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The innate immune response is essential for combating infectious disease. Macrophages and other cells respond to infection by releasing cytokines, such as interleukin-1β (IL-1β), which in turn activate a well-described, [...]

Spray-dried plasma (SDP) is a complex mixture of active proteins that modulates the immune response of gut-associated lymphoid tissue. We examined whether SDP and 1g concentrate (IC) supplementation could modulate cytokine expression and inflammatory mediators in rats challenged with Staphylococcus aureus enterotoxin B (SEB). Wistar-Lewis rats were fed diets supplemented with SDP (8% wt:wt), IC (1.5% wt:wt), or milk proteins (control diet) from weaning (d 21) to d 34 after birth. On d 32 and 35, the rats were given SEB (0.5 mg/kg; intraperitoneal). Six hours after the second SEB dose, jejunal mucosa and Peyer's patches (PP) from the small intestine were collected. The cytokines interferon-[gamma] (IFN[gamma]), tumor necrosis factor-[alpha] (TNF[alpha]), interleukin (IL)-6, IL-10, transforming growth factor-[beta] (TGF[beta]), and leukotrienne [B.sub.4] (LT[B.sub.4]) were analyzed using commercial kits. SEB increased the release of proinflammatory mediators (IFN[gamma], TNF[alpha] IL-6, and LT[B.sub.4]) in PP (P< 0.05) and in the mucosa (P< 0.05). In both tissues, SDP prevented the increase in IFN[gamma], IL-6, and LT[B.sub.4] induced by SEB (P < 0.05). IC reduced the expression of TNF[alpha] and LT[B.sub.4] in PP and mucosa (P < 0.05). SDP supplementation increased IL-10 and mature TGF[beta] concentrations in intestinal mucosa from both inflamed and noninflamed rats. Both SDP and IC increased the mature:total TGF[beta] ratio (all P < 0.05). Both supplements were effective at preventing the SEB-induced increase in proinflammatory:antiinflammatory cytokine ratios in PP and mucosa and in serum. The preventive effects of plasma supplements on intestinal inflammation involve modulation of intestinal cytokines, characterized by an increased expression of antiinflammatory cytokines. J. Nutr. 140: 25-30, 2010. doi: 10.3945/jn.109.112466.

Several lines of studies have suggested that activins are critical mediators of inflammation and tissue repair. As activins and their receptors are expressed in the gastrointestinal tract, we tested the hypothesis that activin signaling is involved in the development of colitis by using two murine models of colitis induced by dextran sodium sulfate (DSS) or in mdr1a-/- mice. By immunohistochemistry, expression of activins was found increased in both models and correlated with the severity of inflammation. Activin expression was observed in macrophages as well as in some nonmacrophage cells. Furthermore, while activin receptors are normally expressed in colonic epithelial cells, their expression was further increased in both epithelial cells and inflammatory cells in inflamed colonic mucosa. Moreover, in vitro studies showed that activin A inhibited proliferation and induced apoptosis of intestinal epithelial cells, and this growth inhibition was largely reversed by administration of the activin inhibitor, follistatin. Because we also observed an increased number of apoptotic epithelial cells in both colitis models, the upregulation of activins occurring in colitis could be involved both in the inflammatory process and in growth inhibition of the intestinal epithelium. Importantly, in vivo administration of follistatin attenuated inflammatory cell infiltration during colitis. Rectal bleeding was reduced, and the integrity of epithelium was preserved in the DSS/follistatin-treated group compared with the group treated with DSS alone. Bromodeoxyuridine incorporation studies showed an increase in proliferative epithelial cells in the DSS/ follistatin-treated group, suggesting that follistatin accelerates epithelial cell proliferation/repair during colitis. Overall, our results reveal that activin signaling may play an important role in the pathogenesis and resolution of colitis. These findings suggest new therapeutic options in inflammatory bowel diseases. follistatin; inflammation; proliferation doi: 10.1152/ajpgi.90631.2008

There is a growing appreciation that endogenously produced mediators may actively promote the resolution of inflammation. Lipoxins (LX) are a group of recently discovered lipid mediators that have been shown to exert anti-inflammatory and proresolution effects on cells of myeloid and nonmyeloid origin. LXs mediate a number of processes, including regression of pro-inflammatory cytokine production, inhibition of cell proliferation, and stimulation of phagocytosis of apoptotic leukocytes by macrophages. Lipoxin [A.sub.4] ([LXA.sub.4]) is one of the principal LXs formed by mammalian cells. Recently, a G protein-coupled receptor that binds [LXA.sub.4]. the lipoxin [A.sub.4] receptor, was identified in astrocytes and microglia, suggesting that these cells may be a target for LX action in the brain. In this study, we have investigated the potential of [LXA.sub.4] to modify inflammatory responses of astrocytes, using the 1321N1 human astrocytoma cell line as a model system. As shown by quantitative RT-PCR, [LXA.sub.4] (10 nM) significantly inhibited (P < 0.05) the IL-1[beta]-induced stimulation of IL-8 and ICAM-1 expression in these cells. Furthermore, [LXA.sub.4] (10 nM) decreased the expression of IL-1[beta]-induced IL-8 protein levels (P < 0.05). [LXA.sub.4] (10 nM) was found to inhibit IL-1[beta]-induced degradation of I[kappa]B[alpha] (P < 0.05), and the activation of an NF[kappa]B regulated reporter gene construct (P < 0.05). Overall, these data suggest that [LXA.sub.4] exerts anti-inflammatory effects in 1321N1 astrocytoma cells at least in part via an NF[kappa]B-dependent mechanism. It is concluded that [LXA.sub.4] may represent a potentially novel therapeutic approach to acute or chronic inflammation in the brain. neuroinflammation; formyl-peptide receptor like-1; alxr

