Targeting granulocyte-macrophage colony-stimulating factor in epithelial and vascular remodeling in experimental eosinophilic esophagitis.

BACKGROUND: Eosinophilic esophagitis (EoE) is a chronic antigen-mediated clinicopathologic disease of the esophagus characterized by an eosinophil-predominant inflammatory infiltrate. A clinical hallmark is extensive tissue remodeling including basal zone hyperplasia, fibrosis, and angiogenesis. However, the cellular mechanisms responsible for these processes are not fully defined. We hypothesized that targeting granulocyte-macrophage colony-stimulating factor (GM-CSF; an agonist cytokine linked with eosinophil survival and activation) would be protective in a preclinical model of EoE. METHODS: Eosinophilic esophagitis-like esophageal inflammation was induced in the L2-IL5OXA EoE mouse model, and GM-CSF production was assessed by mRNA and protein analyses. Granulocyte-macrophage colony-stimulating factor-receptor-alpha expression patterns were examined by flow cytometric and immunofluorescence analysis. L2-IL5OXA EoE mice were treated with anti-GM-CSF neutralizing antibody or isotype control and assessed for histopathological indices of eosinophilia, epithelial hyperplasia, and angiogenesis by immunohistochemistry and RT-PCR. RESULTS: Significantly increased levels of esophageal GM-CSF expression was detected in the L2-IL5OXA mouse EoE model during active inflammation. Granulocyte-macrophage colony-stimulating factor-receptor-alpha was predominantly expressed on esophageal eosinophils during EoE, in addition to select cells within the lamina propria. Anti-GM-CSF neutralization in L2-IL5OXA EoE mice resulted in a significant diminution of epithelial eosinophilia in addition to basal cell hyperplasia and vascular remodeling. This treatment response was independent of effects on esophageal eosinophil maturation or activation. CONCLUSION: Granulocyte-macrophage colony-stimulating factor is a potential therapeutic target to reduce esophageal eosinophilia and remodeling.

Retinoic acids up-regulate functional eosinophil-driving receptor CCR3.

Eotaxins and their receptor CCR3 have a definitive role for tissue accumulation of eosinophils both under homeostatic and pathologic conditions. However, physiological stimuli that can up-regulate CCR3 in blood-derived human eosinophils have not been recognized. As a prior gene microarray study revealed up-regulation of CCR3 in eosinophils stimulated with retinoic acids (RAs), the expression of functional CCR3 was examined. We found that 9-cis RA and all-trans RA (ATRA) significantly induced surface CCR3 expression regardless of the presence of IL-3 or IL-5. Pharmacological manipulations with receptor-specific agonists and antagonists indicated that retinoic acid receptor-α activation is critical for CCR3 up-regulation. RA-induced CCR3 was associated with its functional capacity, in terms of the calcium mobilization and chemotactic response to eotaxin-1 (CCL11). Our study suggests an important role of vitamin A derivatives in the tissue accumulation of eosinophils.

Up-regulation of CCL11 and CCL26 is associated with activated eosinophils in bullous pemphigoid.

Eosinophils contribute to the pathogenesis of bullous pemphigoid (BP) by secretion of proinflammatory cytokines and proteases. Trafficking of eosinophils into tissue in animal models and asthma depends on interleukin-5 and a family of chemokines named eotaxins, comprising CCL11, CCL24 and CCL26. Up-regulation of CCL11 has been described in BP, but the expression of the other two members of the eotaxin-family, CCL24 and CCL26, has not been investigated. In addition to these chemokines, expression of adhesion molecules associated with eosinophil migration to the skin should be analysed. We demonstrate that similar to CCL11, the concentration of CCL26 was up-regulated in serum and blister fluid of BP patients. In contrast, the concentration of CCL24 was not elevated in sera and blister fluid of the same BP patients. In lesional skin, CCL11 and CCL26 were detected in epidermis and dermis by immunohistochemistry. In contrast to CCL11, CCL26 was expressed strongly by endothelial cells. In line with these findings, eosinophils represented the dominating cell population in BP lesional skin outnumbering other leucocytes. The percentage of eosinophils expressing very late antigen (VLA): VLA-4 (CD49d) and CD11c correlated with their quantity in tissue. Macrophage antigen (MAC)-1 (CD11b/CD18) was expressed constitutively by tissue eosinophils. In conclusion, these data link the up-regulation of the eosinophil chemotactic factor CCL26 in BP to the lesional accumulation of activated eosinophils in the skin. Thereby they broaden the understanding of BP pathogenesis and might indicate new options for therapeutic intervention.

