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Interleukin-7 Dose-Dependently Restores Parenteral Nutrition–Induced Gut-Associated Lymphoid Tissue Cell Loss but Does Not Improve Intestinal Immunoglobulin A Levels

Kazuhiko Fukatsu, MD, PhD^*, Tomoyuki Moriya, MD, Fumie Ikezawa, MD^*, Yoshinori Maeshima, MD, Jiro Omata, MD, Yoshihisa Yaguchi, MD, Koichi Okamoto, MD, Hidetaka Mochizuki, MD, PhD and Hoshio Hiraide, MD, PhD^*

From the ^* Division of Basic Traumatology, National Defense Medical College Research Institute, Tokorozawa, Japan; Department of Surgery I, Chiba University, Chiba, Japan; and the Department of Surgery I, National Defense Medical College, Tokorozawa, Japan

Correspondence: Kazuhiko Fukatsu, MD, Division of Basic Traumatology, National Defense Medical College Research Institute, 3-2 Namiki, Tokorozawa, Saitama, Japan, 359-8513. Electronic mail may be sent to fukatsu{at}ndmc.ac.jp.

Background: Without enteral nutrition, the mass and function of gut-associated lymphoid tissue (GALT), a center of systemic mucosal immunity, are reduced. Therefore, new therapeutic methods, designed to preserve mucosal immunity during parenteral nutrition (PN), are needed. Our recent study revealed that exogenous interleukin-7 (IL-7; 1 µg/kg twice a day) restores the GALT cell mass lost during intravenous (IV) PN but does not improve secretory immunoglobulin A (IgA) levels. Herein, we studied the IL-7 dose response to determine the optimal IL-7 dose for recovery of GALT mass and function during IV PN. We hypothesized that a high dose of IL-7 would increase intestinal IgA levels, as well as GALT cell numbers. Methods: Male mice (n = 42) were randomized to chow, IL-7-0, IL-7-0.1, IL-7-0.33, IL-7-1 and IL-7-3.3 groups and underwent jugular vein catheter insertion. The IL-7 groups were fed a standard PN solution and received IV injections of normal saline (IL-7-0), 0.1, 0.33, 1, or 3.3 µg/kg of IL-7 twice a day. The chow group was fed chow ad libitum. After 5 days of treatment, the entire small intestine was harvested and lymphocytes were isolated from Peyer's patches (PPs), intraepithelial (IE) spaces, and the lamina propria (LP). The lymphocytes were counted and phenotypes determined by flow cytometry (βTCR, TCR, CD4, CD8, B cell). IgA levels of small intestinal washings were also examined using ELISA (enzyme-linked immunoabsorbent assay). Results: IL-7 dose-dependently increased total lymphocyte numbers in PPs and the LP. The number of lymphocytes harvested from IE spaces reached a plateau at 1 µg/kg of IL-7. There were no significant differences in any phenotype percentages at any GALT sites among the groups. IgA levels of intestinal washings were significantly higher in the chow group than in any of the IL-7 groups, with similar levels in all IL-7 groups. Conclusions: Exogenous IL-7 dose-dependently reverses PN-induced GALT cell loss, with no major changes in small intestinal IgA levels. IL-7 treatment during PN appears to have beneficial effects on gut immunity, but other therapeutic methods are needed to restore secretory IgA levels.

Gut-associated lymphoid tissue (GALT) is a center of systemic mucosal immunity.¹ Naïve lymphocytes are sensitized in Peyer's patches (PP), travel to mesenteric lymph nodes, are released into the systemic circulation via the thoracic duct, and migrate to intestinal and extraintestinal mucosal sites.² Atrophy and dysfunction of GALT have been demonstrated to reduce the immunologic barriers of the gastrointestinal and respiratory tracts.^3,4 Because various surgical insults may affect gut integrity and function,^5–7 resulting in severe infectious complications and multiple-organ failure, maintenance of GALT is a reasonable preventive and therapeutic strategy in severely injured or critically ill patients.

Experimentally, enteral delivery of nutrients is considered to be an optimal and practical approach to preventing GALT cell loss and dysfunction.^8,9 Moreover, our recent study provided clinical evidence that GALT mass declines in the absence of enteral nutrition in patients, just as it does in animal models.¹⁰ However, there are some patients who should receive PN due to the presence of contraindications to the use of enteral nutrition or intolerance of enteral nutrition. Indeed, higher morbidity with infectious complications has been demonstrated in parenterally fed patients who have sustained severe surgical insults.^11,12 Therefore, new modalities designed to preserve GALT during PN are urgently needed.

