Biochemical and functional characterization of glycosaminoglycans released from degranulating rat peritoneal mast cells: Insights into the physiological role of endogenous heparin.

The properties of commercially prepared heparin as an anticoagulant and antithrombotic agent in medicine are better understood than is the physiological role of heparin in its native form, where it is uniquely found in the secretory granules of mast cells. In the present study we have isolated and characterised the glycosaminoglycans (GAGs) released from degranulating rat peritoneal mast cells. Analysis of the GAGs by NMR spectroscopy showed the presence of both heparin and the galactosaminoglycan dermatan sulphate; heparinase digestion profiles and measurements of anticoagulant activity were consistent with this finding. The rat peritoneal mast cell GAGs significantly inhibited accumulation of leukocytes in the rat peritoneal cavity in response to IL-1β (p < 0.05, n = 6/group), and inhibited adhesion and diapedesis of leukocytes in the inflamed rat cremasteric microcirculation in response to LPS (p < 0.001, n = 4/group). FTIR spectra of human umbilical vein endothelial cells (HUVECs) were altered by treatment of the cells with heparin degrading enzymes, and restored by the addition of exogenous heparin. In conclusion, we have shown that rat peritoneal mast cells contain a mixture of GAGs that possess anticoagulant and anti-inflammatory properties

Platelets Independently Recruit into Asthmatic Lungs and Models of Allergic Inflammation via CCR3.

Platelet activation and pulmonary recruitment occur in patients with asthma and in animal models of allergic asthma, in which leukocyte infiltration, airway remodeling, and hyperresponsiveness are suppressed by experimental platelet depletion. These observations suggest the importance of platelets to various characteristics of allergic disease, but the mechanisms of platelet migration and location are not understood. The aim of this study was to assess the mechanism of platelet recruitment to extravascular compartments of lungs from patients with asthma and after allergen challenge in mice sensitized to house dust mite (HDM) extract (contains the DerP1 [Dermatophagoides pteronyssinus extract peptidase 1] allergen); in addition, we assessed the role of chemokines in this process. Lung sections were immunohistochemically stained for CD42b+ platelets. Intravital microscopy in allergic mice was used to visualize platelets tagged with an anti-mouse CD49b-PE (phycoerythrin) antibody. Platelet-endothelial interactions were measured in response to HDM (DerP1) exposure in the presence of antagonists to CCR3, CCR4, and CXCR4. Extravascular CD42b+ platelets were detected in the epithelium and submucosa in bronchial biopsy specimens taken from subjects with steroid-naive mild asthma. Platelets were significantly raised in the lung parenchyma from patients with fatal asthma compared with postmortem control-lung tissue. Furthermore, in DerP1-sensitized mice, subsequent HDM exposure induced endothelial rolling, endothelial adhesion, and recruitment of platelets into airway walls, compared with sham-sensitized mice, via a CCR3-dependent mechanism in the absence of aggregation or interactions with leukocytes. Localization of singular, nonaggregated platelets occurs in lungs of patients with asthma. In allergic mice, platelet recruitment occurs via recognized vascular adhesive and migratory events, independently of leukocytes via a CCR3-dependent mechanism

Transient receptor potential canonical 5 (TRPC5) protects against pain and vascular inflammation in arthritis and joint inflammation

