Acepromazine

ABSTRACT. Background: Anticancer drugs have been demonstrated to affect gut mucosal morphology and cause gastrointestinal symptoms. We hypothesized that even small doses of 5-fluorouracil (5-FU) would reduce gut-associated lymphoid tissue (GALT) mass and function. Methods: Mice underwent IV cannulation and received continuous infusion of normal saline or 10 mg/kg of 5-FU for 5 days. GALT cell numbers, phenotypes, and mucosal immunoglobulin A (IgA) levels were measured. Results: During the infusion, there were no significant differences in food intake or body weight change between the 2 groups. Cell yields from the intraepithelial space and lamina propria of the small intestine were lower in the 5-FU than the control group. The lamina propria CD4/CD8 ratio was reduced in the 5-FU compared with the control group. Intestinal and respiratory tract IgA levels were lower in the 5-FU than in the control group. Conclusions: A small dose of 5-FU reduces GALT cell number and mucosal IgA levels, regardless of food intake. (Journal of Parenteral and Enteral Nutrition 29:395-400, 2005)

Chemotherapy is an important cancer treatment. However, both clinically and experimentally, anticancer drugs frequently cause a wide range of side effects, including leukopenia, stomatitis, appetite loss, diarrhea, and mild fever.1-4 With regard to gastrointestinal tract changes, mucosal thickness and villous height are decreased, whereas intestinal permeability and mucosal apoptosis are increased.5-7

Gut-associated lymphoid tissue (GALT), a term for lymphoid tissues located in the intestine, has been considered a center of mucosal immunity because of its mass and function.8 GALT is divided into inductive and effector sites.8 Because luminal antigens are sampled, processed, and presented to unprimed T- and B-cells in Peyer's patches (PPs), the PPs are an inductive site for mucosal immunity. The main effector sites of intestinal immune responses are the intestinal lamina propria (LP) and intraepithelial lymphocytes (IEL). After induction in the PPs, primed lymphocytes migrate to these effector sites and extraintestinal mucosal sites.

Although anticancer drugs reportedly cause morphological changes in the intestine, whether or not GALT mass and function are reduced remains unclear. Reduction of GALT mass and function may impair mucosal defense,9 5-Fluorouracil (5-FU), a fluorinated pyrimidine, is widely used in the treatment of several malignancies and is known to rapidly induce gastrointestinal tract mucosal injury.10 Moreover, fluorouracil treatment reportedly reduced salivary immunoglobulin A (IgA) concentrations in patients with neoplastic disorders.11,12 secretory IgA is a primary immunologie component of the extrinsic protective mechanism of the mucosal surface. Therefore, in this study, we examined effects of continuous infusion of 5-FU on GALT (PPs, LP, and IEL) lymphocyte numbers, GALT cell phenotypes, and the levels of secretory IgA.

MATERIALS AND METHODS

Animals

Male Institute of Cancer Research (ICR) mice (Nippon SLC, Hamamatsu, Japan) were housed under controlled temperature and humidity conditions with a 12-hour: 12-hour light:dark cycle. Mice were fed commercial mouse chow with water ad libitum for 1 week before protocol entry. All studies reported herein conform to the guidelines for the care and use of laboratory animals established by the Animal Use and Care Committee of the National Defense Medical College.

Infusion Protocol

Forty mice (6 weeks old) were randomized before cannulation to receive continuous IV infusion of 5-FU (10 mg/kg of body weight/d; n = 22) or normal saline (control group; n = 18). Mice underwent placement of jugular vein catheters for IV infusion after subcutaneous injection of ketamine hydrochloride (100 mg/kg of body weightVacepromazine maleate ( 10 mg/kg of body weight). Via the right jugular vein, a silicon rubber catheter (0.3 mm ID and 0.5 mm OD; Imamura, Tokyo, Japan) was inserted into the vena cava. The proximal end of the catheter was tunneled subcutaneously over the spine, exiting 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 exerts neither physical nor biochemical stress.13

Catheterized mice were immediately connected to infusion pumps (TE331, Terumo, Tokyo, Japan), and received normal saline at 0.2 mL/h for 48 hours with ad libitum access to chow and water. On postoperative day 2, the 5-FU group received 0.2 mL/h of 0.06 mg/mL 5-FU (Kyowa Hakko Kogyo, Tokyo, Japan) solution, ie, 10 mg/kg of 5-FU per day, whereas the control group was given 0.2 mL/h of normal saline (Figure 1).

