Increased Expression of Intestinal P-Selectin and Pulmonary E-Selectin During Intravenous Total Parenteral Nutrition FREE

ENTERAL feeding of critically ill or injured patients increases resistance to infection, particularly pneumonia.^{1 - 3} Our laboratory has focused on IgA-mediated mucosal immunity as a mechanism for this clinical issue, showing that lack of enteral stimulation causes changes in gut-associated lymphoid tissue and loss of respiratory tract immunity.^{4 - 6} Recently, we showed that lack of enteral feeding increased both intestinal intercellular adhesion molecule-1 (ICAM-1) expression and gut polymorphonuclear neutrophil (PMN) accumulation.⁷ The ICAM-1 expressed on endothelial cells is the ligand counterpart of CD11/18 on PMNs. Expression leads to firm adhesion of PMNs to the surface of the endothelium.⁸ Since the vascular bed of gastrointestinal tract primes PMNs and exacerbates pulmonary injury to a subsequent insult,⁹ increased ICAM-1 expression and PMN accumulation in gut during intravenous (IV) total parenteral nutrition (TPN) feeding may unfavorably influence the response to injury or infection.

In addition to ICAM-1, the selectin family of adhesion molecules influences PMN–endothelial cell interaction.^{8 ,10 - 11} The selectins designated L-, P-, and E-selectin are a family of adhesive receptors sharing a common structure of an aminoterminal C-type lectin domain, a single epidermal growth factor–like domain, several short consensus repeats, a transmembrane domain, and a short C-terminal cytoplasmic domain.¹⁰ P- and E-selectin expressed on the surface of endothelial cells regulate leukocyte–endothelial cell interaction via the rolling of leukocytes along the endothelium of postcapillary venules. This is a low-affinity process preceding firm adhesion through CD11/18–ICAM-1 interaction.^{10 - 11}

P-selectin is constitutively expressed and stored in Weibel-Palade bodies.⁸ When stimulated, P-selectin is translocated and up-regulated immediately, initiating the earliest phase of leukocyte recruitment into inflammatory sites.⁸ Moreover, this molecule can be regulated transcriptionally by proinflammatory cytokines, such as tumor necrosis factor (TNF)–α.^{12 - 13} E-selectin is not expressed constitutively in normal tissues but is transcriptionally up-regulated by proinflammatory cytokines, such as interleukin 1 or TNF-α.^{10 ,13 - 14} The kinetics of P- and E-selectin expression after stimulation by endotoxin or cytokines, or after organ ischemia-reperfusion, have been well studied in vitro and in vivo.^{14 - 17} However, no investigations have addressed the influence of nutrition on P- and E-selectin expression. Since 5 days of IV TPN induced intestinal PMN recruitment in association with increased intestinal ICAM-1 expression,⁷ we investigated dietary manipulation of P- and E-selectin expression in this time frame in vivo by means of the dual radiolabeled monoclonal antibody (mAb) technique.

All experimental protocols were approved by the Animal Care and Use Committee of the University of Tennessee, Memphis. Male Institute of Cancer Research mice were purchased (Harlan Laboratories, Indianapolis, Ind) and housed in a conventional facility accredited by the American Association for Accreditation of Laboratory Animal Care. The environment was controlled with regard to temperature and humidity with a 12-hour light-dark cycle. Mice were fed ad libitum chow (RMH3200; Agway Inc, Syracuse, NY) and water for 2 weeks before entry into this study protocol. During feeding protocols, mice were housed in metal metabolism cages with wire grid floors to eliminate coprophagia.

