Adenosine A2A Receptor Agonists Promote More Rapid Wound Healing Than Recombinant Human Platelet–Derived Growth Factor (Becaplermin Gel)

Animal studies of the topical application of adenosine A _2A receptor agonists show that it promotes wound closure. To further confirm the efficacy of adenosine A _2A receptor agonists as promoters of wound healing, we compared the effect of MRE0094, a novel selective adenosine A _2A receptor agonist, to CGS-21680, a reference selective adenosine A _2A receptor agonist, as well as to recombinant human platelet–derived growth factor (0.01% Becaplermin gel), an agent currently used to promote healing of diabetic ulcers, on wound closure in healthy BALB/C mice. Wounds (12 mm diameter) were created on the dorsum of mice (two per mouse) and then treated daily with vehicle, 0.01% Becaplermin gel, or different doses of the adenosine A _2A receptor agonists. The wound margins were traced onto plastic sheets, and the wound areas were digitized, quantitated, and compared. We found that application of MRE0094 (1 g/wound and 10 g/wound) and CGS-21680 (1 g/wound and 5 g/wound) achieved 50% wound closure significantly more rapidly than control application (day 1.9, 1.9, 3.5, 3.2, respectively, versus control day 4, p < 0.05 ANOVA). Surprisingly, neither higher nor lower concentrations of CGS-21680 affected the rate of wound closure, as compared to control. In contrast, Becaplermin gel did not increase the rate at which wounds closed (50% closure by day 7.2, p = NS versus control). These data confirm our prior observations that adenosine A _2A receptor agonists promote wound closure, and they suggest that these agents may be as effective if not more effective than Becaplermin gel for the treatment of poorly healing wounds.     

Importance of Metalloproteinases and Macrophages in Viper Snake Envenomation–Induced Local Inflammation

The inflammatory action of jararhagin, a hemorrhagic metalloproteinase from Bothrops jararaca venom, was studied in mice using dorsal air pouches. The injection of the toxin in 6-day-old air pouches resulted in a leukocyte accumulation comparable to that induced by LPS and whole venom. Polymorphonuclear and mononuclear cells were present in this infiltrate, with a predominance of neutrophils. Treatment of jararhagin with 1,10-phenantroline abolished its proteolytic activity and reduced the pro-inflammatory effect in approximately 50%. Cell influx was not observed when jararhagin was injected into 1-hr air pouches devoid of macrophages, except when it was injected together with 10⁶ syngeneic peritoneal macrophages. Supernatants of macrophages stimulated in vitro with jararhagin did not induce leukocyte influx in 1-hr air pouches; the influx occurred after injection of the pellets of stimulated cultures. In summary, jararhagin is an important pro-inflammatory component of B. jararaca venom, and its activity is dependent upon the proteolytic activity and the presence of macrophages.     

High endothelial-like venules in chronically inflamed periodontal tissues exchange polymorphs

A survey of 58 gingival biopsies revealed the presence of periodontal high endothelial-like venules (PHELVs) in chronically inflamed gingival tissues. PHELVs were found to exchange polymorphonuclear cells (PMNs) almost exclusively in advanced periodontitis, with PMNs greatly exceeding the number of mononuclear cells found in PHELVs (P less than 0.001). Electron microscopy confirmed the emigration of PMNs from these vessels. The enzyme histochemical and ultrastructural features as well as the 35SO4 uptake properties of PHELVs were similar to those of the well-characterized high endothelial venules (HEVs) of rat lymph nodes. It is generally accepted that HEVs in lymphoid tissues and inflammatory sites are specially adapted to assist in the emigration of lymphocytes. However, the observation of preferential PMN emigration in the apparent absence of lymphocyte exchange from PHELVs compels further investigation of other possible functions for HEVs. In relation to this, endothelial cells are capable of producing potent cytokines and inflammatory mediators which may contribute to the development of lesions, and the possibility is discussed that high endothelial cells are functionally adapted to enhance the production of such factors.     

Activation of metabotropic glutamate receptor 3 enhances interleukin (IL)-1beta-stimulated release of IL-6 in cultured human astrocytes

Previous studies have demonstrated that human astrocytes express mRNA and receptor protein for group I and II metabotropic glutamate receptors (mGluRs). Whether these receptors can influence the inflammatory and immune response and can modulate the capacity of astrocytes to produce inflammatory cytokines is still unclear. Inflammatory cytokines can be produced by activated glial cells and play a critical role in several neurological disorders. Astrocyte-enriched human cell cultures growing in a serum-free chemically defined medium were used to study the regulation of IL (interleukin)-1β and IL-6 in response to mGluR activation. Astrocytes cultured in the absence or in the presence of epidermal growth factor (EGF), did not secrete significant IL-1β and IL-6, as determined by specific enzyme-linked immunosorbent assay (ELISA). Activation of mGluRs using (S)-3,5-dihydroxyphenylglycine (DHPG; selective group I agonist) or DCG-IV (selective group II agonist) did not affect the production of interleukins under both growth conditions. On exposure to IL-1β high levels of IL-6 were detected. Activation of mGluR3 with DCG-IV (but not of mGluR5 with DHPG) enhanced, in the presence of IL-1β, the release of IL-6 in a dose dependent manner in astrocytes cultured under conditions (+EGF) in which the mGluR expression is known to be upregulated. The effect of mGluR3 activation on IL-1β stimulated release of IL-6 was prevented by selective group II mGluR antagonists. The capacity of mGluR3 to modulate the release of IL-6 in the presence of IL-1β supports the possible involvement of this receptor subtype in the regulation of the inflammatory and immune response under pathological conditions associated with glial cell activation.     

