Pulmonary inflammation induced by a recombinant Brugia malayi gamma-glutamyl transpeptidase homolog: involvement of humoral autoimmune responses

BACKGROUND: A major allergen from the lymphatic filarial parasite Brugia malayi implicated in the pathogenesis of tropical pulmonary eosinophilia (TPE) has recently been cloned and identified as the homolog of the membrane-bound mammalian enzyme gamma-glutamyl transpeptidase (gamma-GT). Patients with acute TPE show autoreactive antibodies against endogenous gamma-GT from the pulmonary epithelium. MATERIALS AND METHODS: Recombinant B. malayi gamma-GT, alone or adsorbed to aluminium hydroxide (AL), was used in a BALB/c mouse model to analyze its antigenic/allergenic potential, its potential to induce pulmonary inflammation, and its capacity to induce autoreacting antibodies. RESULTS: Mice immunized with B. malayi gamma-GT showed significant levels of gamma-GT-specific IgG1, IgG2a, IgG3, IgA, IgE antibodies, and mild blood eosinophilia, even in the absence of adjuvant. Intranasal challenge with B. malayi gamma-GT induced peribronchial and perivascular inflammation characterized by a mixed infiltrate of lymphocytes, neutrophils, eosinophils, and macrophages. Both IL-4 and IFN-gamma were detected in the peripheral blood and in the bronchoalveolar lavage fluid of immunized and intranasally challenged mice. Histological analysis of murine lungs using affinity-purified antibodies from mice immunized with the parasite's gamma-GT revealed the presence of autoimmune antibodies against pulmonary epithelium. Western blot analysis identified the 55 kDa heavy chain subunit of the murine gamma-GT as the target of autoreactive/crossreacting antibodies. CONCLUSION: Our data from the in vivo mouse model demonstrate the potent allergenicity/antigenicity of B. malayi gamma-GT, and its capacity to induce pulmonary inflammation upon intranasal challenge. This leads to breakdown of tolerance against endogenous murine gamma-GT. Thus, humoral autoimmunity against the airways epithelium may contribute to the pathogenesis of TPE.     

IL-23 in inflammatory bowel diseases and colon cancer

Studies in recent years have identified a pivotal role of the cytokine IL-23 in the pathogenesis of inflammatory bowel diseases (IBD: Crohn´s disease, ulcerative colitis) and colitis-associated colon cancer. Genetic studies revealed that subgroups of IBD patients have single nucleotide polymorphisms in the IL-23R gene suggesting that IL-23R signaling affects disease susceptibility. Furthermore, increased production of IL-23 by macrophages, dendritic cells or granulocytes has been observed in various mouse models of colitis, colitis-associated cancer and IBD patients. Moreover, in several murine models of colitis, suppression of IL-12/IL-23 p40, IL-23 p19 or IL-23R function led to marked suppression of gut inflammation. This finding was associated with reduced activation of IL-23 target cells such as T helper 17 cells, innate lymphoid cells type 3, granulocytes and natural killer cells as well as with impaired production of proinflammatory cytokines. Based on these findings, targeting of IL-23 emerges as important concept for suppression of gut inflammation and inflammation-associated cancer growth. Consistently, neutralizing antibodies against IL-12/IL-23 p40 and IL-23 p19 have been successfully used in clinical trials for therapy of Crohn´s disease and pilot studies in ulcerative colitis are ongoing. These findings underline the crucial regulatory role of IL-23 in chronic intestinal inflammation and colitis-associated cancer and indicate that therapeutic strategies aiming at IL-23 blockade may be of key relevance for future therapy of IBD patients.     

