Proteolytic processing of chemokines: implications in physiological and pathological conditions

Chemokines are small, secreted proteins that orchestrate the migration of cells, which are involved in immune defence, immune surveillance and haematopoiesis. However, chemokines are also implicated in the pathology of various inflammatory diseases, cancers and HIV. The chemokine system is considerably large and has a redundancy in the repertoire of its inflammatory mediators. Therefore, strict regulation of chemokine activity is crucial. Chemokines are the substrate for various proteases including the serine protease CD26/dipeptidyl-peptidase IV and matrix metalloproteinases. Regulation by proteolytic cleavage controls and fine-tunes chemokine function by either enhancing or reducing its chemotactic activity or receptor selectivity. Often chemokines and the proteases that regulate them are produced in the same microenvironment and expression of both may be simultaneously induced by a common stimulus enabling the rapid regulation of chemokine activity. The overall impact of cleaved chemokines in cellular responses is very complex. In this review, we will give an overview on chemokine modification and the respective chemokine modifying proteases. Furthermore, we will summarize the emerging literature describing the consequences in inflammation, haematopoiesis, cancer and HIV infection upon proteolytic chemokine processing.

Gene and protein expression of galectin-3 and galectin-9 in experimental pneumococcal meningitis

Inflammation of the subarachnoid and ventricular space contributes to the development of brain damage i.e. cortical necrosis and hippocampal apoptosis in pneumococcal meningitis (PM). Galectin-3 and -9 are known pro-inflammatory mediators and regulators of apoptosis. Here, the gene and protein expression profile for both galectins was assessed in the disease progression of PM. The mRNA of Lgals3 and Lgals9 increased continuously in the cortex and in the hippocampus from 22 h to 44 h after infection. At 44 h after infection, mRNA levels of Lgals9 in the hippocampus were 7-fold and those of Lgals3 were 30-fold higher than in uninfected controls (P<0.01). Galectin-9 protein did not change, but galectin-3 significantly increased in cortex and hippocampus with the duration of PM. Galectin-3 was localized to polymorphonuclear neutrophils, microglia, monocytes and macrophages, suggesting an involvement of galectin-3 in the neuroinflammatory processes leading to brain damage in PM.

Mechanisms of brain injury in bacterial meningitis: workshop summary

Morbidity and mortality associated with bacterial meningitis remain high, although antibiotic therapy has improved during recent decades. The major intracranial complications of bacterial meningitis are cerebrovascular arterial and venous involvement, brain edema, and hydrocephalus with a subsequent increase of intracranial pressure. Experiments in animal models and cell culture systems have focused on the pathogenesis and pathophysiology of bacterial meningitis in an attempt to identify the bacterial and/or host factors responsible for brain injury during the course of infection. An international workshop entitled "Bacterial Meningitis: Mechanisms of Brain Injury" was organized by the Department of Neurology at the University of Munich and was held in Eibsee, Germany, in June 1993. This conference provided a forum for the exchange of current information on bacterial meningitis, including data on the clinical spectrum of complications, the associated morphological alterations, the role of soluble inflammatory mediators (in particular cytokines) and of leukocyte-endothelial cell interactions in tissue injury, and the molecular mechanisms of neuronal injury, with potential mediators such as reactive oxygen species, reactive nitrogen species, and excitatory amino acids. It is hoped that a better understanding of the pathophysiological events that take place during bacterial meningitis will lead to the development of new therapeutic regimens.

Progression of atherosclerosis in ApoE-deficient mice that express distinct molecular forms of TNF-alpha