Mechanism of sphingosine 1-phosphate- and lysophosphatidic acid-induced up-regulation of adhesion molecules and eosinophil chemoattractant in nerve cells.

The lysophospholipids sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) act via G-protein coupled receptors S1P(1-5) and LPA(1-3) respectively, and are implicated in allergy. Eosinophils accumulate at innervating cholinergic nerves in asthma and adhere to nerve cells via intercellular adhesion molecule-1 (ICAM-1). IMR-32 neuroblastoma cells were used as an in vitro cholinergic nerve cell model. The G(i) coupled receptors S1P(1), S1P(3), LPA(1), LPA(2) and LPA(3) were expressed on IMR-32 cells. Both S1P and LPA induced ERK phosphorylation and ERK- and G(i)-dependent up-regulation of ICAM-1 expression, with differing time courses. LPA also induced ERK- and G(i)-dependent up-regulation of the eosinophil chemoattractant, CCL-26. The eosinophil granule protein eosinophil peroxidase (EPO) induced ERK-dependent up-regulation of transcription of S1P(1), LPA(1), LPA(2) and LPA(3), providing the situation whereby eosinophil granule proteins may enhance S1P- and/or LPA- induced eosinophil accumulation at nerve cells in allergic conditions.

Increase effect of transforming growth factor on eotaxin production by normal cultured dermal fibroblasts stimulated with interleukin-4: inhibitory effect of suplatast tosilate on eotaxin production.

Eotaxin plays a central role in the development of allergic disease, including atopic dermatitis, asthma, and nasal allergy. Interleukin (IL)-4 induces eotaxin production in normal human dermal fibroblasts. On the other hands, Transforming growth factor-beta (TGF-beta), a multifunctional regulatory cytokine, affects many biological functions, including fibroblast growth and differentiation and Th2 cytokine regulation. In this study, we investigated the effect of TGF-beta on IL-4-induced eotaxin production by normal human fibroblasts, as well as the effect of suplatast tosilate, an antiallergic drug that selectively inhibits Th2 cytokine production. Dermal fibroblast treatment with IL-4 and TGF-beta for 24 h increased eotaxin production and expression of eotaxin mRNA, as measured by enzyme-linked immunosorbent assay (ELISA) and reverse-transcriptase polymerase chain reaction (RT-PCR), respectively. TGF-beta synergistically up-regulated eotaxin production and eotaxin mRNA expression when stimulated with IL-4. Suplatast tosilate dose-dependently inhibited eotaxin production induced by IL-4 or IL-4 plus TGF-beta. These results suggest that TGF-beta may regulate skin allergic inflammation by up-regulating eotaxin production in dermal fibroblasts. Suplatast tosilate might suppress this inflammation by inhibiting eotaxin production.

Treatment with a novel chemokine-binding protein or eosinophil lineage-ablation protects mice from experimental colitis.

