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Th2 cytokine balance in mice subjected to operative trauma
Effects of geldanamycin and thalidomide on the Th1/Th2 cytokine balance in mice subjected to operative trauma Takumi Nakano, MD, Keijiro Araki, MD, PhD, Hajime Nakatani, MD, PhD, Michiya Kobayashi, MD, PhD, Takeki Sugimoto, MD, PhD, Yasuo Furuya, MD, PhD, Takanori Matsuoka, MD, PhD, Toufeng Jin, MD, PhD, and Kazuhiro Hanazaki, MD, PhD Nankoku, Japan
Background. Persistence of postoperative immune dysfunction is a critical problem because it increases the risk of serious infectious complications. The mechanisms of the immune dysfunction that occur initially after non-thermal operative injury remain to be fully elucidated. Methods. Two mouse models of operative trauma (simple laparotomy to represent minor operative injury and ileocecal resection to represent major operative injury) were used to define the characteristics of initial cytokine synthesis. Geldanamycin and thalidomide were independently added intraperitoneally before and after operative injury to examine the effect on postoperative immune dysfunction. Mice were sacrificed at scheduled times (3, 6, 12, and 24 h after operative injury) and TNF-␣, IL-2, IL-4, and IL-10 were analyzed. Spleen was used for intracellular cytokines and RT-PCR. Sera were used for ELISA. Results. Major operative injury caused an initial upregulation of IL-10 synthesis with delayed synthesis of TNF-␣ and IL-2. Minor operative injury caused an early induction of IL-2 synthesis preceded by an initial induction of IL-4 synthesis. GA caused a specific early upregulation of TNF-␣ mRNA expression and intracellular TNF-␣ synthesis. The GA and THD groups showed early serum IL-2 production with reduction of IL-10 mRNA expression and intracellular IL-10 synthesis in the early post-operative phase. Conclusions. Major and minor operative injury showed different Th1/Th2 cytokine patterns in the initial post-operative period. Geldanamycin and thalidomide improved the Th1/Th2 imbalance independently after major operative injury. (Surgery 2007;141:490-500.) From the Department of Tumor Surgery, Kochi Medical School, Kochi University, Nankoku, Japan
Studies discussing the immunologic phenomena after operative trauma, other than burns,1-6 sepsis,7-8 and external trauma9-13 are uncommon. We have studied 2 different murine models of nonthermal operative trauma: the first is a simple laparotomy (SL) model to represent minor operative trauma injury and the second is an ileocecal resection (ICR) model to represent major operative trauma. Four basic cytokines, TNF-␣ and IL-2 as Th1 (Type-1 T helper cell) cytokines, and IL-4 and IL-10 as Th2 (Type-2 T helper cell) cytokines, were Accepted for publication October 14, 2006. Reprint requests: Takumi Nakano, MD, Department of Surgery, Kochi Medical School, Kochi University, Okho, Nankoku, Japan. E-mail: [email protected]. 0039-6060/$ - see front matter © 2007 Mosby, Inc. All rights reserved. doi:10.1016/j.surg.2006.10.003
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evaluated. The predominance of Th2 cytokines3,10 (IL-10, IL-4) or suppression of IL-21,5,13 after serious injury has been shown to cause decreased resistance to infection.1,3-6,11,12 This observation suggests that decreasing the inhibition of IL-2 or attenuating the Th2 cytokines is desirable. Heat shock protein 90 (Hsp90) participates in signal transduction14 and is critical in the intracellular signaling pathways that promote inflammatory cytokine production.15 Geldanamycin (GA) is a benzoquinone ansamycin that inhibits the function of Hsp90.14,15 Bucci et al16 have shown that geldanamycin acts as an anti-inflammatory drug in vivo inhibiting carrageenan-induced mouse pawedema. As an antagonist of the glucocorticoid receptor (GR), RU486 has been used to study the receptor activation mechanism17 that prevents the translocation of GR to the nucleus by inhibiting the dissociation of GR from Hsp90.18
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Thalidomide (THD) was synthesized originally in 1954 as a sedative, but soon disappeared due to the high risk of teratogenicity,19,20 however, use of thalidomide has reappeared in as an effective antiinflammatory, immunomodulatory drug19,20 and as a costimulator for IL-2-production.21,22 We have investigated whether geldanamycin or thalidomide could act as an immunomodulator to relieve the initial detrimental cytokine responses that occur postoperatively. MATERIALS AND METHODS Mice. Male C57BL/6 mice 8 to 10 weeks old were purchased from Japan SLC (Shizuoka, Japan) and were maintained under pathogen-free conditions. All mice were fed a liquid food (Calorie Mate; Otsuka Pharmaceutical, Tokyo, Japan) for 1 week before operation and until they were killed. All experiments were approved by the Animal Experimental Committee of Kochi Medical School, Kochi University. Drugs. Thalidomide (⫹) (THD) was obtained from Sigma-Aldrich (St. Louis, MO). Geldanamycin (GA) and RU486 (Mifepristone; LKT Laboratories, St. Paul, MN) were purchased from Wako Pure Chemical Industries (Osaka, Japan). These drugs were dissolved in dimethyl sulfoxide (DMSO; Wako Pure Chemical) at concentrations of 10 mg/ dl, 10 mg/dl, and 2 mg/dl, respectively. Operative procedures. Under Ketamine (80 mg/kg; Veterinary Ketalar 50; Sankyo Yell, Tokyo, Japan) and Xylazine (30 mg/kg; Celactar; Bayer HealthCare, Leverkusen, Germany) anesthesia, minor (simple laparotomy [SL]) or major (ileocecal resection [ICR]) operative trauma was carried out aseptically. Simple laparotomy was accomplished via a 1 cm abdominal incision. The appendix was exteriorized for 5 minutes with occasional moistening with saline-soaked gauze. ICR was also accomplished via a 1 cm incision, and ileocecal resection, which involved a resection spanning 1 cm proximal and 3 mm distal to the appendical origin, was added. Anastomosis was carried out by interrupted suture with 8-0 polyglactin sutures. Abdominal incisions were closed with 6-0 polyglactin suture material. Mean operation times were 13 minutes for SL and 27 minutes for ICR. Experimental design. Two hundred and eighteen mice were divided randomly into the following 4 main groups: (1) SL group, SL ⫹ DMSO intraperitoneally (i.p.); (2) ICR group, ICR ⫹ DMSO i.p.; (3) GA group, ICR ⫹ GA i.p.; 4) THD group, ICR ⫹ THD i.p.; and 4 control groups: (5) AGA group, Anesthesia ⫹ GA i.p.; (6) ATHD group, Anesthesia ⫹ THD i.p.; (7) SGA group, SL⫹
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GA i.p.; (8) STHD group, SL⫹ THD i.p.; and an additional group (9) ICR ⫹ RU486 i.p. (for RTPCR analysis only). In each group, the drug was administered twice intraperitoneally: on the preoperative day and at the time of the peritoneal closure. The administered doses of the drugs were as follows: GA, 1 mg/kg; THD, 1 mg/kg; and RU486, 5 mg/kg. The doses of geldanamycin and RU486 were determined by reference to Bucci et al.16 and the dose of thalidomide was determined by reference to published doses for human use.18 As a control for the SL and ICR groups, DMSO solution, 200 l per mouse, was administered i.p. Mice were killed 3, 6, 12, and 24 h after operative trauma by cervical dislocation under Ketamine and Xylazine anesthesia. Analysis of intracellular cytokines (TNF-␣, IL-2, IL-4, and IL-10). A sample of spleen was removed from each of the mice at each scheduled time point. Splenocytes were suspended at 1 to 2 ⫻ 106/ml in complete RPMI-1640 medium (Invitrogen, Carlsbad, CA) with 2 mmol/L l-glutamine, 50 mmol/L 2-mercaptoethanol, 100 U/ml penicillin, 100 ug/ml streptomycin, 0.25 ug/ml amphotericin B, and 5% heat inactivated fetal calf serum (Invitrogen). The cells were incubated in 6-well plates with a protein transport inhibitor, brefeldin A (1 ul/ml, Golgi Plug; BD Pharmingen, San Diego, CA), Ionomycin (1.6 ug/ml; Sigma-Aldrich, St. Louis, MO) and PMA (0.3 ug/ml, Sigma-Aldrich), for 5 h at 37°C with 5% CO2. After incubation, cells were fixed and permeabilized with Cytofix/Cytoperm (BD Pharmingen) for 20 min, then washed twice with saponin-containing buffer (Perm/Wash, BD Pharmingen). Intracellular cytokines were stained with the following mouse monoclonal antibodies for 30 min: FITC-rat anti-mouse TNF-␣ (BD Pharmingen); FITC-conjugated anti-mouse IL-4; PE-conjugated anti-mouse IL-2; and anti-mouse IL-10 (eBioscience, San Diego, CA). Rat IgG isotype controls were obtained from eBioscience. In flow cytometric analysis, gating for lymphocytes was set for IL-2, IL-4, and TNF-␣ measurements, and the gating for targeting lymphocytes and macrophages was set for IL-10 measurement using forward versus side scatter properties. Analysis was carried out using a FACSCalibur Flow Cytometer (Becton Dickinson) with CellQuest software. Reverse-transcriptase polymerase chain reaction (RT-PCR) for cytokine mRNA expression. Total RNA from spleen was isolated using TRIzol Reagent (Invitrogen), and cDNA was prepared using the RNA PCR Kit (AMV) Ver.2.1 (TaKaRa Biomedicals, Tokyo, Japan). The cDNA was then used in reverse transcription (RT)-PCR with Platinum Taq
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Fig 1. A-1, A-2, Time course of RT-PCR for TNF-␣ mRNA expression in spleen. Results are expressed as means of 2 separate experiments. A-1, The GA and SL groups showed early upregulation of TNF-␣ mRNA expression. A-2, The RU486 group showed strong upregulation of TNF-␣ mRNA expression with no reduction from 3 to 24 hours. B, Serum TNF-␣ production after operative trauma. Results are expressed as mean ⫾ SEM of 3 independent experiments. C, Serum TNF-␣ production in control groups. Results are expressed as mean ⫾ SEM of 2 independent experiments. SL, simple laparotomy ⫹ DMSO i.p.; ICR, ileocecal resection ⫹ DMSO i.p.; GA, ileocecal resection ⫹ geldanamycin i.p.; THD, ileocecal resection ⫹ thalidomide i.p.; RU486, ileocecal resection ⫹ RU486 i.p.; SGA, simple laparotomy ⫹ geldanamycin i.p; STHD, simple laparotomy ⫹ thalidomide i.p.; AGA, anesthesia ⫹ geldanamycin i.p; ATHD, anesthesia ⫹ thalidomide i.p.
