[PubMed] [Google Scholar] 4. observed with IL-12 blockade and STAT4 deficiency on host survival suggest that IL-12 may activate alternate pathways promoting survival. Signal transducers and activators of transcription (STAT) transmit cytokine signals to the nucleus, facilitating Slco2a1 gene transcription (19). Six different STAT proteins have been identified, each with its own array of cytokine specificities and resultant gene activations (19). The STAT factor 4 (STAT4) protein was originally cloned through its homology with previously described STAT proteins (43, 45) and is activated in T cells, natural killer cells, testis, and thymus in response to interleukin-12 (IL-12) (42). Interaction of IL-12 with its cell surface receptor leads to phosphorylation of STAT4 by receptor-associated Janus kinases. Once phosphorylated, STAT4 dimerizes and translocates to the nucleus and promotes gene transcription yielding production of gamma interferon (IFN-), development of the T helper type 1 response, and increased natural killer cell cytotoxicity (25, 39). The host immune response to infections therefore relies to some extent on IL-12 and STAT4. Sepsis is characterized by an inflammatory cytokine response, including production of IL-12 (22). Supplemental IL-12 can improve host bacterial resistance, and deficiency of IL-12 predisposes patients to bacterial infections and sepsis (11, 17). When combined with IL-18 or IL-2, administration of IL-12 can induce a fatal response in mice similar to septic shock (3, 4). Additionally, IL-12 administration in humans has resulted in organ damage, hemodynamic instability, and death (7). These studies suggest an important role for IL-12 Mecamylamine Hydrochloride (and STAT4 by association) in survival, bacterial clearance, and organ failure during bacterial sepsis. The aim of the present study was to examine the role of the STAT4 pathway during sepsis by using the clinically relevant cecal ligation and puncture (CLP) model (31, 33). We demonstrate a survival benefit and impaired bacterial clearance when the host lacks STAT4. Parallel studies of IL-12 neutralization and STAT4 deficiency Mecamylamine Hydrochloride suggest that the antibacterial effects of IL-12 are mediated by STAT4. However, these data also suggest that despite its antibacterial effects, STAT4 activation is detrimental to host survival. (This work was initially presented at the Surgical Infection Society meeting Mecamylamine Hydrochloride at Snowbird, Utah, on 3 May 2001.) MATERIALS AND METHODS Animals. Six- to 8-week-old STAT4?/? mice (BALB/c-mice deficient in the STAT4 protein) and their wild-type controls (BALB/c mice) were used (Jackson Laboratories, Bar Harbor, Maine). Animals were housed Mecamylamine Hydrochloride in a facility approved by the American Association for Accreditation of Laboratory Animal Care and were provided food and water ad libitum. Studies were conducted in accordance with the guidelines of the National Institutes of Health and under the supervision of a veterinarian. CLP. Mice were anesthetized with ketamine (80 mg/kg) and xylazine (16 mg/kg) by intramuscular injection. CLP was performed as follows. The cecum was exposed through a midline laparotomy incision and ligated just below the ileocecal junction with 4-0 silk suture. For survival and 18-h harvest experiments, a single 23-gauge puncture was made in the cecum. For 4-h harvest experiments to yield higher bacterial counts, two 18-gauge punctures were made in the cecum. The cecum was then returned into the peritoneal cavity, and the abdominal incision was closed in layers. Survival. Mice were injected with 1 ml of normal saline subcutaneously for volume resuscitation at the time of CLP. Cefoxitin (100 mg/kg) was given subcutaneously every 12 h. For IL-12 immunoneutralization experiments, 200 g of either IL-12 antibody or control immunoglobulin G (R&D Systems, Minneapolis, Minn.) was injected into the tail vein immediately prior to CLP. Timed harvests. Anesthetized mice were killed at 4 or 18 h after CLP. Peritoneal lavage fluid was acquired for dedication of bacterial counts, cytokine levels, neutrophil counts, and myeloperoxidase levels. Liver, lung, and spleen cells were collected for dedication of bacterial counts and myeloperoxidase levels. Blood was collected by cardiac puncture for evaluation of bacterial and leukocyte counts. Peritoneal lavage. Peritoneal exudate cells were recovered by peritoneal lavage with 4 ml of ice-cold heparinized RPMI 1640 medium (Gibco-BRL, Bethesda, Md.) and were counted by hand by hemacytometer. Myeloperoxidase assay. Myeloperoxidase was used as a measure of neutrophil accumulation. Liver, lung, and spleen cells were homogenized in dilute phosphate buffer and then centrifuged at 10,000.
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