Because interleukin-7 (IL-7), a pleiotropic cytokine, is important in the proliferation and function of GALT lymphocytes^13,14 and intestinal epithelial cell (IEC)–derived IL-7 mRNA expression is reportedly reduced during PN,¹⁵ we previously examined the effects of exogenous IL-7 (1 µg/kg twice a day) on GALT in mice given intravenous parenteral nutrition (IV PN).¹⁶ We found IL-7 treatment to reverse IV PN-induced GALT cell loss but not to improve secretory immunoglobulin A (IgA) levels. In the present study, we examined the IL-7 dose response to determine the optimal IL-7 dose for recovery of GALT mass and function during IV PN. We hypothesized that a high dose of IL-7 would increase intestinal IgA levels, as well as GALT cell numbers.

View larger version (8K): [in this window] [in a new window]   FIGURE 1. Experimental protocol. The chow group received a saline infusion with free access to chow and water. The IL-7-0, -0.1, -0.33, -1, and -3 groups were given identical PN intravenously. The IL-7-0.1, -0.33, -1, and -3 mice were injected with 0.1, 0.33, 1, and 3 µg/kg IL-7 twice a day, respectively, whereas the IL-7-0 and chow animals received a placebo saline injection.

	MATERIALS AND METHODS

Top MATERIALS AND METHODS RESULTS DISCUSSION

Surgical Procedure
The mice (n = 42) were given general anesthesia (ketamine 100 mg/kg and xylazine 10 mg/kg mixture), and all procedures were performed aseptically. After randomization to the chow (n = 6), IL-7-0 (n = 8), IL-7-0.1 (n = 8), IL-7-0.33 (n = 8), IL-7-1 (n = 8) and IL-7-3.3 (n = 4) groups, all mice underwent implantation of silicon rubber catheters (Imamura Co, Tokyo, Japan; 0.014-inch inner diameter/0.026-inch outer diameter) into the right jugular vein. Through a right internal jugular approach, a silicone rubber catheter 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 in metal metabolism cages and partially immobilized by tail restraint to protect the catheter during infusion. This technique is a well-established method of nutrition support that does not induce physical or biochemical stress.¹⁷ Catheterized mice were immediately connected to an infusion pump (TE-331; Terumo, Tokyo, Japan).

Experimental Design and Nutrition Support
The experimental protocol is shown in Figure 1. Catheterized mice were given a 0.9% NaCl solution, 4.8 mL/d, and allowed ad libitum access to chow (CE7) and water for 48 hours to promote recovery from surgical stress. The chow group served as controls and continued to receive a 0.9% NaCl solution intravenously, with free access to chow and water throughout the study period. The PN group received a standard IV PN solution. The compositions of chow and PN are shown in Tables I and II. This PN solution provided 1422 kcal/L, with a nonprotein calorie-nitrogen ratio of 208:1. The IL-7-0.1, -0.33, -1, and -3.3 groups received an identical IV PN solution plus IL-7 injections (0.1, 0.33, 1, and 3.3 µg/kg intravenously twice a day, respectively; Strathmann Biotec AG, Hamburg, Germany). The chow and IL-7-0 groups were given a placebo 0.2 mL saline injection intravenously twice a day. The PN solution rate was advanced from 7.2 mL/d to 14.4 mL/d (20.5 kcal/d) by the third day of feeding (day 1, 0.3 mL/h; day 2, 0.4 mL/h, days 3–5, 0.6 mL/h) because a graded infusion period is necessary to allow the mice to adapt to the glucose and fluid load.¹⁸ The target rate for the PN solution was determined according to our previous data from mice fed chow ad libitum. The chow mice consumed approximately 6–6.5 g of chow (20.4–22.1 kcal) per day in this setting.

GALT Cell Isolation
After receiving their respective diets and treatments for 5 days, the mice were anesthetized with ketamine hydrochloride/xylazine subcutaneously (Figure 1). The mice were exsanguinated by cardiac puncture. The entire small intestine was harvested and flushed with 20 mL of chilled Hanks' balanced salt solution to remove feces. Lymphocytes were isolated from GALT using a modification of the method described by Li et al.⁸ PPs were examined as an inductive site for mucosal immunity, whereas intraepithelial (IE) space and lamina propria (LP) lymphocytes were chosen as effector sites for gut mucosal immunity.

PPs
PPs were excised from the serosal side of the intestine and then teased apart. The fragments were treated with collagenase (Sigma, St. Louis, MO; 40 U/mL) in RPMI1640 for 60 minutes at 37°C with constant shaking. After collagenase digestion, the cell suspensions were passed through nylon filters.