DNA polymerase (Invitrogen). The primer sequences were as follows: TNF-␣U-primer, 5=-CCACATCTCCCTCCAGAAAA-3= and TNF-␣L-primer, 5=-TCCCCTTCATCTTCCTCCTT-3=; IL-2U-primer, 5=-GAGCAGCTGTTGATGGACCT-3= and IL-2Lprimer, 5=-CCCTTGGGGCTTCAAAAAG-3=; IL-
4U-primer, 5=-CGGCACAGAGCTGATGG-3= and IL-4L-primer, 5=-AGTTAAAGCATGGTGGCTCAGT-3=; IL-10U-primer, 5=-TGAATTCCCTGGGTGGAAG-3= and IL-10-L-primer, 5=-TGCTTCCCAAGGGAACC-3=; GAPDHU-primer, 5=-ACCACAGTCCATGCCATCAC-3= and GAPDHL-primer, 5=-GGGTGGTCCAGGGTT-
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Fig 2. Intracellular TNF- ␣ syntheses in spleen. A, Histograms of intracellular TNF-␣ cytokine synthesis at 3 hours after operative trauma. Solid lines indicate the cells stained with mouse monoclonal anti-TNF-␣ Abs; dotted lines indicate cells stained with isotype-matched controls. The marker, M1, represents the indicated percentage of TNF-␣ positive cells. Data shown are representative of 6 separate experiments. B, Intracellular TNF-␣ levels in spleen at 3 to 6 hours after operative trauma. The mean percentage values of TNF-␣ positive cells ⫾ SEM are presented as bar graphs (3 h: *P ⬍ .05 vs ICR, n ⫽ 6; 6 h: #P ⬍ .05 vs ICR, n ⫽ 5). C, Intracellular TNF-␣ levels in spleen at 3 to 6 hours in control groups. The mean percentage values of TNF-␣ positive cells ⫾ SEM are presented as bar graphs (3, 6 h: n ⫽ 2).
TCTTA-3=. Initial denaturation was carried out at 94°C for 2 minutes followed by 30 cycles of 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 90 seconds, using a model PTC100 thermal cycler. PCR products were separated on a 2% agarose gel and visualized by ethidium bromide staining. Cytokine ELISA. At the scheduled postoperative times (3, 6, 12, 24 h), blood samples were obtained from retro-orbital puncture under Ketamine and Xylazine anesthesia just before killing and placed in heparinized capillary tubes. The sera were frozen at ⫺80°C until use. TNF-␣, IL-2, IL4, and IL-10 were measured by enzyme-linked immunosorbent assay (ELISA) with the ELISA Ready-SET-Go! kit (eBioscience) according to the manufacturer’s protocol. Statistical analysis (flow cytometry). In each group, the percentages of cytokine positive values from the histograms were noted (4 to 6 in number), and groups were compared with regard to cytokine synthesis using one-way ANOVA followed by the Bonferroni/Dunn post-hoc test (StatView J-4.5; Abacus Concepts, Berkeley, CA). Data are
presented as mean values ⫾ standard error of the mean (SEM). P less than .05 was considered significant, and n represents the number of mice. RESULTS Twenty-one mice were excluded because of postoperative anastomotic problems (major leakage, hematoma, necrotic changes). Most of these problems occurred at the beginning of the study. TNF-␣ synthesis after operative trauma. The results of RT-PCR in spleen are shown in Fig 1 A-1. The SL group showed early and strong upregulation of TNF-␣ mRNA at 3 to 12 hours compared with the ICR and THD groups. The GA, AGA, and SGA groups showed strong upregulation of TNF-␣ mRNA specifically at 3 hours after operative trauma. The RU486 group was added to observe the relationship between GA and GR on TNF-␣ synthesis. RU486 showed strong upregulation of TNF-␣ synthesis with no reduction from 3 to 24 hours (Fig 1, A-2). Serum TNF-␣ production was observed only in the SL and GA groups at 3 hours
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Fig 3. Postoperative RT-PCR for IL-2 mRNA expression in spleen. Results of RT-PCR for IL-2 mRNA expression at 3 to 24 hours after operative trauma (A) and control groups (B). Results are expressed as means of 2 separate experiments.
after operative trauma. After 24 hours, the level of serum TNF-␣ was maximal in the SL and GA groups, whereas it was still in the low range in the ICR and THD groups (Fig 1, B). In control groups, serum TNF-␣ levels were almost insignificant (Fig 1, C). The GA group showed a significant increase of intracellular TNF-␣ synthesis compared with the other groups at 3 to 6 hours after operative trauma (at 3 h: *P ⬍ .05 GA vs ICR, n ⫽ 6; at 6 h: #P ⬍ .05 GA vs ICR, n ⫽ 6) (Fig 2, A,B). In Fig 2 C, TNF-␣ synthesis was increased in the AGA group at 3 to 6 hours, whereas the SGA group showed high levels of TNF-␣ synthesis at 3 hours and low levels at 6 hours. IL-2 synthesis after operative trauma. Only the SL group showed a strong upregulation of IL2 mRNA expression at 6 to 12 hours after operative trauma in contrast to other groups (Fig 3, A,B). In Fig 4 A, intracellular IL-2 synthesis in spleen increased in the THD group at 3 hours (*P ⬍ .05 THD vs GA, n ⫽ 5) and 24 hours (#P ⬍ .05 THD vs ICR, n ⫽ 4). The SL group showed a persistent increase in intracellular IL-2 synthesis compared with the ICR group. The GA group showed a decrease in IL-2 synthesis at 3 hours. In Fig 4 B, the SGA and STHD groups each showed a persistent increase in intracellular IL-2 synthesis from 3
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hours. In Figs 4 C and D, significant serum IL-2 production was generally below measured levels until 12 hours in all groups. At 24 hours, the SL, GA, and THD groups showed significant increases in serum IL-2 production, whereas the ICR group showed no measurable IL-2 production (Fig 4, C). Serum IL-2 production also reached measured levels at 24 hours in the SGA and STHD groups (Fig 4, D). IL-4 synthesis after operative trauma. In Fig 5 A-1, the SL group showed strong upregulation of IL-4 mRNA expression 3 hours after operative trauma, in contrast to the ICR group that showed weak expression. The GA and THD groups showed strong upregulation of IL-4 mRNA at 12 hours. In control groups, strong upregulation of IL-4 mRNA expression were not observed (Fig 5, A-2). In Fig 5 B, intracellular IL-4 synthesis in spleen increased in the ICR group at 6 hours (*P ⬍ .05 ICR vs GA, n ⫽ 6). The SL and GA groups showed a dominant increase in intracellular IL-4 synthesis at 3 hours. The SGA and STHD groups each showed a persistent increase in IL-4 synthesis from 3 to 24 hours, whereas the AGA and the ATHD groups showed little IL-4 synthesis (Fig 5, C). Serum IL-4 production was increased at 3 hours in both the SL and GA groups (Fig 5, D). In control groups, serum IL-4 production was generally below measured levels in all groups except the SGA group at 6 hours (Fig 5, E). IL-10 synthesis after operative trauma. In Fig 6 A-1, the ICR group showed strong upregulation of IL-10 mRNA expression in spleen at 3 to 6 hours after operative trauma in contrast to the weak induction in the SL group. The GA and THD groups showed less upregulation in IL-10 mRNA expression than the ICR group at 3 to 12 hours. In control groups, upregulation of IL-10 mRNA expression was not observed (Fig 6, A-2). In Fig 6 B, intracellular IL-10 synthesis was increased in the ICR group at 3 to 6 hours after operative trauma (3 h: *P ⬍ .05 ICR vs SL, GA, n ⫽ 6; 6 h: #P ⬍ .05 ICR vs SL, GA, THD, n ⫽ 5). IL-10 synthesis was observed in all control groups from 3 hours (Fig 6, C). The control groups were not directly comparable with the test groups in the positive values as the antibody responses for IL-10 and its matched-isotype controls were different. Serum IL-10 production was increased initially in the ICR group. The GA and THD groups showed low levels of serum IL-10 at 12 hours after operative trauma (Fig 6, D). Serum IL-10 production was generally below measured levels in control groups except the SGA group at 6 hours and the STHD group at 12 hours (Fig 6, E).
