Synergistic cytokine-induced nitric oxide production in human alveolar epithelial cells.

Nitric oxide (NO) is an important mediator molecule in regulating normal airway function, as well as in the pathophysiology of inflammatory airway diseases. In addition, cytokines are potent messenger molecules at sites of inflammation. The specific relationship among IL-1beta, TNF-alpha, and IFN-gamma on iNOS induction and NO synthesis in human alveolar epithelial cells has not been determined. In addition, rigorous methods to determine potential synergistic action between the cytokines have not been employed. We exposed monolayer cultures of A549 cells to a factorial combination of three cytokines (IL-1beta, TNF-alpha, and IFN-gamma) and three concentrations (0, 5, and 100 ng/mL). TNF-alpha alone does not induce NO production directly; however, it does have a stimulatory effect on IL-1beta-induced NO production. IL-1beta and INF-gamma both induce NO production alone, yet at different concentration thresholds, and act synergistically when present together. In the presence of all three cytokines, the net effect of NO production exceeds the predicted additive effect of each individual cytokine and the two-way interactions. Several plausible mechanisms of synergy among IL-1beta, TNF-alpha, and IFN-gamma in NO production from human alveolar epithelial cells (A549) are proposed. In order to verify the proposed mechanisms of synergy, future experimental and theoretical studies must address several molecular steps through which the iNOS gene is expressed and regulated, as well as the expression and regulation of enzyme cofactors and substrates.     

Regulation of inducible nitric oxide synthase in proinflammatory cytokine-stimulated human primary astrocytes

The present study was undertaken to investigate the mechanism of expression of inducible nitric oxide synthase (iNOS) in human primary astrocytes. Among IL-1beta, TNF-alpha, and IFN-gamma, only IL-1beta alone was capable of inducing iNOS. Similarly, among different cytokine combinations, the combinations involving only IL-1beta as a partner were capable of inducing iNOS. The combination of IL-1beta and IFN-gamma (IL-IF) induced the expression of iNOS at the highest level. All three cytokines alone induced the activation of AP-1 while IL-1beta and TNF-alpha but not IFN-gamma induced the activation of NF-kappaB. However, among the three cytokines, only IL-1beta was capable of inducing the activation of CCAAT/enhancer-binding proteinbeta (C/EBPbeta), suggesting an essential role of C/EBPbeta in the expression of iNOS in astrocytes. Although IL-1beta and IFN-gamma alone induced the activation of AP-1, the combination of these two cytokines (IL-IF) markedly inhibited the activation of AP-1. Consistently, JNK-I, a specific inhibitor of JNK, inhibited IL-1beta-mediated activation of AP-1 and expression of iNOS. On the other hand, JNK-I had no effect on (IL-IF)-induced expression of iNOS, suggesting that the activation of AP-1 is involved only during the low level of iNOS induction by IL-1beta but not during the high level of induction by IL-IF. In contrast, the activation of gamma-activation site (GAS) was involved only during the high level of induction by IL-IF but not during the low level of induction by IL-1beta. However, the activation of NF-kappaB and C/EBPbeta was involved in the induction of iNOS by IL-1beta as well as by IL-IF.     

Tumor necrosis factor-alpha production by astrocytes. Induction by lipopolysaccharide, IFN-gamma, and IL-1 beta