Objective Transient receptor potential canonical 5 (TRPC5) is functionally expressed on a range of cells including fibroblast-like synoviocytes, which play an important role in arthritis. A role for TRPC5 in inflammation has not been previously shown in vivo. We investigated the contribution of TRPC5 in arthritis. Methods Male wild-type and TRPC5 knockout (KO) mice were used in a complete Freund's adjuvant (CFA)-induced unilateral arthritis model, assessed over 14 days. Arthritis was determined by measurement of knee joint diameter, hindlimb weightbearing asymmetry and pain behaviour. Separate studies involved chronic pharmacological antagonism of TRPC5 channels. Synovium from human postmortem control and inflammatory arthritis samples were investigated for TRPC5 gene expression. Results At baseline, no differences were observed. CFA-induced arthritis resulted in increased synovitis in TRPC5 KO mice assessed by histology. Additionally, TRPC5 KO mice demonstrated reduced ispilateral weightbearing and nociceptive thresholds (thermal and mechanical) following CFA-induced arthritis. This was associated with increased mRNA expression of inflammatory mediators in the ipsilateral synovium and increased concentration of cytokines in synovial lavage fluid. Chronic treatment with ML204, a TRPC5 antagonist, augmented weightbearing asymmetry, secondary hyperalgesia and cytokine concentrations in the synovial lavage fluid. Synovia from human inflammatory arthritis demonstrated a reduction in TRPC5 mRNA expression. Conclusions Genetic deletion or pharmacological blockade of TRPC5 results in an enhancement in joint inflammation and hyperalgesia. Our results suggest that activation of TRPC5 may be associated with an endogenous anti-inflammatory/analgesic pathway in inflammatory joint conditions

Molecular Blocking of CD23 Supports Its Role in the Pathogenesis of Arthritis

BACKGROUND: CD23 is a differentiation/activation antigen expressed by a variety of hematopoietic and epithelial cells. It can also be detected in soluble forms in biological fluids. Initially known as the low-affinity receptor for immunoglobulin E (Fc epsilonRII), CD23 displays various other physiologic ligands such as CD21, CD11b/c, CD47-vitronectin, and mannose-containing proteins. CD23 mediates numerous immune responses by enhancing IgE-specific antigen presentation, regulating IgE synthesis, influencing cell differentiation and growth of both B- and T-cells. CD23-crosslinking promotes the secretion of pro-inflammatory mediators from human monocytes/macrophages, eosinophils and epithelial cells. Increased CD23 expression is found in patients during allergic reactions and rheumatoid arthritis while its physiopathologic role in these diseases remains to be clarified. METHODOLOGY/PRINCIPAL FINDINGS: We previously generated heptapeptidic countrestructures of human CD23. Based on in vitro studies on healthy and arthritic patients' cells, we showed that CD23-specific peptide addition to human macrophages greatly diminished the transcription of genes encoding inflammatory cytokines. This was also confirmed by significant reduction of mediator levels in cell supernatants. We also show that CD23 peptide decreased IgE-mediated activation of both human and rat CD23(+) macrophages. In vivo studies in rat model of arthritis showed that CD23-blocking peptide ameliorates clinical scores and prevent bone destruction in a dose dependent manner. Ex-vivo analysis of rat macrophages further confirmed the inhibitory effect of peptides on their activation. Taken together our results support the role of CD23 activation and subsequent inflammatory response in arthritis. CONCLUSION: CD23-blocking peptide (p30A) prevents the activation of monocytes/macrophages without cell toxicity. Thus, targeting CD23 by antagonistic peptide decreases inflammatory markers and may have clinical value in the treatment of human arthritis and allergic reactions involving CD23

Regulation of the cd38 promoter in human airway smooth muscle cells by TNF-α and dexamethasone

