Peritonitis

ABSTRACT. Background: The effects of malnutrition on polymorphonuclear neutrophil (PMN) exudation are not well understood. The purpose of this study was to examine the effects of short-term dietary restriction on adhesion molecule expression on circulating PMNs and PMN exudation into the inflamed site in a glycogen-induced peritonitis model. Methods: Twelve mice were randomly assigned to one of two groups. The ad libitum and diet-restricted groups received mouse chow ad libitum (estimated consumption: 132 g/kg per day) and 33 g/kg per day, respectively, for 7 days. Then, 2 mL of a 1% glycogen solution was intraperitoneally administered to all mice. After 4 hours, the animals were killed. Whole blood was drawn by cardiac puncture. Peritoneal exudative cells were harvested by lavaging the peritoneal cavity. Expressions of CDllb, CD 18, and CD62L were measured by flow cytometry. Results: Dietary restriction did not affect the numbers of circulating leukocytes, PMNs, or monocytes. However, CD llb and CD18 expressions on circulating PMNs were significantly lower in the diet-restricted than in the ad libitum group. In contrast, CD62L expression on circulating PMNs was not affected by dietary restriction. The number of exudative PMNs was significantly lower in the diet-restricted group than in the ad libitum group. The expressions of CDllb, CD18 and CD62L on exudative PMNs were unaffected by dietary restriction. There was a significant positive correlation between exudative PMN numbers and CD18 expression on circulating PMNs. Conclusions: Severe dietary restriction in our murine model decreased [32 integrin expression on circulating PMNs and inhibited PMN exudation into inflamed sites in the early phase of inflammation. These events may increase susceptibility to bacterial infection. Nutritional replenishment may improve host defense in part by enhancing PMN adhesion molecule expression. (Journal of Parenteral and Enteral Nutrition 24:276-279, 2000)

Previous studies have demonstrated that malnutrition leads to impaired host defense, resulting in an increased susceptibility to infection.1,2 Polymorphonuclear neutrophils (PMNs) play pivotal roles in the host response to microbial infection. However, the reported effects of malnutrition on PMN function are contradictory. Some investigators have demonstrated that malnutrition impairs chemotaxis and phagocytosis by PMNs,3,4 while other studies have failed to show any substantial effects of malnutrition on PMN functions.5 Moreover, investigations concerning the adverse effects of malnutrition on PMNs are based primarily on circulating PMNs.5 However, exudative PMNs play important roles in host defense at the local site. Interaction with endothelial cells is fundamental to the localized exudation of PMNs into extravascular inflammatory sites.6 Recent studies have revealed that adhesion molecules on both PMNs and endothelial cells contribute to this interaction.7,8 However, few studies have investigated the effects of malnutrition on PMN exudation. Moreover, there are no reports on the effects of malnutrition on adhesion molecule expression on PMNs. We examined the effects of short term dietary restriction on adhesion molecule expression on circulating PMNs, and PMN exudation into inflamed sites, in the early phase of glycogen-induced peritonitis in a murine model.

ANIMAL MODEL

Specific pathogen-free 8-week-old male C57BL/6J mice (Japan SLC, Hamamatsu, Japan) were used for the experiments. The mice were kept in animal facilities for 1 week before experiment initiation to allow acclimation. They were exposed to constant temperature (24C) and humidity (60%) and were fed standard mouse chow (Oriental Koubo, Tokyo, Japan). Regular mouse chow contains protein, fat, carbohydrate, cellulose, minerals, and a vitamin mix (24.6, 5.6, 6.4, 3.1, 3.5, and 0.4 g per 100 g of diet, respectively). All studies were performed in accordance with the Guide for Animal Experimentation, Faculty of Medicine, University of Tokyo. Our institutional review board approved the experimental protocol.

EXPERIMENTAL DESIGN

Twelve 8-week-old male C57BL/6J mice were randomly assigned into one of two groups. The ad libitum and diet-restricted groups received regular mouse chow ad libitum and 33 g/kg per day (120 kcal/kg per day), respectively, for 7 days. Our preliminary experiment revealed that the average chow consumption by mice with free access to chow was 132 g/kg per day, which is equivalent to 480 kcal/kg per day. Then, 2 ml of a 1% glycogen solution (Sigma, St. Louis, MO, USA), which elicits cell exudation, was intraperitoneally administered to all mice. After 4 hours, the animals were killed. A heparinized whole-blood sample was obtained by cardiac puncture. Each 50-(mu)L whole-blood sample was used for counting the number of leukocytes by hemocytometer (Celltac, MEK-6258; Nihon Kouden, Tokyo, Japan). Whole-blood samples were centrifuged at 400g for 5 minutes to remove plasma and then stored in 1-mL quantities with RPMI-1640 (Nikken Biomedical Laboratory, Kyoto, Japan) supplemented with 1% fetal calf serum (FCS) at 4C until sample measurement. Peritoneal exudative cells (PECs) were harvested by lavaging the peritoneal cavity with 5 mL of phosphate-buffered saline (PBS; Nikken Biomedical Laboratory) without Ca^sup 2+^ or Mg^sup 2+^. Before harvesting the peritoneal lavage fluid, the abdominal wall was gently rubbed three times. Contaminating erythrocytes were lysed with distilled water. PECs were then resuspended in RPMI-1640 supplemented with 1% FCS. Live PECs were counted by trypan blue exclusion and adjusted to 1 X 10^sup 6^/mL.

