Tumor necrosis factor (TNF) is a monocyte-derived protein cytotoxic or cytostatic for some tumor cell lines. Here we show that highly purified E. coli-derived recombinant human TNF stimulated the growth of human FS-4 diploid fibroblasts. Stimulation of cell growth was demonstrable at a TNF concentration of 10 pg/ml (3 X 10(-13) M). Maximal stimulation was attained at TNF concentrations of 10 ng/ml (3 X 10(-10) M) or higher. Growth-stimulatory activity of TNF was inhibited by an mAb neutralizing the cytotoxic activity of TNF. Growth stimulation was not inhibited by another mAb specific for TNF, lacking neutralizing activity for the cytotoxic activity of TNF. Growth stimulation by TNF was more marked and more sustained in the presence of greater than or equal to 10% FCS than in medium with less than or equal to 5% FCS. Addition of TNF to confluent FS-4 cultures also produced a marked stimulation of cell growth in the presence of fresh FCS, while a much less marked stimulation was seen in the absence of FCS. Stimulation of confluent cultures by TNF in serum-free medium was enhanced by insulin, suggesting that insulin or insulin-like growth factor(s) in the serum can act synergistically with TNF in producing growth stimulation. While the growth-stimulatory effects of TNF and insulin were synergistic, the actions of TNF and epidermal growth factor (EGF) were less than additive, suggesting that TNF and EGF may activate identical or similar pathways. We conclude that stimulation of cell growth is probably a physiological function of TNF, and that the cytotoxic and cytostatic actions of TNF may be the result of an anomalous growth signal transduction in neoplastic cells lacking the constraints of normal growth control mechanisms.

A 39-nt DNA sequence, the interferon y (IFN-y) response region (GRR), is necessary for the IFN-yinduced transcription of the high-affinity Fc receptor for IgG (FcyRI) and sufficient for the IFN-r-induced transcription of transfected plasmids. By using extracts from IFN--treated cells, three protein complexes will assemble in vitro on a 9-nt core region in the 3' domain of the GRR. The sequence of this core resembles the IFN-y-activated sequence (GAS) described for the GBP gene. Mutations in this GAS core region prevent complex assembly and result in the loss of IFN-y induction of reporter constructs containing the mutation. In addition to the GAS core region, a 5' region of the GRR is necessary for optimal IFN-y induction and for formation of one of the (refs. 8, 9, 22, 24 and K. D. Khan, K.S., G. Lindwall, S. E. Maher, J.E.D., and A. L. M. Bothwell, unpublished results). Since the GBP gene is induced by both IFN-y and IFN-a, interaction of the 91-kDa protein with the GAS may provide a common pathway for transcriptional activation by IFN-a and IFN-y.In previous work we have focused (25) on the mechanism by which IFN-y specifically induces transcription of target genes. FcyRI, in contrast to the GBP, is induced only by IFN-'y. A 39-nt sequence, called the IFN-y response region (GRR), has been identified in the promoter of this gene that is both necessary and sufficient for transcriptional activation by IFN-y (25) and does not respond to IFN-a (unpublished data). These transfection results correlate with the transcriptional induction of FcyRI in macrophages in response to IFN-y that is robust, rapid, and cycloheximide-resistant. In contrast, IFN-a treatment of macrophages results in insignificant FcyRI transcriptional induction (ref. 3 and unpublished observation). Induction of FcyRI accounts for some of the responses attributed to the activated macrophage, such as increased phagocytosis and enhanced antibody-dependent cellular cytotoxicity (26).We now present evidence for the rapid induction of specific protein complexes that assemble on the GRR in IFN--stimulated cells. Mutations that prevent assembly of these complexes in vitro also prevent response to IFN-y induction in transfection experiments with reporter constructs. These complexes interact with a 9-nt core sequence in the GRR that is homologous to the GAS sites found in other IFN-y-induced genes. The 91-kDa DNA binding protein that interacts with the GBP promoter also binds to the GRR of the FcyRI gene. Surprisingly, IFN-a also induces the formation of similar complexes on the GRR yet fails to induce significant transcriptional activation of FcyRI or the GRR. We have begun to reconcile these results by the identification of a second domain within the 5' region of the GRR that is required for optimal IFN-y 4314The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