IE Space and LP
After PP excision, the intestine was turned inside out and cut into 4 segments. The segments were incubated with RPMI1640 containing 5% fetal bovine serum (FBS), 1% glutamine, and a 1% antibiotic mixture (penicillin and streptomycin; GIBCO, Auckland, New Zealand) for 45 minutes at 37°C in a water shaker (150 rpm). Supernatants containing released sloughed epithelial cells and IE lymphocytes were stored on ice. The remaining tissue pieces were incubated 3 times, 45 minutes each, with RPMI1640 containing collagenase (40 U/mL), 5% FBS, glutamine, and an antibiotic mixture at 37°C at 150 rpm in a water shaker. Supernatants containing LP cells from each incubation were pooled on ice.

Supernatants were filtered through a glass wool column (0.2 g of glass wool in a 10-mL syringe). Suspensions were centrifuged, the pellets were resuspended in 40% Percoll (Pharmacia, Piscataway, NJ), and the cell suspensions were overlaid on 75% Percoll. After centrifugation for 20 minutes at 600 x g at 25°C, viable lymphocytes were recovered from the 40%/75% interface and washed in RPMI1640 with 5% FBS, glutamine, and the antibiotic mixture. Viable lymphocytes were counted using the trypan-blue exclusion test. This procedure yields a cell population that is 95%–100% viable.

Flow Cytometry
To determine the phenotypes of lymphocytes isolated from PPs, the IE space, and the LP, 10⁵ cells were suspended in 50 µL of HBSS containing fluorescein isothiocyanate (FITC) antimouse TCR (clone GL3; Caltag, Burlingame, CA) and phycoerythrin (PE)-conjugated antimouse βTCR (clone H57–597; Pharmingen, San Diego, CA) to identify TCR+ T cells and βTCR+ T cells, respectively, or PE–anti-CD4 (clone CT-CD4; Caltag) and FITC–anti-CD8 (clone CT-CD8a; Caltag) to identify CD4+ T cells and CD8+ T cells or FITC–anti-CD45R (B220; clone RA3–6B2; Caltag) to identify B cells. All antibodies were diluted to 1 µg/mL in HBSS containing 1% FBS. Incubations were carried out for 30 minutes on ice. After staining, the cells were washed twice in HBSS/1% FBS and then fixed in paraformaldehyde. Flow cytometric analysis was performed on an Epics XL (Coulter, Hileah, IL). The absolute number of cells with each phenotype was calculated by multiplying total lymphocyte number by the percentage of each phenotype.

IgA Quantification
IgA was measured in intestinal and bronchoalveolar washings in a sandwich enzyme-linked immunosorbent assay using a polyclonal goat antimouse IgA (Sigma) to coat the plate, a purified mouse IgA (Zymed Laboratories, San Francisco, CA) as the standard, and a horseradish peroxidase–conjugated goat antimouse IgA (Sigma).

Statistics
The data are expressed as means ± SE and were analyzed using analysis of variance (ANOVA), followed by the Fisher's protected least significant difference post hoc test. A value of p < .05 was considered to represent statistical significance.

	RESULTS

Top MATERIALS AND METHODS RESULTS DISCUSSION

 
Pre- and Postexperiment Body Weights
There were no significant differences in preexperiment body weights among the groups (Table III). After treatments, the chow group had a significantly higher mean body weight than any of the PN-fed groups, with no significant differences between any 2 PN-fed groups.

GALT Lymphocyte Numbers
NPO status and PN decreased total lymphocyte numbers from PPs, IE spaces, and the LP compared with chow (Figure 2). IL-7 treatment dose-dependently reversed PN-induced GALT lymphocyte loss. PP, IE space, and LP cell yields showed significant positive correlations with IL-7 dosages (simple regression, PP: r = 0.586, p = .0002, n = 36; IE: r = 0.363, p = .0294, n = 36; LP: r = 0.557, p = .0004, n = 36). IL-7 at 3.3 µg/kg increased GALT cell numbers to nearly those of the chow animals. However, the number of lymphocytes from IE spaces reached a plateau at 1 µg/kg of IL-7. By excluding the IL-7-3.3 mice, a better correlation was obtained between IE cell numbers and IL-7 doses (r = 0.632, p = .0001, n = 32).

View larger version (16K): [in this window] [in a new window]   FIGURE 2. GALT lymphocyte numbers. Values are means ± SE. *p < .05 vs chow, p < .05 vs IL-7-0, p < .05 vs IL-7-0.1, p < .05 vs IL-7-0.33. IE, intraepithelial; LP, lamina propria; PP, Peyer's patch.