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Fig 4. A, Intracellular IL-2 syntheses in spleen at 3 to 24 hours after operative trauma. The mean percentage values of IL-2 positive cells ⫾ SEM are shown as bar graphs (n ⫽ 4 to 5) (3 h, *P ⬍ .05 vs GA, n ⫽ 5; 24 h, #P ⬍ .05 vs ICR, n ⫽ 4; ns, not significant). B, Intracellular IL-2 syntheses in spleen at 3 to 24 hours in control groups. The mean percentage values of IL-2 positive cells ⫾ SEM are shown as bar graphs (3 to 24 h; n ⫽ 2). C, Serum IL-2 production after operative trauma. Results are expressed as mean ⫾ SEM of 3 separate experiments. D, Serum IL-2 production in control groups. Results are expressed as mean ⫾ SEM of 2 separate experiments.
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Fig 5. A-1, Postoperative time course of RT-PCR for IL-4 mRNA expression in spleen. A-2, Results of RT-PCR for IL-4 mRNA expression in control groups. B, Intracellular IL-4 syntheses in spleen at 3 to 24 hours after operative trauma. The mean percentage values of IL-4 positive cells ⫾ SEM are presented as bar graphs (n ⫽ 4-6). (3 h: n ⫽ 4; 6 h: *P ⬍ .05 vs GA, n ⫽ 6; 12 h, n ⫽ 5; 24 h, n ⫽ 4; ns, not significant). C, Intracellular IL-4 syntheses in spleen at 3 to 24 hours in control groups. The mean percentage values of IL-4 positive cells ⫾ SEM are presented as bar graphs (n ⫽ 2). D, Serum IL-4 production after operative trauma. Sera were assayed for IL-4 by ELISA. Results are expressed as mean ⫾ SEM of 3 independent experiments. E, Serum IL-4 production in control groups. Results are expressed as mean ⫾ SEM of 2 independent experiments.
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Fig 6. A-1, Post-operative time course of RT-PCR for IL-10 mRNA expression in spleen. Results are expressed as means of 2 separate experiments. A-2, Results of RT-PCR for IL-10 mRNA expression in control groups. Results are expressed as means of 2 separate experiments. B, Intracellular IL-10 syntheses of spleen at 3 to 6 hours after operative trauma are shown. The mean percentage values of IL-10 positive cells ⫾ SEM are presented as bar graphs (3 h: *P ⬍ .05 vs SL, GA, n ⫽ 6; 6 h: *P ⬍ .05 vs SL, GA, THD, n ⫽ 5.) C, Intracellular IL-10 syntheses of spleen at 3 to 6 hours in control groups. The mean percentage values of IL-10 positive cells ⫾ SEM are presented as bar graphs (n ⫽ 2). D, Serum IL-10 production after operative trauma. Sera were assayed for IL-10 by ELISA. Results are expressed as mean ⫾ SEM of 3 independent experiments. E, Serum IL-10 production in control groups. Results are expressed as mean ⫾ SEM of 2 independent experiments.
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DISCUSSION To our knowledge, this is the first study that has evaluated the effects of operative trauma on cytokine synthesis by investigating mRNA levels, intracellular cytokine levels, and serum cytokine levels. In the current study, we found that initial TNF-␣ synthesis was decreased after major operative trauma, whereas TNF-␣ synthesis initially increased after minor operative trauma. Several authors have reported no change in TNF-␣ characteristics after operation or severe trauma,23,24 however, in the current study, the pattern of TNF-␣ synthesis after operative trauma differed depending on the degree of operative trauma. In previous reports, levels of TNF-␣ were measured at daily intervals,23,24 so it may be that the marked variations in TNF-␣ synthesis occur only within the initial postoperative period. The effect of geldanamycin on TNF-␣ synthesis is another intriguing finding. Geldanamycin caused an upregulation of TNF-␣ mRNA expression and intracellular TNF-␣ synthesis during the initial post-operative period in the GA, SGA, and AGA groups. Our results indicate that geldanamycin induces early upregulation of TNF-␣ synthesis under stressed conditions in vivo. The molecular chaperone, heat-shock protein 90 (Hsp90), regulates multiple signal transduction pathways14,25 and has been shown to regulate more than 100 proteins (‘client proteins’ for Hsp90) involved in cellular signaling.26 As geldanamycin is an inhibitor of Hsp90,25 from the current results, it is assumed that the expression of TNF-␣ mRNA is partly related to one or more of the client proteins. The glucocorticoid receptor (GR) is a major client protein of Hsp90,25-27 and RU486 blocks GR function by inhibiting the dissociation of GR from Hsp90.18 We added RU486 after our model of major operative trauma to investigate the interaction of GR and TNF-␣ mRNA expression. Blocking GR by RU486 caused the upregulation of TNF-␣ mRNA expression after major operative trauma. We suggest that under stressed conditions, the expression of TNF-␣ mRNA may be regulated partly by one or more of the Hsp90 client proteins including GR. The elucidation of this mechanism will need further investigation. The significance of the early upregulation of TNF-␣ synthesis is not clear, but because the early upregulation of TNF-␣ synthesis in the GA group was in line with that of the SL group, we assume that the geldanamycin shifted the pattern of TNF-␣ synthesis to the less injured pattern after major operative trauma. Thalidomide did not show significant inhibition of the TNF-␣ effect28 in the current study.