(IFN-a/f3) and type II (IFN-y). Both act through typespecific cell surface receptors to induce the expression of unique, although overlapping, sets of genes. Genes that are induced only by IFN-a/13, such as ISG15 and ISG54, or by both IFN-a/fB and IFN-y, such as the guanylate-binding protein (GBP) and major histocompatibility (MHC) class I genes, appear to share a common mechanism for transcriptional regulation, which is mediated by a conserved sequence, termed the IFN-stimulated response element (ISRE) (2-6). Interaction of IFN-a/p with its receptor activates a positive regulatory factor, ISGF3, that binds to the ISRE (7). IFN-y can enhance this effect by stimulating the synthesis of the DNA-binding subunit ofISGF3 (8). An additional element overlapping the ISRE has been identified in the GBP promoter (4, 9). Termed the gamma-activated sequence (GAS), this element contributes to both IFN-a//3 and IFN-y transcriptional activation by binding distinct trans-acting factors. In contrast, a common mechanism of transcriptional induction has yet to be established for genes that are responsive to IFN-y but not to IFN-a/f3. Included in this set are genes encoding IP-10, monokine induced by IFN-y (MIG), macrophage inhibitory protein 2, MHC class II antigens, the MHC class II-associated invariant chain, and the highaffinity receptor for IgG (Fc,, receptor type I, FcXRI) (10-15).Among the cis-acting elements shown to mediate the IFN-y activation of these genes are the ISRE of the invariant-chain gene promoter and the X, H, and y-IRE sequences of MHC class II gene promoters (16)(17)(18)(19)(20)(21). We are interested in determining the mechanisms by which IFN-y is able to regulate the immune response, and have focused our work on the DNA elements responsible for the transcriptional induction of the human FcXRI gene. In this paper we present the structure of the gene encoding human FcXRI (huFcRI),t focusing on the structure of its promoter and characterization of the DNA sequences responsible for its induction by IFN-y. A 39-base-pair (bp) cis-acting region, termed the IFN-y response region (GRR), is shown to be responsible for IFN-y induction and to be sufficient to confer pronounced IFN-y inducibility upon a heterologous promoter. The GRR contains elements homologous to those defined for MHC class II promoters and several other IFN--responsive genes, indicating that, as in the case of IFN-a/f3-responsive genes, common sequence motifs are involved in transcriptional activation of this class of genes.

mediates pathogenic inflammatory responses to intestinal ischemia-reperfusion injury. Am J Physiol Gastrointest Liver Physiol 299: G833-G843, 2010. First published August 5, 2010 doi:10.1152/ajpgi.00065.2010.-Acute lung injury (ALI) and the development of the multiple organ dysfunction syndrome (MODS) are major causes of death in trauma patients. Gut inflammation and loss of gut barrier function as a consequence of splanchnic ischemia-reperfusion (I/R) have been implicated as the initial triggering events that contribute to the development of the systemic inflammatory response, ALI, and MODS. Since hypoxia-inducible factor (HIF-1) is a key regulator of the physiological and pathophysiological response to hypoxia, we asked whether HIF-1 plays a proximal role in the induction of gut injury and subsequent lung injury. Utilizing partially HIF-1␣-deficient mice in a global trauma hemorrhagic shock (T/HS) model, we found that HIF-1 activation was necessary for the development of gut injury and that the prevention of gut injury was associated with an abrogation of lung injury. Specifically, in vivo studies demonstrated that partial HIF-1␣ deficiency ameliorated T/HS-induced increases in intestinal permeability, bacterial translocation, and caspase-3 activation. Lastly, partial HIF-1␣ deficiency reduced TNF-␣, IL-1␤, cyclooxygenase-2, and inducible nitric oxide synthase levels in the ileal mucosa after T/HS whereas IL-1␤ mRNA levels were reduced in the lung after T/HS. This study indicates that prolonged intestinal HIF-1 activation is a proximal regulator of I/R-induced gut mucosal injury and gut-induced lung injury. Consequently, these results provide unique information on