Local Inflammatory Milieu Research Articles

Mentation on the immunological modulation of gastrointestinal motility

The immunological modulation of gastrointestinal motility is currently one of the most dynamic and fascinating areas of enteric research, as investigators are beginning to focus their studies on the pathophysiology of various gastrointestinal dysmotilities. The new fruits of this investigative initiative has resulted in the appearance of a fascinating series of articles which demonstrate that intestinal inflammatory events alter a distinct population of enteric neurons and that these alterations last long past the apparent resolution of the inciting event. Studies over the past few years have unequivocally demonstrated that the muscularis externa itself is an active and complex immunological compartment with unique features. The rodent muscularis externa is constitutively populated by a dense network of muscularis macrophages throughout the entire gastrointestinal tract. Although few other leukocytes are present in the rodent, the human muscularis is densely populated by both macrophages and mast cells. Postoperative ileus and endotoxin-induced ileus have turned out to be extremely useful rodent models to elucidate the importance of muscularis leukocytes in causing intestinal dysfunction. Using models of ileus, studies have demonstrated that a complex molecular inflammatory scenario is triggered within the muscularis externa, which consists of MAP kinase phosphorylation, transcriptior factor activation and the subsequent induction of various cytokines, chemokines and, importantly, smooth muscle inhibitory substances, such as nitric oxide and prostaglandins from iNOS and COX-2. This local molecular inflammatory milieu leads to leukocyte extravasation. Data suggests that the muscularis macrophage network is the conductor of the molecular and cellular inflammatory responses which causes ileus.

Type I interferon signaling is involved in the spontaneous development of lupus-like disease in B6.Nba2 and (B6.Nba2 × NZW)F1 mice

Several studies have described a role for type I interferons (IFNalphabeta) in the initiation and/or prolongation of autoimmune diseases. Most pronounced has been the association of disease activity with what is now known as 'the interferon signature' of gene expression in peripheral blood mononuclear cells from lupus patients. In correlation, studies have shown that inhibition of IFNalphabeta signaling abrogates disease in various mouse models of lupus. New Zealand black (NZB) and B6.Nba2 congenic mice spontaneously develop elevated levels of serum anti-nuclear autoantibodies (ANAs). Nevertheless, neither of these strains develop fatal renal disease. The female F1 offspring of NZB or B6.Nba2 crossed with New Zealand white (NZW) mice do, however, develop kidney disease. We have previously shown that increases in endogenous IFNalphabeta levels in (B6.Nba2 x NZW)F1 mice leads to accelerated development of renal disease in an IFNalphabeta-dependent manner. We now show that B6.Nba2 and (B6.Nba2 x NZW)F1 mice deficient for the IFNalphabeta-receptor fail to develop ANA and renal disease, although the mice have substantial immune complex deposition in the glomeruli. Thus, endogenous IFNalphabeta might influence disease by affecting B-cell activation and differentiation, as well as the kidneys' susceptibility to damage, the latter perhaps through induction of a local inflammatory milieu.

Omega‐3 fatty acid regulates inflammatory cytokine/mediator messenger RNA expression in Porphyromonas gingivalis‐induced experimental periodontal disease

Porphyromonas gingivalis is strongly implicated in the etiology of adult periodontitis by inducing inflammatory cytokines, resulting in gingival and periodontal tissue inflammation and alveolar bone resorption. This study tested the hypothesis that supplementing the diet with omega-3 fatty acid (omega-3 FA; i.e. fish oil) would exert anti-inflammatory effects in the gingival tissues of P. gingivalis-infected rats. Rats were fed either fish oil or corn oil diets ad libitum for 22 weeks and infected with P. gingivalis strain 381 or strain A7A1-28. After sacrifice, rat gingival tissues were excised and the RNA was isolated and analyzed for proinflammatory mediators [interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), IL-6], T helper type 1 and type 2 cytokines [interferon-gamma (IFN-gamma), IL-4, IL-10), antioxidant enzymes [catalase (CAT), superoxide dismutase (SOD)], and genes critical for eicosanoid mediator production [cyclo-oxygenase-2 (COX-2), 5-lipoxygenase (5-LO)] by reverse transcription-polymerase chain reaction using rat-specific primers. Rats on the omega-3 FA diet exhibited decreased proinflammatory cytokine gene expression (IL-1beta, TNF-alpha) and enhanced IFN-gamma, CAT and SOD messenger RNA expression compared to rats fed a corn oil diet, supporting a diet-induced modulation of host inflammatory reactions. Analyses of alveolar bone resorption in the rats related to gene expression profiles demonstrated significant positive correlations with IL-1beta, IL-6 and COX-2 and negative correlations with CAT and SOD. These findings suggest that diets enriched for omega-3 FA modulate the local gingival inflammatory milieu of the host following oral P. gingivalis infection, which impacts on alveolar bone resorption in rats.