Astrocytes have the capacity to secrete or respond to a variety of cytokines including IL-1, IL-6, IL-3, and TNF-alpha. In this study, we have examined the capacity of astrocytes to secrete TNF-alpha in response to a variety of biologic stimuli, particularly cytokines such as IL-1 and IFN-gamma, which are known to be present in the central nervous system during neurologic diseases associated with inflammation. Rat astrocytes do not constitutively produce TNF-alpha, but have the ability to secrete TNF-alpha in response to LPS, and can be primed by IFN-gamma to respond to a suboptimal dose of LPS. IFN-gamma and IL-1 beta alone do not induce TNF-alpha production, however, the combined treatment of IFN-gamma and IL-1 beta results in a striking synergistic effect on astrocyte TNF-alpha production. Astrocyte TNF-alpha protein production induced by a combined treatment of either IFN-gamma/LPS or IFN-gamma/IL-1 beta occurs in a dose- and time-dependent manner, and appears to require a "priming signal" initiated by IFN-gamma, which then renders the astrocyte responsive to either a suboptimal dose of LPS or IL-1 beta. Astrocyte TNF-alpha production by IFN-gamma/LPS stimulation can be inhibited by the addition of anti-rat IFN-gamma antibody, whereas IFN-gamma/IL-1-induced TNF-alpha production is inhibited by antibody to either IFN-gamma or IL-1 beta. Polyclonal antisera reactive with mouse macrophage-derived TNF-alpha neutralized the cytotoxicity of IFN-gamma/LPS and IFN-gamma/IL-1 beta-induced astrocyte TNF-alpha, demonstrating similarities between these two sources of TNF-alpha. We propose that astrocyte-produced TNF-alpha may have a pivotal role in augmenting intracerebral immune responses and inflammatory demyelination due to its diverse functional effects on glial cells such as oligodendrocytes and astrocytes themselves.     

HLA-B27 misfolding in transgenic rats is associated with activation of the unfolded protein response

The mechanism by which the MHC class I allele, HLA-B27, contributes to spondyloarthritis pathogenesis is unknown. In contrast to other alleles that have been examined, HLA-B27 has a tendency to form high m.w. disulfide-linked H chain complexes in the endoplasmic reticulum (ER), bind the ER chaperone BiP/Grp78, and undergo ER-associated degradation. These aberrant characteristics have provided biochemical evidence that HLA-B27 is prone to misfold. Recently, similar biochemical characteristics of HLA-B27 were reported in cells from HLA-B27/human beta2-microglobulin transgenic (HLA-B27 transgenic) rats, an animal model of spondyloarthritis, and correlated with disease susceptibility. In this study, we demonstrate that the unfolded protein response (UPR) is activated in macrophages derived from the bone marrow of HLA-B27 transgenic rats with inflammatory disease. Microarray analysis of these cells also reveals an IFN response signature. In contrast, macrophages derived from premorbid rats do not exhibit a strong UPR or evidence of IFN exposure. Activation of macrophages from premorbid HLA-B27 transgenic rats with IFN-gamma increases HLA-B27 expression and leads to UPR induction, while no UPR is seen in cells from nondisease-prone HLA-B7 transgenic or wild-type (nontransgenic) animals. This is the first demonstration, to our knowledge, that HLA-B27 misfolding is associated with ER stress that results in activation of the UPR. These observations link HLA-B27 expression with biological effects that are independent of immunological recognition, but nevertheless may play an important role in the pathogenesis of inflammatory diseases associated with this MHC class I allele.     

Efficacy of Setarud (IMod), a novel drug with potent anti-toxic stress potential in rat inflammatory bowel disease and comparison with dexamethasone and infliximab

The inflammatory bowel disease (IBD) is an idiopathic, immune-mediated and chronic intestinal condition. In the present study, the effect of Setarud (IMOD), a novel natural drug with known immunomodulatory, anti-inflammatory and antioxidant properties was investigated in experimental colitis in rats and compared with the dexamethasone and infliximab. Immunologic colitis was induced by intracolonic administration of a mixture of trinitrobenzene sulfonic acid (TNBS) and absolute ethanol in male Wistar rats. Animals were divided into 6 groups of sham (normal group), control (vehicle-treated), positive control (dexamethasone 1 mg/kg/day given orally and infliximab 5 mg/kg/day given subcutaneously) and 3 Setarud-treated groups (13.3, 20, 30 mg/kg/day given intraperitoneally). The treatment continued for 14 consecutive days and then animals were decapitated on the day 15 and distal colons were removed for macroscopic, microscopic, and biochemical assays. Biochemical markers, including TNF-alpha, IL-1beta, ferric reducing/antioxidant power (FRAP), myeloperoxidase (MPO) activity and thiobarbitoric acid-reactive substance (TBARS) were measured in the homogenate of colonic tissue. A remarkable reduction in macroscopic and histological damage scores was observed in the animals treated with Setarud. These findings were confirmed by decreased levels of TNF-alpha, interleukin-1beta, MPO activity and TBARS, and raised levels of FRAP in the colon tissue. These observations confirmed the immunomodulatory, anti-inflammatory and antioxidant properties of Setarud in experimental colitis, which was comparable to those of dexamethasone and infliximab.     