GALT Lymphocyte Phenotypes
There were no significant differences in any phenotype percentages at any GALT sites among the groups (Tables IV, V, VI).

IgA Levels of Intestinal Washings
Intestinal-washing IgA levels were significantly higher in the chow group than in any of the IL-7 groups, with similar levels in all IL-7 groups (Figure 3).

View larger version (15K): [in this window] [in a new window]   FIGURE 3. IgA levels of small intestinal washings. Values are means ± SE. *p < .01 vs chow.

	DISCUSSION

Top MATERIALS AND METHODS RESULTS DISCUSSION

IL-7 is produced by nonhematopoietic stromal cells in various organs. In the intestine, IECs and epithelial goblet cells produce IL-7, and locally produced IL-7 serves as a potent regulatory factor for intestinal mucosal lymphocytes.²² IL-7 is important in the development of IE, LP, and PP lymphocytes.^13,14 Moreover, IL-7 was shown to prevent apoptosis of and β IE lymphocytes.²³ Yang et al¹⁵ reported that IV PN reduced IEC-derived IL-7 mRNA expression and that exogenous IL-7 administration increased IE lymphocyte numbers in normal chow-fed mice.²⁴ Therefore, it would seem to be reasonable to administer IL-7 to IV PN mice with the expectation of GALT mass and function recovery.

In our previous study, we clarified that 1 µg/kg of IL-7 restores GALT cell numbers from parenterally fed mice to nearly the levels of chow-fed mice, but this dose did not reverse IV PN-induced reduction of mucosal IgA levels.¹⁶ Because exogenous IL-7 was demonstrated to change IE lymphocyte-derived cytokine expression,²⁴ it is reasonable to speculate that a high dose of IL-7 improves the gut cytokine milieu, resulting in increased intestinal IgA levels. On the other hand, high IL-7 levels might be associated with autoimmunity or proliferation of neoplastic lymphoid cells.¹⁹ The minimum IL-7 dose with maximal beneficial effects on GALT must be determined. Thus, we performed the present IL-7 dose-response study.

We recognized significant positive correlations between PP, IE, and LP lymphocyte numbers and the administered IL-7 doses. None of the IL-7 doses given in the present study produced any apparent side effects. IL-7 at 3.3 µg/kg appears to have the strongest restorative effects on IV PN-induced GALT cell loss. However, as to the IE spaces, the cell number reached a plateau at 1 µg/kg of IL-7. Thus, 1–3.3 µg/kg of IL-7 may be the optimal dose for mice receiving PN. The mechanism underlying the different patterns of GALT cell recovery at each GALT site is not clear from the present results. Because IE lymphocytes are located very close to IECs that produce IL-7, even a relatively small dose of exogenous IL-7 might increase local IL-7 levels, reaching saturation in terms of IE lymphocyte number maintenance.

Contrary to the GALT cell numbers, IgA levels in small intestinal washings did not change in response to IL-7 therapy and remained lower in all PN-fed mice than in the chow group. IgA production in the gut is regulated via the gut cytokine milieu.²¹ Th1 cytokines inhibit, whereas Th2 cytokines stimulate, IgA production. Lack of enteral nutrition was demonstrated to have no effect on gut IFN levels (a Th1 cytokine) while decreasing gut IL-4 and -10 levels (Th2 cytokines), which may be an important mechanism by which intestinal IgA levels are reduced during parenteral feeding.²¹ Although IL-7 was shown to enhance peripheral T-cell expression of IL-4 mRNA and IE lymphocyte-derived IL-10^24,25 in our previous study, we found no significant differences in IFN or IL-10 levels in gut homogenates from PN-fed mice with vs without IL-7 administration.¹⁶ Cytokine production by IE lymphocytes might not have a strong influence on IgA production in the LP.

With regard to the percentages of GALT cell phenotypes, there were no significant differences among the groups. Although IL-7 expression by IECs has been shown to trigger the development of extrathymic TCR cells in the IE spaces,²⁶ exogenous IL-7 appears to increase lymphocytes with various phenotypes. This may be reasonable because IL-7 contributes to lymphopoiesis of T and B cells, inhibition of β and T-cell apoptosis, generation of CD4+ and CD8+ cells, and trafficking of thymic emigrants to lymphoid tissues.¹⁹

The present data clearly show that IL-7 therapy alone cannot fully restore the changes in GALT induced by IV PN. Despite recovery of GALT cell mass, exogenous IL-7 does not improve intestinal IgA levels during IV PN. Absence of enteral nutrition disturbs GALT homeostasis, for example, by decreasing (1) expression of PP MAdCAM-1, an important adhesion molecule for lymphocyte homing to GALT,²⁷ (2) gut Th2 IgA-stimulating cytokine levels,²¹ and (3) IEC IL-7 production.¹⁵ Therefore, combinations of exogenous IL-7 and other therapies normalizing the gut cytokine milieu need to be tested in future studies.