The aim of the present study was to evaluate the effect of overstimulation of [beta]-adrenoceptors on vascular inflammatory mediators. Wistar rats were treated with the [beta]-adrenoceptor agonist isoproterenol (0.3 mg*[kg.sup.-1]*[day.sup.-1] sc) or vehicle (control) for 7 days. At the end of treatment, the right carotid artery was catheterized for arterial and left ventricular (LV) hemodynamic evaluation. Isoproterenol treatment increased LV weight but did not change hemodynamic parameters. Aortic mRNA and protein expression were quantified by real-time RT-PCR and Western blot analysis, respectively. Isoproterenol enhanced aortic mRNA and protein expression of IL-1[beta] (124% and 125%) and IL-6 (231% and 40%) compared with controls but did not change TNF-[alpha] expression. The nuclear-to-cytoplasmatic protein expression ration of the NF-[kappa]B p65 subunit was increased by isoproterenol treatment (51%); in addition, it reduced the cytoplasmatic expression of I[kappa]B-[alpha] (52%) in aortas. An electrophoretic mobility shift assay was performed using the aorta, and increased NF-[kappa]B DNA binding (31%) was observed in isoproterenol-treated rats compared with controls (P < 0.05). Isoproterenol treatment increased phenylephrine-induced contraction in aortic rigs (P < 0.05), which was significantly reduced by superoxide dismutase (150 U/ml) and sodium salicylate (5 mM). Cotreatment with thalidomide (150 mg*[kg.sup.-1]*[day.sup.-1] for 7 days) also reduced hyperreactivity to phenylephrine induced by isoproterenol. In conclusion, overstimulation of [beta]-adrenoceptors increased proinflammatory cytokines and upregulated NF-[kappa]B in the rat aorta. Moreover, local oxidative stress and the proinflammatory state seem to play key roles in the altered vascular reactivity of the rat aorta induced by chronic [beta]-adrenergic stimulation. [beta]-adrenoceptor; blood vessels; cytokines; nuclear factor-[kappa]B

High-mobility group box 1 (HMGB 1) released from necrotic cells or macrophages functions as a late inflammatory mediator and has been shown to induce cardiovascular collapse during sepsis. Thus far, however, the effect(s) of HMGB1 in the heart are not known. We determined the effects of HMGB 1 on isolated feline cardiac myocytes by measuring sarcomere shortening in contracting cardiac myocytes, intracellular [Ca.sup.2+] transients by using fluo-3, and L-type calcium currents by using whole cell perforate configuration of the patch-clamp technique. Treatment of isolated myocytes with HMGB1 (100 ng/ml) resulted in a 70% decrease in sarcomere shortening and a 50% decrease in the height of the peak [Ca.sup.2+] transient within 5 rain (P < 0.01). The immediate negative inotropic effects of HMGB1 on cell contractility and calcium homeostasis were partially reversible upon washout of HMGB1. A significant inhibition of the inward L-type calcium currents was also documented by the patch-clamp technique. HMGB1 induced the PKC-[epsilon] translocation, and a PKC inhibitor significantly attenuated the negative inotropic effects of HMGB1. These studies show for the first time that HMGB1 impairs sarcomere shortening by decreasing calcium availability in cardiac myocytes through modulating membrane calcium influx and suggest that HMGB1 maybe acts as a novel myocardial depressant factor during cardiac injury. inflammation; innate immunity; myocardial function; sepsis

Altered function of [Na.sup.+] channels is responsible for increased hyperexcitability of primary afferent neurons that may underlie pathological pain states. Recent evidence suggests that the Navl.9 subunit is implicated in inflammatory but not acute pain. However, the contribution of Nav1.9 channels to the cellular events underlying nociceptor hyperexcitability is still unknown, and there remains much uncertainty as to the biophysical properties of Nav1.9 current and its modulation by inflammatory mediators. Here, we use gene targeting strategy and computer modeling to identify Nav1.9 channel current signature and its impact on nociceptors' firing patterns. Recordings using internal fluoride in small DRG neurons from wild-type and Nav1.9-null mutant mice demonstrated that Nav1.9 subunits carry the TTX-resistant 'persistent' [Na.sup.+] current called NaN. Nav1.9-/- nociceptors showed no significant change in the properties of the slowly inactivating TTX-resistant SNS/Nav1.8 current. The loss in Nav1.9-mediated [Na.sup.+] currents was associated with the inability of small DRG neurons to generate a large variety of electrophysiological behaviors, including subthreshold regenerative depolarizations, plateau potentials, active hyperpolarizing responses, oscillatory bursting discharges, and bistable membrane behaviors. We further investigated, using CsC1- and KCl-based pipette solutions, whether C-protein signaling pathways and inflammatory mediators upregulate the NaN/Nav1.9 current. Bradykinin, ATE histamine, prostaglandin-E2, and norepinephrine, applied separately at maximal concentrations, all failed to modulate the Nav1.9 current. However, when applied conjointly as a soup of inflammatory mediators they rapidly potentiated Nav1.9 channel activity, generating subthreshold amplification and increased excitability. We conclude that Nav1.9 channel, the molecular correlate of the NaN current, is potentiated by the concerted action of inflammatory mediators that may contribute to nociceptors' hyperexcitability during peripheral inflammation.