Metalloproteinase-dependent transforming growth factor-alpha release mediates neurotensin-stimulated MAP kinase activation in human colonic epithelial cells

Expression of the neuropeptide neurotensin (NT) and its high affinity receptor (NTR1) is increased during the course of Clostridium difficile toxin A-induced acute colitis, and NTR1 antagonism attenuates the severity of toxin A-induced inflammation. We recently demonstrated in non-transformed human colonic epithelial NCM460 cells that NT treatment caused activation of a Ras-mediated MAP kinase pathway that significantly contributes to NT-induced interleukin-8 (IL-8) secretion. Here we used NCM460 cells, which normally express low levels of NTR1, and NCM460 cells stably transfected with NTR1 to identify the upstream signaling molecules involved in NT-NTR1-mediated MAP kinase activation. We found that inhibition of the epidermal growth factor receptor (EGFR) by either an EGFR neutralizing antibody or by its specific inhibitor AG1478 (0.2 microm) blocked NT-induced MAP kinase activation. Moreover, NT stimulated tyrosine phosphorylation of the EGFR, and pretreatment with a broad spectrum metalloproteinase inhibitor batimastat reduced NT-induced MAP kinase activation. Using neutralizing antibodies against the EGFR ligands EGF, heparin-binding-EGF, transforming growth factor-alpha (TGFalpha), or amphiregulin we have shown that only the anti-TGFalpha antibody significantly decreases NT-induced phosphorylation of EGFR and MAP kinases. Furthermore, inhibition of the EGF receptor by AG1478 significantly reduced NT-induced IL-8 promoter activity and IL-8 secretion. This is the first report demonstrating that NT binding to NTR1 transactivates the EGFR and that this response is linked to NT-mediated proinflammatory signaling. Our findings indicate that matrix metalloproteinase-mediated release of TGFalpha and subsequent EGFR transactivation triggers a NT-mediated MAP kinase pathway that leads to IL-8 gene expression in human colonic epithelial cells.     

Gene therapy for rheumatoid arthritis

Rheumatoid arthritis (RA) is a severe autoimmune systemic disease. Chronic synovial inflammation results in destruction of the joints. No conventional treatment is efficient in RA. Gene therapy of RA targets mainly the players of inflammation or articular destruction: TNF-alpha or IL-1 blocking agents (such as anti-TNF-alpha monoclonal antibodies, soluble TNF-alpha receptor, type II soluble receptor of IL-1, IL-1 receptor antagonist), antiinflammatory cytokines (such as IL-4, IL-10, IL-1), and growth factors. In this polyarticular disease, the vector expressing the therapeutic protein can be administered as a local (intra-articular injection) or a systemic treatment (extra-articular injection). All the main vectors have been used in experimental models, including the more recent lentivirus and adeno-associated virus. Ex vivo gene transfer was performed with synovial cells, fibroblasts, T cells, dendritic cells, and different cells from xenogeneic origin. In vivo gene therapy is simpler, although a less controlled method. Clinical trials in human RA have started with ex vivo retrovirus-expressing IL-1 receptor antagonists and have demonstrated the feasibility of the strategy of gene therapy. The best target remains to be determined and extensive research has to be conducted in preclinical studies.     

Treatment with bindarit, a blocker of MCP-1 synthesis, protects mice against acute pancreatitis

Chemokines are believed to play a key role in the pathogenesis of acute pancreatitis. We have earlier shown that pancreatic acinar cells produce the chemokine monocyte chemotactic protein (MCP)-1 in response to caerulein hyperstimulation, demonstrating that acinar-derived MCP-1 is an early mediator of inflammation in acute pancreatitis. Blocking chemokine production or action is a major target for pharmacological intervention in a variety of inflammatory diseases, such as acute pancreatitis. 2-Methyl-2-[[1-(phenylmethyl)-1H-indazol-3yl]methoxy]propanoic acid (bindarit) has been shown to preferentially inhibit MCP-1 production in vitro in monocytes and in vivo without affecting the production of the cytokines IL-1, IL-6, or the chemokines IL-8, protein macrophage inflammatory-1alpha, and RANTES. The present study aimed to define the role of MCP-1 in acute pancreatitis with the use of bindarit. In a model of acute pancreatitis induced by caerulein hyperstimulation, prophylactic as well as therapeutic treatment with bindarit significantly reduced MCP-1 levels in the pancreas. Also, this treatment significantly protected mice against acute pancreatitis as evident by attenuated hyperamylasemia neutrophil sequestration in the pancreas (pancreatic MPO activity), and pancreatic acinar cell injury/necrosis on histological examination of pancreas sections.     

