Soluble transforming growth factor-beta type III receptor gene transfection inhibits fibrous airway obliteration in a rat model of Bronchiolitis obliterans

Post-transplant bronchiolitis obliterans (BO) is characterized by fibroproliferation and fibrous obliteration of distal airways in chronically rejected lungs. In this study, using a rat heterotopic allogeneic tracheal transplant model of BO, we evaluated the expression of transforming growth factor-beta (TGFbeta) during the development of airway fibrous obliteration. Immunohistochemical analysis revealed TGFbeta staining in infiltrating mononuclear cells at Days 2 and 7, and in the fibrous tissues until Day 21. Soluble TGFbeta receptor type III (TGFBIIIR), by blocking TGFbeta binding to its membrane receptors, functions as a TGFbeta antagonist. To study the role of TGFbeta in the development of BO, adenoviral-mediated soluble TGFBIIIR gene transfection (5 x 10(9) particles) was performed topically at the site of transplant on Day 5 after transplantation, which leads to inhibition of fibrous airway obliteration. In contrast, empty vector gene delivered through intramuscular injection, or given locally at Days 0 or 10 after tracheal transplantation had no significant effect. These results suggest that TGFbeta expressed in the allografts plays a pivotal role in the pathogenesis of BO. Soluble TGFBIIIR may competitively inhibit TGFbeta activity locally. Adenoviral-mediated soluble TGFBIIIR gene transfection should be further explored as a potential therapeutic modality for BO and other conditions involving chronic fibrosis.     

Upregulation of T-helper 1 cytokines and chemokine expression in post-transplant airway obliteration.

The major obstacle to long-term survival after lung transplantation is chronic graft dysfunction manifest as bronchiolitis obliterans. Since the early stages are characterized by proliferation of itinerant cells (lymphocytes and macrophages), we hypothesized that cytokines and chemokines may play a role in the development of the fibroproliferative process. In a heterotopic rat tracheal transplant model, we studied isografts and allografts 3, 7, and 21 d after transplantation as representative time points for the triphasic time course in the evolution of allograft airway obliteration. Using a semiquantitative RT-PCR technique, intragraft gene expression of T-helper 1 (Th1)- and Th2-type cytokines and of C-C and C-X-C chemokines was examined. The results of our study show a distinct pattern of cytokine and chemokine gene expression in the development of post-transplant airway obliteration. Allografts, in contrast to isografts, showed a strong and persistent Th1-type response (expression of interleukin-2 and interferon-gamma genes), even after fibrous airway obliteration was complete, suggesting an ongoing allo-immune process until late in the fibroproliferative stage. RANTES and MCP-1 were also upregulated late after transplantation, whereas MIP-2 upregulation occurred early post-transplant and was not restricted to allografts alone, which might reflect alloantigen-independent processes after transplantation that are present in both allografts and isografts.     

Role of platelet-derived growth factor and vascular endothelial growth factor in obliterative airway disease

RATIONALE: Platelet-derived growth factor (PDGF) is an important smooth muscle cell mitogen, and vascular endothelial growth factor (VEGF) is a known angiogenic and proinflammatory growth factor. We hypothesized that specific therapy aimed at these growth factors might inhibit the development of experimental obliterative airway disease (OAD). METHODS: In fully mismatched rat tracheal allografts, we used imatinib and PTK/ZK, either alone or in combination, to block PDGF and VEGF receptor protein tyrosine kinase (RTK) action, respectively. Prophylaxis was initiated at the time of transplantation. Early treatment was commenced on Day 7 during the inflammatory phase and late treatment on Day 14 during the fibroproliferative phase of OAD. No immunosuppression was administered. MEASUREMENTS AND MAIN RESULTS: Prophylaxis with either PTK/ZK or imatinib alone significantly reduced OAD, and combined prophylaxis completely prevented its development. Early treatment with PTK/ZK and imatinib also effectively reduced the development of OAD. Late treatment failed to show significant efficacy. Blocking VEGF RTK action with PTK/ZK reduced the activation of allograft blood vessels and the number of lymph vessels in the allograft airway wall, and significantly diminished allograft inflammation, whereas PDGF blockade with imatinib inhibited the growth of smooth muscle cells in the proliferating lesion. CONCLUSIONS: Combined prophylactic PDGF and VEGF RTK blockade completely prevents the development of OAD. Also, when early treatment with PTK/ZK and imatinib is commenced during the inflammatory phase of OAD development, it significantly attenuates the development of tracheal occlusion, suggesting that these drugs could potentially be used to treat bronchiolitis obliterans syndrome in its early phase.     