Inhibition of IFN-gamma-inducible protein-10 abrogates colitis in IL-10-/- mice

A deficiency in understanding the steps responsible for colitis is the lack of comprehension for the role chemokines play in mucosal inflammation. IFN-gamma-inducible protein-10 (IP-10) and CXCR3 are highly expressed at sites of colitis. Our findings show that IP-10 significantly contributes to the development of Th1 and inflammatory responses. Specifically, IP-10 inhibition in IL-10(-/-) mice attenuates the associated increases in serum and/or local amyloid A, IL-2, IL-6, TNF-alpha, IFN-gamma, IL-1alpha, and IL-1beta with colitis as compared with IL-10(-/-) mice that develop colitis similar to human Crohn's disease. Correspondingly, the rate or intensity of inflammation in IL-10(-/-) mice treated with anti-IP-10 Abs showed improved scoring of inflammation, compared with control IL-10(-/-) mice. This study provides important and novel information regarding IP-10 as a target for the treatment of colitis.     

The degenerative effect of a single intranigral injection of LPS on the dopaminergic system is prevented by dexamethasone,and not mimicked by rh-TNF-alpha,IL-1beta and IFN-gamma

It is becoming widely accepted that the inflammatory response is involved in neurodegenerative disease. In this context, we have developed an animal model of dopaminergic system degeneration by the intranigral injection of lipopolysaccharide (LPS), a potent inductor of inflammation. To address the importance of the inflammatory response in the LPS-induced degeneration of nigral dopaminergic neurones, we carried out two different kinds of studies: (i) the possible protective effect of an anti-inflammatory compound, and (ii) the effect of the intranigral injection of inflammatory cytokines (TNF-alpha, IL-1beta and IFN-gamma) on dopaminergic neurones viability. Present results show that dexamethasone, a potent anti-inflammatory drug that interferes with many of the features characterizing pro-inflammatory glial activation, prevented the loss of catecholamine content, Tyrosine hydroxylase (TH) activity and TH immunostaining induced by LPS-injection and also the bulk activation of microglia/macrophages. Surprisingly, injection of the pro-inflammatory cytokines failed to reproduce the LPS effect. Taken together, our results suggest that inflammatory response is implicated in LPS-induced neurodegeneration. This damage may be due, at least in part, to a cascade of events independent of that described for TNF-alpha/IL-1 beta/IFN-gamma.     

Estrogen receptor-β signaling modulates epithelial barrier function

Impaired epithelial barrier function and estrogens are recognized as factors influencing inflammatory bowel disease (IBD) pathology and disease course. Estrogen receptor-β (ERβ) is the most abundant estrogen receptor in the colon and a complete absence of ERβ expression is associated with disrupted tight-junction formation and abnormal colonic architecture. The aim of this study was to determine whether ERβ signaling has a role in the maintenance of epithelial permeability in the colon. ERβ mRNA levels and colonic permeability were assessed in IL-10-deficient mice and HLA-B27 rats by RT-PCR and Ussing chambers. ERβ expression and monolayer resistance were measured in HT-29 and T84 colonic epithelial monolayers by RT-PCR and electric cell-substrate impedance sensing. The effect of 17β-estradiol and an estrogen agonist [diarylpropionitrile (DPN)] and antagonist (ICI 182780) on epithelial resistance in T84 cells was measured. Expression of ERβ and proinflammatory cytokines was investigated in colonic biopsies from IBD patients. Levels of ERβ mRNA were decreased, whereas colonic permeability was increased, in IL-10-deficient mice and HLA-B27 transgenic rats prior to the onset of colitis. T84 cells demonstrated higher resistance and increased levels of ERβ mRNA compared with HT-29 cells. 17β-estradiol and DPN induced increased epithelial resistance in T84 cells, whereas an ERβ blocker prevented the increased resistance. Decreased ERβ mRNA levels were observed in colonic biopsies from IBD patients. This study suggests a potential role for ERβ signaling in the modulation of epithelial permeability and demonstrates reduced ERβ mRNA in animal models of colitis and colon of patients with inflammatory bowel disease.     