After 5 days of chemotherapy, the first set of mice (5-FU group: n = 14, control group: n = 10) was anesthetized with ketamine hydrochloride/acepromazine maleate subcutaneously. The mice were then weighed and exsanguinated by cardiac puncture. The entire small intestine was harvested for GALT lymphocyte isolation. Daily food intake was also monitored during the 5-FU treatment. The second set of mice (5-FU group: n = 8, control group: n = 8) was used for measurement of mucosal IgA levels. Under anesthesia, nasal and bronchoalveolar washings were obtained by lavage with 1 mL of phosphate-buffered saline solution. The entire small intestine was harvested and flushed twice with a total of 20 mL of chilled Hanks' balanced salt solution. The washings were stored in a -80°C freezer for IgA analysis.

Cell Isolation

Lymphocytes were isolated from GALT using a modification of the method described by Li et al.9 PPs were examined as an inductive site for mucosal immunity. The IE spaces and LP were chosen as effector sites of gut mucosal immunity.

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.

After excision of the PPs, 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 time, with RPMI1640 containing collagenase (Sigma; 40 U/mL), 5% FBS, glutamine, and an antibiotic mixture 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. 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 × g at 25°C, viable lymphocytes were recovered from the 40/75% interface and washed in RPMI1640. The lymphocytes were resuspended in RPMI1640 with 5% FBS, 1% glutamine, and a 1% antibiotic mixture and then counted.

Flow Cytometry

To determine the phenotypes of lymphocytes isolated from PPs, IE spaces, and the LP, 10 cells were suspended in 50 µL HBSS containing fluorescein isothiocyanate (FITC) antimouse γδSTCR (clone GL3; Caltag, Burlingame, CA) and phycoerythrin (PE) conjugated antimouse αβTCR (clone H57-597; Pharmingen, San Diego, CA) to identify γδTCR+ T-cells and αβTCR+ T-cells, respectively, or PE-anti-CD4 (clone CT-CD4, Caltag) and FITC-anti-CD8aα (clone CT-CDSa, Caltag) to identify the 2 T-cell subsets, 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 1% paraformaldehyde. Flowcytometric analysis was performed on an Epics XL (Coulter, Hileah, IL).

IgA Quantification

Immunoglobulin A was measured in intestinal and respiratory tract washings by 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).

Statistical Analysis

Data are expressed as means ± SEM. Differences between groups were evaluated with Student's t test. A value of p

RESULTS

Body Weight Change

There were no significant differences in body weight at the beginning of the experiment, or in body weight change, between the 5-FU and control groups (Table I).

Food Intake

We found no significant difference in daily food intake between the 5-FU and control groups during the course of continuous infusion, suggesting 5-FU to have no major impact on food intake (Figure 2).

Numbers of PPs Excised and Total Cell Yield From GALT

Numbers of PPs excised were similar in the 5-FU and control groups (Figure 3). The number of lymphocytes isolated from PPs did not differ significantly between the 2 groups (Figure 3). However, 5-FU infusion significantly decreased IE and LP lymphocyte numbers compared with the controls.

GALT Phenotype

There were no significant differences in the percentages of αβTCR, γδTCR, CD4, CD8 or B220 positive cells, at any of the GALT sites examined, between the 2 groups (Table II).

CD4/CD8 Ratio

When we calculated the CD4/CD8 ratios, LP lymphocytes from the 5-FU group had a significantly lower ratio than those from the control group (Figure 4). There were no significant differences in CD4/CD8 ratios of PP or IE lymphocytes between the 5-FU and control groups.

Secretory IgA Levels

5-FU infusion significantly reduced intestinal and bronchoalveolar IgA levels compared with the control group (Figure 5). However, nasal IgA levels did not differ significantly.

DISCUSSION

The present study revealed the influence of chemotherapy on GALT mass and function. A small dose of 5-FU reduces lymphocyte numbers at GALT effector sites but does not significantly influence PP lymphocyte numbers. In association with the GALT mass reduction, intestinal and respiratory tract IgA levels were decreased in the 5-FU group.