Fifty-three mice (6-8 weeks old) were randomized to receive chow (n = 16), IV TPN (n = 21), or intragastric (IG) TPN (n = 16). Mice randomized to the chow and IV TPN groups received internal jugular catheters under anesthesia with ketamine hydrochloride (100 mg/kg of body weight) and acepromazine maleate (10 mg/kg of body weight). Through a right jugular approach, a silicone rubber catheter (0.3-mm inner diameter and 0.6-mm outer diameter; Baxter Healthcare Corp, Chicago, Ill) was inserted into the vena cava. The proximal end of the catheter was tunneled subcutaneously over the spine and exited the tail at its midpoint. The mice were placed into metal metabolism cages and partially immobilized by tail restraint to protect the catheter during infusion. The technique is an acceptable method of nutritional support that does not induce physical or biochemical stress.¹⁸ Mice randomized to IG TPN received gastrostomy tubes. Through a vertical midline incision, the stomach was delivered into the wound. A 7-0 silk purse-string suture was placed on the posterior wall of the stomach followed by a gastrotomy with the use of a 25-gauge needle. The silicone catheter was inserted into the gastrotomy. Omentum was mobilized and wrapped around the insertion of the gastrostomy tube with the use of 7-0 silk suture. The proximalmal end of the catheter was tunneledsubcutaneously over the spine and exited the tail at its midpoint.

Catheterized mice were immediately connected to infusion pumps (Instech Laboratories, Plymouth Meeting, Pa) and received 0.9% saline at 4 mL/d for 48 hours with ad libitum access to chow and water. On postoperative day 2, mice received their respective feeds. Chow-fed animals received 4 mL of 0.9% saline IV along with ad libitum chow and water throughout the study. The IV TPN– and IG TPN–fed animals initially received 4 mL of TPN per day and were advanced to a goal rate of 10 mL/d by the third day of feeding. The TPN solution contained 4.1% amino acids, 34.3% glucose (4878 kJ/L), electrolytes, and multivitamins with a nonprotein energy-nitrogen ratio of 743 kJ/g of nitrogen. This feeding met the calculated nutritional requirement of mice used in the present study. The TPN-fed mice received 1951 kJ of nonprotein energy intake per kilogram per day and 16.4 g of protein per kilogram per day.

After receiving their respective diets for 5 days, 29 animals were used for quantification of P-selectin expression in the lung, liver, kidney, small intestine, colon, pancreas, mesentery, and heart, and 24 were used for E-selectin quantification in the organs. P-selectin expression in the stomach and gastrocnemius muscle was also quantified.

The mAbs used for the assessment of P-selectin expression were RB40.34, a rat IgG1 directed against mouse P-selectin, and R3-34, a nonbinding rat IgG1 control antibody. To evaluate E-selectin expression, 10E9.6, a rat IgG2a directed against mouse E-selectin, and R35-95, a nonbindingrat IgG2a control antibody (PharMingen Inc, San Diego, Calif) were used.

The binding mAbs and the nonbinding mAbs were radiolabeled with iodine 125 (¹²⁵ I) and iodine 131 (¹³¹ I), respectively, by means of the iodogen method. Briefly, 250 µg of protein was incubated with 9.25 MBq of sodium iodide (¹²⁵ I) (or sodium iodide [¹³¹ I]) and 125 µg of iodogen (1,3,4,6-tetrachloro-3α,6α-DI-phenylglycouril; Sigma-Aldrich Chemical Co, St Louis, Mo) at 4°C for 12 minutes. Phosphate-buffered saline was then added to bring the total volume to 2.5 mL. The radiolabeled mAb was then separated from free ¹²⁵ I (or ¹³¹ I) by gel filtration on a gel-based matrix column (Sephadex PD-10; Pharmacia LKB, Uppsala, Sweden). Phosphate buffer with 1% bovine serum albumin was used to equilibrate the column and to elute the radiolabeled antibody. Two 2.5-mL fractions were collected, with the second fraction containing the labeled mAb. Radiolabeled mAbs were stored at 4°C.

After anesthesia with ketamine and acepromazine, the left jugular vein and the right carotid artery were cannulated with a silicone rubber catheter and polyethylene tubing (PE10; Becton Dickinson, Sparks, Md).