Proinflammatory properties of IL-4 in the intestinal microenvironment

IL-4 is involved in type 2 T helper cell (Th)2-type immune responses and, in some cases, can promote Th1 responses. However, the proinflammatory potential of IL-4 alone is unclear. In this study, we examined the ability of IL-4 to induce colitis after its overexpression in the colon using an adenoviral vector (Ad5) and compared results with those obtained after overexpression of IL-12, a cytokine implicated in several models of colitis. Overexpression of IL-4 or IL-12 caused a fatal colitis within 24 h in 60% of animals and was dose and strain dependent. IL-12-induced colitis was accompanied by the local expression of IFN-gamma and TNF-alpha but not IL-4 mRNA and protein. Conversely, IL-4-induced colitis was accompanied by the local expression of IL-4 and TNF-alpha but not IFN-gamma mRNA and protein. The Ad5-IL4-induced colitis did not persist beyond 3 days and was present in recombinase activation gene-2 (RAG-2)-/- mice but not in STAT6-/- mice. Acute lethal colitis induced by Ad5IL12 was T cell mediated and IFN-gamma receptor (IFN-gamma R) dependent. Furthermore, TNF-alpha was found to be important in the pathogenesis of Ad5IL-4 and Ad5IL-12-induced colitis. Results of this study indicate that IL-4 alone can act as a proinflammatory cytokine in the gut of normal mice, inducing a rapid onset and short-lived colonic injury while maintaining a Th2-type cytokine profile that functions via a local T cell-independent mechanism involving TNF-alpha.     

CD8 alpha beta T cells are not essential to the pathogenesis of arthritis or colitis in HLA-B27 transgenic rats

The class I MHC allele HLA-B27 is highly associated with the human spondyloarthropathies, but the basis for this association remains poorly understood. Transgenic rats with high expression of HLA-B27 develop a multisystem inflammatory disease that includes arthritis and colitis. To investigate whether CD8alphabeta T cells are needed in this disease, we depleted these cells in B27 transgenic rats before the onset of disease by adult thymectomy plus short-term anti-CD8alpha mAb treatment. This treatment induced profound, sustained depletion of CD8alphabeta T cells, but failed to suppress either colitis or arthritis. To address the role of CD8alpha(+)beta(-) cells, we studied four additional groups of B27 transgenic rats treated with: 1) continuous anti-CD8alpha mAb, 2) continuous isotype-matched control mAb, 3) the thymectomy/pulse anti-CD8alpha regimen, or 4) no treatment. Arthritis occurred in approximately 40% of each group, but was most significantly reduced in severity in the anti-CD8alpha-treated group. In addition to CD8alphabeta T cells, two sizeable CD8alpha(+)beta(-) non-T cell populations were also reduced by the anti-CD8alpha treatment: 1) NK cells, and 2) a CD4(+)CD8(+)CD11b/c(+)CD161a(+)CD172a(+) monocyte population that became expanded in diseased B27 transgenic rats. These data indicate that HLA-B27-retricted CD8(+) T cells are unlikely to serve as effector cells in the transgenic rat model of HLA-B27-associated disease, in opposition to a commonly invoked hypothesis concerning the role of B27 in the spondyloarthropathies. The data also suggest that one or more populations of CD8alpha(+)beta(-) non-T cells may play a role in the arthritis that occurs in these rats.     