Expression of interleukin-16 by tumor-associated macrophages/activated microglia in high-grade astrocytic brain tumors

.IntroductionMacrophages/microglial cells are considered as immune cells in the central nervous system. Interleukin (IL)-16 is a proinflammatory cytokine produced by activated monocytic cells.Materials and MethodsExpression of IL-16 was analyzed by immunohistochemistry in human astrocytic brain tumors and the rat C6 glioblastoma tumor model. IL-16 was detected in both human astrocytic brain tumors and rat C6 glioma.ResultsCompared with human control brains, a significant increase in the percentages of parenchymal IL-16+ macrophages/microglia was observed already in grade II astrocytomas, indicating that IL-16+ immunostaining could be a descriptor of a macrophage/microglia subset in astrocytic brain tumors. A further increase was observed at the transition from grade II to III astrocytomas. This increase in IL-16 immunoreactivity correlated with WHO grades of human astrocytic brain tumors.ConclusionsTherefore, IL-16 might be a so far unknown factor in the regulation of the local inflammatory milieu of human and experimental astrocytomas.

High Mobility Group B1 Protein Suppresses the Human Plasmacytoid Dendritic Cell Response to TLR9 Agonists

Plasmacytoid dendritic cells (PDC) are innate immune effector cells that are recruited to sites of chronic inflammation, where they modify the quality and nature of the adaptive immune response. PDCs modulate adaptive immunity in response to signals delivered within the local inflammatory milieu by pathogen- or damage-associated molecular pattern, molecules, and activated immune cells (including NK, T, and myeloid dendritic cells). High mobility group B1 (HMGB1) is a recently identified damage-associated molecular pattern that is released during necrotic cell death and also secreted from activated macrophages, NK cells, and mature myeloid dendritic cells. We have investigated the effect of HMGB1 on the function of PDCs. In this study, we demonstrate that HMGB1 suppresses PDC cytokine secretion and maturation in response to TLR9 agonists including the hypomethylated oligodeoxynucleotide CpG- and DNA-containing viruses. HMGB1-inhibited secretion of several proinflammatory cytokines including IFN-alpha, IL-6, TNF-alpha, inducible protein-10, and IL-12. In addition, HMGB1 prevented the CpG induced up-regulation of costimulatory molecules on the surface of PDC and potently suppressed their ability to drive generation of IFN-gamma-secreting T cells. Our observations suggest that HMGB1 may play a critical role in regulating the immune response during chronic inflammation and tissue damage through modulation of PDC function.

Metabolic Inactivation of Resolvin E1 and Stabilization of Its Anti-inflammatory Actions

The resolvins (Rv) are lipid mediators derived from omega-3 polyunsaturated fatty acids that act within a local inflammatory milieu to stop leukocyte recruitment and promote resolution. Resolvin E1 (RvE1; (5S,12R,18R)-trihydroxy-6Z,8E,10E,14Z,16E-eicosapentaenoic acid) is an oxygenase product derived from omega-3 eicosapentaenoic acid that displays potent anti-inflammation/pro-resolution actions in vivo. Here, we determined whether oxidoreductase enzymes catalyze the conversion of RvE1 and assessed the biological activity of the RvE1 metabolite. With NAD+ as a cofactor, recombinant 15-hydroxyprostaglandin dehydrogenase acted as an 18-hydroxyl dehydrogenase to form 18-oxo-RvE1. In the murine lung, dehydrogenation of the hydroxyl group at carbon 18 position to form 18-oxo-RvE1 represented the major initial metabolic route for RvE1. At a concentration where RvE1 potently reduced polymorphonuclear leukocyte (PMN) recruitment in zymosan-induced peritonitis, 18-oxo-RvE1 was devoid of activity. In human neutrophils, carbon 20 hydroxylation of RvE1 was the main route of conversion. An RvE1 analog, i.e. 19-(p-fluorophenoxy)-RvE1, was synthesized that resisted rapid metabolic inactivation and proved to retain biological activity reducing PMN infiltration and pro-inflammatory cytokine/chemokine production in vivo. These results established the structure of a novel RvE1 initial metabolite, indicating that conversion of RvE1 to the oxo product represents a mode of RvE1 inactivation. Moreover, the designed RvE1 analog, which resisted further metabolism/inactivation, could be a useful tool to evaluate the actions of RvE1 in complex disease models.