Sepsis is characterized by a systemic response to severe infection. Although the inflammatory phase of sepsis helps eradicate the infection, it can have detrimental consequences if left unchecked. Therapy directed against inflammatory mediators of sepsis has shown little success and has the potential to impair innate antimicrobial defenses. Heme oxygenase-1 (HO-1) and the product of its enzymatic reaction, CO, have beneficial antiinflammatory properties, but little is known about their effects on microbial sepsis. Here, we have demonstrated that during microbial sepsis, HO-1-derived CO plays an important role in the antimicrobial process without inhibiting the inflammatory response. HO-1-deficient mice suffered exaggerated lethality from polymicrobial sepsis. Targeting HO-1 to SMCs and myofibroblasts of blood vessels and bowel ameliorated sepsis-induced death associated with Enterococcus faecalis, but not Escherichia coli, infection. The increase in HO-1 expression did not suppress circulating inflammatory cells or their accumulation at the site of injury but did enhance bacterial clearance by increasing phagocytosis and the endogenous antimicrobial response. Furthermore, injection of a CO-releasing molecule into WT mice increased phagocytosis and rescued HO-1-deficient mice from sepsis-induced lethality. These data advocate HO-1-derived CO as an important mediator of the host defense response to sepsis and suggest CO administration as a possible treatment for the disease., Introduction Sepsis represents a complex process of cellular events induced by a severe underlying infection (1), (2). Sepsis has been defined clinically as a syndrome characterized by both the presence [...]

Eicosanoids are generally considered to be potent pro-inflammatory mediators, and their suppression has, therefore, been a desirable therapeutic goal. However, analysis of the literature reveals that inhibition of specific eicosanoids per se is a simplistic approach because it overlooks the fact that net pathophysiological effects of these lipid mediators arise from a complex balance between eicosanoids derived from different pathways, which might exhibit both pro-and anti-inflammatory activities (depending on organs and disease stage), or which might have essential physiological roles. An alternative strategy, discussed in this review, might be to control inflammatory lipid mediators in such a way as to avoid disrupting this intricate inter-eicosanoid balance and its physiological sequelae.

The silent information regulator 2 (Sir2) family of proteins (sirtuins or SIRTs), which belong to class III histone/protein deacetylases, have been implicated in calorie restriction, aging, and inflammation. We hypothesized that cigarette smoke-mediated proinflammatory cytokine release is regulated by SIRT1 by its interaction with NF-[kappa]B in a monocyte-macrophage cell line (MonoMac6) and in inflammatory cells of rat lungs. Cigarette smoke extract (CSE) exposure to MonoMac6 cells caused dose- and time-dependent decreases in SIRT1 activity and levels, which was concomitant to increased NF-[kappa]B-dependent proinflammatory mediator release. Similar decrements in SIRT1 were also observed in inflammatory cells in the lungs of rats exposed to cigarette smoke as well as with increased levels of several NF-[kappa]B-dependent proinflammatory mediators in bronchoalveolar lavage fluid and in lungs. Sirtinol, an inhibitor of SIRT1, augmented, whereas resveratrol, an activator of SIRT1, inhibited CSE-mediated proinflammatory cytokine release. CSE-mediated inhibition of SIRT1 was associated with increased NF-[kappa]B levels. Furthermore, we showed that SIRT1 interacts with the RelA/p65 subunit of NF-[kappa]B, which was disrupted by cigarette smoke, leading to increased acetylation RelA/ p65 in MonoMac6 cells. Thus our data show that SIRT1 regulates cigarette smoke-mediated proinflammatory mediator release via NF[kappa]B, implicating a role of SIRT1 in sustained inflammation and aging of the lungs. oxidants; resveratrol; MonoMac6 cells; chronic obstructive pulmonary disease; histone; acetylation; deacetylase

Abstract: Muramyl peptides derived from bacterial peptidoglycan are detected intracellularly by Nod1 and Nod2, 2 members of the newly characterized nod-like receptor (NLR) family of pattern recognition molecules. In the [...]

Following the sequencing of the human genome, data-mining efforts have revealed the existence of a new histamine receptor that is expressed at high levels in mast cells and leukocytes. The histamine [H.sub.4] receptor has a distinct pharmacological profile and the first compounds that act selectively on the [H.sub.4] receptor have been developed. Initial experiments in vivo with H4 receptor antagonists indicate a role for the [H.sub.4] receptor in inflammatory conditions.

Kinins are vasoactive peptides released from precursors called kininogens, and serum levels of both T- and K-kinmogens increase dramatically as rats age. Kinin release is tightly regulated, and here we show that serum kinin levels also increase with age, from 63 [+ or -] 16 nmol/L in young Fisher 344 rats to 398 [+ or -] 102 nmol/L in old animals. Both K- and T-kininogens contribute sequentially to this increase, with the increase in middle-aged animals being driven primarily by K-kininogen, whereas the further augmentation in older rats occurs by increasing T-kininogen. By measuring ERK activation, we show that aorta endothelial cells from old animals are hyporesponsive to exogenous bradykinin. However, if serum kinin levels are experimentally decreased by lipopolysaccharide treatment, then the endothelial response to bradykinin is reestablished. These results indicate that serum levels of kinins increase with age, whereas the responsiveness of target cells to kinins is reduced in these same animals.

