Peritonitis

Diet Restriction Impairs Extracellular Signal-Regulated Kinase Activation of Peritoneal Exudative Cells After N-Formyl-MethionylLeucyl-Phenylalanine Stimulation in a Murine Peritonitis Model*

ABSTRACT. Background: Phosphorylation of extracellular signal-regulated kinase (ERK) enhances various inflammatory responses in immune cells. It is unknown whether dysfunction of immune cells during malnutrition is attributed to derangement of ERK activation. Methods: Male Institute of Cancer Research (ICR) mice received chow (146 g/kg per day, ad libitum or 36.5 g/kg per day, diet-restricted) for 7 days. Mice (n = 55) were given 6.5 mg/kg of an ERK inhibitor (PD98059) or vehicle intraperitoneally (IP), at 2 hours before cecal ligation and puncture (CLP). Survival was observed up to 60 hours. Detection of phosphorylated ERK (pERK) in the peritoneal exudative cells (PECs) was done as follows. In a separate study, PECs were harvested by peritoneal lavage 2 hours after an IP injection of 1% glycogen. PECs were incubated with or without 100 nmol/L N-formyl-methionyl-leucyl- phenylalanine (fMLP) for 1 minute. PEC ERK activation was detected with Western blot analysis (n = 38), by densitometric quantification, and with a laser scanning cytometer (LSC; n = 13). Subpopulations of PECs were determined by WrightGiemsa staining. Unstimulated pERK expression was nor

malized to 100% for Western blot analysis. Results: Diet restriction reduced survival after CLP compared with the ad libitum mice. ERK inhibition showed no effect on survival in diet-restricted mice but reduced survival in ad libitum mice. There were no differences in subpopulations of PECs 2 hours after glycogen injection between the groups. Western blot analysis revealed that fMLP stimulation significantly increased the phosphorylation of ERK1/2 in PECs from the ad libitum group (ERK1, 199 +/- 41%; ERK2, 211 +/- 49%; p

Malnutrition induces dysfunction of immune cells such as macrophages, polymorphonuclear neutrophils (PMNs), and lymphocytes, resulting in impaired host defense.1,2 We have demonstrated that severe diet restriction reduces the phagocyte functions in a glycogen-induced peritonitis model.1 Dietary restriction impairs PMN exudation into local inflammatory sites in murine peritonitis by reducing cytokine and chemokine production. Phagocytosis by the exudative PMNs is also decreased by severe diet restriction. In addition, apoptosis in peritoneal resident macrophages is reportedly accelerated in mice with protein-calorie malnutrition.3

Intracellular transduction is responsible for cytokine production,4 migration,5 phagocytosis,6 and antiapoptosis of phagocytes.7 There are a variety of intracellular signaling pathways in the phagocytes. The pathway from tyrosine kinase activation to mitogenactivated protein kinase (MAPK) activation is regarded as a principal route to the inflammatory responses of PMNs (Fig. 1). Phosphorylation of protein tyrosine kinase in phagocytes does not increase after inflammatory stimulation during diet restriction. However, the effects of diet restriction on MAPK activation in phagocytes are not known. MAPKs include extracellular signal-regulated kinase (ERK) 1/2, p38MAPK, and c-Jun N-terminal kinase (JNK). ERK and p38MAPK play a central role in mediating inflammatory responses.9

The aim of this study was to examine the effect of ERK inhibition on survival of ad libitum and dietrestricted mice in a model of polymicrobial sepsis, induced by cecal ligation and puncture (CLP). Furthermore, we investigated whether diet restriction would influence ERK activation of peritoneal exudative cells (PECs) after stimulation with a bacterial tripeptide, N-formyl-methionyl-leucyl-phenylalanine (fMLP).

METHODS

Animal Model

Specific pathogen-free 5-week-old male Institute of Cancer Research (ICR) 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 (24 deg C) 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 (23.8, 5.1, 54.0, 3.2, 3.8, and 0.7 g per 100 g diet, respectively). All studies were performed in accordance with the Guide for Animal Experimentation, Faculty of Medicine, The University of Tokyo. Our institutional review board approved the protocol.

Experiment 1: Effects of ERK Inhibition on Survival in Peritonitis

Mice (n = 55) were randomly assigned to 2 groups. The ad libitum and diet-restricted groups received mouse chow ad libitum and 36.5 g/kg (133 kcal/kg) per day for 7 days, respectively. Our preliminary experiment revealed that the average chow consumption by mice with free access to chow was 146 g/kg (532 kcal/kg) per day.

After 7 days of dietary restriction, all mice underwent CLP. In brief, mice were anesthetized with pentobarbital sodium (100 mg/kg) and a 1-cm midline incision was made. The cecum was exposed, ligated proximal to the ileocecal valve, punctured once with an 18-gauge needle, and returned to the abdominal cavity. The abdominal cavity was closed in layers and the animals were resuscitated with 30 mL/kg of saline solution.