Simultaneous donor marrow cell transplantation with reduced intensity conditioning prevents tracheal allograft obliteration in a bronchiolitis obliterans murine model

STUDY OBJECTIVES: Prolonged survival of transplanted kidney or liver allografts has been associated with prolonged chimerism resulting from donor-origin leukocytes carried within the allograft parenchyma. The present study, performed in a murine model, examined whether simultaneous administration of donor bone marrow cells after reduced intensity conditioning allows acceptance of heterotopic tracheal allografts and prevents the formation of the airway fibroproliferative lesion, which mimics bronchiolitis obliterans (BO). METHODS: Allogeneic tracheal allografts from C57BL/6 mice were grafted subcutaneously into BALB/c mice (n = 6) [day 0]. Conditioning consisted of total lymphoid irradiation (200 cGy) at day - 1, donor marrow cells (3 x 10(7)) administered IV on day 0, intraperitoneal cyclophosphamide (200 mg/kg) on day 1 to eliminate alloreactive marrow cells, followed by a repeated dose of donor marrow cells on day 2. Control groups consisted of one group (n = 4) that underwent similar conditioning without donor marrow cells, and another group (n = 4) that received syngeneic BALB/c marrow cells. None of these groups were administered maintenance immunosuppression. Grafts were harvested and histopathology findings were evaluated semiquantitatively at day 28, day 55, and day 95. RESULTS: Tracheal allografts from donor marrow cell recipients still maintained a patent airway with intact airway epithelium at 95 days after transplant. However, grafts from control animals not receiving donor marrow cells or mice administered syngeneic marrow cells had lumen obliteration by 28 days after transplant. Chimerism in animals receiving allogeneic bone marrow was confirmed. Graft vs host disease did not develop in animals receiving allogeneic marrow cells. CONCLUSIONS: Further investigation may verify this approach to be applicable for the prevention of posttransplantation BO.     

RANTES plays an important role in the evolution of allograft transplant-induced fibrous airway obliteration

Although lung transplantation is a widely applied therapeutic modality for end-stage pulmonary disease, the long-term survival following this procedure is limited by the development of bronchiolitis obliterans (BO). We investigated the role of RANTES, a C-C chemokine, in the evolution of fibrous airway obliteration (FAO) using a rat heterotopic tracheal transplant model. RANTES was highly expressed in infiltrating mononuclear cells in both allogeneic and isogeneic grafts as revealed by immunohistochemistry. Using a miniosmotic pump, neutralizing anti-RANTES antibody was locally and continuously infused to allografts, whereas recombinant rat RANTES was administered to isografts. Anti-RANTES antibody treatment decreased the number of CD4(+) infiltrating cells in allotracheas and preserved luminal patency compared with those of allocontrols. However, RANTES infusion in isografts did not induce FAO, even though CD4(+) cell migration was increased by this treatment. It appears that RANTES is relevant to the recruitment of CD4(+) cells and the development of FAO in the process of allorejection. Local administration of anti-RANTES might be a therapeutic option for BO following lung transplantation.     

Immune mechanisms of lung allograft rejection

Extended survival after lung transplantation is primarily limited by progressive airflow obstruction and fibrotic obliteration of the small airways, termed bronchiolitis obliterans syndrome (BOS) and bronchiolitis obliterans (BO), respectively. BO is thought to represent the pulmonary-specific manifestation of chronic allograft rejection and the end result of a spectrum of different immunological insults to the allograft. Historically, research has focused on the adaptive immune system and its cellular-based rejection as the driving factor in the development of BO. Recent research in animal lung transplant models and human lung transplant recipients has identified that chemokines, humoral immunity, autoimmunity, and innate immunity also contribute to lung allograft rejection and BO. This review explores the complex immunological mechanisms that promote the high rate of pulmonary allograft failure and significantly impair survival after lung transplantation. We also identify areas for further research critical to improving transplant outcomes.     