A mixture of 10 µg of ¹²⁵ I-labeled anti–P-selectin mAb or anti–E-selectin mAb was given with an appropriate amount of ¹³¹ I-labeled nonbinding antibody (400,000-600,000 cpm) through the jugular vein catheter (total volume, 200 µL). Binding mAb labeled with ¹²⁵ I that is directed against P- (or E-) selectin was injected to evaluate P- (or E-) selectin expression on the endothelial cells, while ¹³¹ I-labeled nonbinding mAb was given to eliminate the influence of nonspecific binding of anti–P- (or E-) selectin antibody to endothelial cells. A blood sample was obtained through the carotid artery catheter 5 minutes after injection of the mAb mixture. The animals were then treated with 40 U of heparin sodium and rapidly exsanguinated by perfusion of bicarbonate-buffered saline through the jugular vein catheter with simultaneous blood withdrawal through the carotid artery catheter. This was followed by perfusion of 15 mL of bicarbonate-buffered saline through the carotid artery catheter after severing of the inferior vena cava at the thoracic level. Thus, after removal of all mAbs that did not bind to the endothelial cells, the lungs, liver, kidney, and small intestine were harvested and weighed.

An automated counting system (Cobra Automated Gamma Counting System; Packard Instrument, Meriden, Conn) was used to count ¹²⁵ I (binding mAb) and ¹³¹ I (nonbinding mAb) activities in each organ and in a 50-µL plasma sample. A 2-µL aliquot of the preinjection mixture of radiolabeled mAbs was measured to determine total injected activity of each labeled mAb. The amount of radioactivity remaining in the tube used to mix the mAbs and the syringe used to inject the mAb mixture was subtracted from the total calculated injected activity. Intercellular adhesion molecule-1 expression was determined by subtracting the accumulated activity of the nonbinding mAb from that of binding mAb, and expressed as micrograms of mAb per gram of tissue in the following equation:

Grahic Jump Location

A section of lung tissue was taken from animals and dried at 60°C for 48 hours to evaluate wet-to-dry lung weight ratios.

Results are presented as means ± SEs. Statistical analysis was performed by means of analysis of variance, followed by Fisher protected least significant difference post hoc test or t test. Differences were considered statistically significant at P<.05.

The preexperiment weights of all groups were similar (Table 1). The mice in the IV TPN and IG TPN groups lost more body weight than chow-fed mice (P = .04), but there were no differences between the IV TPN and IG TPN groups.

P-selectin expression in small intestine, colon, stomach, and pancreas was significantly higher in the IV TPN group than in the chow or the IG TPN groups, with no significant differences between the chow and the IG TPN groups (Figure 1 and Table 2). No significant differences were observed in the other organs among the 3 groups.

The animals in the IV TPN group showed significantly higher E-selectin expression in the lung than did the animals in the chow or IG TPN group (Figure 2). In other organs, no significant changes were observed among all groups (Figure 2; and Table 3).

Wet-to-dry lung weight ratios (±SEM) were 5.45 ± 0.16 in the chow group, 5.15 ± 0.13 in the IV TPN group, and 5.38 ± 0.09 in the IG TPN group. There were no significant differences among all groups.

Adhesion molecules mediate leukocyte–endothelial cell adhesion and participate in PMN accumulation in tissues.^{8 ,10} E- and P-selectin expressed on the endothelial cells cause leukocyte rolling, which should precede ICAM-1–mediated firm adhesion.^{8 ,10 - 11} Regulated and coordinated expression of adhesion molecules is essential for appropriate tissue repair and host defense against infection.^{9 ,19}

The PMNs are the major effector of the nonspecific immune response in host resistance to infection.^{20 - 21} Under some conditions, PMNs could be mediators of tissue-destructive events in a variety of systemic disorders, because they have little intrinsic ability to differentiate between foreign and host antigens.^{22 - 23} The PMNs have been implicated as a major mediator for organ injury, particularly lung injury such as adult respiratory distress syndrome, in critically ill or severely injured patients.²⁴ Therefore, the changes in adhesion molecule expression may be associated with PMN-mediated organ injury or, at least, modulate subsequent response after an insult.