Two hours before CLP, one-half of the mice in each group were given 6.5 mg/kg of an ERK inhibitor, PD98059 (Calbiochem, La Jolla, CA), intraperitoneally (IP), and the others received an equal volume of the vehicle dimethyl sulfoxide/saline. Survival was observed up to 60 hours after CLP.

Experiment 2: Measurement of Phosphorylated ERKs (pERKs) of PECs

As in experiment 1, mice (n = 51) were randomly assigned to the ad libitum or diet-restricted, ie, 75% restricted food intake, group for 7 days.

Cell Preparations

After 7 days of dietary restriction, all the mice were administered 2 mL of a 1% glycogen solution (Sigma, St. Louis, MO) IP. Two hours after glycogen injection, the animals were anesthetized with pentobarbital sodium, and the peritoneal cavity was lavaged with 5 mL of cold Hanks' balanced salt solution (HBSS; without Ca^sup 2+^; Sigma), containing 20 U/mL of heparin sodium. Cell subpopulations of peritoneal exudates may depend on the severity and time course of inflammation and infection in the peritoneal cavity.10,11 Our previous study demonstrated that the exudative neutrophil number peak in ad libitum mice was found at 2 hours after glycogen injection.1

Cell Subpopulation Analysis

After centrifugation at 1600 rpm for 10 minutes, the PECs were washed and resuspended in phosphate buffered saline (PBS; without Ca^sup 2+^) containing 0.1% bovine serum albumin (BSA). Cytocentrifuged PECs were fixed in methanol. The differential cell counts were performed with the Wright-Giemsa staining technique (n = 20).

Western Blot Analysis for pERKs

The PECs harvested by peritoneal lavage were resuspended in ice-cold HBSS containing 10 mmol/L HEPES (Sigma), pH 7.4, and 4 mmol/L NaHCO^sub 3^ (Sigma) and kept in an ice bath before use. The PECs were preincubated at 37 deg C for 30 minutes and stimulated with 100 nmol/L fMLP plus 5 (mu)g/mL dihydrocytochalasin B (CB; Sigma), or vehicle, for 1 minute at 37 deg C in a water bath. After washing with ice-cold HBSS containing 10 mmol/L HEPES and 4 mmol/L NaHCO^sub 3^, the PECs were resuspended in a stopping solution containing 20% trichloroacetic acid (Sigma), 1 mmol/L phenylmethyl sulfonyl fluoride (PMSF) (Sigma), 2 mmol/L N-ethylmaleimide (Sigma), 10 mmol/L NaF (Sigma), 2 mmol/L Na^sub 3^VO^sub 4^ (Sigma), 2 mmol/L p-nitrophenyl phosphate (Sigma), 7 (mu)g/mL of leupeptin (Sigma), and 7 (mu)g/mL of pepstatin (Sigma). The PECs were sonicated and centrifuged at 12,000 rpm in an Eppendorf centrifuge for 10 minutes at 4 deg C. The supernatants were collected, electrophoresed on 10% sodium dodecylsulfate (SDS)-polyacrylamide gel, and transferred onto a nitrocellulose membrane. The blot membrane was blocked with Tris-buffered saline solution (TBS) containing 10 mmol/L Tris-HCl (Sigma), pH 7.6, and 150 mmol/L NaCl (Sigma) with 0.1% Tween (Bio-Rad, Hercules, CA) for 5 minutes, and with 5% nonfat dry milk in TBS-0.1% Tween for i hour at room temperature. Subsequently, the membrane was incubated with rabbit polyclonal antiphospho-p44/42 MAPK antibodies (1:1000; Cell Signaling, Beverly, MA) followed by horseradish peroxidase-conjugated antirabbit IgG (1:1000) (Cell Signaling) in 2.5% nonfat dry milk in TBS-0.1% Tween. Protein bands were visualized using the ECL detection system (Amersham Pharmacia Biotech, Buckinghamshire, England). To quantify the differences present in various samples, the band intensities were assessed by densitometry using the public domain National Institutes of Health (NIH) Image program (version 1.61). pERK expression without fMLP stimulation was normalized to 100%. Results are shown as a percentage of fMLP-stimulated to unstimulated pERK expression.