the initiating events in trauma-hemorrhagic shock-induced ALI and MODS as well as potential therapeutic insights. hemorrhagic shock; inflammation; multiple organ dysfunction syndrome; acute lung injury IN PATIENTS SUSTAINING major trauma, the development of the systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction (MODS) is a major clinical problem resulting in 50 -80% of all deaths in surgical intensive care units. Since the pathophysiology of this syndrome remains incompletely understood and therapy remains largely supportive (16), studies focusing on the basic biology of traumainduced SIRS, organ injury/dysfunction, and MODS have been major areas of investigation. These mechanistic studies have generated several working hypotheses, one of which is the gut hypothesis of MODS. A key element in the gut hypothesis of MODS is that a splanchnic ischemia-reperfusion (I/R) insult leading to gut inflammation and loss of barrier function is the initial triggering event that turns the gut into the "motor" of MODS (19). However, the exact mechanisms by which gut I/R leads to intestinal injury and how an intestinal ischemic insult is transduced into a systemic inflammatory response remains incomplete. To date, the majority of the molecular and cellular studies investigating shock-induced gut injury and gut-induced MODS have focused pr...

. The female intestine is more resistant than the male intestine to gut injury and inflammation when subjected to conditions associated with shock states. Am J Physiol Gastrointest Liver Physiol 288: G466 -G472, 2005. First published October 21, 2004; doi:10.1152/ajpgi.00036.2004.-Having documented that proestrus female rats are more resistant to shock-induced acute gut and hence lung injury than male rats, we tested the hypothesis that the female gut is more resistant to injury and produces less of an inflammatory response than the male gut when exposed to conditions associated with shock states (hypoxia and acidosis) utilizing the ex vivo Ussing chamber system. Ileal mucosal membranes harvested from normal male and female rats mounted in Ussing chamber systems were exposed to normoxia or 40 min of hypoxia at a normal pH (pH 7.3) or acidosis (pH 6.8). Cytokine and nitric oxide levels in the serosal compartment of the Ussing chamber were measured at the end of the 3-h experimental period to assess the immunoinflammatory response, whereas FITC-dextran (mol wt 4,300) was employed to assess barrier function. Histomorphological changes were used to quantitate gut mucosal injury. Hypoxia, acidosis, or hypoxia plus acidosis was associated with a significant increase in proinflammatory cytokine production [interleukin (IL)-6, tumor necrosis factor, and macrophage inflammatory protein (MIP)-2] by the male compared with the female intestinal segments. In contrast, the female gut manifested a higher anti-inflammatory response (nitric oxide and IL-10) and improved intestinal barrier function as well as less evidence of mucosal injury than the male intestinal segments. Administration of estradiol or the testosterone receptor antagonist, flutamide, to male rats abrogated the increase in gut injury and the increased IL-6 and MIP-2 response observed after hypoxia plus acidosis. These results suggest that gender differences in the ex vivo intestinal response to stresses, such as hypoxia and acidosis, exist and that the administration of estradiol or blockade of the testosterone receptor to male rats mitigates these gender differences.Ussing chamber; gender; ileal membrane; hypoxia/low pH; nitric oxide ALTHOUGH MULTIPLE ORGAN DYSFUNCTION syndrome (MODS) is the leading cause of death in intensive care units today (14), effective therapies have been slow to be developed, at least in part, because of an incomplete understanding of its basic biology (14). Consequently, studies directed at elucidating the pathophysiology of MODS have assumed major clinical importance. Over the years, we and others have focused on the role of gut ischemia/injury as a factor in the subsequent development of MODS (7, 13). Most recently, these studies have documented that, after trauma-hemorrhagic shock (T/ HS), the gut becomes a cytokine-generating organ (17) and that factors exiting the gut via the mesenteric lymphatics contribute to lung injury, endothelial cell dysfunction, and neutrophil activation (2, 14 -16, 19, 41). Although these studies illust...