Novel insights into lung transplant rejection by microarray analysis

Gene expression microarrays can estimate the prevalence of mRNA for thousands of genes in a small sample of cells or tissue. Organ transplant researchers are increasingly using microarrays to identify specific patterns of gene expression that predict and characterize acute and chronic rejection, and to improve our understanding of the mechanisms underlying organ allograft dysfunction. We used microarrays to assess gene expression in bronchoalveolar lavage cell samples from lung transplant recipients with and without acute rejection on simultaneous lung biopsies. These studies showed increased expression during acute rejection of genes involved in inflammation, apoptosis, and T-cell activation and proliferation. We also studied gene expression during the evolution of airway obliteration in a murine heterotopic tracheal transplant model of chronic rejection. These studies demonstrated specific patterns of gene expression at defined time points after transplantation in allografts, whereas gene expression in isografts reverted back to that of native tracheas within 2 wk after transplantation. These studies demonstrate the potential power of microarrays to identify biomarkers of acute and chronic lung rejection. The application of new genetic, genomic, and proteomic technologies is in its infancy, and the microarray-based studies described here are clearly only the beginning of their application to lung transplantation. The massive amount of data generated per tissue or cell sample has spawned an outpouring of invention in the bioinformatics field, which is developing methodologies to turn data into meaningful and reproducible clinical and mechanistic inferences.     

骨化三醇在小鼠气管移植排斥反应中的作用

目的:研究骨化三醇对小鼠异位移植气管排斥反应的作用及其与环孢素有无协同性。方法:建立小鼠异位气管移植模型。术后分为4组,每组10只。对照组给予生理盐水25mL.kg-1.d-1灌胃;骨化三醇(cal)组给予骨化三醇2.5μg.kg-1.d-1灌胃;环孢素组(Csa)给予环孢素25mg.kg-1.d-1。灌胃;混合用药组(mix)给予骨化三醇2.5μg.kg-1.d-1和环孢素25mg.kg-1.d-1灌胃。术后4周取出移植气管,行HE染色,观察每组气管的阻塞率、上皮细胞缺失率、有无软骨破坏等指标。同时测定小鼠血清中白细胞介素-12(IL-12)的浓度及计算小鼠体重增长值。结果:对照组气管阻塞率、上皮细胞缺失率与环孢素、骨化三醇、混合用药组3组相比有明显增高(P<0.05)。混合用药组的气管阻塞率与其他3组相比明显降低(P<0.05)。环孢素组的上皮细胞缺失率与骨化三醇组、混合用药组2组相比明显升高(P<0.05)。对照组90%观察到软骨破坏,环孢素组20%观察到有软骨破坏,骨化三醇、混合用药2组没有观察到软骨破坏。对照组血清IL-12的浓度较其他3组血清IL-12浓度明显增高(P<0.05),混合用药组的IL-12浓度明显较其他3组降低(P<0.05)。环孢素组和混合用药组体重增长值与其他2组相比明显降低(P<0.05)。结论:骨化三醇对小鼠异位气管移植的排斥反应有抑制作用,在此过程中骨化三醇与环孢素表现出协同性。     

Lung transplantation in the fischer 344-wistar kyoto strain combination is a relevant experimental model to study the development of bronchiolitis obliterans in the rat

Chronic allograft rejection and bronchiolitis obliterans (BO) limited successful long-term outcome after lung transplantation (LTX). Reliable animal models are needed to study the pathogenesis of BO and to develop effective therapeutic strategies. The relevance of an available experimental LTX model without immunosuppression-the Fischer (F344)-Wistar Kyoto (WKY) rat strain combination-was analyzed. The kinetics of acute and chronic rejection and respective graft histopathology were described in the F344-to-WKY rat strain combination after allogeneic LTX. A modified classification of chronic lung allograft rejection was introduced to describe obliterative changes in the airways after rat LTX. Animals from Harlan Winkelmann (HW) and Charles River (CR) were examined. Within 14 days after LTX, allografts showed moderate to severe acute vascular and bronchiolar inflammation. In contrast to rats from CR, animals from HW showed a delayed acute rejection process. Mid-term phase after LTX (days 20-40) presented a "borderline form" consisting of both acute and first signs of chronic airway rejection. On postoperative day (POD) 60, first signs of airway remodelling and distinct BO were diagnosed in 80% of animals from HW. At the same time, the allografts with BO-like lesions increased up to 100% in rats from CR. The F344-to-WKY rat LTX model allows detailed assessment of all features of acute and chronic pulmonary rejection representing a clinically relevant model. However, due to breeding differences resulting in various sublines of the same rat strain, the source and husbandary history of the animals is important for analysis of immuno-mediated processes.     