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The ICR group showed persistent suppression of serum IL-2 production until 24 hours after operative trauma, whereas the other groups that underwent operative trauma showed significant serum IL-2 increase at 24 hours. It seems that major operative trauma (ICR) causes persistent suppression of IL-2 synthesis in contrast with minor operative trauma (SL). Furthermore, our results also suggest that geldanamycin and thalidomide may relieve independently the persistent suppression of IL-2 synthesis. In the current study, addition of thalidomide was associated with a significant increase in IL-2 synthesis after major operative trauma. This is the first in vivo report that shows that thalidomide may act as a stimulator for IL-2 synthesis after operative trauma. The ELISA results showed a stimulatory effect of geldanamycin on serum IL-2 production after operative trauma. This characteristic of geldanamycin parallels the results of intracellular IL-2 synthesis from 6 hours onward. The mechanism of the stimulatory effect on IL-2 synthesis is not clear, however, as IL-10 suppresses IL-2 synthesis,29-32 it is possible that the concomitant decrease in IL-10 synthesis in the early postoperative period may have played a part in enhancing IL-2 synthesis in the later postoperative phase. Geldanamycin showed unexpectedly the reverse effect on IL-2 synthesis in the GA and the SGA groups at 3 hours (Fig 4, A). The exact reason is not clear. Further investigation will be needed to address this problem. We also showed a significant increase in intracellular IL-4 synthesis and serum IL-4 production from 6 hours after major operative trauma. These results are in accordance with previous reports.4,10 In contrast, minor operative trauma showed a different expression profile. At 3 hours, minor operative trauma showed strong upregulation of IL-4 mRNA expression with concomitant increases in intracellular IL-4 synthesis and serum IL-4 production (Fig 5, A-D). The significance of early upregulation of IL-4 synthesis after operative trauma is not clear. In recent reports,33,34 however, IL-4 has been shown paradoxically to induce Th1 responses. This paradoxic role of IL-4 for positive Th1 induction might account for the initial increase in IL-4 synthesis after minor operative trauma. The initial increase in IL-4 synthesis may reflect the lesser amount of damage in minor operative trauma. In the current study, the initial response after major operative trauma was characterized by an increase in IL-10 synthesis in contrast with a lesser induction of IL-10 synthesis after minor operative trauma. Conversely, at the later time periods, serum IL-10 production was predominant in the mi-
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nor operative trauma model (Fig 6, D). Minor and major operative trauma caused different transitional patterns for induction of IL-10 synthesis. It is not clear what factor is controlling directly the induction of IL-10 synthesis after major operative trauma. Because geldanamycin attenuated the IL-10 synthesis after major operative trauma, we assume that one or more client proteins for Hsp 90 are associated with the control of IL-10 synthesis. In contrast, the exact role of IL-10 in post-traumatic immunosuppression remains unclear.13 Some previous investigators have shown the beneficial effect of anti-IL-10 treatment after injury,4,6,8,29 whereas others have shown the protective effect of IL-10 after injury.30,35-37 In the current study, both geldanamycin and thalidomide showed early upregulation of IL-2 synthesis after decreased IL-10 synthesis after major operative trauma. Our results support the beneficial effect of early inhibition of IL-10 synthesis after operative trauma. These drugs might be useful as immunomodulators to improve the Th1/Th2 cytokine imbalance after major operative trauma. In the current study, the results of intracellular cytokine synthesis and cytokine mRNA expression were often not parallel with the levels of serum cytokines. According to Huse et al,38 T cells release IL-2 through an immunologic response to APC (antigen presenting cell) in an antigen-specific way. This secretion method suggests a longer latent time for serum IL-2 to reach increased levels at distant circulations. In our RT-PCR results, SL showed strong upregulation of IL-2 mRNA expression at 6 to 12 hours (Fig 3, A), but the serum IL-2 levels were below measured levels (Fig 4, C). Similarly in control groups, serum IL-2 levels were almost below measured levels, although the percentage of positive values of intracellular IL-2 synthesis were increased in the SGA and STHD groups at the same time (Fig 4, B,D). In addition, the upregulation of TNF-␣ mRNA expression in control groups (Fig 1, A-1) was not reflected directly in the elevation of serum levels (Fig 1, C) though the samples were taken from the same two mice. The same discrepancies were also observed in IL-4 and IL-10 in control groups. There seems to be a time lag between the response of intracellular cytokine synthesis in spleen and the levels of serum cytokine at distant microcirculations. The level of intracellular cytokine synthesis may reflect a more latent immune condition than the serum cytokine levels, at these early time points after operative trauma. Furthermore, the SGA group showed measurable serum TNF-␣ elevation at 6 hours (Fig 1, C), although the SGA group showed a
lower intracellular TNF-␣ synthesis at the same time (Fig 2, C). Certain feed back loops may exist between serum cytokine levels and intracellular mRNA cytokine levels. At present, however, there is insufficient evidence to address this issue and further investigation is required. In conclusion, we created mouse surgical models and assessed postoperative measure of immunologic changes for 24 hours. The current data give new insights on the initial variation in cytokine synthesis after models of major and minor operative trauma in mice. Geldanamycin and thalidomide relieved the persistent suppression of Th1 (IL-2) synthesis after major operative trauma. To validate the therapeutic benefits of these drugs, however, further investigations including infectious resistance, survival, and activity will need to be pursued. The authors wish to express great thanks to Dr Yoshiya Watanabe (SOPHY, Inc., Kochi, Japan) for his excellent work in ELISA. Thanks also to research student Kaoru Orihashi (Laboratory of Animal Experiments for Regeneration, Institute for Frontier Medical Science, Kyoto University) and Miss Yuka Takezaki for their expert assistance in RT-PCR, and to Miss Motoko Miyata for her excellent assistance in experimental preparations, and finally great thanks to Kenji Okajima (Department of Biodefense, Nagoya City University Graduated School of Medical Science) for assistance with this manuscript. REFERENCES 1. Wood JJ, Rodrick ML, O’Mahony JB, et al. Inadequate interleukin 2 production: a fundamental immunological deficiency in patients with major burns. Ann Surg 1984;200:311-20. 2. Horgan AF, Mendez MV, O’Riordain DS, et al. Altered gene transcription after burn injury results in depressed T lymphocyte activation. Ann Surg 1994;220:342-52. 3. O’Sullivan ST, Lederer JA, Horgan AF, et al. Major injury leads to predominance of the T helper-2 lymphocyte phenotype and diminished interleukin-12 production associated with decreased resistance to infection. Ann Surg 1995;222:482-92. 4. Lyons A, Kelly JL, Rodrick ML, et al. Major injury increases production of interleukin-10 by cells of the immune system with a negative impact on resistance to infection. Ann Surg 1997;226:450-60. 5. O’Riordain DS, Mendez MV, O’Riordain MG, et al. Molecular mechanism of decreased interleukin-2 production after thermal injury. Surgery 1993;114:407-15. 6. Lyons A, Goebel A, Mannick JA, et al. Protective effects of early interleukin 10 antagonism on injury-induced immune dysfunction. Arch Surg 1999;134:1317-24. 7. Keith RW, Walley NW, Theodore JS, et al. Balance of inflammatory cytokines related to severity and mortality of murine sepsis. Infect Immun 1996;64:4733-8. 8. Song GY, Chung CS, Chaudry IH, et al. What is the role of interleukin 10 in polymicrobial sepsis: anti-inflammatory agent or immunosuppressant? Surgery 1999;126:378-83. 9. Faist E, Mewes A, Strasser T, et al. Alteration of monocyte function following major injury. Arch Surg 1988;123:287-92.
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10. Mack VE, McCarter MD, Naama HA, et al. Dominance of T-helper 2-type cytokines after severe injury. Arch Surg 1996;131:1303-9. 11. De AK, Kodys KM, Pellegrini J, et al. Induction of global anergy rather than inhibitory Th2 lymphokines mediates post trauma T cell immunodepression. Clin Immunol 2000; 96:52-66. 12. Murphy T, Paterson H, Roger S, et al. Use of intracellular cytokine staining and bacterial superantigen to document suppression of the adaptive immune system in injured patients. Ann Surg 2003;238:401-11. 13. Schäffer M, Barbul A. Lymphocyte function in wound healing and following injury. Br J Surg 1998;85:444-60. 14. Pratt WB. The role of the hsp90-based chaperon system in signal transduction by nuclear receptors and receptors signaling via MAP-kinase. Annu Rev Pharmacol Toxicol 1997;37:297-326. 15. Wax S, Piecyk M, Maritim B, et al. Geldanamycin inhibits the production of inflammatory cytokines in activated macrophages by reducing the stability and translation of cytokine transcripts. Arthritis Rheum 2003;48:541-50. 16. Bucci M, Roviezzo F, Cicala C, et al. Geldanamycin, an inhibitor of heat shock protein 90 (Hsp90) mediated signal transduction has anti-inflammatory effects and interacts with glucocorticoid receptor in vivo. Br J Pharmacol 2000;131:13-6. 17. Beck CA, Estes PA, Bona BJ, et al. The steroid antagonist RU486 exerts different effects on the glucocorticoid and progesterone receptors. Endocrinology 1993;133:728-40. 18. Cronstein BN, Kimmel SC, Levin RI, et al. A mechanism for the antiinflammatory effects of corticosteroids: the glucocorticoid receptor regulates leukocyte adhesion to endothelial cells and expression of endothelial-leukocyte adhesion molecule 1 and intercellular adhesion molecule 1. Proc Natl Acad Sci USA 1992;89:9991-5. 19. Franks ME, Macpherson GR, Figg WD. Thalidomide. Lancet 2004;363:1802-11. 20. Kumar S, Witzig TE, Rajkumar V. Thalidomide: current role in the treatment of non-plasma cell malignancies. J Clin Oncol 22:2477-88. 21. Haslett PAJ, Corral LG, Albert M, et al. Thalidomide costimulates primary human T lymphocytes, preferentially inducing proliferation, cytokine production, and cytotoxic responses in the CD8⫹ subset. J Exp Med 1998;187:1885-92. 22. Davies FE, Raje N, Hideshima T, et al. Thalidomide and immunomodulatory derivatives augment natural killer cell cytotoxicity in multiple myeloma. Blood 2001;98:210-6. 23. Mokart D, Capo C, Blache JL, et al. Early postoperative compensatory anti-inflammatory response syndrome is asso-
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24. 25.