<p>Abstract</p> <p>Background</p> <p>CD38 is expressed in human airway smooth muscle (HASM) cells, regulates intracellular calcium, and its expression is augmented by tumor necrosis factor alpha (TNF-α). CD38 has a role in airway hyperresponsiveness, a hallmark of asthma, since deficient mice develop attenuated airway hyperresponsiveness compared to wild-type mice following intranasal challenges with cytokines such as IL-13 and TNF-α. Regulation of CD38 expression in HASM cells involves the transcription factor NF-κB, and glucocorticoids inhibit this expression through NF-κB-dependent and -independent mechanisms. In this study, we determined whether the transcriptional regulation of CD38 expression in HASM cells involves response elements within the promoter region of this gene.</p> <p>Methods</p> <p>We cloned a putative 3 kb promoter fragment of the human <it>cd38 </it>gene into pGL3 basic vector in front of a luciferase reporter gene. Sequence analysis of the putative <it>cd38 </it>promoter region revealed one NF-κB and several AP-1 and glucocorticoid response element (GRE) motifs. HASM cells were transfected with the 3 kb promoter, a 1.8 kb truncated promoter that lacks the NF-κB and some of the AP-1 sites, or the promoter with mutations of the NF-κB and/or AP-1 sites. Using the electrophoretic mobility shift assays, we determined the binding of nuclear proteins to oligonucleotides encoding the putative <it>cd38 </it>NF-κB, AP-1, and GRE sites, and the specificity of this binding was confirmed by gel supershift analysis with appropriate antibodies.</p> <p>Results</p> <p>TNF-α induced a two-fold activation of the 3 kb promoter following its transfection into HASM cells. In cells transfected with the 1.8 kb promoter or promoter constructs lacking NF-κB and/or AP-1 sites or in the presence of dexamethasone, there was no induction in the presence of TNF-α. The binding of nuclear proteins to oligonucleotides encoding the putative <it>cd38 </it>NF-κB site and some of the six AP-1 sites was increased by TNF-α, and to some of the putative <it>cd38 </it>GREs by dexamethasone.</p> <p>Conclusion</p> <p>The EMSA results and the cd38 promoter-reporter assays confirm the functional role of NF-κB, AP-1 and GREs in the cd38 promoter in the transcriptional regulation of CD38.</p

XXVI Congreso Nacional y II Congreso Internacional de SEDEM

Organizan: Sociedad Española de Educación Médica y Facultad de Medicina y Enfermería, Universidad del País Vasco (UPV/EHU)Comunicaciones aceptadas en el XXVI Congreso de la Sociedad Española de Educación Médica, celebrado en Bilbao del 28 al 30 de noviembre de 2024

Role of cytokines and chemokines in bronchial hyperresponsiveness and airway inflammation

Over the last decade there has been an intense interest in the potential role of cytokines and chemokines as important mediators in various atopic diseases, including asthma and the mechanisms by which these mediators regulate airway inflammation and bronchial hyperresponsiveness. This research effort has recently culminated in the publication of clinical studies that have assessed the role of interleukin (IL)-4 [Borish et al., Am J Respir Crit Care Med 160, 1816-1823 (1999)], IL-5 [Leckie et al., Lancet 356, 2144-2148 (2000)], and IL-12 [Bryan et al., Lancet 356, 2149-2153 (2000)] in allergic asthma, and the results have been disappointing. This is not surprising given the pleiotropic role cytokines play in the allergic response confirmed by numerous animal studies providing evidence of functional redundancy. The alternative view is that our current concepts in asthma pathogenesis need significant revision. This review will summarise the evidence for the role of cytokines and chemokines in various aspects of asthma pathophysiology; namely, bronchial hyperresponsiveness, eosinophil recruitment to the airways, mucus secretion, and airway remodelling. (C) 2002 Elsevier Science Inc. All rights reserved

Murine models of inflammation:role of CD23

The role of IgE in eosinophil recruitment and bronchial hyperresponsiveness has been extensively studied with murine models of inflammation. Many investigators using various knockout models have clearly shown that both IgE-dependent and -independent pathways play a role in eosinophil recruitment and bronchial hyperresponsiveness after allergen challenge, illustrating the complexity of airways inflammation. The expression of this response is likely to involve many interacting pathways, and it will be a considerable challenge to determine key points within these pathways that will yield novel targets for future therapeutic strategies

Cytokines in airway inflammation

With over 50 potential asthma mediators. cytokines are the latest group of substances which have been investigated for their potential role in this disease. The use of murine models of allergic inflammation has facilitated the investigation of the role of individual cytokines in this response. The use of targeted gene disruption, overexpression of genes and monoclonal antibodies directed against cytokines have allowed a detailed examination of the role cytokines play in IgE production. eosinophil recruitment and bronchial hyperresponsiveness, which are the characteristic features of the asthma phenotype. Despite the introduction of this new methodology, conflicting reports relating to the role of cytokines in allergic inflammation, highlight the complexity of allergic inflammation and challenge the notion that a single cytokine can explain the asthma phenotype. (C) 2000 Elsevier Science Ltd. All rights reserved