MEASUREMENT OF ADHESION MOLECULE EXPRESSION ON PHAGOCYTES

The monoclonal antibodies (mAb) used were R-phycoerythrin (R-PE)-conjugated rat antimouse CD11b (integrin alpha^sub M^beta^sub 2^ chain), fluorescein isothiocyanate (FITC)-conjugated rat antimouse CD18 (integrin Beta^sub 2^ chain), and CD62L mAbs (r.-selectin) (Pharmingen, San Diego, CA). One hundred microliters of the PEC suspension and washed whole-blood samples were incubated with saturating amounts of anti-CDllb, anti-CD18, anti-CD62L mAbs, or the related isotype antibodies for 30 minutes at 4degC. At the end of incubation, the cells were washed twice with cold PBS followed by fixation with 0.5 mL of 1% paraformaldehyde.

FLOW CYTOMETRY

The flow cytometric analysis was performed using FACScan (Becton Dickinson Immunocytometry Systems, San Jose, CA). In each PEC sample, 10,000 leukocytes, and in whole blood samples, 20,000 leukocytes were counted. PMNs and monocytes/macrophages were gated using morphologic characteristics displayed on a dot plot of forward light scatter Us side scatter. The results of adhesion molecule expression on PMNs and macrophages are expressed as mean channel fluorescence intensity.

LIGHT MICROSCOPY

Cytocentrifuged cells were fixed in methanol. Then, using the Wright-Giemsa staining technique (Muto Pure Chemicals, Tokyo, Japan), the percentages of PMNs, macrophages, and lymphocytes in both circulating leukocytes and PECs were determined by light microscopy at 40OX magnification. Sample identity was blinded before counting. Two hundred cells per slide were examined.

STATISTICAL ANALYSIS

Data are presented as means +/- SE. To achieve a normal distribution, levels of PEC, exudated PMN, and macrophage numbers were transformed, if necessary, to a logarithmic scale before statistical analysis. Statistical analyses were done using the unpaired t test. Linear regression analysis was also performed. Differences were defined as statistically significant when the p value was

RESULTS

Body Weight Change

Percent body weight (BW at sacrifice/predietary restriction) was significantly lower in the diet-restricted than in the ud libitum group (73 +/- 2 and 107% +/-1%, respectively, p

Changes in Total and Differential Counts of Circulating and Exudative Cells

Dietary restriction did not affect the numbers of circulating leukocytes, PMNs, monocytes, or lymphocytes (Table I). However, after 4 hours of intraperitoneal glycogen injection, the total PEC number was significantly lower in the diet-restricted than in the ad libitum group (Fig. 1). Moreover, the numbers of exudative PMNs and macrophages were significantly lower in the diet-restricted than in the ad libitum group.

Adhesion Molecule Expression on Circulating and Exudative Phagocytes

CD11b and CD18 expressions on circulating PMNs were both significantly lower in the diet-restricted group than in the ad libitum group (Table II). In contrast, CD62L expression on circulating PMNs was not affected by dietary restriction. CD11b and CD18 expressions on peritoneal exudative PMNs were higher than those on circulating PMNs in both groups. On the contrary, CD62L expression was lower on peritoneal exudative PMNs than on circulating PMNs in both groups. Expressions of CDllb, CD18, and CD62L on exudative PMNs were unaffected by dietary restriction. There was a significant positive correlation between exudative PMN numbers and CD18 expression on circulating PMNs (r = .73, p

CD18 expression on circulating monocytes was also significantly lower in the diet-restricted than in the ad libitum group (Table II). CDllb and CD62L expressions on circulating monocytes were lower in the dietrestricted than in the ad libitum group, but the difference was not significant. There was a significant positive correlation between the number of exudative macrophages and CD18 expression on circulating monocytes (r = .68, p

DISCUSSION

Exudative leukocytes, particularly PMNs, serve to eliminate pathogens in the peritoneal cavity during peritonitis. Expression of adhesion molecules, such as CDllb, CD18, and CD62L on leukocytes regulates leukocyte-endothelial interaction, resulting in leukocyte exudation.6 In patients with leukocyte adhesion deficiency-1 syndrome, which is characterized by a deficiency of Beta2 integrin molecules, there is impaired migration of PMNs, resulting in recurrent bacterial infection.9 Clinically, patients with malnutrition are more susceptible to infection than well-nourished patients.1,2 Therefore, we hypothesized that malnutrition may cause deleterious effects on PMN adhesion molecule expression and subsequent migration into the peritoneal cavity in a peritonitis model. No studies to date have focused on adhesion molecule expression on circulating and exudative PMNs simultaneously.