Effects of six different cytokines on lymphocyte adherence to microvascular endothelium and in vivo lymphocyte migration in the rat

The first step in the migration of lymphocytes out of the blood is adherence of lymphocytes to endothelial cells (EC) in the postcapillary venule. It is thought that in inflammatory reactions cytokines activate the endothelium to promote lymphocyte adherence and migration into the inflammatory site. Injection of IFN-gamma, IFN-alpha/beta, and TNF-alpha into the skin of rats stimulated the migration of small peritoneal exudate lymphocytes (sPEL) into the injection site, and these cytokines mediated lymphocyte recruitment to delayed-type hypersensitivity, sites of virus injection, and in part to LPS. The effect of cytokines on lymphocyte adherence to rat microvascular EC was examined. IFN-gamma, IFN-alpha/beta, IL-1, TNF-alpha, and TNF-beta increased the binding of small peritoneal exudate lymphocyte (sPEL) to EC. IFN-gamma was more effective and stimulated adherence at much lower concentrations than the other cytokines. IL-2 did not increase lymphocyte adherence. LPS strongly stimulated lymphocyte binding. Treatment of EC, but not sPEL, enhanced adhesion, and 24 h of treatment with IFN-gamma and IL-1 induced near maximal adhesion. Lymph node lymphocytes, which migrate poorly to inflammatory sites, adhered poorly to unstimulated and stimulated EC, whereas sPEL demonstrated significant spontaneous adhesion which was markedly increased by IFN-gamma, IL-1, and LPS. Spleen lymphocytes showed an intermediate pattern of adherence. Combinations of IFN-gamma and TNF-alpha were additive in stimulating sPEL-EC adhesion. Depletion of sPEL and spleen T cells by adherence to IFN-gamma stimulated EC decreased the in vivo migration of the lymphocytes to skin sites injected with IFN-gamma, IFN-alpha/beta, TNF-alpha, poly I:C, LPS, and to delayed-type hypersensitivity reactions by 50%, and significantly increased the migration of these cells to normal lymph nodes, as compared to unfractionated lymphocytes. Thus the cytokines and lymphocytes involved in migration to cutaneous inflammation in the rat stimulate lymphocyte adhesion to rat EC in vitro, and IFN-gamma stimulated EC appear to promote the selective adhesion of inflammatory site-seeking lymphocytes.     

Protective role of arginase in a mouse model of colitis

Arginase is the endogenous inhibitor of inducible NO synthase (iNOS), because both enzymes use the same substrate, l-arginine (Arg). Importantly, arginase synthesizes ornithine, which is metabolized by the enzyme ornithine decarboxylase (ODC) to produce polyamines. We investigated the role of these enzymes in the Citrobacter rodentium model of colitis. Arginase I, iNOS, and ODC were induced in the colon during the infection, while arginase II was not up-regulated. l-Arg supplementation of wild-type mice or iNOS deletion significantly improved colitis, and l-Arg treatment of iNOS(-/-) mice led to an additive improvement. There was a significant induction of IFN-gamma, IL-1, and TNF-alpha mRNA expression in colitis tissues that was markedly attenuated with l-Arg treatment or iNOS deletion. Treatment with the arginase inhibitor S-(2-boronoethyl)-l-cysteine worsened colitis in both wild-type and iNOS(-/-) mice. Polyamine levels were increased in colitis tissues, and were further increased by l-Arg. In addition, in vivo inhibition of ODC with alpha-difluoromethylornithine also exacerbated the colitis. Taken together, these data indicate that arginase is protective in C. rodentium colitis by enhancing the generation of polyamines in addition to competitive inhibition of iNOS. Modulation of the balance of iNOS and arginase, and of the arginase-ODC metabolic pathway may represent a new strategy for regulating intestinal inflammation.     

Role of M-CSF-dependent macrophages in colitis is driven by the nature of the inflammatory stimulus