Abundant expression of chemokines in malignant and infective human lymphadenopathies.

Lymph nodes can be the primary target of infection or malignant transformation and may exhibit characteristic patterns of leukocyte infiltration analogous to those seen in inflammation of other tissues. Leukocyte migration to lymph nodes in vivo is a highly regulated, multi-step process that depends upon adhesion molecules and as yet, uncharacterized chemotactic signals. Chemokines are a key part of the orchestrated code of signals that directs leukocyte subsets to sites of inflammation or immune response. The potential role of these chemoattractants in selective trafficking of leukocyte subsets into lymph nodes was assessed by determining the expression of chemokines on a range of pathological and normal human lymph nodes and by evaluating the cellular composition of each lymph node. In situ hybridization using chemokine riboprobes and immunohistochemistry using specific antibodies were performed in order to correlate the mRNA and protein expression of the chemokines. The cellular source(s) of each chemokine was assessed by immunohistochemical staining of adjacent sections using antibodies directed against distinctive cellular markers. Substantial, but varied, expression of macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, RANTES, macrophage chemotactic protein (MCP)-1, eotaxin, and interleukin 8 (IL-8) were detected in the pathological lymph nodes by diverse cell types. Control lymph nodes showed expression only of RANTES, mainly by high endothelial venules. In all lymph nodes, except the nodes infiltrated with breast cancer, chemokine mRNA expression was highly concordant with the corresponding protein. In contrast with in vitro studies that have suggested discrete target cell specificity of chemokines, this study showed that with the possible exception of the neutrophil chemoattractant, IL-8, no chemokine appeared to be uniquely associated with the accumulation of a specific leukocyte subset. These data implicate chemokines in the recruitment of leukocytes to lymph nodes affected by diverse disease states.     

Identification of a key pathway required for the sterile inflammatory response triggered by dying cells

Dying cells stimulate inflammation, and this response is thought to contribute to the pathogenesis of many diseases. Very little has been known, however, about how cell death triggers inflammation. We found here that the acute neutrophilic inflammatory response to cell injury requires the signaling protein myeloid differentiation primary response gene 88 (Myd88). Analysis of the contribution of Myd88-dependent receptors to this response revealed only a minor reduction in mice doubly deficient in Toll-like receptor 2 (Tlr2) and Tlr4 and normal responses in mice lacking Tlr1, Tlr3, Tlr6, Tlr7, Tlr9, Tlr11 or the interleukin-18 receptor (IL-18R). However, mice lacking IL-1R showed a markedly reduced neutrophilic inflammatory response to dead cells and tissue injury in vivo as well as greatly decreased collateral damage from inflammation. This inflammatory response required IL-1alpha, and IL-1R function was required on non-bone-marrow-derived cells. Notably, the acute monocyte response to cell death, which is thought to be important for tissue repair, was much less dependent on the IL-1R-Myd88 pathway. Also, this pathway was not required for the neutrophil response to a microbial stimulus. These findings suggest that inhibiting the IL-1R-Myd88 pathway in vivo could block the damage from acute inflammation that occurs in response to sterile cell death, and do so in a way that might not compromise tissue repair or host defense against pathogens.     