Laser Scanning Cytometric Analysis for pERKs

The PECs harvested by peritoneal lavage were washed with ice-cold PBS (without calcium; Sigma) containing 4% fetal calf serum (FCS) and resuspended in RPMI (Roswell Park Memorial Institute) 1640 containing 0.1% FCS. The PECs were preincubated at 37 deg C for 30 minutes before stimulation with 100 nmol/L fMLP plus 5 (mu)g/mL CB or without stimulation. One minute later, the PECs were washed with ice-cold PBS containing 4% FCS and fixed using 2% formaldehyde at 37 deg C for 10 minutes. PECs were then washed and resuspended in PBS containing 4% FCS at 1 x 10^sup 6^ cells/mL. Then, the PECs were cytocentrifuged (500 rpm for 5 minutes) onto microscope slides and permeabilized in 100% methanol for 3 minutes. The PECs were washed in PBS containing 0.1% BSA and incubated in blocking buffer (20% normal goat serum in PBS with 0.1% BSA) for 45 minutes at room temperature. For detection of activated ERKs, cells were incubated with antiphospho-p44/42 MAPK antibodies (1:100; Cell Signaling) in PBS with 0.1% BSA for 50 minutes at room temperature. After washing in PBS with 0.1% BSA, cells were incubated with an Alexa 488-conjugated secondary antibody (goat anti rabbit IgG; Molecular Probes, Eugene, OR) diluted 1:200 in PBS with 0.1% BSA. The cells were washed and photographed using a cooled charge-coupled device (CCD) digital camera (Sensys 1400; Olympus, Tokyo, Japan) adapted to the laser scanning cytometry (LSC) microscope. The samples were incubated with 50 (mu)g/mL propidium iodide (PI; Sigma) solution and 200 (mu)g/mL ribonuclease A (Sigma) for 10 minutes. Slides were examined using LSC (LSC101; Olympus) with WinCyte 3.3 data analysis software (CompuCyte, Cambrige, MA). The slides were scanned under a 20X objective lens using the instrument's 488-nm wavelength argon ion laser with red and green fluorescence detectors. The primary contouring parameter, used to detect and quantify cells, was set on red fluorescence of PI-- stained nuclei. In each PECs sample, 1000 leukocytes were counted. The results of phosphorylation of ERKs in the PECs are expressed as a mean fluorescence intensity (MFI).

Statistical Analysis R esults are presented as means +/- SE. Analysis of variance, followed by the Fisher protected least-signif icant difference post hoc test, was used for body weight comparisons. The Log rank test was used for survival time comparisons. The Wilcoxon signed rank test was used in the Western blot study. The paired t test was used in the LSC study. Differences were defined as statistically significant when p

RESULTS

Body Weight Change

Mean initial body weights of each group were similar. Mean body weight change after 1 week of pairfeeding was significantly different between the ad libiturn (+4.2 +/- 0.2 g) and diet-restricted group (-10.3 +/- 0.6 g; p

Survival Study

Diet restriction significantly reduced survival time after CLP, compared with the ad libitum group (Fig. 2). PD98059 treatment did not affect the survival of dietrestricted mice, but significantly decreased the survival of ad libitum mice.

Subpopulation of PECs

There were no differences in subpopulations of PECs at 2 hours after glycogen injection: PMNs, 70 +/- 2% versus 68 +/- 4%; macrophages, 17 +/- 2% versus 19 +/-4%; and lymphocytes, 12 +/- 2% versus 13 +/-4% in the ad libitum and diet-restricted mice, respectively.

Measurement of pERK

Western blot analysis. fMLP stimulation dramatically increased the phosphorylation of ERK1/2 in PECs harvested from the ad libitum mice, whereas the induction was abrogated in those from diet-restricted mice (Fig. 3). When unstimulated pERK expression was normalized to 100%, the percentage of fMLP-stimulated to unstimulated pERK expression was significantly increased in the ad libitum mice (ERK1, 199 +/- 41%; ERK2, 211 +/- 49%). In contrast, the percentage did not change significantly in the diet-restricted mice (ERK1, 98 +/- 10%; ERK2, 91 +/- 9%).

Laser scanning cytometric analysis. Figure 4 shows the representative LSC images of PECs with Alexa 488-- conjugated antibody. fMLP-stimulation remarkably increased green fluorescence (phosphorylation of ERKs) of the cells from an ad libitum mouse (Fig. 4, A versus B). In contrast, fMLP stimulation had no signif icant effect on fluorescence intensity in the cells from a diet-restricted animal (Fig. 4, C versus D).

fMLP unstimulated MFI was similar between the ad libitum and the diet-restricted groups (Fig. 5). fMLP stimulation significantly elevated MFI in pERK of PECs harvested from the ad libitum mice (from 19.4 +/1.5 MFI to 22.4 +/-1.2 MFI; p

DISCUSSION

The present study demonstrated that ERK inhibition decreased the survival of ad libitum but not diet-- restricted mice in the CLP model. fMLP stimulation up-regulated ERK activation of PECs from the ad libitum mice. In contrast, diet restriction abrogated ERK activation of PECs after fMLP stimulation in the glycogen-induced peritonitis model.