Role of osteopontin in amplification and perpetuation of rheumatoid synovitis

Osteopontin (OPN) is an extracellular matrix protein of pleiotropic properties and has been recently recognized as a potential inflammatory cytokine. In this study, we demonstrate, for the first time to our knowledge, that overexpression of OPN in synovial T cells is associated with local inflammatory milieu and that OPN acts as an important mediator in amplification and perpetuation of rheumatoid synovitis. The study revealed that mRNA expression of OPN was highly elevated in CD4(+) synovial T cells derived from patients with RA, which correlated with increased OPN concentrations in synovial fluid (SF). The pattern of OPN overexpression was confined to rheumatoid synovium and correlated with coexpression of selected OPN receptors in synovial T cells, including integrins alphav and beta1 and CD44. RA-derived SF stimulated the expression of OPN in T cells, which was attributable to IL-10 present in SF and abrogated by anti-IL-10 antibody. Among the more than 300 autoimmune and inflammatory response genes examined, OPN selectively induced the expression of proinflammatory cytokines and chemokines known to promote migration and recruitment of inflammatory cells. Furthermore, it was evident that OPN activated transcription factor NF-kappaB in mononuclear cells. The study has important implications for understanding the role of OPN in rheumatoid synovitis and other inflammatory conditions.

Histamine H4 antagonism: a therapy for chronic allergy?

Chronic inflammatory diseases such as bronchial asthma, allergic gastrointestinal disease, and atopic dermatitis are characterized by the selective recruitment and activation of distinct subtypes of leukocytes, particularly eosinophils, into the tissue from peripheral blood (Rothenberg, 1998). Eosinophils, along with mast cells, are postulated to play a key role in the pathophysiology of these chronic diseases. The striking accumulation of eosinophils in allergic disease has stimulated intense scientific examination leading to the discovery of numerous molecular mechanisms responsible for this process. Eosinophils, like all leukocytes, migrate from the vascular lumen, across the endothelial cell surface into the appropriate tissue sites. This elaborate mechanism, known as transendothelial migration, has been shown to be a highly orchestrated process (Springer, 1994). The migratory pathway, or chemotaxis, of the leukocyte is directly influenced by soluble molecules known as chemoattractants. These molecules reside along a tissue gradient, and bind to and activate G-protein coupled receptors (GPCRs) on the leukocyte cell surface. Several eosinophil chemoattractants have been extensively studied. Some of these molecules, the classical chemoattractants, include the complement cleavage fragments, leukotrienes and platelet-activating factor (PAF). These chemoattractants have been shown to stimulate the recruitment of a wide variety of leukocyte subtypes, and are therefore nonselective. In contrast, chemotactic cytokines (or chemokines) such as eotaxin-1, -2, -3, and monocyte chemoattractant protein (MCP)-4 are more selective for the recruitment of leukocytes associated with allergic inflammation, such as eosinophils, mast cells, and basophils.

Human ALX receptor regulates neutrophil recruitment in transgenic mice: roles in inflammation and host defense.