Eosinophils are multifunctional leukocytes implicated in numerous inflammatory diseases. The present study was conducted to clarify the precise role of eosinophils in the development of colitis by using eosinophil-depleted mice and a novel chemokine-binding protein that neutralizes CCL11 action. Colitis was induced by administration of dextran sodium sulfate (DSS) to wild-type and eosinophil-deficient DeltadblGATA-1 mice. Accumulation of eosinophils in the gut of mice given DSS paralleled worsening of clinical score and weight loss. In response to DSS, DeltadblGATA-1 mice showed virtual absence of eosinophil recruitment, amelioration of clinical score, weight loss, and tissue destruction, and no lethality. There was a decrease in CXCL1 and CCL3 production and decreased neutrophil influx in the intestine of DeltadblGATA-1 mice. Transfer of bone marrow cells from wild-type mice reconstituted disease manifestation in DSS-treated DeltadblGATA-1 mice, and levels of CCL11 were increased after DSS treatment and localized to inflammatory cells. Treatment with the chemokine-binding protein evasin-4 at a dose that prevented the function of CCL11 greatly ameliorated clinical score, weight loss, overall tissue destruction, and death rates. In conclusion, the influx of eosinophils is critical for the induction of colitis by DSS. Treatment with a novel chemokine-binding protein decreased eosinophil influx and greatly ameliorated colitis, suggesting that strategies that interfere with the recruitment of eosinophils may be useful as therapy for colitis.

Clinical data and inflammation parameters in patients with cypress allergy treated with sublingual swallow therapy and subcutaneous immunotherapy.

The clinical efficacy of immunotherapy, either by high dose sublingual-swallow therapy (SLIT) or subcutaneous immunotherapy (SCIT), has been demonstrated in patients with pollinosis but few studies have been carried out analysing differences in these treatments in terms of an improvement of clinical and allergic phlogosis parameters. The aim of this double-blind placebo-controlled study is to investigate the efficacy of high dose SLIT and SCIT using a purified standardized Juniperus ashei extract in a population of allergic patients monosensitized to cypress. Forty patients with cypress-allergic rhino conjunctivitis were administered therapeutic or placebo SLIT or SCIT for 12 months. Laboratory parameters were studied, namely the eosinophil cationic protein (ECP) level in nasal lavage and in serum, as well as the number of eosinophils (EOS) in peripheral blood and in nasal lavage and the level of eosinophil chemotactic activity (ECA). These parameters were correlated with clinical symptoms, evaluated by means of the clinical symptoms score (CSS). After SCIT and SLIT the levels of ECP and ECA were reduced in nasal lavage. We also observed a significant reduction in the values of ECP in serum in the patients treated with SLIT. EOS were unchanged in peripheral blood, but significantly reduced in nasal lavage. These data were in accordance with the improvement of clinical symptoms, supported by the close correlation between CSS and laboratory parameters. Our data confirm a clinical improvement correlated with a decline in inflammation parameters after one year of immunotherapy, supporting the hypothesis that treatment with a major allergen of cypress is able to change the course of allergic rhinitis.

Mechanism of eosinophilic esophagitis.

Eosinophilic esophagitis (EoE) is a newly recognized disease and is an emerging entity throughout developing and developed countries, including the United States. Therefore, understanding the causes, natural history, diagnosis, and management is important for future therapeutic interventions. The pathogenesis of EoE is still not clear, but a growing body of evidence has established that this condition represents a T-cell-mediated immune response involving several proinflammatory mediators and chemoattractants known to regulate eosinophilic accumulation in the esophagus, such as IL-4, IL-5, IL-3 and eotaxin-1, -2, and -3. Determining the mechanism or mechanisms through which human esophageal-derived factors ultimately induce the functional abnormalities observed, and to which antigens patients who have EoE are sensitized that lead to the manifestation of symptoms, is of significant interest.

Exploring the role of mast cells in eosinophilic esophagitis.

The mast cell plays a critical role in allergic responses in the gastrointestinal tract and other sites. Emerging evidence indicates that mast cells also participate in the pathogenesis of eosinophilic esophagitis, although their precise role has not been defined. This article reviews the biology of mast cells and examines the potential involvement of the cell as an effector of the inflammatory response and tissue remodeling, and as a cell that has the potential to function as an immunomodulator and limit inflammation.

Relationships between eosinophilic inflammation, tissue remodeling, and fibrosis in eosinophilic esophagitis.

