The Complex


of Cytokines in Sepsis

Association Between Prostaglandins, Cachectin, and Interleukins


Although the cytokines tumor necrosis factor (TNF), interleukin1 (IL-1), and interleukin-6 (IL-6) are important mediators of hemodynamic, metabolic, and immunologic alterations in the host during sepsis, it is not known whether there is any association between the release of these cytokines and prostanoids during sepsis. Sepsis induced by cecal ligation and puncture in rats led to a persistent elevation (p < 0.05) of plasma TNF until 10 hours, steadily increasing (p < 0.05) IL-1 plasma levels, and enhanced (p < 0.05) IL-6 plasma levels at all time points compared to the sham group. Prostaglandin E2 plasma levels were elevated (p < 0.05) at 5 hours (153 ± 29 pg/mL; control: 47 ± 11 pg/mL) and 10 hours (96 ± 16 pg/mL; control: 21 ± 5 pg/ mL). Prostaglandin E2 production by splenic macrophages (sM4) from septic animals was increased (p < 0.05) at 5 hours (9.1 ± 2.2 ng/mL) and 10 hours (5.6 ± 1.5 ng/mL) compared to controls (3.3 ± 0.3 ng/mL at 5 hours; 1.3 ± 1.3 ng/mL at 10 hours). Incubation of sMq from septic animals with ibuprofen enhanced (p < 0.05) IL-1 and TNF synthesis, while IL-6 production was reduced (p < 0.05). These results indicate that the alterations in prostanoid release and elevated plasma prostanoids may regulate the release and consequently the circulating levels of cytokines during sepsis.

From the Departments of Surgery,* Microbiology and Public Health,t and Physiology,f Michigan State University, East Lansing, Michigan

prevented the development of lethal septic shock syndrome.9-'.' Tumor necrosis factor induces the synthesis of monokines, such as IL-f1'2 and IL-6,'3 which have been implicated in the pathophysiology of infection.4",5 Passive immunization against TNF attenuated the appearance of IL-I1 and IL-6,16 indicating that TNF is the initial stimulus for the release of these cytokines during the septic syndrome. Furthermore administration of recombinant IL- 1 or IL-6 induced some of the characteristic physiologic derangements associated with injury,'7"18 including fever and acute-phase protein responses. In addition to enhanced cytokine release, macrophages produce large amounts of arachidonic acid metabolites in response to endotoxin, IL- 1, and TNF. 19-2' Prostaglandins are potent immunosuppressive mediators.22 In vitro studies23125 using macrophages from unmanipulated animals have shown that prostaglandins, in particular prostaglandin E2 (PGE2), downregulate their inducer cytokines, IL- 1 and TNF, through a negative feedback mechanism. The effect of prostaglandins on the synthesis and release of IL-6 during sepsis, however, is not known. Although the above-referenced in vitro studies clearly indicate a regulatory role of prostaglandins on cytokine release, the kinetics of circulating PGE2 in the early and the late stages of sepsis, its synthesis by macrophages, and its role in altering the release of cytokines remain unknown. It was, therefore, the aim of this study to correlate the alterations in plasma TNF, IL- 1, and IL-6 levels with plasma PGE2 levels in sepsis. Furthermore PGE2 synthesis by splenic macrophages was measured and the effect of cyclooxygenase blockade by ibuprofen on splenic macrophage cytokine release was determined.

T| a HE RELEASE OF cytokines such as interleukin-1

(IL-1), interleukin-6 (IL-6), and cachectin (tumor necrosis factor [TNF]) by macrophages is significantly altered during endotoxemia, sepsis, and septic shock. 1-6 Tumor necrosis factor, in particular, appears to play a pivotal role in lethal endotoxemia.7-8 Anti-TNF antibodies attenuated the lethal effect of endotoxemia and

Supported by National Institutes of Health grants ROI GM 37127 and GM 39519. Dr. Ertel's present address is Chirurgische Klinik und Poliklinik der, Ludwig-Maximilians-Universitaet, Muenchen, Klinikum Grosshadern, Marchioninistrasse 15, D-8000 Munich 70, Germany. Address reprint requests to Irshad H. Chaudry, Ph.D., Department of Surgery, B424 Clinical Center, Michigan State University, East Lansing, MI 48824-1315. Accepted for publication September 1990.