Granzyme K activates protease-activated receptor-1

Granzyme K (GrK) is a trypsin-like serine protease that is elevated in patients with sepsis and acute lung inflammation. While GrK was originally believed to function exclusively as a pro-apoptotic protease, recent studies now suggest that GrK may possess other non-cytotoxic functions. In the context of acute lung inflammation, we hypothesized that GrK induces pro-inflammatory cytokine release through the activation of protease-activated receptors. The direct effect of extracellular GrK on PAR activation, intracellular signaling and cytokine was assessed using cultured human lung fibroblasts. Extracellular GrK induced secretion of IL-6, IL-8 and MCP-1 in a dose- and time-dependent manner in lung fibroblasts. Heat-inactivated GrK did not induce cytokine release indicating that protease activity is required. Furthermore, GrK induced activation of both the ERK1/2 and p38 MAP kinase signaling pathways, and significantly increased fibroblast proliferation. Inhibition of ERK1/2 abrogated the GrK-mediated cytokine release. Through the use of PAR-1 and PAR-2 neutralizing antibodies, it was determined that PAR-1 is essential for GrK-induced IL-6, IL-8 and MCP-1 release. In summary, extracellular GrK is capable of activating PAR-1 and inducing fibroblast cytokine secretion and proliferation.     

Catecholamines induce IL-10 release in patients suffering from acute myocardial infarction by transactivating its promoter in monocytic but not in T-cells

The anti-inflammatory cytokine IL-10 is up-regulated in response to TNF- suggesting a control mechanism of inflammation. In addition, we recently found systemic IL-10 release in response to acute stress reactions in the absence of any systemic inflammation. In vitro and in vivo studies in experimental models suggest that catecholamines induce IL-10 release via a cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) dependent pathway. Here we studied patients for plasma IL-10 after acute myocardial infarction, a very stressful event without significant signs of systemic inflammation. In fact, the activation of the sympathetic system initiated by cardiac infarction was accompanied by a temporary systemic release of IL-10. Catecholamine induced IL-10 may be released by different cells. Recently, we demonstrated that catecholamines directly stimulate the IL-10 promoter/enhancer via a cAMP/PKA pathway in monocytic cells. A cAMP responsive element (CRE) was identified as major target. Here we show that there is no influence of catecholamines on the IL-10 promoter activity in T-cells. In contrast to monocytic cells, in T-cells cAMP-induced PKA-dependent phosphorylation of the CRE-binding protein 1 (CREB-1) seems to play a marginal role in IL-10 induction, which was reflected by a low cAMP-dependent IL-10-promoter/enhancer stimulation in reporter gene assays. Thus, catecholamines are directly involved in the regulation of IL-10 expression in monocytic but not in T-cells after acute stressful conditions.     

Up-regulation of microRNA-335-5p reduces inflammation via negative regulation of the TPX2-mediated AKT/GSK3β signaling pathway in a chronic rhinosinusitis mouse model

Chronic rhinosinusitis (CRS) is featured with chronic symptoms of inflammation or infection in the nasal and sinus tissues. MicroRNAs (miRNAs/miRs), such as dysregulated expression of miR-125b and miR-26a, has been previously demonstrated to be related to CRS. The present study is intended to define the role of miR-335-5p in inflammation and the related mechanism in a mouse model of CRS. The differentially expressed genes associated with CRS were screened by microarray analysis. The targeting relationship between miR-335-5p and TPX2 was analyzed by target prediction program and dual luciferase reporter gene assay. The mouse model of CRS was established, and mice were introduced with miR-335-5p mimics, miR-335-5p inhibitors, or siRNA against TPX2 to explore the regulatory functions of miR-335-5p. The regulatory effect of miR-335-5p on inflammation with the involvement of the AKT signaling pathway was also analyzed with the expression of inflammatory cytokines and AKT signaling pathway-related factors measured. It was indicated that miR-335-5p regulated the TPX2 gene-mediated AKT signaling pathway. TPX2 was identified as a target gene of miR-335-5p, and miR-335-5p elevation inhibited the activation of the AKT signaling pathway. In mice with CRS, up-regulation of miR-335-5p or silence of TPX2 inhibited the inflammation, as evidenced by decreased levels of TNF-α, IL-6 and IL-8, and higher levels of GSK3β and IL-10. Collectively, miR-335-5p inhibits the activation of AKT signaling pathway by negatively mediating TPX2, which may confer anti-inflammatory protection in CRS.     