The intestinal mucosa is the barrier between nonharmful and harmful molecules. Uncontrolled penetration of harmful molecules could initiate pathologic processes, leading to a gastrointestinal disease state and, moreover, systemic inflammatory disorders.14 Under normal conditions, antigen entry is prevented by nonspecific and immunologic mechanisms in the gastrointestinal tract and by the physical structure of the epithelium.14

GALT provides an immunologic barrier in the intestine. Secretory IgA produced by plasma cells in the LP is a major immunologic component of the extrinsic protective mechanism in the intestinal lumen.15 LP CD4+ T-cells secrete high levels of cytokines that stimulate (IL-4, -5, and -10) or inhibit (IFN-γ) IgA production.16 The LP CD8+ T-cell population contains cytolytic effector cells.8 Although the role of IELs is still unclear, IELs may lyse infected epithelial cells and eliminate infection by eliminating the cellular substrate necessary for pathologic invasion.8 IELs, particularly those expressing γδTCR, may also be involved in homeostasis of the intestinal epithelium and of the resident immune system through chemokine production.17

Continuous infusion of 5-FU decreased cell numbers at these effector sites, suggesting impairment of the gut immunologic barrier. Indeed, IgA levels in small intestinal washings were significantly lower in the 5-FU than in the control group. Therefore, gastrointestinal symptoms and the systemic inflammatory response accompanying 5-FU treatment may be associated at least partly with these GALT changes. Although there were no significant differences in lymphocyte phenotypes between the 2 groups, the CD4/CD8 ratio in the LP was decreased in the 5-FU group, which likely contributed to the intestinal IgA level reduction.

The mechanisms underlying decreased GALT cell number in the 5-FU group were not clarified by the present results. Because 5-FU exerts its anticancer effects through inhibition of thymidylate synthase and incorporation of its metabolites into RNA and DNA,18 proliferation of GALT lymphocytes may be inhibited, whereas apoptosis may be increased. Because cell numbers at effector sites (IE and LP) were reduced by 5-FU infusion without marked changes in PP cell numbers, expression of homing receptors on primed lymphocytes or endothelial cells at effector sites might be altered in response to 5-FU.

Interestingly, respiratory tract IgA levels, particularly bronchoalveolar IgA levels, were also decreased in the 5-FU mice compared with the controls. Generally, GALT changes affect systemic mucosal immunity because the primed lymphocytes migrate from PPs to intestinal and extraintestinal mucosal systems.15 However, PP cell yields were similar in the 5-FU and control groups in this study, suggesting that this 5-FU does not impair inductive sites of mucosal immunity. 5-FU treatment may directly impair effector sites of extraintestinal mucosal immunity and intestinal LP and IE lymphocytes. Otherwise, despite unchanged PP lymphocyte numbers, 5-FU infusion might interfere with antigen sampling, processing or presentation in PPs.

Chemotherapy is associated with a variety of disturbances that can profoundly affect nutrition status.19 Malnutrition affects GALT, reducing secretory IgA responses and GALT lymphocyte numbers.20 However, our 5-FU mice ate almost the same amount of chow as the control mice. In addition, body weight change did not differ significantly between the 2 groups. Thus, GALT alteration observed in this study did not arise from nutrition factors.

Oral 5-FU at 10 mg/kg reportedly prolongs survival time in a metastatic mouse model.21 Although we administered this dose IV, 10 mg/kg is a relatively small dose, and we observed no gastrointestinal symptoms such as diarrhea.

To learn whether the observations exist in humans with cancer, we need further studies using human GALT samples. We previously examined influences of lack of enterai nutrition on GALT with human terminal ileum specimens.22 A similar approach should be performed in clinical settings. Nonetheless, we believe that the present study revealed a possible mechanism for chemotherapy-related impairment of host defense. Clinically, during chemotherapy with 5-FU, special considerations aimed at preserving mucosal immunity may be needed. Because a normal diet failed to maintain GALT mass and function in this study, supplementation of some certain immune-enhancing nutrients should be taken into consideration as a strategy for avoiding 5-FU-induced impairment of gut mucosal immunity in clinical settings.

In summary, a 5-day infusion of 5-FU at 10 mg/kg/d resulted in GALT cell loss and mucosal IgA reduction, regardless of food intake. There were no significant differences in GALT lymphocyte phenotypes, but the LP CD4/CD8 ratio was decreased in the 5-FU group. It is important to recognize GALT changes during the course of chemotherapy. New approaches aimed at replenishing GALT mass and function during chemotherapy warrant further study.

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Hidetoshi Nagayoshi, MD*; Kazuhiko Fukatsu, MD[dagger]; Chikara Ueno, MD*; Etsuko Kara, MT[dagger]; Yoshinori Maeshima, MD*; Jiro Omata, MD*; Hoshio Hiraide, MD[dagger]; and Hidetaka Mochizuki, MD*

From the * Department of Surgery I, National Defense Medical College, and the [dagger] Division of Basic Traumatology, National Defense Medical College Research Institute, Saitama, Japan

Received for publication March 17, 2005.

Accepted for publication July 21, 2005.

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@ndmc.ac.jp.

Presented at Nutrition Week 2004, February 9-12, 2004, Las Vegas, Nevada.

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