To clarify a mechanism for the increased septic complication in patients fed parenterally, we have focused on IgA-mediated mucosal immunity.^{4 - 6} Intravenous TPN fails to maintain respiratory antibacterial or antiviral immunity with decreases in IgA levels in both the respiratory tract and the intestine.^{4 - 6} Changes in gut-associated lymphoid tissue presumably lead to decreased respiratory immunity through depression of common mucosal immunity, establishing a link between the intestine and lung injury after a traumatic insult, as others have reported.⁹ The intestinal vascular bed has been shown to serve as a priming bed for PMNs after traumatic events.^{9 ,25} Our work strengthens this linkage, since lack of enteral feeding increases ICAM-1 expression and PMN accumulation in intestine.⁷

In this work, we postulated that the type and route of nutrition may affect intestinal expression of other adhesion molecules within the selectin family, since intestinal PMN accumulation with parenteral feeding may have resulted from up-regulated expression of P- or E-selectin as well as increased ICAM-1 expression. Second, we were interested in the changes that occur in nonsplanchnic organs. Since immunohistochemical staining cannot provide accurate quantification and the measurement of messenger RNA expression generates only indirect evidence, we used the dual radiolabeled antibody technique to quantify E- and P-selectin expression in vivo. Otherwise, quantification of the expression would be difficult in uninjured mice.^{16 ,26}

P-selectin expression was increased only in the gastrointestinal tract, ie, small intestine, stomach, colon, and pancreas. Although constitutive expression was observed in the other organs and tissues, there were no significant differences in expression within those organs among the 3 diet groups. Increased P-selectin is likely to cooperate with ICAM-1 in recruiting PMNs to the intestinal vascular bed. Although we studied uninjured animals manipulated with diet alone, dietary effects may have implications on responsiveness to subsequent injury. P-selectin has also been implicated in the increase in complement deposition after intestinal ischemia reperfusion.²⁷ PB1.3, a monoclonal antibody directed against P-selectin, reportedly limits deposition of the C5b-9 membrane attack complex and reduces the complement-dependent intestinal injury in a rat intestinal ischemia-reperfusion model.²⁷ Consequently, increased intestinal P-selectin in the animals fed parenterally might lead not only to PMN-mediated organ injury, but also to non–PMN-mediated intestinal injury after insult.

Generally, the expression of P-selectin is increased temporarily after stimulation by translocation from Weibel-Palade bodies.⁸ Rapid internalization and degradation allow the molecule to be expressed on the surface of the endothelium for a short time.^{8 ,10} Although P-selectin can also be regulated transcriptionally on cell activation by TNF-α, the elevation of expression may last only 24 hours after a single dose of TNF-α.¹⁰ Since gut expression of P-selectin remained up-regulated in the IV TPN group in a longer time frame (5 days), it is possible that failure of rapid degradation occurred or that the cytokine milieu in the intestine changed during lack of enteral feeding. We have no evidence that IV TPN increases the levels of the intestinal proinflammatory cytokines interleukin 1 and TNF-α in our mouse feeding model, but we have noted changes in cytokines related to IgA production within the gut-associated lymphoid tissue. Nevertheless, Ogle et al²⁸ reported that IV TPN up-regulates messenger RNA levels of these cytokines.

The pattern of E-selectin expression was quite different from that of P-selectin. E-selectin was detectable only in lung, liver, and kidney, and only a few animals had any detectable and very low levels in the small intestine, colon, and stomach. Enhanced pulmonary E-selectin expression in mice in the IV TPN group may reflect pulmonary inflammation or the activation of pulmonary endothelium. While IV TPN does impair respiratory IgA-mediated immunity,^{4 - 6} the animals did not show any symptom of lung inflammation. It remains unknown whether this increased expression of E-selectin is beneficial or detrimental to the host during parenteral feeding, but this observation warrants further exploration.

In summary, even though P- and E-selectin are believed to be expressed on the endothelium temporarily after stimulation, the uninjured mice in the IV TPN group had higher P-selectin expression in the gastrointestinal tract and increased E-selectin expression in the lung when compared with mice fed with IG TPN and chow. The mechanism by which each selectin increases differently in specific organs is still unclear. Nonetheless, we can speculate that the changes in P- and E-selectin expression during lack of enteral nutrition may be associated with the changes in subsequent response, particularly PMN-mediated host defense or organ injury, to surgical insults or infectious events.

This study was supported by grant 5 R01 GM53439 from the National Institutes of Health, Bethesda, Md.

We thank Doris Parsons for help in preparing and editing the manuscript.

Reprints: Kenneth A. Kudsk, MD, 956 Court Ave, Suite E228, Memphis, TN 38163.