The clinical and pathologic features of eosinophilic esophagitis (EE) include extensive tissue remodeling. Increasing evidence supports a key role for the eosinophil in multiple aspects of the esophageal remodeling and fibrosis seen in this allergic disease. This article reviews the clinical implications of esophageal remodeling and fibrosis in EE and discusses the possible pathogenic mechanisms inducing and regulating these responses. The focus is specifically on eosinophil and cytokine interactions with the esophageal epithelium, vascular endothelium, resident fibroblasts, and smooth muscle. Current and potential therapeutic interventions are discussed that may impact the development or resolution of chronic esophageal remodeling and fibrosis in EE.

Characterization of a galectin-like activity from the parasitic nematode, Haemonchus contortus, which modulates ovine eosinophil migration in vitro.

The development of eosinophilia is a characteristic feature of helminth infection, although the exact nature of the interaction between eosinophils and parasites remains to be fully defined. Previously, it has been reported that Haemonchus contortus and other nematodes produce eosinophil-specific chemoattractants. This paper describes studies aimed at isolating and identifying the factor(s) responsible. Initial studies showed that soluble extracts of infective larvae (L3) of H. contortus provoked a chemokinetic, rather than chemotactic, response in ovine bone marrow eosinophils in vitro. This activity was inhibited by lactose to a markedly greater extent than sucrose suggesting a galectin-like identity. Lactose affinity chromatography of soluble H. contortus extracts resulted in the isolation a specific bound fraction which retained biological activity. SDS-PAGE gel electrophoresis indicated a single Coomassie-stained band at between 31 and 41kDa. Subsequent, mass spectrometric analysis confirmed that the bound fraction contained a mixture of nematode galectins. The results confirm that H. contortus larvae produce several galectin-like proteins, at least one of which demonstrates eosinophil chemokinetic activity in vitro. The possibility of the parasite-derived factor mimicking the mammalian galectin-9, a known eosinophil chemokine, is discussed.

Identification of an eosinophil chemotactic factor from anopheline mosquitoes as a chitinase family protein.

Anopheline mosquitoes play an essential role in malaria transmission. The mosquito salivates copiously when probing for the location of a blood vessel. We found that the saliva of anopheline mosquitoes has chemotactic activity for naive eosinophils or neutrophils. The major eosinophil chemotactic component in saliva was shown to be one of the chitinase family proteins. A similar chitinase family protein was found also in the midgut of the anopheline mosquito. Production of antibodies to the chitinase family protein was generally observed in the sera of residents of a malaria endemic area. Both Plasmodium falciparum-infected and uninfected individuals had antibodies to chitinases. These results suggest that the chitinase family protein in mosquito saliva contributes to eliciting an inflammatory response of eosinophils in the host skin followed by antibody production in the host.

Mechanisms of eosinophilia in the pathogenesis of hypereosinophilic disorders.

The increased numbers of activated eosinophils in the blood and tissues that typically accompany hypereosinophilic disorders result from a variety of mechanisms. Exciting advances in translating discoveries achieved from mouse models and molecular strategies to the clinic have led to a flurry of new therapeutics specifically designed to target eosinophil-associated diseases. So far, this form of hypothesis testing in humans in vivo through pharmacology generally has supported the paradigms generated in vitro and in animal models, raising hopes that a spectrum of novel therapies soon may become available to help those who have eosinophil-associated diseases.

[Morphological and proinflammatory expression of the experimental autoimmune prostatitis in the rat model].

OBJECTIVE: To establish a Wistar rat model of chronic abacterial prostatitis (CAP) by injecting purified prostate protein and Freund's complete adjuvant, and to study the influence on the morphology and proinflammatory expression. METHODS: Male rats were injected with the Pertussis-Diphteria-Tetanus vaccine into the abdominal cavity and purified prostate protein and Freund's complete adjuvant intradermally at 0 and 30 days. At 60 days, the rats were sacrificed, and then the prostate specimens were observed, under the light microscope and electron microscope, and the changes of proinflammatory expression was observed too, using PCR technique. RESULTS: The products of proinflammatory expression, such as eotaxin, iNOS and IL-4 increased markedly. The change of chronic inflammation was shown by light microscope and electron microscope. CONCLUSION: Chronic prostatitis is associated with autoimmunity.