Cytokine pattern in allergic and non-allergic chronic rhinosinusitis in asthmatic children

BACKGROUND: Rhinosinusitis represents one of the most common chronic diseases. The association of rhinosinusitis with asthma has been frequently reported. Eosinophils and Th2 cells play a pathogenic mechanism in asthma. OBJECTIVE: The aims of the study were to evaluate the cytokine pattern in chronic rhinosinusitis in asthmatic children and to compare the findings in allergic vs. non-allergic asthmatics. METHODS: Thirty-five asthmatic children were evaluated, 19 males and 16 females, with an average age of 8.7 years. All children were asthmatic and suffered from chronic rhinosinusitis. Twenty were allergic and 15 were non-allergic. Ten healthy children were studied as normal controls. Evaluated parameters were the levels of the following cytokines: IL-1beta, IL-4, IL-6, IL-8, IL-12, IFN-gamma and TNF-alpha. Cytokines were recovered from rhinosinusal lavage and measured by immunoassays. Nasal cytology was also performed in all subjects and inflammatory cells were counted by conventional staining. RESULTS: Allergic subjects showed a significant increase of IL-4 (P < 0.01) and TNF-alpha (P < 0.05) and a significant decrease of IL-12 (P < 0.05) and of IFN-gamma (P < 0.0001), whereas IL-1beta, IL-6 and IL-8 were not significantly increased. Non-allergic children showed a significant increase of IL-4 (P < 0.05) and a significant decrease of IFN-gamma (P < 0.0001), IL-12 was not significantly decreased, and IL-1beta, IL-6 and IL-8 were not significantly increased. A significant inflammatory infiltrate was present in all asthmatic children. Significant correlations were demonstrated between IL-4 and IL-12 (P < 0.001), IL-12 and IFN-gamma (P < 0.001), IL-8 and neutrophils (P < 0.01), and TNF-alpha and monocytes/macrophages (P < 0.05), in allergic asthmatics. IL-4 and IL-12 were significantly correlated (P < 0.05) as well as IL-8 and neutrophils (P < 0.01) in non-allergic asthmatics. CONCLUSION: This study shows that allergic asthmatic children with chronic rhinosinusitis have a typical Th2 cytokine pattern, but also non-allergic asthmatic children share a similar pattern. These findings would suggest the existence of a common pathophysiological mechanism shared by upper and lower airways and are consistent with the concept of united airways disease.     

Accuracy of eosinophils and eosinophil cationic protein to predict steroid improvement in asthma

BACKGROUND: There is a large variability in clinical response to corticosteroid treatment in patients with asthma. Several markers of inflammation like eosinophils and eosinophil cationic protein (ECP), as well as exhaled nitric oxide (NO), are good candidates to predict clinical response. AIM: We wanted to determine whether we could actually predict a favourable response to inhaled corticosteroids in individual patients. METHODS: One hundred and twenty patients with unstable asthma were treated with either prednisolone 30 mg/day, fluticasone propionate 1000 microg/day b.i.d. or fluticasone propionate 250 microg/day b.i.d., both via Diskhaler. They were treated during 2 weeks, in a double-blind, parallel group, double dummy design. We measured eosinophils and ECP in blood and sputum, and exhaled nitric oxide as inflammatory parameters before and after 2 weeks in order to predict the changes in forced expiratory volume in 1 s (FEV1), provocative concentration of methacholine causing a 20% fall in FEV1 (PC20 Mch), and asthma quality of life (QOL). Secondly, to test whether these results were applicable in clinical practice we determined the individual prediction of corticosteroid response. RESULTS: We found that changes in FEV1, PC20 Mch and QOL with corticosteroids were predominantly predicted by their respective baseline value and to a smaller extent by eosinophils in blood or sputum. ECP, measured in blood or sputum, was certainly not better than eosinophils in predicting clinical response to corticosteroids. Smoking status was an additional predictor for change in FEV1, but not for change in PC20 Mch or QOL. Prediction of a good clinical response was poor. For instance, high sputum eosinophils (> or = 3%) correctly predicted an improvement in PC20 Mch in only 65% of the patients. CONCLUSION: Our findings show that baseline values of the clinical parameters used as outcome parameters are the major predictors of clinical response to corticosteroids. Eosinophil percentage in blood or sputum adds to this, whereas ECP provides no additional information. Correct prediction of clinical response in an individual patient, however, remains poor with our currently used clinical and inflammatory parameters.     