Although macrophages are considered a critical factor in determining the severity of acute inflammatory responses in the gut, recent evidence has indicated that macrophages may also play a counterinflammatory role. In this study, we examined the role of a macrophage subset in two models of colitis. Macrophage colony-stimulating factor (M-CSF)-deficient osteopetrotic mice (op/op) and M-CSF-expressing heterozygote (+/?) mice were studied following the induction of colitis by either dinitrobenzene sulfonic acid (DNBS) or dextran sulfate sodium (DSS). DNBS induced a severe colitis in M-CSF-deficient op/op mice compared with +/? mice. This was associated with increased mortality and more severe macroscopic and microscopic injury. Colonic tissue myeloperoxidase (MPO) activity as well as concentrations of TNF-alpha, IL-1beta, and IL-6 were higher and IL-10 lower in op/op mice with DNBS colitis. The severity of inflammation and mortality was attenuated in op/op mice that had received human recombinant M-CSF prior to the induction of colitis. In contrast, op/op mice appeared less vulnerable to colitis induced by DSS. Macroscopic damage, microscopic injury, MPO activity, and tissue concentrations of TNF-alpha, IL-1beta, and IL-6 were all lower in op/op mice compared with +/? mice with DSS colitis, and no changes were seen in IL-10. Macrophage inflammatory protein-1alpha concentrations were increased in op/op but not +/? mice following colitis induced by DNBS but not DSS. These results indicate that M-CSF-dependent macrophages may play either a pro- or counterinflammatory role in acute experimental colitis, depending on the stimulus used to induce colitis.     

Inhibition of inducible NO synthase by TH2 cytokines and TGF beta in rheumatoid arthritic synoviocytes: effects on nitrosothiol production.

The aim of this study was to compare the effects on NO production of IL-4, IL-10, and IL-13 with those of TGF-beta. RA synovial cells were stimulated for 24 h with IL-1 beta (1 ng/ml), TNF-alpha (500 pg/ml), IFN-gamma (10(-4)IU/ml) alone or in combination. Nitrite was determined by the Griess reaction, S-nitrosothiols by fluorescence, and inducible NO synthase (iNOS) by immunofluorescence and fluorescence activated cell sorter analysis (FACS). In other experiments, IL-4, IL-10, IL-13, and TGF beta were used at various concentrations and were added in combination with proinflammatory cytokines. The addition of IL-1 beta, TNF-alpha, and IFN-gamma together increased nitrite production: 257.5 +/- 35.8 % and S-nitrosothiol production : 413 +/- 29%, P < 0.001. None of these cytokines added alone had any significant effect. iNOS synthesis increased with NO production. IL-4, IL-10, IL-13, and TGF beta strongly decreased the NO production caused by the combination of IL-1 beta, TNF-alpha, and IFN-gamma. These results demonstrate that stimulated RA synoviocytes produce S-nitrosothiols, bioactive NO* compounds, in similar quantities to nitrite. IL-4, IL-10, IL-13, and TGF-beta decrease NO production by RA synovial cells. The anti-inflammatory properties of these cytokines may thus be due at least in part to their effect on NO metabolism.     

HLA-B27 rats develop osteopaenia through increased bone resorption without any change in bone formation

Osteopaenia is a common complication of inflammatory bowel diseases (IBD). However, the mechanisms of bone loss are still the subject of debate. The aims of this study were to investigate bone loss in HLA-B27 transgenic rats, a spontaneous model of colitis and to compare the results provided by the usual markers of bone remodelling and by direct measurement of bone protein synthesis. Systemic inflammation was evaluated in HLA-B27 rats and control rats from 18 to 27 months of age. Then bone mineral density, femoral failure load, biochemical markers of bone remodelling and protein synthesis in tibial epiphysis were measured. Bone mineral density was lower in HLA-B27 rats than in controls. Plasma osteocalcin, a marker of bone formation, and fractional protein synthesis rate in tibial epiphysis did not differ between the two groups of rats. In contrast, urinary excretion of deoxypyridinoline, a marker of bone resorption, was significantly increased in HLA-B27 rats. The present results indicate that bone fragility occurs in HLA-B27 rats and mainly results from an increase in bone resorption. Systemic inflammation may be the major cause of the disruption in bone remodelling homeostasis observed in this experimental model of human IBD.     