The intravenous injection of LPS rapidly evokes fever. We have hypothesized that its onset is mediated by prostaglandin (PG)[E.sub.2] quickly released by Kupffer cells (Kc). LPS, however, does not stimulate PG[E.sub.2] production by Kc as rapidly as it induces fever; but complement (C) activated by LPS could be the exciting agent. To test this hypothesis, we injected LPS (2 or 8 [micro]g/kg) or cobra venom factor (CVF, an immediate activator of the C cascade that depletes its substrate, ultimately causing hypocomplementemia; 25 U/animal) into the portal vein of anesthetized guinea pigs and measured the appearance of PG[E.sub.2], TNF-[alpha], IL-1[beta], and IL-6 in the inferior vena cava (IVC) over the following 60 min. LPS (at both doses) and CVF induced similar rises in PG[E.sub.2] within the first 5 min after treatment; the rises in PG[E.sub.2] due to CVF returned to control in 15 min, whereas PG[E.sub.2] rises due to LPS increased further, then stabilized. LPS given 3 h after CVF to the same animals also elevated PG[E.sub.2], but after a 30- to 45-min delay. CVF per se did not alter basal PG[E.sub.2] and cytokine levels and their responses to LPS. These in vivo effects were substantiated by the in vitro responses of primary Kc from guinea pigs to C (0.116 U/ml) and LPS (200 ng/ml). These results indicate that LPS-activated C rather than LPS itself triggers the early release of PG[E.sub.2] by Kc. liver; fever; portal vein cannulation; cobra venom factor; pyrogenic cytokines

We previously showed that testosterone, administered in vivo, increases the tone of cerebral arteries. A possible underlying mechanism is increased vasoconstriction through the thromboxane [A.sub.2] (Tx[A.sub.2]) pathway. Therefore, we investigated the effect of chronic testosterone treatment (4 wk) on Tx[A.sub.2] synthase levels and the contribution of Tx[A.sub.2] to vascular tone in rat middle cerebral arteries (MCAs). Using immunofluorescence and confocal microscopy, we demonstrated that Tx[A.sub.2] synthase is present in MCA segments in both smooth muscle and endothelial layers. Using Western blot analysis, we found that Tx[A.sub.2] synthase protein levels are higher in cerebral vessel homogenates from testosteronetreated orchiectomized (ORX+T) rats compared with orchiectomized (ORX) control animals. Functional consequences of changes in cerebrovascular Tx[A.sub.2] synthase were determined using cannulated, pressurized MCA segments in vitro. Constrictor responses to the Tx[A.sub.2] mimetic U-46619 were not different between the ORX+T and ORX groups. However, dilator responses to either the selective Tx[A.sub.2] synthase inhibitor furegrelate or the Tx[A.sub.2]-endoperoxide receptor (TP) antagonist SQ-29548 were greater in the ORX+T compared with ORX group. In endothelium-denuded arteries, the dilation to furegrelate was attenuated in both the ORX and ORX+T groups, and the difference between the groups was abolished. These data suggest that chronic testosterone treatment enhances Tx[A.sub.2]-mediated tone in rat cerebral arteries by increasing endothelial Tx[A.sub.2] synthesis without altering the TP receptors mediating constriction. The effect of in vivo testosterone on cerebrovascular Tx[A.sub.2] synthase, observed here after chronic hormone administration, may contribute to the risk of vasospasm and thrombosis related to cerebrovascular disease. thromboxane synthase; cerebral circulation; vascular smooth muscle; endothelium

Leukotriene [A.sub.4] (LT[A.sub.4]) hydrolase catalyzes the final step in leukotriene [B.sub.4] (LT[B.sub.4]) synthesis. In addition to its role in LT[B.sub.4] synthesis, the enzyme possesses aminopeptidase activity. In this study, we sought to define the subcellular distribution of LT[A.sub.4] hydrolase in alveolar epithelial cells, which lack 5-lipoxygenase and do not synthesize LT[A.sub.4]. Immunohistochemical staining localized LT[A.sub.4] hydrolase in the nucleus of type II but not type I alveolar epithelial cells of normal mouse, human, and rat lungs. Nuclear localization of LT[A.sub.4] hydrolase was also demonstrated in proliferating type II-like A549 cells. The apparent redistribution of LT[A.sub.4] hydrolase from the nucleus to the cytoplasm during type II-to-type I cell differentiation in vivo was recapitulated in vitro. Surprisingly, this change in localization of LT[A.sub.4] hydrolase did not affect the capacity of isolated cells to convert LT[A.sub.4] to LT[B.sub.4]. However, proliferation of A549 cells was inhibited by the aminopeptidase inhibitor bestatin. Nuclear accumulation of LT[A.sub.4] hydrolase was also conspicuous in epithelial cells during alveolar repair following bleomycin-induced acute lung injury in mice, as well as in hyperplastic type II cells associated with fibrotic lung tissues from patients with idiopathic pulmonary fibrosis. These results show for the first time that LT[A.sub.4] hydrolase can be accumulated in the nucleus of type II alveolar epithelial cells and that redistribution of the enzyme to the cytoplasm occurs with differentiation to the type I phenotype. Furthermore, the aminopeptidase activity of LT[A.sub.4] hydrolase within the nucleus may play a role in promoting epithelial cell growth. aminopeptidase; bleomycin; fibrosis; leukotriene [B.sub.4]