TNFalpha (TNF) critically regulates inflammation-driven atherosclerosis. Because the transmembrane (tmTNF) and soluble (sTNF) forms of TNF possess distinct immuno-modulatory properties, we hypothesized that they might differentially regulate atherosclerosis progression. Three groups of male ApoE(-/-) mice were studied: one expressing wild-type TNF (WT-TNF); one expressing exclusively a mutated non-cleavable form of TNF (KI-TNF); and one deficient in TNF (KO-TNF). Mice aged 5 weeks were fed the high-fat diet for 5 (T5) and 15 weeks (T15) or a standard chow diet for 15 weeks. At T5, in mice fed the high-fat diet, no significant differences in lesion area were observed among the three groups, either in valves or in aortas. At T15, lesion areas in valves were significantly lower in KO-TNF mice compared with those in WT-TNF mice, whereas in KI-TNF mice, they were intermediate between KO- and WT-TNF mice but not significantly different from these two groups. In aortas, lesions in KI-TNF were comparable to those of KO-TNF, both being significantly lower than those in WT-TNF. Theses differences were not linked to circulating lipids, or to macrophage, actin, and collagen contents of lesions. At T15, in mice fed the chow diet, lesion areas in valves and the aortic arch were not significantly different between the three groups. Levels of IL-6, IFNgamma, IL-10, and Foxp3 mRNAs in spleens and production of IL-6, IL-10, MCP-1, RANTES, and TNFR-2 by peritoneal macrophages at T15 of the high-fat diet showed a decrease in pro-inflammatory status, more marked in KO-TNF than in KI-TNF mice. Apoptosis was reduced only in KO-TNF mice. In conclusion, these data show that TNF effects on atherosclerosis development are detectable at stages succeeding fatty streaks and that wild-type TNF is superior to tmTNF alone in promoting atherosclerosis. TNF-dependent progression of atherosclerosis is probably linked to the differential production of pro-inflammatory mediators whether tmTNF is preponderant or essentially cleaved. Copyright (c) 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley ; Sons, Ltd.

Eicosanoids in exhaled breath condensates in the assessment of childhood asthma

The value of measurements of eicosanoids in exhaled breath condensate (EBC) for the evaluation of childhood asthma is still inconclusive most likely because of the limited value of the methods used. In this case-control study in 48 asthmatic and 20 healthy children, we aimed to characterize the baseline profile of the inflammatory mediators cysteinyl leukotrienes (cysLTs), 9(alpha)11(beta)PGF(2), PGE(2), PGF(2alpha), 8-isoprostane (8-iso-PGF(2alpha)) within EBC in asthmatic compared with healthy children using new methods. In addition, we investigated their relation to other inflammatory markers. The assessment included collection of EBC, measurement of fractional exhaled nitric oxide (FE(NO)) and evaluation of urinary excretion of leukotriene E(4.) cysLTs were measured directly in EBC by radioimmunoassay and prostanoids were measured using gas chromatography negative-ion chemical ionization mass spectrometry. Only cysLT levels were significantly higher in asthmatic compared with healthy children (p = 0.002). No significant differences in cysLTs were found between steroid naïve and patients receiving inhaled corticosteroids. In contrast, FE(NO) was significantly higher in steroid naïve compared with steroid-treated asthmatic and healthy children (p = 0.04 and 0.024, respectively). The diagnostic accuracy of cysLTs in EBC for asthma was 73.6% for the whole group and 78.2% for steroid-naïve asthmatic children. The accuracy to classify asthmatic for FE(NO) was poor (62.9%) for the whole group, but improved to 79.9% when only steroid-naïve asthmatic children were taken into consideration. cysLTs in EBC is an inflammatory marker which distinguishes asthmatics, as a whole group, from healthy children.

Gene expression profiling in anti-CD11d mAb-treated spinal cord-injured rats

Acute administration of a mononclonal antibody (mAb) raised against the CD11d subunit of the leukocyte CD11d/CD18 integrin after spinal cord injury (SCI) in the rat greatly improves neurological outcomes. We have profiled gene expression in anti-CD11d and isotyped-matched control mAb-treated rats after SCI. Microarray analysis demonstrated reduced expression of pro-inflammatory cytokines and increased expression of inflammatory mediators that promote wound healing and the expression of scar proteins predicted to improve nerve growth. These changes in gene expression may reflect changes in the types of macrophages that populate the lesions in anti-CD11d mAb-treated rats.

[Adsorption therapy in sepsis.]

BACKGROUND The activation of multiple pro- and anti-inflammatory mediators is a key feature in the pathophysiology of sepsis. Many of these mediators may directly contribute to organ dysfunction and determine disease severity. So far our ability to modulate these upregulated mediator pathways is very limited. Therefore the adsorption of such mediators via an extracorporeal circuit may be a beneficial intervention during sepsis. OBJECTIVES Recent technical innovations have made this intervention feasible. Both systems for exclusive mediator adsorption and for adsorption beside a conventional renal replacement therapy are now available. Some of the membranes can adsorb a broad range of mediators by rather unspecific binding, whereas others specifically adsorb endotoxin or mediators. DISCUSSION Whilst biochemical efficacy could be demonstrated by some of the systems, controlled and randomized studies demonstrating improved clinical endpoints are still lacking. Therefore the use of such therapies outside clinical studies cannot yet be recommended.

Caffeine affects the biological responses of human hematopoietic cells of myeloid lineage via downregulation of the mTOR pathway and xanthine oxidase activity.