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ciated with septic complications after major surgical trauma in patients with cancer. Br J Surg 2002;89:1450-6. Baigrie RJ, Lamont PM, Kwiatkowski D, et al. Systemic cytokine response after major surgery. Br J Surg 1992;79:757-60. Zhang H, Burrows F. Targeting multiple signal transduction pathways through inhibition of Hsp90. J Mol Med 2004;82: 488-99. Pratt WB, Galigniana MD, Harrell JM, et al. Role of hsp90 and the hsp90-binding immunophilins in signaling protein movement. Cell Signal 2004;16:857-72. Pratt WB, Silverstein AM, Galigniana MD. A model for the cytoplasmic trafficking of signaling proteins involving the hsp90-binding immunophilins and p50cdc37. Topical review. Cell Signal 1999;11;839-51. Sampaio EP, Sarno EN, Galilly R, et al. Thalidomide selectively inhibits tumor necrosis factor ␣ production by stimulated human monocytes. J Exp Med 1991;173:699-703. Kelly JL, Lyons Ann, Soberg CC, et al. Anti-interleukin-10 antibody restores burn-induced defects in T-cell function. Surgery 1997;122:146-52. Shneider CP, Schwacha MG, Chaudry IH. The role of interleukin-10 in the regulation of the systemic inflammatory response following trauma-hemorrhage. Biochim Biophys Acta 2004;1689:22-32. Pestka S, Krause CD, Sarkar D, et al. Interleukin-10 and related cytokines and receptors. Ann Rev Immunol 2004;22:929-79. Fiorentino DF, Bond MW, Mosmann TR. Two types of mouse T helper cell: IV. Th2 clones secrete a factor that inhibits cytokine production by Th1 clones. J Exp Med 1989;170:2081-95. Biedermann T, Zimmermann S, Himmelrich H, et al. IL-4 instructs TH1 responses and resistance to Leishmania major in susceptible BALB/c mice. Nat. Immunol 2001;2:1054-60. Yao Y, Li W, Kaplan MH, et al. Interleukin (IL)-4 inhibits IL-10 to promote IL-12 production by dendritic cells. J Exp Med 2005;201:1889-903. Farivar AS, Krishnadasan B, Naidu BV, et al. Endogenous interleukin-4 and interleukin-10 regulate experimental lung ischemia reperfusion injury. Ann Thorac Surg 2003;76: 253-9. Welborn MB III, Moldawer LL, Seeger JM, et al. Role of endogenous interleukin-10 in local and distant organ injury after visceral ischemia-reperfusion. Shock 2003;20:35-40. Zimmerman MA, Reznikov LL, Raeburn CD, et al. Interleukin-10 attenuates the response to vascular injury. J Surg Res 2004;121:206-13. Huse M, Lillemeier BF, Kuhns MS, et al. T cells use two directionally distinct pathways for cytokine secretion. Nat Immunol 2006;7:247-55.
Background. Persistence of postoperative immune dysfunction is a critical problem because it increases the risk of serious infectious complications. The mechanisms of the immune dysfunction that occur initially after non-thermal operative injury remain to be fully elucidated. Methods. Two mouse models of operative trauma (simple laparotomy to represent minor operative injury and ileocecal resection to represent major operative injury) were used to define the characteristics of initial cytokine synthesis. Geldanamycin and thalidomide were independently added intraperitoneally before and after operative injury to examine the effect on postoperative immune dysfunction. Mice were sacrificed at scheduled times (3, 6, 12, and 24 h after operative injury) and TNF-␣, IL-2, IL-4, and IL-10 were analyzed. Spleen was used for intracellular cytokines and RT-PCR. Sera were used for ELISA. Results. Major operative injury caused an initial upregulation of IL-10 synthesis with delayed synthesis of TNF-␣ and IL-2. Minor operative injury caused an early induction of IL-2 synthesis preceded by an initial induction of IL-4 synthesis. GA caused a specific early upregulation of TNF-␣ mRNA expression and intracellular TNF-␣ synthesis. The GA and THD groups showed early serum IL-2 production with reduction of IL-10 mRNA expression and intracellular IL-10 synthesis in the early post-operative phase. Conclusions. Major and minor operative injury showed different Th1/Th2 cytokine patterns in the initial post-operative period. Geldanamycin and thalidomide improved the Th1/Th2 imbalance independently after major operative injury. (Surgery 2007;141:490-500.) From the Department of Tumor Surgery, Kochi Medical School, Kochi University, Nankoku, Japan
Studies discussing the immunologic phenomena after operative trauma, other than burns,1-6 sepsis,7-8 and external trauma9-13 are uncommon. We have studied 2 different murine models of nonthermal operative trauma: the first is a simple laparotomy (SL) model to represent minor operative trauma injury and the second is an ileocecal resection (ICR) model to represent major operative trauma. Four basic cytokines, TNF-␣ and IL-2 as Th1 (Type-1 T helper cell) cytokines, and IL-4 and IL-10 as Th2 (Type-2 T helper cell) cytokines, were Accepted for publication October 14, 2006. Reprint requests: Takumi Nakano, MD, Department of Surgery, Kochi Medical School, Kochi University, Okho, Nankoku, Japan. E-mail: [email protected]. 0039-6060/$ - see front matter © 2007 Mosby, Inc. All rights reserved. doi:10.1016/j.surg.2006.10.003
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evaluated. The predominance of Th2 cytokines3,10 (IL-10, IL-4) or suppression of IL-21,5,13 after serious injury has been shown to cause decreased resistance to infection.1,3-6,11,12 This observation suggests that decreasing the inhibition of IL-2 or attenuating the Th2 cytokines is desirable. Heat shock protein 90 (Hsp90) participates in signal transduction14 and is critical in the intracellular signaling pathways that promote inflammatory cytokine production.15 Geldanamycin (GA) is a benzoquinone ansamycin that inhibits the function of Hsp90.14,15 Bucci et al16 have shown that geldanamycin acts as an anti-inflammatory drug in vivo inhibiting carrageenan-induced mouse pawedema. As an antagonist of the glucocorticoid receptor (GR), RU486 has been used to study the receptor activation mechanism17 that prevents the translocation of GR to the nucleus by inhibiting the dissociation of GR from Hsp90.18
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Thalidomide (THD) was synthesized originally in 1954 as a sedative, but soon disappeared due to the high risk of teratogenicity,19,20 however, use of thalidomide has reappeared in as an effective antiinflammatory, immunomodulatory drug19,20 and as a costimulator for IL-2-production.21,22 We have investigated whether geldanamycin or thalidomide could act as an immunomodulator to relieve the initial detrimental cytokine responses that occur postoperatively. MATERIALS AND METHODS Mice. Male C57BL/6 mice 8 to 10 weeks old were purchased from Japan SLC (Shizuoka, Japan) and were maintained under pathogen-free conditions. All mice were fed a liquid food (Calorie Mate; Otsuka Pharmaceutical, Tokyo, Japan) for 1 week before operation and until they were killed. All experiments were approved by the Animal Experimental Committee of Kochi Medical School, Kochi University. Drugs. Thalidomide (⫹) (THD) was obtained from Sigma-Aldrich (St. Louis, MO). Geldanamycin (GA) and RU486 (Mifepristone; LKT Laboratories, St. Paul, MN) were purchased from Wako Pure Chemical Industries (Osaka, Japan). These drugs were dissolved in dimethyl sulfoxide (DMSO; Wako Pure Chemical) at concentrations of 10 mg/ dl, 10 mg/dl, and 2 mg/dl, respectively. Operative procedures. Under Ketamine (80 mg/kg; Veterinary Ketalar 50; Sankyo Yell, Tokyo, Japan) and Xylazine (30 mg/kg; Celactar; Bayer HealthCare, Leverkusen, Germany) anesthesia, minor (simple laparotomy [SL]) or major (ileocecal resection [ICR]) operative trauma was carried out aseptically. Simple laparotomy was accomplished via a 1 cm abdominal incision. The appendix was exteriorized for 5 minutes with occasional moistening with saline-soaked gauze. ICR was also accomplished via a 1 cm incision, and ileocecal resection, which involved a resection spanning 1 cm proximal and 3 mm distal to the appendical origin, was added. Anastomosis was carried out by interrupted suture with 8-0 polyglactin sutures. Abdominal incisions were closed with 6-0 polyglactin suture material. Mean operation times were 13 minutes for SL and 27 minutes for ICR. Experimental design. Two hundred and eighteen mice were divided randomly into the following 4 main groups: (1) SL group, SL ⫹ DMSO intraperitoneally (i.p.); (2) ICR group, ICR ⫹ DMSO i.p.; (3) GA group, ICR ⫹ GA i.p.; 4) THD group, ICR ⫹ THD i.p.; and 4 control groups: (5) AGA group, Anesthesia ⫹ GA i.p.; (6) ATHD group, Anesthesia ⫹ THD i.p.; (7) SGA group, SL⫹