We have used normal rat kidney (NRK) fibroblasts as an in vitro model system to study cell transformation. These cells obtain a transformed phenotype upon stimulation with growth-modulating factors such as retinoic acid (RA) or transforming growth factor-[beta] (TGF-[beta]). Patch-clamp experiments showed that transformation is paralleled by a profound membrane depolarization from around -70 to -20 mV. This depolarization is caused by a compound in the medium conditioned by transformed NRK cells, which enhances intracellular [Ca.sup.2+] levels and thereby activates [Ca.sup.2+]-dependent [Cl.sup.-] channels. This compound was identified as prostaglandin [F.sub.2.sub.[alpha]] (PG[F.sub.2.sub.[alpha]]) using electrospray ionization mass spectrometry. The active concentration in the medium conditioned by transformed NRK cells as determined using an enzyme immunoassay was 19.7 [+ or -] 2.5 nM (n = 6), compared with 1.5 [+ or -] 0.1 nM (n = 3) conditioned by nontransformed NRK cells. Externally added PG[F.sub.2.sub.[alpha]] was able to trigger NRK cells that had grown to density arrest to restart their proliferation. This proliferation was inhibited when the FP receptor (i.e., natural receptor for PG[F.sub.2.sub.[alpha]]) was blocked by AL-8810. RA-induced phenotypic transformation of NRK cells was partially (~25%) suppressed by AL-8810. Our results demonstrate that PG[F.sub.2.sub.alpha]] acts as an autocrine enhancer and paracrine inducer of cell transformation and suggest that it may play a crucial role in carcinogenesis in general. membrane potential; intracellular calcium; mass spectrometry; FP receptor

Objectives: To analyse the cerebrospinal fluid (CSF) values of the proinflammatory cytokines, interleukin 1β (IL1β), tumour necrosis factor a (TNFα), GM-CSF, of the anti-inflammatory cytokine TGFβ, of tau protein, a [...]

IL-17 is a T cell-derived, proinflammatory cytokine that is suspected to be involved in the development of various inflammatory diseases. Although there are elevated levels of IL-17 in synovial fluid of patients with rheumatoid arthritis, the pathogenic role of IL-17 in the development of rheumatoid arthritis remains to be elucidated. In this report, the effects of IL-17 deficiency were examined in IL-1 receptor antagonist-deficient (IL-1[Ra.sup.-/-]) mice that spontaneously develop an inflammatory and destructive arthritis due to unopposed excess IL-1 signaling. IL-17 expression is greatly enhanced in IL-1[Ra.sup.-/-] mice, suggesting that IL-17 activity is involved in the pathogenesis of arthritis in these mice. Indeed, the spontaneous development of arthritis did not occur in IL-1[Ra.sup.-/-] mice also deficient in IL-17. The proliferative response of ovalbumin-specific T cells from DO11.10 mice against ovalbumin cocultured with antigen-presenting cells from either IL-1[Ra.sup.-/-] mice or wild-type mice was reduced by IL-17 deficiency, indicating insufficient T cell activation. Cross-linking OX40, a cosignaling molecule on CD[4.sup.+] T cells that plays an important role in T cell antigen-presenting cell interaction, with anti-OX40 Ab accelerated the production of IL-17 induced by CD3 stimulation. Because OX40 is induced by IL-1 signaling, IL-17 induction is likely to be downstream of IL-1 through activation of OX40. These observations suggest that IL-17 plays a crucial role in T cell activation, downstream of IL-1, causing the development of autoimmune arthritis.

Weight loss can decrease blood levels of inflammatory markers that increase a person's risk of cardiovascular disease, according to a study of 120 obese women. These inflammatory markers include interleukin-6, interleukin-18, and C-reactive protein. This could explain why obesity is a risk factor for cardiovascular diseases, since fat cells can synthesize and secrete these inflammatory markers.

In this study, we examined the effect of [lambda]-carrageenan-induced inflammatory pain on the functional and structural properties of the rat blood-brain barrier (BBB) over a 72-h time period. Systemic inflammation was induced by an intraplantar injection of 3% [lambda]-carrageenan into the right hind paw of female Sprague-Dawley rats. In situ brain perfusion and Western blot analyses were performed at 1, 3, 6, 12, 24, 48, and 72 h. In situ brain perfusion showed [lambda]-carrageenan significantly increased brain uptake of [[sup.14]C]sucrose at 1, 3, 6, and 48 h (139 [+ or -] 9%, 166 [+ or -] 19%, 138 [+ or -] 13%, and 146 [+ or -] 7% compared with control, respectively). Capillary depletion analysis insured the increased brain uptake was due to increased BBB permeability and not vascular trapping. Western blot analyses for zonula occludens-1 (ZO-1) and occludin were performed on isolated cerebral microvessels. ZO-1 expression was significantly increased at 1, 3, and 6 h and returned to control expression levels by 12 h. Total occludin expression was significantly reduced at 1, 3, 6, 12, and 48 h. This investigation demonstrated that [lambda]-carrageenan-induced inflammatory pain elicits a biphasic increase in BBB permeability with the first phase occurring from 1-6 h and the second phase occuring at 48 h. Furthermore, changes in BBB function are correlated with altered tight junctional protein expression of occludin and ZO-1. Changes in the structure of tight junctions may have important clinical ramifications concerning central nervous system homeostasis and therapeutic drug delivery. inflammation; ZO-1; ZO-2; membrane-associated guanylate kinase; occludin; immunoprecipitation