Signaling pathways instrumental in the temporal and spatial progression of acute inflammation toward resolution are of wide interest. Here a transgenic mouse with myeloid-selective expression of human lipoxin A4 receptor (hALX) was prepared and used to evaluate in vivo the effect of hALX expression. hALX-transfected HEK293 cells transmitted LXA4 signals that inhibit TNFalpha-induced NFkappaB activation. Transgenic FvB mice were generated by DNA injections of a 3.8 kb transgene consisting of the full-length hALX cDNA driven by a fragment of the hCD11b promoter. When topically challenged via dermal ear skin, hALX transgenic mice gave attenuated neutrophil infiltration (approximately 80% reduction) in response to leukotriene B4 (LTB4) plus prostaglandin E2 (PGE2) as well as approximately 50% reduction in PMN infiltrates (P<0.02) to receptor-bypass inflammation evoked by phorbol ester. The hALX transgenic mice gave markedly decreased PMN infiltrates to the peritoneum with zymosan and altered the dynamics of this response. Transgenic hALX mice displayed increased sensitivity with >50% reduction in PMN infiltrates to suboptimal doses (10 ng/mouse) of the ligand lipoxin A4 stable analog compared with <10% reduction of PMN in nontransgenic littermates. Soluble mediators generated within the local inflammatory milieu of hALX mice showed diminished ability to activate the proinflammatory transcription factor NFkappaB. Analyses of the lipid-derived mediators from exudates using LC-MS tandem mass spectroscopy indicated an altered profile in hALX transgenic mice that included lower levels of LTB4 and increased amounts of lipoxin A4 compared with nontransgenic littermates. Together these results demonstrate a gain-of-function with hALX transgenic mouse and indicate that ALX is a key receptor and sensor in formation of acute exudates and their resolution.

High serum levels of additional IL-18 forms may be reciprocally correlated with IgE levels in patients with atopic dermatitis

We established an ELISA system for determination of as yet unidentified species of interleukin 18 (IL-18), named IL-18 type 2, in human serum. Serum IL-18 levels and their effect on IgE levels were examined in 18 patients with atopic dermatitis (AD) with no other allergic symptoms. Three of these patients showed high IL-18 type 2 concentrations (25–100 ng/ml) in their blood serum, and this IL-18 type 2 was detectable only with our established ELISA system. In contrast, the level of the conventional form of IL-18 (type 1) was found to be 50–400 pg/ml in all patients by the commercially available ELISA. The levels of type 1 IL-18 showed no correlation with those of type 2 and ∼2-fold higher in AD patients than in normal subjects. IL-12 p40 and IgE levels were correlated in the patients with no IL-18 type 2, and interestingly, relatively low IgE concentrations were detected in the three IL-18 type 2-positive patients. They showed considerable levels of IL-12 p40 unlike normal subjects. The IFNγ-inducing activity of IL-18 type 2 was >100-fold less potent by weight ratio than that of a recombinant ‘active’ IL-18 preparation, even after the treatment with Caspase 1. Although the relationship between AD and serum IgE levels is not clear cut, IL-18 type 2 appears to play some roles in the Th2-polarization involving IgE production in association with immune responses occurring in local inflammatory milieu such as atopic lesions.

Oxidoreductases in Lipoxin A4 Metabolic Inactivation: A NOVEL ROLE FOR 15-OXOPROSTAGLANDIN 13-REDUCTASE/LEUKOTRIENE B4 12-HYDROXYDEHYDROGENASE IN INFLAMMATION

The lipoxins (LX) are autacoids that act within a local inflammatory milieu to dampen neutrophil recruitment and promote resolution. 15-Hydroxyprostaglandin dehydrogenase (15-PGDH) and 15-oxoprostaglandin 13-reductase, also termed leukotriene B(4) 12-hydroxydehydrogenase (PGR/LTB(4)DH), are two enzymatic activities appreciated for their roles in the metabolism of prostaglandins and LTB(4). Here, we determined whether these oxidoreductases also catalyze the conversion of lipoxin A(4) (LXA(4)) and assessed the activities of these LXA(4) metabolites. 15-Oxo-LXA(4) was generated by incubating LXA(4) with 15-PGDH and NAD(+) for studies of its further conversion. PGR/LTB(4)DH catalyzed the NADH-dependent reduction of 15-oxo-LXA(4) to yield 13,14-dihydro-15-oxo-LXA(4). With NADH as a cofactor, 15-PGDH acted as a 15-carbonyl reductase and catalyzed the conversion of 13,14-dihydro-15-oxo-LXA(4) to 13, 14-dihydro-LXA(4). Human polymorphonuclear leukocytes (PMN) exposed to native LXA(4), 15-oxo-LXA(4), or 13,14-dihydro-LXA(4) did not produce superoxide anions. At concentrations where LXA(4) and a metabolically stable LXA(4) analog potently inhibited leukotriene B(4)-induced superoxide anion generation, the further metabolites were devoid of activity. Neither 15-oxo-LXA(4) nor 13, 14-dihydro-LXA(4) effectively competed with (3)H-labeled LXA(4) for specific binding to recombinant LXA(4) receptor (ALXR). In addition, introducing recombinant PGR/LTB(4)DH into a murine exudative model of inflammation increased PMN number by approximately 2-fold, suggesting that this enzyme participates in the regulation of PMN trafficking. These results establish the structures of LXA(4) further metabolites and indicate that conversion of LXA(4) to oxo- and dihydro- products represents a mode of LXA(4) inactivation in inflammation. Moreover, they suggest that these eicosanoid oxidoreductases have multifaceted roles controlling the levels of specific eicosanoids involved in the regulation of inflammation.