In the current study, we did not examine the effects of exogenous IL-7 on extraintestinal mucosal immunity. Because PPs are inductive sites for systemic mucosal immunity and IL-7 administration reversed IV PN-induced PP cell loss, respiratory tract immunity against pathogens could be maintained with exogenous IL-7. It is also possible that IgA production is enhanced by IL-7 therapy under stimulation with toxins or pathogens. These questions also need to be addressed in future studies.

In conclusion, exogenous IL-7 dose-dependently reverses PN-induced GALT atrophy, with no marked effect on small-intestinal IgA levels. IL-7 treatment during PN may have beneficial effects on gut immunity, but other therapeutic strategies are needed to restore secretory IgA levels.

Top MATERIALS AND METHODS RESULTS DISCUSSION

 
Presented in the Premier Paper Session at the American Society for Parenteral and Enteral Nutrition Clinical Nutrition Week, February 12–15, 2006.

Received for publication February 27, 2006. Accepted for publication April 27, 2006.

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Discussant

University of Auckland

This is an interesting follow-up study to earlier work from a multi-center Japanese research group. The presented data will add to our knowledge of the role of cytokines, especially interleukin-7 (IL-7) in the proliferation and function of gut associated lymphoid tissue (GALT).

The authors have attempted to determine the optimal IL-7 dose for restoring GALT function during parenteral nutrition (PN) using a mouse model. Groups of mice receiving PN via a central catheter were given twice daily IV doses of IL-7 ranging from zero to 3.3 µg/kg, with an ad libitum chow group acting as controls.

The compositions of the PN and chow diets were reasonably similar, with the exception of L-glutamine and fat (Tables I and II). L-Glutamine is an important amino acid for the immune system and would have been present in the chow protein, but is unfortunately only included in the total Glutamic Acid value, at >30 times the PN content. Fat was totally missing from the PN formulation but provided approximately 10% of energy in the chow, which may be important for the immune system. The PN and oral diets were therefore not precisely comparable.

The results from these laboratory experiments confirm that PN decreases total lymphocyte count in Peyer's patches, intraepithelial spaces and lamina propria. Moreover the data indicates that IL-7 at the highest IV dose (3.3 µg/kg) increased GALT cell numbers to almost the same levels as the chow group. In contrast, lymphocyte counts from the intraepithelial spaces plateaued at a lower concentration of IL-7. There were no significant differences in any phenotype percentages at those GALT sites examined, although the CD4:CD8 ratios appeared to range from 1:2 for chow vs 1:4 for the highest IL-7 dose, but this is not discussed by the authors. IgA levels disappointingly did not change in any of the PN-supplemented groups, remaining lower than levels in the control group.

GALT dysfunction is known to decrease the immunologic defenses of the gastrointestinal tract which may be accentuated in patients who cannot receive oral or enteral nutrition. This manuscript demonstrates some benefits from cytokine supplementation, but under these experimental conditions, exogenous IL-7 cannot improve intestinal IgA levels and consequently cannot fully restore PN-induced GALT changes. The title of the paper [originally, Interleukin-7 Dose-Dependently Restores Parenteral Nutrition-Induced Gut Associated Lymphoid Tissue Cell Loss] should therefore be changed to better reflect this dichotomy.

Nevertheless, novel therapeutic approaches to supplement standard PN in order to normalize cytokine levels and improve mucosal immunity for critically ill patients are much needed. Further investigation by these and other researchers are to be welcomed.

Author's Response

With regard to the CD4/CD8 ratios, because we did not obtain significant differences among the groups nor significant IL-7 dose dependent changes, we did not discuss that here.

As you pointed out, it was disappointing that IL-7 treatment did not improve intestinal IgA levels. To maintain IgA levels, other therapeutic methods normalizing intestinal gut cytokine milieu may be needed. And as you suggested, our title was changed to reflect the dichotomy.

Journal of Parenteral and Enteral Nutrition, Vol. 30, No. 5, 388-394 (2006)
DOI: 10.1177/0148607106030005388


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