Md), and anesthesia was maintained by supplemental injections of ketamine as required. After attainment of an adequate plane of anesthesia, the animal was shaved along the midline of the abdomen. The mesenteric lymphatic was cannulated as described later (13). The skin was opened from the pubis to the xyphoid process. The muscle layers of the abdomen were opened along the linea alba, with a lateral extension of the incision toward the left flank at the level of the stomach. The intestines were eviscerated beginning at the cecum and wrapped in saline-moistened gauze. Great care was taken to avoid putting any pressure on the intestines or pancreas. The peritoneal covering surrounding the mesenteric artery was sharp-dissected using Dumont 3c forceps. After gentle cleaning, the lymphatic was cut part way through with iris scissors, cannulated with silastic tubing (Dow Corning CAT no. 508-002), and secured with super glue. After the super glue dried (2 min) and the cannula was secure, the distal end was externalized through a hole in the right flank, and lymph was collected on ice. The intestines were then replaced in the abdomen, and the abdominal cavity was closed with sutures in two layers (muscle and skin). Thereafter, the femoral artery and vein were cannulated (Renepulse, RPT-040, Braintree Scientific, Braintree, Mass). Core temperatures were monitored and maintained at 37°C by a feedback control unit that monitors rectal temperature (Homeothermic Blanket, Harvard Apparatus, Holliston, Mass). The animals received no heparin. The arterial catheter was connected to a blood pressure transducer (CDX III transducer, Argon Medical, Athens, Tex) and analyzer (BPA-400, Micromed, Louisville, Ky), which were used to monitor blood pressure. The arterial pressure wave was displayed using a custom Labview program (14) and used to assess catheter patency. The venous catheter was used for blood withdrawal and resuscitation. An additional side hole was placed near the tip of the venous catheter to facilitate blood withdrawal in a nonheparinized animal.
After a control period of 20 min, the animals were hemorrhaged over 15 min to a MAP of 40 mmHg. They were maintained at that pressure by intermittent blood withdrawal. Resuscitation was initiated when blood pressure could no longer be maintained by blood withdrawal, and when the MAP began to fall less than 40 mmHg (approximately 40 min). Thereafter, the animals were resuscitated with a volume of Ringer's lactate solution equal to three times the shed blood volume, infused over 1 h. No blood was reinfused into the animal. Lymph was collected in three periods: (a) control (Pre-Hemo); (b) during hemorrhage, which includes the 15 min required to lower arterial pressure and the period when the animal was maintained at 40 mmHg (Hemo); and (c) during resuscitation (i.e., Hemo + LR). The lymph was centrifuged to remove formed elements and frozen in liquid nitrogen until subsequent analysis.
Blots were performed as previously described (15). One hundred micrograms of each sample was dissolved on sodium dodecyl sulfate-polyacrylamide slab gels (precast 10% gels; Invitrogen, Carlsbad, Calif). Protein was then blotted onto a nitrocellulose membrane (type NC, 0.45 μm; Schleicher and Schuell) using a Novex blotting apparatus and the manufacturer's protocol. The nitrocellulose membrane was blocked by incubation for 90 min at room temperature in Tris-buffered saline containing 0.1% Tween 20 (TBST) and 3% nonfat dried milk. The blot was then incubated for 60 min at room temperature with antibodies against α-defensin 4, Reg 2, or sPLA2 at 1 μg/mL in TBST with 3% nonfat dried milk. The blot was washed three times (10 min each) in TBST before incubating for 60 min at room temperature with a 1,000× dilution of species-specific immunoglobulin G (IgG) peroxidase conjugate (Santa Cruz Biotechnology, Inc) in TBST with 3% nonfat dried milk. The blot was washed six times (5 min each) in TBST before detection of the peroxidase activity using the enhanced chemiluminescence kit (Amersham Life Science Products, Arlington Heights, Ill). The protein band of interest was quantitated with Gel Doc Work Station (BioRad, Richmond, Calif).
Asymmetric dimethylarginine was measured by using ADMA direct ELISA kit. The assay is based on the methods of competitive enzyme-linked immunoassay, with the sample ADMA coupling to an acylation reagent. In brief, 20 μL of samples in a precoated 96-well plate was incubated with 10 μL of acylation reagent for 5 min at 24°C. Then ADMA antibody (final concentration, 1 μg/mL) was added to the sample, and the mixture was incubated for 5 h at 24°C. The wells were washed five times with wash buffer and then incubated with the secondary antibody (1:1,000) for 1 h. The wells were washed again with wash buffer five times. Tetramethylbenzidine substrate (200 μL) was added into each well and incubated for 20 min in the dark at 24°C. Then the reaction was terminated by addition of 100 μL of stop solution. Asymmetric dimethylarginine absorption was read at 405 nm with Spectromax M5e plate reader and SoftMax Pro v5 (Molecular Devices, Sunnyvale, Calif).