Cytokines and other mediators whose induction in inflammatory lung disease is attenuated by glucocorticoids are potential targets for development of selective anti-inflammatory treatments. We refer to genes with these regulatory characteristics as glucocorticoid-attenuated response genes, or GARGs. Systematic identification of GARGs has not been attempted previously in vivo. Using an endotoxemia model in adrenalectomized mice, we constructed a subtracted lung library enriched in endotoxemia-induced genes and identified candidate GARGs by differential hybridization screening. Northern analysis confirmed induction in the lung during endotoxemia and attenuation by glucocorticoids of 36 genes of diverse types. The majority were genes of unknown function not previously implicated in the pulmonary response to inflammation, including a new member of a 2'-5'-oligoadenylate synthetase-like family and a novel lung inducible Neuralized-related C3HC4 RING protein. Our results suggest that a full understanding of glucocorticoid effects on lung inflammation will require elucidation of the roles of an extensive network of glucocorticoid-modulated genes. gene expression; inflammation; lipopolysaccharide; molecular cloning; mouse model

Mechanical forces affect both the function and phenotype of cells in the lung. In this symposium, recent studies were presented that examined several aspects of biomechanics in lung cells and their relationship to disease. Wound healing and recovery from injury in the airways involve epithelial cell spreading and migration on a substrate that undergoes cyclic mechanical deformation; enhanced green fluorescent protein-actin was used in a stable cell line to examine cytoskeletal changes in airway epithelial cells during wound healing. Eosinophils migrate into the airways during asthmatic attacks and can also be exposed to cyclic mechanical deformation; cyclic mechanical stretch caused a decrease in leukotriene [C.sub.4] synthesis that may be dependent on mechanotransduction mechanisms involving the production of reactive oxygen species. Recent studies have suggested that proinflammatory cytokines are increased in ventilator-induced lung injury and may be elevated by overdistention of the lung tissue; microarray analysis of human lung epithelial cells demonstrated that cyclic mechanical stretch alone profoundly affects gene expression. Finally, airway hyperresponsiveness is a basic feature of asthma, but the relationship between airway hyperresponsiveness and changes in airway smooth muscle (ASM) function remain unclear. New analysis of the behavior of the ASM cytoskeleton (CSK) suggests, however, that the CSK may behave as a glassy material and that glassy behavior may account for the extensive ASM plasticity and remodeling that contribute to airway hyperresponsiveness. Together, the presentations at this symposium demonstrated the remarkable and varied roles that mechanical forces may play in both normal lung physiology as well as pathophysiology. actin remodeling; 5-lipoxygenase; inflammatory cytokines; mechanical properties

A study of genetic variations in the toll-like receptor supports the theory that atherosclerosis may involve inflammation. In a study of 810 people, those who had two particular genetic variations of this receptor were 50% less likely to have atherosclerosis in the carotid arteries. The toll-like receptors are involved in the immune response to bacteria.

Results show that CXCL1 induces PAK1 kinase activation through the Rho GTPase subfamily signal cascade member, cdc42. The cdc42-PAK1 cascade in turn mediates the CXCL1-induced chemotaxis.

Most previous stress-immune research focused on the immunosuppressive effects of stress on acquired immunity. More recently, it has become clear that acute stressor exposure can potentiate innate, as well as suppress acquired, immunity. For example, acute stress improves recovery from bacterial inflammation, a classic in vivo measure of innate immunity. The previous work was done in sedentary organisms. Physical activity status can modulate the impact of stress on immune function. The following studies tested the hypothesis that the effect of stress on inflammation after subcutaneous challenge with bacteria (Escherichia coli) is facilitated by physical activity. The results were that sedentary, stressed rats resolved their inflammation 1-2 days faster and have increased circulating neutrophils compared with their nonstressed, sedentary counterparts. In contrast, physically active, stressed rats resolve their inflammation 3-4 days faster and have increased circulating and inflammatory site neutrophils compared with their nonstressed counterparts. Importantly, the beneficial impact of stress on inflammation recovery and neutrophil migration was greater in the physically active, than sedentary, stressed rats. Thus physical activity status facilitates the positive effect of acute stress on innate immunity. physical activity; neutrophil; inflammation; Escherichia coli

The influence of stress and diazepam treatment on airway inflammation was investigated in ovalbumin (OVA)-sensitized rats. Animals were injected with OVA plus aluminum hydroxide intraperitoneally (day 0) and boosted with OVA subcutaneously (day 7). From the first to 13th day after sensitization, rats were treated with diazepam, and 1 h later they were placed in a shuttle box where they received 50 mild escapable foot shocks/day preceded by a sound signal (S). Response during the warning (S) canceled shock delivery and terminated the S. On day 14, rats were submitted to a single session of 50 inescapable foot shocks preceded by S and then were challenged with OVA. High levels of stress were detected in shocked animals, manifested as ultrasonic vocalizations. Morphometric analysis of stressed animals revealed a significant increase in both edema and lymphomononucleated cells in airways compared with controls. Diazepam treatment reduced edema in stressed and nonstressed rats. No differences were found in polymorphonucleated cell infiltration. Diazepam treatment reduced lymphomononucleated cell infiltration in stressed animals. These data suggest that stress and diazepam treatment play relevant roles in edema and lymphomononucleated airway inflammation in OVA-sensitized rats. ovalbumin; allergic inflammation; peribronchial edema; lymphomononucleated cells; aversive stimulation; asthma