Correction of human myeloid cell function is crucial for the prevention of inflammatory and allergic reactions as well as leukaemia progression. Caffeine, a naturally occurring food component, is known to display anti-inflammatory effects which have previously been ascribed largely to its inhibitory actions on phosphodiesterase. However, more recent studies suggest an additional role in affecting the activity of the mammalian target of rapamycin (mTOR), a master regulator of myeloid cell translational pathways, although detailed molecular events underlying its mode of action have not been elucidated. Here, we report the cellular uptake of caffeine, without metabolisation, by healthy and malignant hematopoietic myeloid cells including monocytes, basophils and primary acute myeloid leukaemia mononuclear blasts. Unmodified caffeine downregulated mTOR signalling, which affected glycolysis and the release of pro-inflammatory/pro-angiogenic cytokines as well as other inflammatory mediators. In monocytes, the effects of caffeine were potentiated by its ability to inhibit xanthine oxidase, an enzyme which plays a central role in human purine catabolism by generating uric acid. In basophils, caffeine also increased intracellular cyclic adenosine monophosphate (cAMP) levels which further enhanced its inhibitory action on mTOR. These results demonstrate an important mode of pharmacological action of caffeine with potentially wide-ranging therapeutic impact for treating non-infectious disorders of the human immune system, where it could be applied directly to inflammatory cells.

Phospholipid oxidation generates potent anti-inflammatory lipid mediators that mimic structurally related pro-resolving eicosanoids by activating Nrf2.

Exposure of biological membranes to reactive oxygen species creates a complex mixture of distinct oxidized phospholipid (OxPL) species, which contribute to the development of chronic inflammatory diseases and metabolic disorders. While the ability of OxPL to modulate biological processes is increasingly recognized, the nature of the biologically active OxPL species and the molecular mechanisms underlying their signaling remain largely unknown. We have employed a combination of mass spectrometry, synthetic chemistry, and immunobiology approaches to characterize the OxPL generated from the abundant phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) and investigated their bioactivities and signaling pathways in vitro and in vivo. Our study defines epoxycyclopentenones as potent anti-inflammatory lipid mediators that mimic the signaling of endogenous, pro-resolving prostanoids by activating the transcription factor nuclear factor E2-related factor 2 (Nrf2). Using a library of OxPL variants, we identified a synthetic OxPL derivative, which alleviated endotoxin-induced lung injury and inhibited development of pro-inflammatory T helper (Th) 1 cells. These findings provide a molecular basis for the negative regulation of inflammation by lipid peroxidation products and propose a novel class of highly bioactive compounds for the treatment of inflammatory diseases.

Surfactant protein D modulates allergen particle uptake and inflammatory response in a human epithelial airway model

Background Allergen-containing subpollen particles (SPP) are released from whole plant pollen upon contact with water or even high humidity. Because of their size SPP can preferentially reach the lower airways where they come into contact with surfactant protein (SP)-D. The aim of the present study was to investigate the influence of SP-D in a complex three-dimensional human epithelial airway model, which simulates the most important barrier functions of the epithelial airway. The uptake of SPP as well as the secretion of pro-inflammatory cytokines was investigated. Methods SPP were isolated from timothy grass and subsequently fluorescently labeled. A human epithelial airway model was built by using human Type II-pneumocyte like cells (A549 cells), human monocyte derived macrophages as well as human monocyte derived dendritic cells. The epithelial cell model was incubated with SPP in the presence and absence of surfactant protein D. Particle uptake was evaluated by confocal microscopy and advanced computer-controlled analysis. Finally, human primary CD4+ T-Cells were added to the epithelial airway model and soluble mediators were measured by enzyme linked immunosorbent assay or bead array. Results SPP were taken up by epithelial cells, macrophages, and dendritic cells. This uptake coincided with secretion of pro-inflammatory cytokines and chemokines. SP-D modulated the uptake of SPP in a cell type specific way (e.g. increased number of macrophages and epithelial cells, which participated in allergen particle uptake) and led to a decreased secretion of pro-inflammatory cytokines. Conclusion These results display a possible mechanism of how SP-D can modulate the inflammatory response to inhaled allergen.