PGD2 metabolism in plasma: kinetics and relationship with bioactivity on DP1 and CRTH2 receptors.

Prostaglandin (PG)D(2), an important mediator in allergic diseases, is rapidly transformed in plasma to active metabolites that bind and activate two distinct receptors, DP1 and CRTH2. Since the rate of PGD(2) degradation and the bioactivity of the resulting metabolites are still unclear, the aim of our study was to analyze the kinetics and biological effects of PGD(2) metabolites formed in plasma. Eosinophil shape change was taken as a parameter of chemotactic activation mediated by CRTH2 whereas inhibition of platelet aggregation served as a measure of DP1 activity. PGD(2) was degraded in plasma with an apparent half-life of approximately 30 min, accompanied by a loss of potency in inhibiting platelet aggregation as well as inducing eosinophil stimulation. Incubation of PGD(2) in plasma for 120 min caused an increase in the IC(50) for platelet aggregation by a factor of 6.5 and an increase of the EC(50) for eosinophil shape change by a factor of 7.2. However, tandem mass spectrometry analysis showed that incubation of PGD(2) in plasma for 120 min resulted in clearance of PGD(2) of more than 92%, which was mirrored by a continuous formation of Delta(12)-PGD(2) and Delta(12)-PGJ(2), whereas only small amounts of 15d-PGD(2) and 15d-PGJ(2) were detected. Interestingly, a rapid degradation of PGD(2) was also observed in serum, which was not prevented by pepsin digestion of serum preceding the addition of PGD(2). Therefore, despite extensive non-enzymatic metabolization of PGD(2) in plasma, its biological activity with respect to DP1 and CRTH2 is maintained through the formation of bioactive metabolites.

Expressions of the VLA-4, LFA-1 and Mac-1 integrins in eosinophil migration in a case of chronic eosinophilic leukaemia.

Migration of eosinophil (Eo) into tissues is a hallmark of chronic eosinophilic leukaemia (CEL), but the exact mechanism involved in cell migration is unknown. We report on a patient with CEL who presented high expressions of VLA-4, LFA-1 and Mac-1 integrins on the Eo surface, increased chemotaxis of Eo to eotaxin, decreased chemotaxis of Eo after inhibition of the cyclic guanosine monophosphate (cGMP), and a high level of intracellular cGMP. These findings suggest that an upregulation of expression of these integrins on Eo surface and a high intracellular level of cGMP may be involved in the increased Eo migration observed in our CEL patient. However, the definitive roles of these molecules in the proliferation and migration of Eo in CEL disease require wider confirmation by analysis of additional patients with the disease.

Elevated levels of antimicrobial peptides in bronchoalveolar lavage fluid in patients with chronic eosinophilic pneumonia.

BACKGROUND: Chronic eosinophilic pneumonia (CEP) is an idiopathic pulmonary disease. As the lung is in direct communication with the environment, inhaled antigen may activate immune mechanisms in the airway that may participate in the pathogenesis of idiopathic pulmonary diseases. Defensins are antimicrobial peptides that consist of alpha-defensin (HAD) in neutrophils and beta-defensin (HBD) in epithelial cells. Defensins act as innate immunity against pathogens acquired from the environment and as mediators to induce local inflammation. OBJECTIVES: The aim of the present study was to determine whether immune mechanisms in the airway are induced in CEP patients. METHODS: We measured BALF defensin levels in patients with CEP, acute EP (AEP) and drug-induced eosinophilic pneumonia (drug-EP). We also measured BALF levels of IL-5, GM-CSF, eotaxin and RANTES. These substances can recruit eosinophils. RESULTS: BALF HAD levels were higher in patients with CEP than in those with drug-EP and normal controls. HBD-2 was detected in BALF of 10 of 11 CEP patients and in 3 of 5 AEP patients while its level was below detection in drug-EP patients and normal controls. BALF HBD-2 levels correlated with the proportion of lymphocytes in CEP patients. CONCLUSION: The defensin-linked immune system is activated in CEP but not in drug-EP. This suggests that inhaled antigen(s) may be involved in the pathogenesis of CEP.