Immunofluorescence techniques and image analysis
The 5-μm cryosections of the obtained tissue samples were fixed on glass slides and processed for immunofluorescence analysis as described previously (16). Briefly, tissue specimens were incubated in phosphate buffered saline solution (PBS) containing 2% paraformaldehyde and 0.1% Triton X-100 for 20 min, washed three times with PBS, and then PBS containing 0.5% bovine serum albumin and 0.15% glycine (Buffer A). Any nonspecific binding was blocked with purified donkey normal serum (Santa Cruz Biotechnology, Inc; http://www.scbt.com) diluted 1:20 in buffer A. Primary goat polyclonal antibody against α-defensin 4 and mouse monoclonal antibody against CD15 adhesion molecules (Santa Cruz Biotechnology, Inc) were used with 1:250 dilution and 1:150, respectively, in the buffer A. The selected dilution levels were tested in a preliminary set of experiments using the dilution range 1:50 through 1:500 as recommended by the manufacturer. That was followed by the washing procedure as described previously and incubation with a secondary fluorochrome-conjugated antibody and counterstaining with Hoechst 33342 (Molecular Probes, Inc, Eugene, Oreg) diluted 1:1,000. The secondary antibodies used were (a) ALEXA 488 conjugated donkey antigoat IgG (Molecular Probes, Inc), (b) Cy3-conjugated donkey antimouse IgG (VWR International Inc; http://www.vwr.com).
The specimens for immunofluorescence negative control (i.e., assessment of nonspecific binding and background fluorescence) included those treated with normal donkey serum and secondary antibody alone or primary antibody alone in the presence of Hoechst 33342. The labeled specimens were rinsed, mounted in Gelvatol (Monsanto Corp, St Louis, Mo), and coverslipped for fluorescence microscopy. The specimens were analyzed with an Olympus AX 80 microscope equipped with Hamamatsu digital camera. Processing of the digital fluorescence images and analysis of signal interactions (i.e., Pearson correlation, r) of green (α-defensin 4) and red (CD15) channels in the captured fluorescent images were conducted using SimplePCI High Performance Imaging software (Hamamatsu Co; www.cimaging.net) and ImageJ software (http://rsb.info.nih.gov).
All data are expressed as mean ± SEM. One-way ANOVA and studentized range test are used for comparison of groups with 5% as a significant level.
Mesenteric lymph protein concentrations and flow rates
Protein concentrations in the mesenteric lymph averaged 27 ± 4 μg/μL during the control period, 27 ± 3 μg/μL during the hemorrhage period, and 11 ± 2 μg/μL during the resuscitation period (n = 5). The decrease in protein concentration during resuscitation was significant (P = 0.006, F2,12 = 8.21, hemorrhage versus resuscitation) and approximated 2.7-fold. Lymph flow rates averaged 0.02 ± 0.001 mL/min per 100 g body weight during the control period, 0.02 ± 0.003 mL/min per 100 g body weight during the hemorrhage period, and 0.04 ± 0.01 mL/min per 100 g body weight during the resuscitation period (n = 3). The increase in flow during resuscitation did not reach significance, but there was a tendency for flow to increase approximately 2-fold during the resuscitation.
Levels of AMPs in mesenteric lymph
Figure 1 shows that no appreciable level of α-defensin 4 was detected during the control period (lane 1), but it significantly increased during hemorrhage (lane 2, P < 0.05 Pre-Hemo vs. Hemo). Resuscitation with LR further increased α-defensin 4 by 200% ± 40% (lane 3, P = 0.0002, F2,12 = 52.94, n = 5 per group). Expressed as optical density units per microliter, these values were: Pre-Hemo, 43 ± 4; Hemo, 557 ± 61; and Hemo + LR, 547 ± 114. This indicates that there was no further increase in the concentration of α-defensin 4 during resuscitation, although the tendency for lymph flow to increase 2-fold suggests that there may have been an increase in mass flow. Secretory phospholipase A2 was not detectable in any samples from the control period, the hemorrhage period, or the resuscitation period. Abundant Reg 2 was present in lymph during the control period (lane 1). Hemorrhage did not alter it (lane 2). However, the subsequent resuscitation with LR decreased it by 36% (lane 3, P = 0.012, F2,12 = 10.05, n = 5 per group). Expressed as optical density per microliter, these values were: Pre-Hemo, 2206 ± 271; Hemo, 2271 ± 419; and Hemo + LR 683 ± 172.