The present study indicated that circulating PMNs from the diet-restricted group had lower CD11b/CD18 expression, although only six mice per group were studied in this experiment leaving the potential for type I statistical errors. However, the SEMs for this experiment are relatively tight. CD62L expression on circulating PMNs did not differ significantly between the diet-restricted and ad libitum groups. Exudative PMN number in the diet-restricted group was decreased in association with decreased in CD11b/CD18 expression. The results suggest that malnutrition impairs host defense during peritonitis, at least in part, by decreasing exudative PMN numbers that result from a reduction of CD11b/CD18 expression on circulating PMNs. Moreover, endothelial transmigration regulates neutrophil function through cross-linking of adhesion molecules.10,11CD18 reportedly plays an important role in adhesion-dependent priming of neutrophil functions.11,12 Thus, it is possible that functions of exudative PMNs may be different between the ad libitum and dietrestricted groups. Further study will be needed to clarify the functional alteration of exudative PMNs by diet restriction.

CD11b/CD18, an adhesion-promoting (32 integrin, is constitutively expressed on the plasma membranes of PMNs.9 On activation, additional CD11b/CD18 molecules are rapidly mobilized from intracellular specific granules to the cell surface membrane.13 Although we did not investigate the mechanisms underlying decreased adhesion molecule expression on circulating PMNs in this study, it is possible that malnutrition depressed the synthesis or mobilization of adhesion molecules on circulating PMNs.

This type of malnutrition may decrease circulating insulin-like growth factor-1 (IGF-1) levels,14 elevate circulating free cortisol,15 and blunt the activation of complement systems and proinflammatory cytokine production in response to an inflammatory stimulus.16,17 previous work demonstrated IGF-1 treatment increases beta2 integrin expression on PMNs.18 Cortisol inhibits CD18 expression on leukocytes.19 Activated complement components and tumor necrosis factor (TNF)-alpha stimulate beta2 integrin expression on PMNs and enhance PMN adhesion.20-22

CD62L expressed on the leukocyte surface is also an adhesion molecule regulating leukocyte trafficking in the systemic microcirculation.7 The diet restriction applied herein did not change CD62L expression on circulating PMNs in response to intraperitoneal glycogen installation, regardless of decreased PMN exudation. This observation suggests that CD62L expression on circulating PMNs does not impair host immunity in this malnutrition model. Decreased CD62L expression on exudative PMNs, compared with circulating PMNs, in both the diet-restricted and the ad libitum group may be caused by shedding through transmigration.6

We studied the influence of 1 week of dietary restriction on leukocyte adhesion molecule expression and exudative leukocyte numbers at 4 hours after glycogen installation. Intraperitoneal inoculation of a glycogen solution has been demonstrated to be an adequate model for investigating inflammatory responses.2 One week of dietary restriction in mice reportedly leads to impaired host immunity.24,25 peritoneal macrophages from mice subjected to one week of protein-caloriemalnourishment produced less interleukin-6 or TNF-a in response to lipopolysaccharide stimulation than macrophages from control mice.24 Mice fed a proteinfree diet for 7 days showed significantly reduced survival compared with control mice with a 24% casein diet after Candida albicans challenge.25 With regard to the timing of sacrifice, our preliminary experiments revealed maximum PMN influx into the peritoneal cavity to occur at 4 hours after glycogen injection.

In addition to its effects on PMNs, dietary restriction also decreased circulating monocyte CD18 expression and the number of peritoneal macrophages. Malnutrition appears to affect monocyte function and macrophage influx into the peritoneal cavity.24 However, maximum macrophage influx into the peritoneal cavity reportedly occurs at 12 hours after glycogen administration.23 Further studies focusing on the late phase of inflammation are required to examine the influence of malnutrition on the monocyte/macrophage axis.

In summary, ours is a preliminary study that for the first time has demonstrated that 1 week of dietary restriction reduces the numbers of exudative PMNs at the inflammatory site with decreased expression of beta2-integrin on circulating PMNs. Many questions remain to be answered, including the mechanisms underlying changes in adhesion molecule expression, poststimulation kinetics, and the effects of different durations and degrees of dietary restriction. Functional effects of malnutrition on leukocytes must be clarified in future studies. Nevertheless, our findings imply that nutritional replenishment may improve host defense, at least in part, via enhancing PMN adhesion molecule expression and subsequent PMN exudation into inflammatory sites.

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Shigeo Ikeda, MD*; Hideaki Saito, MDt; Tomomi moue, MD*; Kazuhiko Fukatsu, MD*; Ilsoo Han, MD*; Satoshi Furukawa, MD*; Takeaki Matsuda, MD*; and Akio Hidemura, MD*

From the *Department of Sugery, Faculty of Medicine, ^Surgical Center, The University of Tokyo, Japan

Received for publication, October 4, 1999. Accepted for publication, April 6, 2000.

Correspondence and reprint requests: Shigeo Ikeda, MD, Department of Surgery, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. Electronic mail may be sent to ikeda-lsu@h.u.-tokyo.ac.jp.

*Presented in part and rewarded as an ESPEN travel fellowship at ESPEN 99 Congress, on September 5, 1999, Stockholm, Sweden.

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