142 Materials and Methods

Experimental Protocol The experimental protocol and the care of the animals were carried out according to the guidelines set forth by the Animal Welfare Act and with the Guide for the Care and Use of Laboratory Animals, National Institutes of Health Publications. Male Sprague-Dawley rats, weighing 260 to 360 g, were fasted overnight. After administration of light ether anesthesia, sepsis was induced by cecal ligation and puncture (CLP), as described in detail previously.26 Briefly, after a 2-cm midline incision was made, the cecum was isolated and a 3-0 silk ligature was placed around it, ligating the cecum just below the ileocecal valve (without interrupting the continuation of the small bowel and the colon). The cecum was then punctured twice with an 18-gauge needle, replaced in the peritoneal cavity, and the abdominal incision closed in two layers. Saline (3 mL/ 100 g body weight) was given subcutaneously at this time. Laparotomy without CLP was performed in control (sham) animals. Animals from both groups were killed at different time points (2, 5, 10, and 20 hours) after CLP or sham laparotomy. Plasma for cytokine determinations was collected and the spleen harvested. To obtain cytokine and endotoxin baseline values in plasma, blood was collected from unmanipulated rats by cardiac stick.

Preparation of Splenic Macrophages

Spleens were harvested, gently ground between frosted slides, and resuspended in Click's medium (Irvine Scientific, Santa Ana, CA). Erythrocytes were lysed by hypotonic shock. The remaining cells were washed twice and resuspended in Click's medium. Cell viability, tested by Trypan Blue exclusion, was .95% and did not reveal any differences between the septic and the sham-operated group. After determination of splenic macrophage (sMk) numbers present in the cultures, 1 X 106 sMk/mL Click's medium was transferred to 24-well plates and incubated for 4 hours (at 37 C, 5% C02). Nonadherent and nonviable cells were removed by five repeated washings of the well. This protocol provided cultures that were more than 95% M4s, as determined by nonspecific esterase staining, and exhibited typical MO morphology. The remaining adherent sM4 were incubated further for 24 hours in Click's medium containing 10% fetal bovine serum (Biologos Inc., Naperville, IL) with 10 ,g/mL (for cytokine measurement) or 1 ,ug/mL (for PGE2 measurement) lipopolysaccharide W (LPS, from Escherichia coli 055:B5, Difco Labs., Detroit, MI). For studying the in vitro effects of prostaglandins on cytokine production, the cyclooxygenase inhibitor ibuprofen (10 ,ug/mL, Sigma Chemical Co., St. Louis, MO) was added to sMo cultures. Supernatants

Ann. Surg. * August 1991

of sMo were harvested, filtered, aliquoted, and frozen at -70 C until assayed.

Interleukin-l Assay The activity of IL-1 in plasma samples and sMo supernatants was determined by incubating serial dilutions of samples with Dl0.G4. 1 cells (2 X 104 cells/well)27 in the presence of Concanavalin A (Pharmacia Fine Chemicals, Piscataway, NJ). Recent studies28 have shown that DlO.G4. 1 cells do not react with recombinant IL-6. This observation is important for the determination of IL- 1 in plasma because following endotoxemia, significant amounts of IL-6 are present in plasma.'6 However we cannot preclude the possibility that there might be interference with other cytokines, such as IL-2, IL-4, or IL-7, in this assay.28 After an incubation period of 48 hours, DIO.G4.1 proliferation was measured by incorporation of 3H-thymidine. The unit(s) of IL-1 activity was calculated, comparing the slope of the sample curve to that of the standard curve according to the method of Mizel.29 One unit was defined as the amount of the cytokine required to produce one-half maximal proliferation. Interleukin-6 Assay The activity of IL-6 was measured by the proliferation of the 7TD1 hybridoma.30 Briefly, serial dilutions of plasma or supernatant were incubated in duplicate with 4 X 104 7TDl cells/mL for 72 hours in 96-well plates and then MTT tetrazolium (Sigma Chemical Co.) was added. Four hours later the supernatants were removed and the cells lysed with 0.04 N HCl in isopropanol. The degree ofcell proliferation was determined colorimetrically in an enzyme-linked immunoadsorbent assay plate reader (620 nm). The units of activity in the samples were calculated as described above.29 Tumor Necrosis Factor Assay Tumor necrosis factor activity was determined by its cytotoxic effect on the fibrosarcoma cell line WEHI 164 subclone 13.3' The degree of lysis induced by TNF was measured by proliferation of nonlysed WEHI cells using the MTT assay as described above. It should be noted that the IL-6 and TNF assay have been shown to react in a highly specific fashion to TNF or IL-6, respectively.32'33

PGE2 Radioimmunoassay Prostaglandin E2 levels in plasma and sMo supernatants were measured using a radioimmunoassay technique (NEN, DuPont, Boston, MA; cross-reactivity with PGE,: 3.7%, DHKPGE2:0.4%, PGA2:0.04%, other prostaglandins and arachidonic acid:

The complex pattern of cytokines in sepsis. Association between prostaglandins, cachectin, and interleukins.

Although the cytokines tumor necrosis factor (TNF), interleukin-1 (IL-1), and interleukin-6 (IL-6) are important mediators of hemodynamic, metabolic, ...
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