Eosinophil chemotactic factor-L (ECF-L) enhances osteoclast formation by increasing in osteoclast precursors expression of LFA-1 and ICAM-1.

ECF-L is a novel autocrine stimulator of osteoclast (OCL) formation that enhances the effects of 1,25-(OH)2D3 and RANK ligand (RANKL) and is increased in inflammatory conditions such as rheumatoid arthritis. ECF-L acts at the later stages of OCL formation and does not increase RANKL expression. Thus, its mechanism of action is unclear. Therefore, RAW 264.7 cells and M-CSF-dependent murine bone marrow macrophage (MDBM) cells were treated with RANKL and/or with recombinant ECF-L expressed as a Fc fusion protein (ECF-L-Fc) to determine their effects on NF-kappaB, AP-1 and JNK activity, and on the expression of the adhesion molecules that have been implicated in OCL formation. These parameters were measured by semiquantitative and PCR and Western blot analysis. In addition, the role of ICAM-1 was further assessed by treating normal mouse marrow cultures with ECF-L-Fc and 10(-10) M 1,25-(OH)2D3 in the presence or absence of a blocking ICAM-1 antibody or treating marrow cultures from ICAM-1 knockout mice with ECF-L and 1,25-(OH)2D3. ECF-L-Fc by itself only modestly increased NF-kappaB binding and JNK activity in RAW 264.7 cells, which was further enhanced by RANKL. In contrast, ECF-L-Fc increased LFA-1alpha and ICAM-1 mRNA levels 1.8-fold in mouse marrow cultures, and anti-ICAM-1 almost completely inhibited OCL formation induced by 10(-10) M 1,25-(OH)2D3 and ECF-L. Furthermore, ECF-L did not increase OCL formation in marrow cultures from ICAM-1 knockout mice. Taken together, these results demonstrate that ECF-L enhances RANKL and 1,25-(OH)2D3-induced OCL formation by increasing adhesive interactions between OCL precursors through increased expression of ICAM-1 and LFA-1.

Toll-like receptor-2-mediated C-C chemokine receptor 3 and eotaxin-driven eosinophil influx induced by Mycobacterium bovis BCG pleurisy.

An acute and persistent eosinophil infiltration is observed during Mycobacterium bovis BCG pleural infection in mice. Eosinophil accumulation, lipid body formation, and eotaxin production were significantly reduced in BCG-infected Toll-like receptor-2 (TLR2)-deficient mice compared to wild-type mice. Neutralization of eotaxin or CCR3 drastically inhibited BCG-induced eosinophil accumulation and lipid body formation, indicating that BCG-induced eosinophil recruitment and activation is largely dependent of TLR2-mediated eotaxin generation.

Morphological and proinflammatory expression of the experimental autoimmune prostatitis in the rat model / 中华男科学杂志

<p><b>OBJECTIVE</b>To establish a Wistar rat model of chronic abacterial prostatitis (CAP) by injecting purified prostate protein and Freund's complete adjuvant, and to study the influence on the morphology and proinflammatory expression.</p><p><b>METHODS</b>Male rats were injected with the Pertussis-Diphteria-Tetanus vaccine into the abdominal cavity and purified prostate protein and Freund's complete adjuvant intradermally at 0 and 30 days. At 60 days, the rats were sacrificed, and then the prostate specimens were observed, under the light microscope and electron microscope, and the changes of proinflammatory expression was observed too, using PCR technique.</p><p><b>RESULTS</b>The products of proinflammatory expression, such as eotaxin, iNOS and IL-4 increased markedly. The change of chronic inflammation was shown by light microscope and electron microscope.</p><p><b>CONCLUSION</b>Chronic prostatitis is associated with autoimmunity.</p>