Levels of ADMA in mesenteric lymph
Asymmetric dimethylarginine is normally present in lymph (Fig. 2). The basal level was 0.101 ± 0.021 pmol/μg protein. It increased significantly with hemorrhage by 193%. During the resuscitation period, ADMA increased further by 327% to a level of 0.33 ± 0.075 pmol/μg protein. (P = 0.0088, F2,12 = 11.51, n = 5 per group). Expressed as picomole per microliter, these values were: Pre-Hemo, 0.044 ± 0.007; Hemo, 0.086 ± 0.029; and Hemo + LR, 0.064 ± 0.008. This indicates that there was no further increase in the concentration of ADMA during resuscitation.
Immunofluorescence of the ileum
Immunohistochemistry of the ileum before hemorrhage demonstrated the presence of a significant immunoreactivity for α-defensin 4 in the lumen of the intestine with low specific immunoreactivity for CD15 (Fig. 3, A-C). As shown in Figure 3G, at this stage, immunoreactivity of CD15 was mostly located in the interstitial space and was manifested by a relatively high correlation with immunoreactivity for α-defensin 4 (Pearson r = 0.57). As shown in Figure 3, D to F, and H, hemorrhage followed by resuscitation resulted in dramatic alterations in the deposition of α-defensin 4 and CD15. This was characterized by substantial increase in the immunoreactivity of α-defensin 4, along the villus-crypt axis, and visualization of CD15 in circular structures consistent with the Paneth cells (Fig. 3, D and E). As shown in Figure 3, D and E, colocalization of α-defensin 4 and CD15 appeared in yellow because of interference of green and orange colors (indicated with white arrow). However, despite an overall increase in the immunoreactivity of α-defensin 4 and CD15 after hemorrhage/resuscitation, their 2-dimensional spatial correlation declined (Pearson r = 0.31), apparently because of the increase in the immunoreactivity of α-defensin 4 in the lumen of the intestine.
This study is the first to demonstrate the presence of the antimicrobial peptide α-defensin 4 and the antimicrobial protein Reg 2 in mesenteric lymph (Fig. 1). These AMPs are known to be produced in the Paneth cells of the intestine (α-defensin 4, sPLA2, and Reg 2) and in the pancreas (Reg 2) (17, 18), but other sites of production are possible. The immunohistology showing α-defensin 4 in the lumen of the intestine and colocalized with CD15 (Fig. 3D), a Paneth cell marker (19), indicates that the antibody used in the present study recognizes the Paneth cell secretory product, but it does not exclude additional sites of origin.
The AMPs porate the outer wall of microbes, which is thought to be their principal action within the lumen of the intestine (4), but the AMPs are also immunomodulatory (5). As an example, the application of human Paneth cell product HD5 to the luminal surface of enterocytes stimulates the enterocytes to secrete IL-8 (7). α-Defensin 5 is also a potent chemotaxin for macrophages (20). Luminal application of the mouse Paneth cell defensinlike cryptdin 3 to the human intestinal cell line T84 induces basolateral secretion of IL-8, macrophage inflammatory protein 1α (MIP-1α), MIP-1β, MIP-1δ, IL-12 p70, and IL-17 (21). It is likely that the AMPs in mesenteric lymph will have similar immunomodulatory roles in the mesenteric lymph nodes.
α-Defensin 4 increases in the mesenteric lymph during hemorrhage (Fig. 1). In vitro assays of mesenteric lymph collected during hemorrhage in two species have shown it to be more proinflammatory than lymph collected before hemorrhage (22, 23). The increase in α-defensin 4 may contribute to this finding, and it may be an example of the cross talk described by Clark and Coopersmith (3).
The time course suggests that this increase results from the secretion of preformed product, activation of a prodefensin, or changes in transport or metabolism of the AMP rather than de novo peptide synthesis. Preformed prodefensin is normally stored in the secretory granules of the Paneth cells of the intestine, which are found in the highest density in the ileum. The studies of Condon et al. (6) have shown that hemorrhage provides a potent stimulus for the Paneth cells of the ileum, causing a 10-fold increase in the expression of the messenger RNA for HD5. The immunofluorescence of the ileum suggests that there is increased concentration of α-defensin 4 in the lumen after hemorrhage/resuscitation (Fig. 3C versus Fig. 3F). However, the findings that there was no detectable sPLA2 in mesenteric lymph and that Reg 2 was unchanged by hemorrhage suggest that either the Paneth cells did not increase secretion during hemorrhage or that the secretion was specific to certain products. Further studies are required to ascertain the source of the α-defensin 4 found in mesenteric lymph after hemorrhage.