Hepatocyte gap junction proteins, connexins (Cxs) 26 and 32, are downregulated during obstructive cholestasis (OC) and lipopolysaccharide hepatocellular cholestasis (LPS-HC). We investigated rat hepatic Cxs during ethynylestradiol hepatocellular cholestasis (EE-HC) and choledochocaval fistula (CCF) and compared them with OC and LPS-HC. Levels (immunoblotting) and cellular distribution (immunofluorescence) of Cx26, -32, and -43, as well as macrophage infiltration, were studied in livers of rats under each condition. Cx26 and -32 were reduced in LPS-HC, OC, and CCF. However, in EE-HC, Cx26 did not change and Cx32 was increased. Prominent inflammation occurred in LPS-HC, OC, and CCF, which was associated with increased levels of Cx43 in LPS-HC and OC but not CCF. No inflammation nor changes in Cx43 levels occurred during EE-HC. In cultured hepatocytes, dye coupling was reduced by tumor necrosis factor-[alpha] and interleukins-1[beta] and -6, whereas reduction induced by LPS required coculture with Kupffer cells. Thus hepatocyte gap junctions are down-regulated in forms of cholestasis associated with inflammation, and reduced intercellular communication might be induced in part by proinflammatory mediators. obstructive cholestasis; hepatocellular cholestasis; cytokines; macrophages.

By pharmacological manipulation of endogenous adenosine, using chemically distinct methods, we tested the hypothesis that endogenous adenosine tempers proinflammatory cytokine responses and oxyradical-mediated tissue damage during endotoxemia and sepsis. Rats were pretreated with varying doses of pentostatin (PNT; adenosine deaminase inhibitor) or 8-sulfophenyltheophylline (8-SPT; adenosine receptor antagonist) and then received either E. coli endotoxin (lipopolysaccharide; 0.01 or 2.0 mg/kg) or a slurry of cecal matter in 5% dextrose in water (200 mg/kg). Resultant levels of tumor necrosis factor (TNF)-[alpha], interleukin (IL)-1[beta], and IL-10 were measured in serum and in liver and spleen. Untreated, 2 mg/kg lipopolysaccharide elevated serum TNF-[alpha], IL-1[beta], and IL-10. PNT dose dependently attenuated, without ablating, the elevation in serum TNF-[alpha] and IL-1[beta] and raised liver and spleen IL-10. PNT also attenuated elevation of TNF-[alpha] in serum, liver, and spleen at 4 and 24 h after sepsis induction, and 8-SPT resulted in higher proinflammatory cytokines. Modulating endogenous adenosine was also effective in exacerbated (8-SPT) or diminished (PNT) tissue peroxidation. Survival from sepsis was also improved when PNT was used as a posttreatment. These data indicate that endogenous adenosine is an important modulatory component of systemic inflammatory response syndromes. These data also indicate that inhibition of adenosine deaminase may be a novel and viable therapeutic approach to managing the systemic inflammatory response syndrome without ablating important physiological functions. shock; cytokines; oxyradical

Endothelial cells (EC) play a central role in inflammatory immune responses and efficiently induce effector functions in T cells, despite lacking the classical costimulatory ligands CD80 and CD86. By using the mAb HIL-131 we now demonstrate that human inducible costimulator-ligand (ICOS-L), a molecule related to CD80/ CD86, is constitutively expressed on human EC in vivo. In vitro, ICOS-L expression was strongly enhanced on human umbilical vein EC and microvascular EC by the inflammatory cytokines tumor necrosis factor [alpha] and IL-1[beta], and to a lower extent by stimulation of EC by CD40 or lipopolysaccharide. Coculture of MHC class [II.sup.+] EC with resting memory CD[4.sup.+] T cells in the presence of superantigen led to a marked up-regulation of ICOS on T cells and to the production of Th1 (IFN-[gamma], IL-2) and Th2 cytokines (IL-4, IL-10, IL-13). When these cocultures were performed in the presence of the inhibitory mAb HIL-131, secretion of all cytokines was reduced by about 50-80%, indicating that ICOS-L is a major costimulator in EC-mediated T cell activation. Taken together, our data suggest an important physiological role of ICOS-L in the reactivation of effector/memory T cells on the endothelium controlling the entry of immune cells into inflamed tissue.