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GA i.p.; (8) STHD group, SL⫹ THD i.p.; and an additional group (9) ICR ⫹ RU486 i.p. (for RTPCR analysis only). In each group, the drug was administered twice intraperitoneally: on the preoperative day and at the time of the peritoneal closure. The administered doses of the drugs were as follows: GA, 1 mg/kg; THD, 1 mg/kg; and RU486, 5 mg/kg. The doses of geldanamycin and RU486 were determined by reference to Bucci et al.16 and the dose of thalidomide was determined by reference to published doses for human use.18 As a control for the SL and ICR groups, DMSO solution, 200 l per mouse, was administered i.p. Mice were killed 3, 6, 12, and 24 h after operative trauma by cervical dislocation under Ketamine and Xylazine anesthesia. Analysis of intracellular cytokines (TNF-␣, IL-2, IL-4, and IL-10). A sample of spleen was removed from each of the mice at each scheduled time point. Splenocytes were suspended at 1 to 2 ⫻ 106/ml in complete RPMI-1640 medium (Invitrogen, Carlsbad, CA) with 2 mmol/L l-glutamine, 50 mmol/L 2-mercaptoethanol, 100 U/ml penicillin, 100 ug/ml streptomycin, 0.25 ug/ml amphotericin B, and 5% heat inactivated fetal calf serum (Invitrogen). The cells were incubated in 6-well plates with a protein transport inhibitor, brefeldin A (1 ul/ml, Golgi Plug; BD Pharmingen, San Diego, CA), Ionomycin (1.6 ug/ml; Sigma-Aldrich, St. Louis, MO) and PMA (0.3 ug/ml, Sigma-Aldrich), for 5 h at 37°C with 5% CO2. After incubation, cells were fixed and permeabilized with Cytofix/Cytoperm (BD Pharmingen) for 20 min, then washed twice with saponin-containing buffer (Perm/Wash, BD Pharmingen). Intracellular cytokines were stained with the following mouse monoclonal antibodies for 30 min: FITC-rat anti-mouse TNF-␣ (BD Pharmingen); FITC-conjugated anti-mouse IL-4; PE-conjugated anti-mouse IL-2; and anti-mouse IL-10 (eBioscience, San Diego, CA). Rat IgG isotype controls were obtained from eBioscience. In flow cytometric analysis, gating for lymphocytes was set for IL-2, IL-4, and TNF-␣ measurements, and the gating for targeting lymphocytes and macrophages was set for IL-10 measurement using forward versus side scatter properties. Analysis was carried out using a FACSCalibur Flow Cytometer (Becton Dickinson) with CellQuest software. Reverse-transcriptase polymerase chain reaction (RT-PCR) for cytokine mRNA expression. Total RNA from spleen was isolated using TRIzol Reagent (Invitrogen), and cDNA was prepared using the RNA PCR Kit (AMV) Ver.2.1 (TaKaRa Biomedicals, Tokyo, Japan). The cDNA was then used in reverse transcription (RT)-PCR with Platinum Taq
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Fig 1. A-1, A-2, Time course of RT-PCR for TNF-␣ mRNA expression in spleen. Results are expressed as means of 2 separate experiments. A-1, The GA and SL groups showed early upregulation of TNF-␣ mRNA expression. A-2, The RU486 group showed strong upregulation of TNF-␣ mRNA expression with no reduction from 3 to 24 hours. B, Serum TNF-␣ production after operative trauma. Results are expressed as mean ⫾ SEM of 3 independent experiments. C, Serum TNF-␣ production in control groups. Results are expressed as mean ⫾ SEM of 2 independent experiments. SL, simple laparotomy ⫹ DMSO i.p.; ICR, ileocecal resection ⫹ DMSO i.p.; GA, ileocecal resection ⫹ geldanamycin i.p.; THD, ileocecal resection ⫹ thalidomide i.p.; RU486, ileocecal resection ⫹ RU486 i.p.; SGA, simple laparotomy ⫹ geldanamycin i.p; STHD, simple laparotomy ⫹ thalidomide i.p.; AGA, anesthesia ⫹ geldanamycin i.p; ATHD, anesthesia ⫹ thalidomide i.p.
DNA polymerase (Invitrogen). The primer sequences were as follows: TNF-␣U-primer, 5=-CCACATCTCCCTCCAGAAAA-3= and TNF-␣L-primer, 5=-TCCCCTTCATCTTCCTCCTT-3=; IL-2U-primer, 5=-GAGCAGCTGTTGATGGACCT-3= and IL-2Lprimer, 5=-CCCTTGGGGCTTCAAAAAG-3=; IL-
4U-primer, 5=-CGGCACAGAGCTGATGG-3= and IL-4L-primer, 5=-AGTTAAAGCATGGTGGCTCAGT-3=; IL-10U-primer, 5=-TGAATTCCCTGGGTGGAAG-3= and IL-10-L-primer, 5=-TGCTTCCCAAGGGAACC-3=; GAPDHU-primer, 5=-ACCACAGTCCATGCCATCAC-3= and GAPDHL-primer, 5=-GGGTGGTCCAGGGTT-
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Fig 2. Intracellular TNF- ␣ syntheses in spleen. A, Histograms of intracellular TNF-␣ cytokine synthesis at 3 hours after operative trauma. Solid lines indicate the cells stained with mouse monoclonal anti-TNF-␣ Abs; dotted lines indicate cells stained with isotype-matched controls. The marker, M1, represents the indicated percentage of TNF-␣ positive cells. Data shown are representative of 6 separate experiments. B, Intracellular TNF-␣ levels in spleen at 3 to 6 hours after operative trauma. The mean percentage values of TNF-␣ positive cells ⫾ SEM are presented as bar graphs (3 h: *P ⬍ .05 vs ICR, n ⫽ 6; 6 h: #P ⬍ .05 vs ICR, n ⫽ 5). C, Intracellular TNF-␣ levels in spleen at 3 to 6 hours in control groups. The mean percentage values of TNF-␣ positive cells ⫾ SEM are presented as bar graphs (3, 6 h: n ⫽ 2).
TCTTA-3=. Initial denaturation was carried out at 94°C for 2 minutes followed by 30 cycles of 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 90 seconds, using a model PTC100 thermal cycler. PCR products were separated on a 2% agarose gel and visualized by ethidium bromide staining. Cytokine ELISA. At the scheduled postoperative times (3, 6, 12, 24 h), blood samples were obtained from retro-orbital puncture under Ketamine and Xylazine anesthesia just before killing and placed in heparinized capillary tubes. The sera were frozen at ⫺80°C until use. TNF-␣, IL-2, IL4, and IL-10 were measured by enzyme-linked immunosorbent assay (ELISA) with the ELISA Ready-SET-Go! kit (eBioscience) according to the manufacturer’s protocol. Statistical analysis (flow cytometry). In each group, the percentages of cytokine positive values from the histograms were noted (4 to 6 in number), and groups were compared with regard to cytokine synthesis using one-way ANOVA followed by the Bonferroni/Dunn post-hoc test (StatView J-4.5; Abacus Concepts, Berkeley, CA). Data are
presented as mean values ⫾ standard error of the mean (SEM). P less than .05 was considered significant, and n represents the number of mice. RESULTS Twenty-one mice were excluded because of postoperative anastomotic problems (major leakage, hematoma, necrotic changes). Most of these problems occurred at the beginning of the study. TNF-␣ synthesis after operative trauma. The results of RT-PCR in spleen are shown in Fig 1 A-1. The SL group showed early and strong upregulation of TNF-␣ mRNA at 3 to 12 hours compared with the ICR and THD groups. The GA, AGA, and SGA groups showed strong upregulation of TNF-␣ mRNA specifically at 3 hours after operative trauma. The RU486 group was added to observe the relationship between GA and GR on TNF-␣ synthesis. RU486 showed strong upregulation of TNF-␣ synthesis with no reduction from 3 to 24 hours (Fig 1, A-2). Serum TNF-␣ production was observed only in the SL and GA groups at 3 hours
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Fig 3. Postoperative RT-PCR for IL-2 mRNA expression in spleen. Results of RT-PCR for IL-2 mRNA expression at 3 to 24 hours after operative trauma (A) and control groups (B). Results are expressed as means of 2 separate experiments.