During resuscitation, there is a tendency for lymph flow to increase, but there is no further increase in the concentration of α-defensin 4 per volume. An increase in mass flow could contribute to physiologic effects of resuscitation in vivo, but the results of the present study do not account for the in vitro findings that a set volume of postresuscitation lymph is more cytotoxic than preresuscitation lymph (2). The list of possible sources of α-defensin 4 during resuscitation must be expanded to include secretion from invading cells, de novo synthesis from stimulated cells (24), or increased absorption of previously secreted product. The cause of the decrease in Reg 2 with resuscitation is unknown, but it may have functional significance because Reg 2 (also named PAP I) seems to have anti-inflammatory properties (25).
This study is the first to demonstrate the presence of ADMA in mesenteric lymph. Nijveldt et al. (12) have shown that normal gut is a source of ADMA, and that the production of ADMA in the gut increases after the administration of LPS. It is likely that the study by Nijveldt et al. (12) underestimated the production of ADMA in the gut because it did not measure ADMA in mesenteric lymph. Asymmetric dimethylarginine could be produced in the cells of the gut or absorbed from the lumen of the intestine. Asymmetric dimethylarginine is produced by the hydrolysis of proteins containing dimethylarginine residues (26). In mammalian tissues, the formation of proteins with these residues is catalyzed by a group of protein-arginine methyltransferases (27). Some bacteria have similar enzymes, and it has been shown that the lysis and subsequent trypsin digestion of Helicobacter pylori produces ADMA (28). Asymmetric dimethylarginine is carried on the cationic transporters (29) and should be easily taken up by the enterocyte.
The increase in ADMA during hemorrhage may contribute to the increased cytotoxicity seen in mesenteric lymph during hemorrhage (22, 23) because ADMA has been shown to cause increased oxidative stress in cultured endothelial cells (8) and cytokine-stimulated lung epithelial cells (9).
The increase in ADMA may be the result of decreased metabolism. Asymmetric dimethylarginine is degraded by the enzyme dimethylarginine dimethylaminohydrolase. Dimethylarginine dimethylaminohydrolase activity can be decreased by oxidative injury (30), which may occur in the gut during hemorrhage. The increase in ADMA could also be explained by increased destruction of bacteria that would be expected to occur if there was increased luminal secretion of α-defensins by the Paneth cells.
The liver expresses high levels of dimethylarginine dimethylaminohydrolase, and the liver has been shown to remove significant amounts of ADMA (12, 31). Therefore, the ADMA produced in the gut and targeted to mesenteric lymph may have special importance because it avoids first-pass metabolism by the liver. The plasma levels of ADMA are increased during hemorrhage (32). Elevated ADMA levels is a strong independent predictor of intensive care unit death from multiorgan failure (10) and death in patients with sepsis (11).
As with α-defensin 4, there is no further increase in ADMA (concentration by volume) after resuscitation. This suggests that there are additional proinflammatory mediators in postresuscitation mesenteric lymph, which is consistent with the findings that both the aqueous (33) and lipid (23, 34) fractions are cytotoxic.
We have recently demonstrated that there is brisk production of NO during hemorrhage deriving from two sources (a) constitutive NOS and (b) conversion of nitrite to NO (14). This results in a linear increase in hemoglobin-NO during hemorrhage. The steady production of NO in the face of continuous delivery of ADMA through mesenteric lymph suggests that either plasma levels of ADMA do not reach inhibitory concentrations during hemorrhage or more likely that nitrite has a progressively larger role in NO production in the later stages of hemorrhage when the increasing tissue hypoxia would favor increased production of NO from nitrite (35, 36).
This study has several limitations: (a) because of the limited availability of antibodies to rat defensins, only one of the α-defensins was studied; (b) the lack of measurements of concentrations of ADMA and α-defensin 4 in portal blood precluded determination if these mediators appear in higher concentration in lymph than portal blood, as previously shown for the proinflammatory mediators in postresuscitation lymph (37).
In summary, hemorrhage results in an increase in the concentration of ADMA and α-defensin 4 in mesenteric lymph, which may contribute to the increase in inflammatory activity of mesenteric lymph during hemorrhage. There is no further increase in the concentration of ADMA or α-defensin 4 during resuscitation, so it is unlikely that they are responsible for the further increase in the inflammatory activity seen in postresuscitation lymph. Reg 2 protein decreases during resuscitation, which may alter the inflammatory potential of postresuscitation lymph because Reg 2 is normally anti-inflammatory.
The authors thank Dr Edwin A. Deitch for advice on the technique for the cannulation of mesenteric lymphatics and Sara Smith for her excellent technical assistance.
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