Lipids as targets for novel anti-inflammatory therapies

Lipids serve important functions as membrane constituents and also as energy storing molecules. Besides these functions certain lipid species have now been recognized as signalling molecules that regulate a multitude of cellular responses including cell growth and death, and also inflammatory reactions. Bioactive lipids are generated by hydrolysis from membrane lipids mainly by phospholipases giving rise to fatty acids and lysophospholipids that either directly exert their function or are further converted to active mediators. This review will summarize the present knowledge about bioactive lipids that either promote or attenuate inflammatory reactions. These lipids include polyunsaturated fatty acids (PUFA), eicosanoids including the epoxyeicosatrienoic acids (EET), peroxisome proliferation activating receptor (PPAR) activators, cannabinoids and the sphingolipids ceramide, sphingosine 1-phosphate and sphingosylphosphorylcholine.

Enhanced inflammatory activity of endometriotic lesions from the rectovaginal septum.

Endometriosis is characterised by the growth of ectopic lesions at multiple locations outside the uterine cavity and may be considered a collection of distinct but related conditions. The exact aetiology of endometriosis is still not clear although a role for inflammation is increasingly accepted. We therefore investigated the inflammatory activity of eutopic tissue and that of the matching ectopic lesions from different locations by measuring the genetic expression of inflammatory chemokines and cytokines. The gene expression in matching eutopic and ectopic tissue was compared, as was the gene expression in lesions from different locations. A significantly higher mRNA expression of the chemokines ENA-78 and RANTES and the cytokines IL-6 and TNF α was observed in endometriotic lesions of the rectovaginal septum (RVS) compared to that of matching eutopic tissue. Comparisons across lesion locations showed a significantly higher expression of IL-6 and TNF α in the RVS compared to lesions from either the ovaries or the peritoneum. These results show that the production of some inflammatory chemokines and cytokines is significantly increased in the ectopic endometrial tissue compared to matching eutopic tissue. Furthermore, IL-6 and TNF α are produced in significantly higher quantities in RVS lesions compared to other lesions.

Correlation between altered central pain processing and concentration of peritoneal fluid inflammatory cytokines in endometriosis patients with chronic pelvic pain

Translational research has not yet elucidated whether alterations in central pain processes are related to peripheral inflammatory processes in chronic pain patients. We tested the hypothesis that the concentration of cytokines in the peritoneal fluid of endometriosis patients with chronic pain correlate with parameters of hyperexcitability of the nociceptive system. The concentrations of 15 peritoneal fluid cytokines were measured in 11 patients with chronic pelvic pain and a diagnosis of endometriosis. Six parameters assessing central pain processes were recorded. Positive correlations between concentration of some cytokines in the peritoneal fluid and amplification of central pain processing were found. The results suggest that inflammatory mechanisms may be important in the pathophysiology of altered central pain processes and that cytokines produced in the environment of endometriosis could act as mediators between the peripheral lesion and changes in central nociceptive processes.

Effects of stimulus with proinflammatory mediators on nitric oxide production and matrix metalloproteinase activity in explants of cranial cruciate ligaments obtained from dogs

OBJECTIVE To evaluate the origin and degree of activity of nitric oxide (NO) and matrix metalloproteinase (MMP) in explants of cranial cruciate ligaments (CCLs) obtained from dogs and cultured with and without inflammatory activators. SAMPLE POPULATION Tissue specimens obtained from 7 healthy adult Beagles that were (mean +/- SD) 4.5 +/- 0.5 years old and weighed 12.5 +/- 0.8 kg. PROCEDURE The CCLs were harvested immediately after dogs were euthanatized, and specimens were submitted for explant culture. Cultures were stimulated by incubation with a combination of interleukin-1, tumor necrosis factor-alpha, and lipopolysaccharide, or they were not stimulated. Culture supernatants were examined for production of NO nitrite-nitrate metabolites (NOts) and activity of MMP Cultured specimens were evaluated by use of immunohistochemical analysis to detect activity of inducible NO synthase (iNOS). RESULTS All ligament explants produced measurable amounts of NOts. Stimulated cultures produced significantly more NOts after incubation for 24 and 48 hours, compared with nonstimulated cultures. Production of MMP in supernatants after incubation for 48 hours was significantly higher in stimulated cultures than in nonstimulated cultures. Cells with positive staining for iNOS were detected on all slides. Positively stained cells were predominantly chondroid metaplastic. There was a significant difference in intensity of cell staining between stimulated and non-stimulated cultures. CONCLUSIONS AND CLINICAL RELEVANCE Explant cultures of intact CCLs obtained from dogs produce iNOS-induced NO. Stimulation of chondroid metaplastic cells in CCL of dogs by use of inflammatory activators can increase production of iNOS, NOts, and MMP.