Corticosteroids and interferons inhibit cytokine-induced production of IL-8 by human endothelial cells

IL-8, secreted by endothelial cells at the site of inflammation, participates in recruitment and transmigration of leukocytes. IL-8 may also have pathophysiological consequences in inflammatory and immunological disorders. We have investigated the effect of interferons (IFNs) and glucocorticosteroids (GCs) on cytokine induced secretion and production of IL-8 by human umbilical endothelial cells (HUVEC). There was a low spontaneous secretion of IL-8 by unstimulated HUVEC which increased after 6 or 24 h of stimulation with the pro-inflammatory cytokines TNF-alpha or IL-1beta. IFN-gamma as well as the GCs, Dexamethasone and Budesonide, inhibited TNF-alpha induced IL-8 secretion in a dose-dependent manner. IFNs may have a general modulating effect, since IFN-alpha also inhibited the TNF-alpha-induced IL-8 secretion. There was a slight, but significant, increase in the content of intracellular IL-8 in stimulated HUVEC. However, there was no difference between stimulation with IL-1beta or TNF-alpha alone or in combination with IFNs or GCs, whereas inhibition of IL-8 secretion with monensin increased IL-8 content suggesting that IFNs and GCs inhibit synthesis rather than secretion of IL-8. In conclusion, IFNs or GCs may be useful for inhibiting IL-8 production by endothelial cells and could thus be used for therapeutic modulation of the inflammatory response.     

Acute and chronic responses associated with adrenomedullin administration in experimental colitis

Adrenomedullin (AM) is a 52 amino acid peptide and member of the calcitonin gene-related peptide (CGRP) super family. Given that AM has emerged as a potential immuno-regulatory and anti-inflammatory agent in various experimental models, this study has deepened into its possible therapeutic effect in intestinal inflammation analyzing the responses in both acute and chronic (14 and 21 days) phases of TNBS-induced colitis in rats. In the acute model, AM treatment reduced the incidence of diarrhea and the severity of colonic damage, and improved the survival rate at the three doses assayed (50, 100, and 200ng/kg animal). AM administration was able to reduce the early production of TNF-alpha and collaborated to maintaining basal levels of IFN-gamma and IL-10. In the chronic studies the peptide attenuated the extent of the damage with lesser incidence of weight loss and diarrhea (50 and 100ng/kg animal). Cellular neutrophil infiltration, with the subsequent increase in myeloperoxidase (MPO) levels caused by TNBS, was reduced after chronic AM administration. The peptide played a role in the evolution of Th1/Th2 cytokines balance and chronic disease recuperation: levels of proinflammatory TNF-alpha and IFN-gamma decreased and anti-inflammatory IL-10 increased significantly. Cyclooxygenase-2 (COX-2) and nitric oxide synthase (iNOS) protein expression were not modified by AM administration, although a reduction of nitric oxide (NO) production could be detected in the chronic model. These results support a role of AM as an anti-inflammatory factor with beneficial effects in intestinal inflammatory colitis.     

Differential regulation of human monocyte programmed cell death (apoptosis) by chemotactic factors and pro-inflammatory cytokines

In the absence of appropriate stimuli, monocytes undergo programmed cell death (PCD) or apoptosis. IL-1 beta and TNF-alpha prevent monocyte PCD, which suggests that viability may be regulated by biologically active peptides released during inflammation. To explore this possibility, we evaluated several chemotactic factors and pro-inflammatory cytokines for their ability to regulate PCD. The recruitment factors, FMLP, C fragment C5a, monocyte chemotactic protein-1, or transforming growth factor-beta 1, were incapable of rescuing monocytes from PCD nor did they enhance PCD, whereas several inflammatory cytokines in addition to IL-1 beta and TNF-alpha, including granulocyte-monocyte-CSF and IFN-gamma, prevented monocyte PCD provided that sufficient levels of these cytokines were continuously maintained in the cultures. Cytokine-mediated inhibition of PCD could be blocked by specific antisera, ruling out potential effects caused by LPS contamination. When tested at equivalent concentrations, IL-2, IL-4, and IL-6 had no effect on PCD indicating selectivity in cytokine modulation of monocyte PCD. Because monocytes produce IL-1 beta, TNF-alpha, and granulocyte-monocyte CSF when activated, the data suggest autocrine as well as paracrine control of cell survival and accumulation. The results also suggest that monocytes recruited to a site of inflammation will undergo PCD in the absence of specific cytokines and/or other stimuli that block this process.