Role of connective tissue autoimmunity in Graves' ophthalmopathy.

Histologic similarities exist between the tissues involved in the extrathyroidal manifestations of Graves' disease, namely ophthalmopathy and pretibial dermopathy. Both conditions are characterized by an accumulation of glycosaminoglycans (GAGs) and an infiltration of lymphocytes. We have shown that interleukin-1 and transforming growth factor-beta, cytokines released by the local inflammatory cell infiltrate, are capable of stimulating GAG synthesis by retroocular and pretibial fibroblasts. Additionally, affected retroocular and pretibial fibroblasts demonstrate an enhanced induction of HLA-DR in response to interferon-gamma treatment and both cell types express the 72 kDa heat shock protein in vivo and in vitro. Retroocular and pretibial fibroblasts from patients with Graves' ophthalmopathy and pretibial dermopathy thus seem to possess unique immunological features that may render them more susceptible to the autoimmune process in Graves' disease. Chronic stimulation of fibroblasts by cytokines released in the local inflammatory milieu may result in excessive GAG production. The accumulation of these hydrophilic mucopolysaccharides, with its associated edema, leads to the clinical manifestations of Graves' ophthalmopathy and pretibial dermopathy.

Retroocular fibroblasts: important effector cells in Graves' ophthalmopathy.

Retroocular and pretibial fibroblasts are likely important effector cells in Graves' ophthalmopathy and pretibial dermopathy. Histologic similarities exist between the tissues involved in these clinically diverse extrathyroidal manifestations of Graves' disease. Both conditions are characterized by an accumulation of glycosaminoglycans (GAGs) and an infiltration of lymphocytes. We have shown that particular cytokines, probably released by the local inflammatory cell infiltrate, are capable of stimulating GAG synthesis by retroocular and pretibial fibroblasts. In order to explain the site-selective involvement of these anatomically distinct areas in Graves' disease, we sought to identify unique characteristics shared by retroocular and pretibial fibroblasts. We have shown that affected retroocular and pretibial fibroblasts demonstrate an enhanced HLA-DR response to interferon-gamma treatment and that retroocular fibroblasts are especially sensitive to the GAG-stimulating effect of this cytokine. In addition, we have shown that only affected retroocular and pretibial fibroblasts express the 72 kDa heat shock protein. Therefore, affected retroocular and pretibial fibroblasts possess unique immunologic features that may render them more susceptible to the autoimmune process in Graves' disease. Chronic stimulation of fibroblasts by cytokines released in the local inflammatory milieu may result in excessive GAG production by these cells. The accumulation of these hydrophilic mucopolysaccharides, with attendant edema, leads to the clinical manifestations of Graves' ophthalmopathy and pretibial dermopathy.

Inside the Skin: the Local Immune and Inflammatory Milieu in Leprosy

Skin lesions of leprosy have become a rich source of new information about the mechanisms involved in the uniquely broad spectrum of human responsiveness to M. leprae. Recent technological advances in immunology and molecular biology have been applied to the study of skin lesions using 3 approaches: immunohistologic studies of skin biopsies from leprosy lesions, correlated assessment of cell subsets and soluble immunologic mediators, and studies of the effects of the inoculation of exogenous lymphokines into the lesions. Results from these studies suggest that an immunologic equilibrium may exist among long-established lesions across the spectrum so that, although T-helper and -suppressor cells are present in different proportions, immunologic activity is at a low, similar level in all types of lesions. Exogenous lymphokines can alter this equilibrium and temporarily change the histologic picture. Spontaneous immunologic changes occurring in acute leprosy reactions may also lead to changes in T cell subsets and quantities of lymphokines.