Cutting edge: IL-6 is a marker of inflammation with no direct role in inflammasome-mediated mouse models

IL-6 is a known downstream target of IL-1β and is consistently increased in serum from patients with NLRP3 inflammasome-mediated conditions. Therefore, IL-6 could be a therapeutic target in the treatment of IL-1β-provoked inflammation. IL-6 was increased in serum with accompanying neutrophilia in tissues of an inducible mouse model of Muckle-Wells syndrome. However, an IL-6-null background failed to provide phenotypic rescue and did not significantly impact inflammatory cytokine levels. In a second model of IL-1β-driven inflammation, NLRP3 activation by monosodium urate crystals similarly increased IL-6. Consistent with our Muckle-Wells syndrome model, ablation of IL-6 did not impact an acute neutrophilic response in this in vivo evaluation of gouty arthritis. Taken together, our results indicate that IL-6 is a reliable marker of inflammation, with no direct role in inflammasome-mediated disease.     

Shp2 plays an important role in acute cigarette smoke-mediated lung inflammation

Cigarette smoke (CS), the major cause of chronic obstructive pulmonary disease, contains a variety of oxidative components that were implicated in the regulation of Src homology domain 2-containing protein tyrosine phosphatase 2 (Shp2) activity. However, the contribution of Shp2 enzyme to chronic obstructive pulmonary disease pathogenesis remains unclear. We investigated the role of Shp2 enzyme in blockading CS-induced pulmonary inflammation. Shp2 levels were assessed in vivo and in vitro. Mice (C57BL/6) or pulmonary epithelial cells (NCI-H292) were exposed to CS or cigarette smoke extract (CSE) to induce acute injury and inflammation. Lungs of smoking mice showed increased levels of Shp2, compared with those of controls. Treatment of lung epithelial cells with CSE showed elevated levels of Shp2 associated with the increased release of IL-8. Selective inhibition or knockdown of Shp2 resulted in decreased IL-8 release in response to CSE treatment in pulmonary epithelial cells. In comparison with CS-exposed wild-type mice, selective inhibition or conditional knockout of Shp2 in lung epithelia reduced IL-8 release and pulmonary inflammation in CS-exposed mice. In vitro biochemical data correlate CSE-mediated IL-8 release with Shp2-regulated epidermal growth factor receptor/Grb-2-associated binders/MAPK signaling. Our data suggest an important role for Shp2 in the pathological alteration associated with CS-mediated inflammation. Shp2 may be a potential target for therapeutic intervention for inflammation in CS-induced pulmonary diseases.     

Depletion of pulmonary intravascular macrophages inhibits acute lung inflammation

Pulmonary intravascular macrophages (PIMs) are present in ruminants and horses. These species are highly sensitive to acute lung inflammation compared with non-PIM-containing species such as rats and humans. There is evidence that rats and humans may also recruit PIMs under certain conditions. We investigated precise contributions of PIMs to acute lung inflammation in a calf model. First, PIMs were recognized with a combination of in vivo phagocytic tracer Monastral blue and postembedding immunohistology with anti-CD68 monoclonal antibody. Second, gadolinium chloride depleted PIMs within 48 h of treatment (P < 0.05). Finally, PIMs contain TNF-alpha, and their depletion reduces cells positive for IL-8 (P < 0.05) and TNF-alpha (P < 0.05) and histopathological signs of acute lung inflammation in calves infected with Mannheimia hemolytica. The majority of IL-8-positive inflammatory cells in lung septa of infected calves were platelets. Platelets from normal cattle contained preformed IL-8 that was released upon in vitro exposure to thrombin (P < 0.05). These novel data show that PIMs, as the source of TNF-alpha, promote recruitment of inflammatory cells including IL-8-containing platelets to stimulate acute inflammation and pathology in lungs. These data may also be relevant to humans due to our ability to recruit PIMs.     