after operative trauma. After 24 hours, the level of serum TNF-␣ was maximal in the SL and GA groups, whereas it was still in the low range in the ICR and THD groups (Fig 1, B). In control groups, serum TNF-␣ levels were almost insignificant (Fig 1, C). The GA group showed a significant increase of intracellular TNF-␣ synthesis compared with the other groups at 3 to 6 hours after operative trauma (at 3 h: *P ⬍ .05 GA vs ICR, n ⫽ 6; at 6 h: #P ⬍ .05 GA vs ICR, n ⫽ 6) (Fig 2, A,B). In Fig 2 C, TNF-␣ synthesis was increased in the AGA group at 3 to 6 hours, whereas the SGA group showed high levels of TNF-␣ synthesis at 3 hours and low levels at 6 hours. IL-2 synthesis after operative trauma. Only the SL group showed a strong upregulation of IL2 mRNA expression at 6 to 12 hours after operative trauma in contrast to other groups (Fig 3, A,B). In Fig 4 A, intracellular IL-2 synthesis in spleen increased in the THD group at 3 hours (*P ⬍ .05 THD vs GA, n ⫽ 5) and 24 hours (#P ⬍ .05 THD vs ICR, n ⫽ 4). The SL group showed a persistent increase in intracellular IL-2 synthesis compared with the ICR group. The GA group showed a decrease in IL-2 synthesis at 3 hours. In Fig 4 B, the SGA and STHD groups each showed a persistent increase in intracellular IL-2 synthesis from 3
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hours. In Figs 4 C and D, significant serum IL-2 production was generally below measured levels until 12 hours in all groups. At 24 hours, the SL, GA, and THD groups showed significant increases in serum IL-2 production, whereas the ICR group showed no measurable IL-2 production (Fig 4, C). Serum IL-2 production also reached measured levels at 24 hours in the SGA and STHD groups (Fig 4, D). IL-4 synthesis after operative trauma. In Fig 5 A-1, the SL group showed strong upregulation of IL-4 mRNA expression 3 hours after operative trauma, in contrast to the ICR group that showed weak expression. The GA and THD groups showed strong upregulation of IL-4 mRNA at 12 hours. In control groups, strong upregulation of IL-4 mRNA expression were not observed (Fig 5, A-2). In Fig 5 B, intracellular IL-4 synthesis in spleen increased in the ICR group at 6 hours (*P ⬍ .05 ICR vs GA, n ⫽ 6). The SL and GA groups showed a dominant increase in intracellular IL-4 synthesis at 3 hours. The SGA and STHD groups each showed a persistent increase in IL-4 synthesis from 3 to 24 hours, whereas the AGA and the ATHD groups showed little IL-4 synthesis (Fig 5, C). Serum IL-4 production was increased at 3 hours in both the SL and GA groups (Fig 5, D). In control groups, serum IL-4 production was generally below measured levels in all groups except the SGA group at 6 hours (Fig 5, E). IL-10 synthesis after operative trauma. In Fig 6 A-1, the ICR group showed strong upregulation of IL-10 mRNA expression in spleen at 3 to 6 hours after operative trauma in contrast to the weak induction in the SL group. The GA and THD groups showed less upregulation in IL-10 mRNA expression than the ICR group at 3 to 12 hours. In control groups, upregulation of IL-10 mRNA expression was not observed (Fig 6, A-2). In Fig 6 B, intracellular IL-10 synthesis was increased in the ICR group at 3 to 6 hours after operative trauma (3 h: *P ⬍ .05 ICR vs SL, GA, n ⫽ 6; 6 h: #P ⬍ .05 ICR vs SL, GA, THD, n ⫽ 5). IL-10 synthesis was observed in all control groups from 3 hours (Fig 6, C). The control groups were not directly comparable with the test groups in the positive values as the antibody responses for IL-10 and its matched-isotype controls were different. Serum IL-10 production was increased initially in the ICR group. The GA and THD groups showed low levels of serum IL-10 at 12 hours after operative trauma (Fig 6, D). Serum IL-10 production was generally below measured levels in control groups except the SGA group at 6 hours and the STHD group at 12 hours (Fig 6, E).
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Fig 4. A, Intracellular IL-2 syntheses in spleen at 3 to 24 hours after operative trauma. The mean percentage values of IL-2 positive cells ⫾ SEM are shown as bar graphs (n ⫽ 4 to 5) (3 h, *P ⬍ .05 vs GA, n ⫽ 5; 24 h, #P ⬍ .05 vs ICR, n ⫽ 4; ns, not significant). B, Intracellular IL-2 syntheses in spleen at 3 to 24 hours in control groups. The mean percentage values of IL-2 positive cells ⫾ SEM are shown as bar graphs (3 to 24 h; n ⫽ 2). C, Serum IL-2 production after operative trauma. Results are expressed as mean ⫾ SEM of 3 separate experiments. D, Serum IL-2 production in control groups. Results are expressed as mean ⫾ SEM of 2 separate experiments.
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Fig 5. A-1, Postoperative time course of RT-PCR for IL-4 mRNA expression in spleen. A-2, Results of RT-PCR for IL-4 mRNA expression in control groups. B, Intracellular IL-4 syntheses in spleen at 3 to 24 hours after operative trauma. The mean percentage values of IL-4 positive cells ⫾ SEM are presented as bar graphs (n ⫽ 4-6). (3 h: n ⫽ 4; 6 h: *P ⬍ .05 vs GA, n ⫽ 6; 12 h, n ⫽ 5; 24 h, n ⫽ 4; ns, not significant). C, Intracellular IL-4 syntheses in spleen at 3 to 24 hours in control groups. The mean percentage values of IL-4 positive cells ⫾ SEM are presented as bar graphs (n ⫽ 2). D, Serum IL-4 production after operative trauma. Sera were assayed for IL-4 by ELISA. Results are expressed as mean ⫾ SEM of 3 independent experiments. E, Serum IL-4 production in control groups. Results are expressed as mean ⫾ SEM of 2 independent experiments.
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Fig 6. A-1, Post-operative time course of RT-PCR for IL-10 mRNA expression in spleen. Results are expressed as means of 2 separate experiments. A-2, Results of RT-PCR for IL-10 mRNA expression in control groups. Results are expressed as means of 2 separate experiments. B, Intracellular IL-10 syntheses of spleen at 3 to 6 hours after operative trauma are shown. The mean percentage values of IL-10 positive cells ⫾ SEM are presented as bar graphs (3 h: *P ⬍ .05 vs SL, GA, n ⫽ 6; 6 h: *P ⬍ .05 vs SL, GA, THD, n ⫽ 5.) C, Intracellular IL-10 syntheses of spleen at 3 to 6 hours in control groups. The mean percentage values of IL-10 positive cells ⫾ SEM are presented as bar graphs (n ⫽ 2). D, Serum IL-10 production after operative trauma. Sera were assayed for IL-10 by ELISA. Results are expressed as mean ⫾ SEM of 3 independent experiments. E, Serum IL-10 production in control groups. Results are expressed as mean ⫾ SEM of 2 independent experiments.
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DISCUSSION To our knowledge, this is the first study that has evaluated the effects of operative trauma on cytokine synthesis by investigating mRNA levels, intracellular cytokine levels, and serum cytokine levels. In the current study, we found that initial TNF-␣ synthesis was decreased after major operative trauma, whereas TNF-␣ synthesis initially increased after minor operative trauma. Several authors have reported no change in TNF-␣ characteristics after operation or severe trauma,23,24 however, in the current study, the pattern of TNF-␣ synthesis after operative trauma differed depending on the degree of operative trauma. In previous reports, levels of TNF-␣ were measured at daily intervals,23,24 so it may be that the marked variations in TNF-␣ synthesis occur only within the initial postoperative period. The effect of geldanamycin on TNF-␣ synthesis is another intriguing finding. Geldanamycin caused an upregulation of TNF-␣ mRNA expression and intracellular TNF-␣ synthesis during the initial post-operative period in the GA, SGA, and AGA groups. Our results indicate that geldanamycin induces early upregulation of TNF-␣ synthesis under stressed conditions in vivo. The molecular chaperone, heat-shock protein 90 (Hsp90), regulates multiple signal transduction pathways14,25 and has been shown to regulate more than 100 proteins (‘client proteins’ for Hsp90) involved in cellular signaling.26 As geldanamycin is an inhibitor of Hsp90,25 from the current results, it is assumed that the expression of TNF-␣ mRNA is partly related to one or more of the client proteins. The glucocorticoid receptor (GR) is a major client protein of Hsp90,25-27 and RU486 blocks GR function by inhibiting the dissociation of GR from Hsp90.18 We added RU486 after our model of major operative trauma to investigate the interaction of GR and TNF-␣ mRNA expression. Blocking GR by RU486 caused the upregulation of TNF-␣ mRNA expression after major operative trauma. We suggest that under stressed conditions, the expression of TNF-␣ mRNA may be regulated partly by one or more of the Hsp90 client proteins including GR. The elucidation of this mechanism will need further investigation. The significance of the early upregulation of TNF-␣ synthesis is not clear, but because the early upregulation of TNF-␣ synthesis in the GA group was in line with that of the SL group, we assume that the geldanamycin shifted the pattern of TNF-␣ synthesis to the less injured pattern after major operative trauma. Thalidomide did not show significant inhibition of the TNF-␣ effect28 in the current study.