Direct modulatory effect of C-reactive protein on primary human monocyte adhesion to human endothelial cells

C-reactive protein (CRP) is the prototypic acute phase serum protein in humans. The effects of CRP on primary human monocyte adhesion molecule expression and interaction with the endothelium have not been studied. Herein, we describe an investigation into the phenotypic and functional consequences of CRP binding to peripheral blood monocytes ex vivo. Peripheral whole blood was collected from healthy, non-smoking males. Mononuclear cells (MNC) and monocytes were isolated by differential centrifugation using lymphoprep and Dynal negative isolation kit, respectively. Cells were exposed to CRP from 0 to 250 micro g/ml for 0-60 min at 37 degrees C and analysed for (a) CD11b, PECAM-1 (CD31) and CD32 expression by flow cytometry and (b) adhesion to LPS (1 micro g/ml; 0-24 h) treated human umbilical vein endothelial cells (HUVEC). CD14+ monocyte expression of CD11b increased significantly up to twofold when exposed to CRP, compared to controls. There was no significant difference in CD32 expression, whereas CD31 expression decreased after exposure to CRP. CRP treatment of monocytes inhibited their adhesion to early LPS-activated HUVEC (0-5 h). However, the adhesion of CRP-treated monocytes to HUVEC was significantly greater to late activation antigens on HUVEC (24 h, LPS) compared to controls. We have shown that CRP can affect monocyte activation ex vivo and induce phenotypic changes that result in an altered recruitment to endothelial cells. This study provides the first evidence for a further role for C-reactive protein in both monocyte activation and adhesion, which may be of importance during an inflammatory event.     

Effector and regulatory events during natural killer–dendritic cell interactions

Summary:  The different cell types of the innate immune system can interact with each other and influence the quality and strength of an immune response. The cross talk between natural killer (NK) cells and myeloid dendritic cells (DCs) leads to NK cell activation and DC maturation. Activated NK cells are capable of killing DCs that fail to undergo proper maturation ('DC editing'). Encounters between NK cells and DCs occur in both inflamed peripheral tissues and lymph nodes, where both cell types are recruited by chemokines released in the early phases of inflammatory responses. Different NK cell subsets (CD56^brightCD16⁻ versus CD56⁺CD16⁺) differ in their homing capabilities. In particular, CD56^brightCD16⁻ NK cells largely predominate the lymph nodes. In addition, these two subsets display major functional differences in their cytolytic activity, cytokine production, and ability to undergo proliferation. NK cell functions are also greatly influenced by the presence of polarizing cytokines such as interleukin (IL)-12 and IL-4. The cytokine microenvironment reflects the presence of different cell types that secrete such cytokines in response to microbial products acting on different Toll-like receptors (TLRs). Moreover, NK cells themselves can respond directly to microbial products by means of TLR3 and TLR9. Thus, it appears that the final outcome of a response to microbial infection may greatly vary as a result of the interactions occurring between different pathogen-derived products and different cell types of the innate immunity system. These interactions also determine the quality and strength of the subsequent adaptive responses. Remarkably, NK cells appear to play a key role in this complex network.     

Mast cell tryptase may modulate endothelial cell phenotype in healing myocardial infarcts

Mast cells and macrophages infiltrate healing myocardial infarcts and may play an important role in regulating fibrous tissue deposition and extracellular matrix remodelling. This study examined the time-course of macrophage and mast cell accumulation in healing infarcts and studied the histological characteristics and protease expression profile of mast cells in a canine model of experimental infarction. Although macrophages were more numerous than mast cells in infarct granulation tissue, macrophage density decreased during maturation of the scar, whereas mast cell numbers remained persistently elevated. During the inflammatory phase of infarction, newly recruited leucocytes infiltrated the injured myocardium and appeared to be clustered in close proximity to degranulating cardiac mast cells. During the proliferative phase of healing, mast cells had decreased granular content and were localized close to infarct neovessels. In contrast, macrophages showed no selective localization. Mast cells in healing canine infarcts were alcian blue/safranin-positive cells that expressed both tryptase and chymase. In order to explain the pro-inflammatory and angiogenic actions of tryptase--the major secretory protein of mast cells--its effects on endothelial chemokine expression were examined. Chemokines are chemotactic cytokines that play an important role in leucocyte trafficking and angiogenesis and are highly induced in infarcts. Tryptase, a proteinase-activated receptor (PAR)-2 agonist, induced endothelial expression of the angiogenic chemokines CCL2/MCP-1 and CXCL8/IL-8, but not the angiostatic chemokine CXCL10/IP-10. Endothelial PAR-2 stimulation with the agonist peptide SLIGKV induced a similar chemokine expression profile. Mast cell tryptase may exert its angiogenic effects in part through selective stimulation of angiogenic chemokines.