Calcitonin gene-related peptide induces IL-8 synthesis in human corneal epithelial cells

Calcitonin gene-related peptide (CGRP), a neuropeptide with proinflammatory activities, is released from termini of corneal sensory neurons in response to pain stimuli. Because neutrophil infiltration of the clear corneal surface is a hallmark of corneal inflammation in the human eye, we determined whether CGRP can bind to human corneal epithelial cells (HCEC) and induce expression of the neutrophil chemotactic protein IL-8. It was found that HCEC specifically bound CGRP in a saturable manner with a Kd of 2.0 x 10-9 M. Exposure of HCEC to CGRP induced a significant increase in intracellular cAMP levels and enhanced IL-8 synthesis nearly 4-fold. The capacity of CGRP to stimulate cAMP and IL-8 synthesis was abrogated in the presence of the CGRP receptor antagonist CGRP8-37. CGRP stimulation had no effect on the half-life of IL-8 mRNA while increasing IL-8 pre-mRNA synthesis >2-fold. In contrast to IL-8, CGRP did not induce monocyte chemotactic protein-1 or RANTES synthesis, nor did the neuropeptide enhance detectable increases in steady state levels of mRNA specific for these two beta-chemokines. The results suggest that HCEC possess CGRP receptors capable of initiating a signal transduction cascade that differentially activates expression of the IL-8 gene but not the genes for monocyte chemotactic protein-1 or RANTES. The capacity of CGRP to stimulate IL-8 synthesis in HCEC suggests that sensory neurons are involved in induction of acute inflammation at the eye surface.     

IL-7R blockade reduces post-myocardial infarction-induced atherosclerotic plaque inflammation in ApoE−/- mice

Modulating inflammation by targeting IL-1β reduces recurrent athero-thrombotic cardiovascular events without lipid lowering. This presents an opportunity to explore other pathways associated with the IL-1β signaling cascade to modulate the inflammatory response post-myocardial infarction (MI). IL-7 is a mediator of the inflammatory pathway involved in monocyte trafficking into atherosclerotic plaques and levels of IL-7 have been shown to be elevated in patients with acute MI. Recurrent athero-thrombotic events are believed to be mediated in part by index MI-induced exacerbation of inflammation in atherosclerotic plaques. The objective of the study was to assess the feasibility of IL-7R blockade to modulate atherosclerotic plaque inflammation following acute MI in ApoE^?/- mice. Mice were fed Western diet for 12 weeks and then subjected to coronary occlusion to induce an acute MI. IL-7 expression was determined using qRT-PCR and immuno-staining, and IL-7R was assessed using flow cytometry. Plaque inflammation was evaluated using immunohistochemistry. IL-7R blockade was accomplished with monoclonal antibody to IL-7R. IL-7 mRNA expression was significantly increased in the cardiac tissue of mice subjected to MI but not in controls. IL-7 staining was observed in the coronary artery. Plaque macrophage and lipid content were significantly increased after MI. IL-7R antibody treatment but not control IgG significantly reduced macrophage and lipid content in atherosclerotic plaques. The results show that IL-7R antibody treatment reduces monocyte/macrophage and lipid content in the atherosclerotic plaque following MI suggesting a potential new target to mitigate increased plaque inflammation post-MI.     

Homeostatic regulation of Salmonella-induced mucosal inflammation and injury by IL-23

IL-12 and IL-23 regulate innate and adaptive immunity to microbial pathogens through influencing the expression of IFN-γ, IL-17, and IL-22. Herein we define the roles of IL-12 and IL-23 in regulating host resistance and intestinal inflammation during acute Salmonella infection. We find that IL-23 alone is dispensable for protection against systemic spread of bacteria, but synergizes with IL-12 for optimal protection. IL-12 promotes the production of IFN-γ by NK cells, which is required for resistance against Salmonella and also for induction of intestinal inflammation and epithelial injury. In contrast, IL-23 controls the severity of inflammation by inhibiting IL-12A expression, reducing IFN-γ and preventing excessive mucosal injury. Our studies demonstrate that IL-23 is a homeostatic regulator of IL-12-dependent, IFN-γ-mediated intestinal inflammation.