Lung injury induced by acute endotoxemia is associated with increased generation of inflammatory mediators such as nitric oxide and eicosanoids, which have been implicated in the pathophysiological process. Although production of these mediators by alveolar macrophages (AM) has been characterized, the response of type II cells is unknown and was assessed in the present studies. Acute endotoxemia caused a rapid (within 1 h) and prolonged (up to 48 h) induction of nitric oxide synthase-2 (NOS-2) in type II cells but a delayed response in AM (12-24 h). In both cell types, this was associated with increased nitric oxide production. Although type II cells, and to a lesser extent AM, constitutively expressed cyclooxygenase-2, acute endotoxemia did not alter this activity. Endotoxin administration had no effect on mitogen-activated protein kinase or protein kinase B-[alpha] (PKB-[alpha]) expression. However, increases in phosphoinositide 3-kinase and phospho-PKB-[alpha] were observed in type II cells. The finding that this was delayed for 12-24 h suggests that these proteins do not play a significant role in the regulation of NOS-2 in this model. After endotoxin administration to rats, a rapid (within 1-2 h) activation of nuclear factor-KB was observed. This response was transient in type II cells but was sustained in AM. Interferon regulatory factor-1 (IRF-1) was also activated rapidly in type II cells. In contrast, IRF-1 activation was delayed in AM. These data demonstrate that type II cells, like AM, are highly responsive during acute endotoxemia and may contribute to pulmonary inflammation. type II cells; alveolar macrophages; nitric oxide synthase-2; cycloxygenase-2; nuclear factor-KB; interferon regulatory factor-1

Mice deficient in inducible nitric oxide synthase (iNOS; C57Bl/6Ai-[KO]NOS2 N5) or wild-type C57Bl/6 mice were exposed to 1 part/million of ozone 8 h/night or to filtered air for three consecutive nights. Endpoints measured included lavagable total protein, macrophage inflammatory protein (MIP)-2, matrix metalloproteinase (MMP)-9, cell content, and tyrosine nitration of whole lung proteins. Ozone exposure caused acute edema and an inflammatory response in the lungs of wild-type mice, as indicated by significant increases in lavage protein content, MIP-2 and MMP-9 content, and polymorphonuclear leukocytes. The iNOS knockout mice showed significantly greater levels of lung injury by all of these criteria than did the wild-type mice. We conclude that iNOS knockout mice are more susceptible to acute lung damage induced by exposure to ozone than are wild-type C57Bl/6 mice and that protein nitration is associated with the degree of inflammation and not dependent on iNOS-derived nitric oxide. nitrotyrosine; nitric oxide; inflammation; matrix metalloproteinase-9; macrophage inflammatory protein-2

Anorexia and weight loss are frequent complications of acute and chronic infections and result from induction of cytokines, prostaglandins, and other inflammatory mediators that are critical for pathogen elimination. Selective attenuation of the hypophagic response to infection and maintenance of the production of factors essential for infection control would be a useful addition to antimicrobial therapy in the treatment of human disease. Here, we evaluate the relative contribution of cyclooxygenase (COX)-1- and COX-2-derived prostaglandins to anorexia and weight loss precipitated by systemic immune activation by lipopolysaccharide (LPS). Using COX isoform-selective pharmacological inhibitors and gene knockout mice, we found that COX-2 inhibition during LPS-induced inflammation results in preserved food intake and maintenance of body weight, whereas COX-1 inhibition results in augmented and prolonged weight loss. Regulation of neuropeptide Y, corticotropin-releasing hormone, leptin, and interleukin-6 does not change as a function of COX-2 inhibition after LPS administration. Our data implicate COX-2 inhibition as a therapeutic target to maintain nutritional status while still allowing a normal cytokine response during infection. nonsteroidal anti-inflammatory drugs; knockout mice; appetite; cyclooxygenase-2

The function of urokinase and its receptor is essential for cell migration in pathological conditions, as shown by the analysis of knockout mice phenotypes. How a protease of a fibrinolytic pathway can induce migration is not understood and no link between this protease and migration-promoting G protein-coupled receptors has been described. We now show that FPRL1/LXA4R, a G protein-coupled receptor for a number of polypeptides and for the endogenous lipoxin A4 (LXA4), is the link between urokinase-type plasminogen activator (uPA) and migration as it directly interacts with an activated, soluble, cleaved form of uPA receptor (uPAR) (D2[D3.sub.88-274]) to induce chemotaxis. In this article we show that (i) both uPAR and FPRL1/LXA4R are necessary for the chemotactic activity of uPA whereas FPRL1/LXA4R is sufficient to mediate D2[D3.sub.88-274]-induced cell migration. (ii) Inhibition or desensitization of FPRL1/LXA4R by antibodies or specific ligands specifically prevents chemotaxis induced by D2[D3.sub.88-274] in THP-1 cells and human peripheral blood monocytes. (iii) Desensitization of FPRL1/ LXA4R prevents the activation of tyrosine kinase Hck induced by D2[D3.sub.88-274]. (iv) D2[D3.sub.88-274] directly binds to FPRL1/LXA4R and is competed by two specific FPRL1/LXA4R agonists, the synthetic MMK-1 peptide and a stable analog of LXA4. Thus, a naturally produced cleaved form of uPAR is a unique endogenous chemotactic agonist for FPRL1/LXA4R receptor and its activity can be antagonized by specific ligands. These results provide the first direct link, to our knowledge, between the fibrinolytic machinery and the inflammatory response, demonstrating that uPA-derived peptide fragments can activate a specific chemotactic receptor.

Research demonstrates that dermal injections of the Pseudomonas aeruginosa quorum sensing molecule N-(3-oxododecanoyl)homoserine lacton activate T cells to produce inflammatory cytokine gamma interferon, which causes influx of white blood cells and tissue destruction in mice.

The genomic information contained in the AlgU sigma factor-dependent promoters in Pseudomonas aeruginosa can be used for uncovering the genes under a defined sigma factor at a global level. Data also suggest a relationship between mucoidy and lipoprotein expression with proinflammatory implication of the former in cystic fibrosis.