MAP kinases play important roles in transcytoplasmic signaling to the nucleus by activating gene expression.12 Three subfamilies of MAP kinases, ie, the ERK, the p38 kinase, and the JNK, have been identified in mammalian cells.13 In this study, the main subpopulation of PECs in both the ad libitum and diet-restricted groups was PMNs. Among the three MAP kinases, ERK has crucial signaling functions in PMNs, including migration,5 degranulation,14 cytokine and chemokine production,4 phagocytosis,6 respiratory burst activity,15 and antiapoptosis.7 In the present study, inhibition of ERK by PD98059 decreased survival time after CLP in the ad libitum group. This result suggests that activation of ERK is essential for host survival during bacterial contamination.

Severe diet restriction worsened survival compared with normally fed animals, as expected. Interestingly, ERK inhibition did not show any significant effects on survival in diet-restricted mice. To elucidate the underlying mechanism for the unchanged survival of diet-- restricted animals with an ERK inhibitor, we evaluated the degree of ERK activation in harvested PECs after IP glycogen injection using Western blotting and a sophisticated tool, laser scanning cytometry, with or without fMLP stimulation. Both methods revealed that ERK was activated in PECs from ad libitum mice after fMLP stimulation, whereas those from diet-restricted animals did not show any increase in ERK phosphorylation. Thus, this type of malnutrition may impair host defense, at least partly, by decreasing activation of ERK signaling in PECs.

There are some possible mechanisms for the blunted ERK activation of PECs by diet restriction. One possible mechanism is that unresponsiveness of upstream tyrosine kinase signaling reduces ERK activity in the diet-restricted group. We previously found that phosphorylation of tyrosine kinase was blunted after fMLP stimulation in PECs from diet-restricted mice.8 It is also possible that the reduced amount of total ERK proteins in PECs is associated with decreased phosphorylated ERK in diet-restricted mice. We did not measure total amount of ERK proteins in our study. However, Liu et al 16 reported that 40% dietary energy restriction was accompanied by reduced levels of total ERK proteins in the epidermis of mice. Moreover, ERK activity is known to be down-regulated by tyrosine phosphatases and serine-threonine-tyrosine phosphatases.17 Severe diet restriction may increase activity of these phosphatases.

p38MAPK, another MAP kinase subfamily, also enhances the inflammatory response through an increase in proinflammatory cytokine production,as phagocytosis,19 adhesion,18 and elastase release20 from PMNs. This study did not clarify the effects of diet restriction on p38MAPK activation. Although delayed treatment with an IP p38 MAPK inhibitor, SB203580, reportedly improves survival in a CLP mouse model,4 our preliminary study demonstrated that pretreatment with the p38 MAPK inhibitor did not exert any significant effect on survival in our CLP mouse model. This result suggests that p38MAPK does not play an important role for host defense at the early proinflammatory phase in our CLP model. Further studies are warranted to elucidate the effects of diet restriction on p38MAPK activity of PECs.

In this study, it is unclear which component of nutrition mainly affects ERK phosphorylation after stimulation. Because the diet-restricted mice received only 25% of the food intake compared with the ad libitum mice, the animals were likely to be short of almost all nutritional components. Caloric restriction has been reported to inhibit ERK activity in the epidermis of mice.16 In addition, refeeding for only 1 day recovered host defense responses in our previous study.1 Taken together, it is suggested that energy malnutrition is an important factor for reduced ERK activity. However, the supplementation of protein and micronutrients and energy may be important to restore host defense responses on the cellular level. To isolate these shortterm benefits solely on the basis of energy, further studies are needed to clarify whether carbohydrateonly supplementation could restore the host defense responses.

Malnutrition induces immunosuppression and predisposes the patient to septic complications. Detailed information concerning the molecular mechanisms involved in regulation of host defense by malnutrition may help to develop new strategies for nutrition support. We believe that the present data provide a basis for future work for the development of new strategies aimed at enhancing host defense in malnourished patients.

In summary, ERK activation is essential for survival during a polymicrobial sepsis. Unresponsiveness of ERK and blunted tyrosine kinase activation in PECs after stimulation may be an important mechanism for impaired host defense caused by malnutrition.

*This paper was presented at the Premier Scientific Paper session of the 26th A.S.P.E.N. Clinical Congress, February 23-27, 2002, San Diego, CA.

Received for publication, February 13, 2002.

Accepted for publication, May 29, 2002.

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Woodae Kang, MD*; Hideaki Saito, MD*; Kazuhiko Fukatsu, MD^; Akio Hidemura, MD^; and Takeaki Matsuda, MD^

From the *Surgical Center and ^Department of Surgery, The University of Tokyo, Japan

Correspondence and reprint requests: Hideaki Saito, MD, Surgical Center, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. Electronic mail may be sent to saito-ope@h.utokyo.ac.jp.

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