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The ICR group showed persistent suppression of serum IL-2 production until 24 hours after operative trauma, whereas the other groups that underwent operative trauma showed significant serum IL-2 increase at 24 hours. It seems that major operative trauma (ICR) causes persistent suppression of IL-2 synthesis in contrast with minor operative trauma (SL). Furthermore, our results also suggest that geldanamycin and thalidomide may relieve independently the persistent suppression of IL-2 synthesis. In the current study, addition of thalidomide was associated with a significant increase in IL-2 synthesis after major operative trauma. This is the first in vivo report that shows that thalidomide may act as a stimulator for IL-2 synthesis after operative trauma. The ELISA results showed a stimulatory effect of geldanamycin on serum IL-2 production after operative trauma. This characteristic of geldanamycin parallels the results of intracellular IL-2 synthesis from 6 hours onward. The mechanism of the stimulatory effect on IL-2 synthesis is not clear, however, as IL-10 suppresses IL-2 synthesis,29-32 it is possible that the concomitant decrease in IL-10 synthesis in the early postoperative period may have played a part in enhancing IL-2 synthesis in the later postoperative phase. Geldanamycin showed unexpectedly the reverse effect on IL-2 synthesis in the GA and the SGA groups at 3 hours (Fig 4, A). The exact reason is not clear. Further investigation will be needed to address this problem. We also showed a significant increase in intracellular IL-4 synthesis and serum IL-4 production from 6 hours after major operative trauma. These results are in accordance with previous reports.4,10 In contrast, minor operative trauma showed a different expression profile. At 3 hours, minor operative trauma showed strong upregulation of IL-4 mRNA expression with concomitant increases in intracellular IL-4 synthesis and serum IL-4 production (Fig 5, A-D). The significance of early upregulation of IL-4 synthesis after operative trauma is not clear. In recent reports,33,34 however, IL-4 has been shown paradoxically to induce Th1 responses. This paradoxic role of IL-4 for positive Th1 induction might account for the initial increase in IL-4 synthesis after minor operative trauma. The initial increase in IL-4 synthesis may reflect the lesser amount of damage in minor operative trauma. In the current study, the initial response after major operative trauma was characterized by an increase in IL-10 synthesis in contrast with a lesser induction of IL-10 synthesis after minor operative trauma. Conversely, at the later time periods, serum IL-10 production was predominant in the mi-
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nor operative trauma model (Fig 6, D). Minor and major operative trauma caused different transitional patterns for induction of IL-10 synthesis. It is not clear what factor is controlling directly the induction of IL-10 synthesis after major operative trauma. Because geldanamycin attenuated the IL-10 synthesis after major operative trauma, we assume that one or more client proteins for Hsp 90 are associated with the control of IL-10 synthesis. In contrast, the exact role of IL-10 in post-traumatic immunosuppression remains unclear.13 Some previous investigators have shown the beneficial effect of anti-IL-10 treatment after injury,4,6,8,29 whereas others have shown the protective effect of IL-10 after injury.30,35-37 In the current study, both geldanamycin and thalidomide showed early upregulation of IL-2 synthesis after decreased IL-10 synthesis after major operative trauma. Our results support the beneficial effect of early inhibition of IL-10 synthesis after operative trauma. These drugs might be useful as immunomodulators to improve the Th1/Th2 cytokine imbalance after major operative trauma. In the current study, the results of intracellular cytokine synthesis and cytokine mRNA expression were often not parallel with the levels of serum cytokines. According to Huse et al,38 T cells release IL-2 through an immunologic response to APC (antigen presenting cell) in an antigen-specific way. This secretion method suggests a longer latent time for serum IL-2 to reach increased levels at distant circulations. In our RT-PCR results, SL showed strong upregulation of IL-2 mRNA expression at 6 to 12 hours (Fig 3, A), but the serum IL-2 levels were below measured levels (Fig 4, C). Similarly in control groups, serum IL-2 levels were almost below measured levels, although the percentage of positive values of intracellular IL-2 synthesis were increased in the SGA and STHD groups at the same time (Fig 4, B,D). In addition, the upregulation of TNF-␣ mRNA expression in control groups (Fig 1, A-1) was not reflected directly in the elevation of serum levels (Fig 1, C) though the samples were taken from the same two mice. The same discrepancies were also observed in IL-4 and IL-10 in control groups. There seems to be a time lag between the response of intracellular cytokine synthesis in spleen and the levels of serum cytokine at distant microcirculations. The level of intracellular cytokine synthesis may reflect a more latent immune condition than the serum cytokine levels, at these early time points after operative trauma. Furthermore, the SGA group showed measurable serum TNF-␣ elevation at 6 hours (Fig 1, C), although the SGA group showed a
lower intracellular TNF-␣ synthesis at the same time (Fig 2, C). Certain feed back loops may exist between serum cytokine levels and intracellular mRNA cytokine levels. At present, however, there is insufficient evidence to address this issue and further investigation is required. In conclusion, we created mouse surgical models and assessed postoperative measure of immunologic changes for 24 hours. The current data give new insights on the initial variation in cytokine synthesis after models of major and minor operative trauma in mice. Geldanamycin and thalidomide relieved the persistent suppression of Th1 (IL-2) synthesis after major operative trauma. To validate the therapeutic benefits of these drugs, however, further investigations including infectious resistance, survival, and activity will need to be pursued. The authors wish to express great thanks to Dr Yoshiya Watanabe (SOPHY, Inc., Kochi, Japan) for his excellent work in ELISA. Thanks also to research student Kaoru Orihashi (Laboratory of Animal Experiments for Regeneration, Institute for Frontier Medical Science, Kyoto University) and Miss Yuka Takezaki for their expert assistance in RT-PCR, and to Miss Motoko Miyata for her excellent assistance in experimental preparations, and finally great thanks to Kenji Okajima (Department of Biodefense, Nagoya City University Graduated School of Medical Science) for assistance with this manuscript. REFERENCES 1. Wood JJ, Rodrick ML, O’Mahony JB, et al. Inadequate interleukin 2 production: a fundamental immunological deficiency in patients with major burns. Ann Surg 1984;200:311-20. 2. Horgan AF, Mendez MV, O’Riordain DS, et al. Altered gene transcription after burn injury results in depressed T lymphocyte activation. Ann Surg 1994;220:342-52. 3. O’Sullivan ST, Lederer JA, Horgan AF, et al. Major injury leads to predominance of the T helper-2 lymphocyte phenotype and diminished interleukin-12 production associated with decreased resistance to infection. Ann Surg 1995;222:482-92. 4. Lyons A, Kelly JL, Rodrick ML, et al. Major injury increases production of interleukin-10 by cells of the immune system with a negative impact on resistance to infection. Ann Surg 1997;226:450-60. 5. O’Riordain DS, Mendez MV, O’Riordain MG, et al. Molecular mechanism of decreased interleukin-2 production after thermal injury. Surgery 1993;114:407-15. 6. Lyons A, Goebel A, Mannick JA, et al. Protective effects of early interleukin 10 antagonism on injury-induced immune dysfunction. Arch Surg 1999;134:1317-24. 7. Keith RW, Walley NW, Theodore JS, et al. Balance of inflammatory cytokines related to severity and mortality of murine sepsis. Infect Immun 1996;64:4733-8. 8. Song GY, Chung CS, Chaudry IH, et al. What is the role of interleukin 10 in polymicrobial sepsis: anti-inflammatory agent or immunosuppressant? Surgery 1999;126:378-83. 9. Faist E, Mewes A, Strasser T, et al. Alteration of monocyte function following major injury. Arch Surg 1988;123:287-92.
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