小鼠闭塞性细支气管炎动物模型的建立

目的通过异位气管移植建立小鼠的闭塞性细支气管炎动物模型,观察其病理改变及免疫因素对模型的影响。方法 C57BL/6小鼠30只作为供体,取出气管分别植入10只C57BL/6小鼠、20只BALB/c小鼠颈部皮下。术后分3组。同质组：10只C57BL/6小鼠。对照组和实验组：每组10只BALB/c小鼠。同质组、对照组小鼠给予生理盐水25mL·kg^-1·d^-1灌胃。实验组小鼠25mg·kg^-1·d^-1环孢素A灌胃。术后4周处死小鼠,取出移植气管。观察移植气管的阻塞率、上皮细胞缺失率等指标。结果同质组移植气管管腔通畅,上皮细胞连续完整。对照组移植气管管腔阻塞,气管上皮细胞全部脱落。实验组气管有部分阻塞,部分上皮细胞脱落,气管的阻塞率、上皮细胞缺失率明显低于对照组（P〈0.05）。结论小鼠的异位气管移植在术后4周可以观察到与闭塞性细支气管炎相同的病理改变。免疫药物对该病理过程有抑制作用。     

Effect of atrial natriuretic factor on angiotensin converting enzyme

We studied the effects of atrial natriuretic factor (ANF) on the conversion of angiotensin I to angiotensin II. Pulmonary artery endothelial cells converted 1.22 nmol/min per dish [125I]angiotensin I to angiotensin II, but ANF suppressed the conversion by 0.475 nmol/min per dish. The maximum rate of inhibition of angiotensin converting enzyme (ACE) activity by ANF was 60% at 10(-6) mol/l ANF compared with control conditions. The amino acid fragments of ANF were studied to determine whether its specific structure was necessary for inhibiting ACE in endothelial cells. We found that atriopeptin II (fragment 5-27) inhibited ACE activity as much as ANF. Fragment 7-25 had the same rate of inhibition, but fragment 13-27 had no effect on the conversion of angiotensin I to angiotensin II. These results suggest that the amino acid sequence of Cys (7)-Cys (23) in ANF is important for the inhibition of ACE in pulmonary endothelial cells.     

Hierarchical contributions of allorecognition pathways in chronic lung rejection

The role of allorecognition in initiating lung graft rejection is not clearly defined. Using the heterotopic tracheal transplantation model, we examined the contributions of the indirect and direct allorecognition pathways in chronic airway rejection. Fully mismatched, wild-type grafts were transplanted into major histocompatibility complex (MHC) II-/-, class II-like accessory molecule (H2-DMalpha)-/- using MHC I-/- and wild-type allorecipients as control subjects. Similarly, MHC I-/-, MHC II-/-, or MHC I/II-/- allografts were transplanted into wild-type mice with appropriate control subjects. Grafts from nonimmunosuppressed recipients were evaluated at Weeks 2, 4, and 6. Grafts transplanted into MHC II-/- and H2-DMalpha-/- allorecipients showed a more intact epithelium and reduced lumen obliteration compared with grafts transplanted into wild-type or MHC I-/- allorecipients (p < 0.05 for each). These grafts exhibited abundant CD4+ and CD8+ cell infiltrates similar to control allografts. MHC I-/- and MHC I/II-/- but not MHC II-/- allografts placed in wild-type animals demonstrated less severe rejection compared with allograft control subjects (p < 0.05 for each). Although the indirect allorecognition pathway has the strongest influence on rejection, the direct pathway is sufficient to ultimately cause chronic airway rejection. In addition, these results suggest that MHC class I molecules are the principal alloantigens in the mouse heterotopic tracheal model of obliterative bronchiolitis.     

骨化三醇对小鼠异位气管移植后胸腺T细胞的影响

目的研究骨化三醇对小鼠异位移植气管动物模型胸腺T细胞的免疫作用及其与环孢菌素有无协同作用。方法建立小鼠异位气管移植模型。术后将小鼠分为四组,每组18支。对照组给予生理盐水0.5ml·d-1灌胃;Cal组给予骨化三醇2.5μg·kg-1·d-1灌胃;Csa组给予环孢菌素25mg·kg-1·d-1灌胃;混合组给予骨化三醇2.5μg·kg-1·d-1和环孢菌素25mg·kg-1·d-1灌胃。移植术后第3、10、30天,每组分别处死6只小鼠。取出移植气管,行HE染色,观察移植气管的阻塞率、上皮缺失率。流式细胞仪测定受体小鼠胸腺中CD4＋、CD8＋细胞数目。结果术后第3天：各组气管阻塞率、上皮缺损率无明显差异,各组胸腺CD4＋、CD8＋淋巴细胞、CD4＋/CD8＋计数无明显差别。术后第10天：对照组移植气管阻塞率、上皮缺失率明显高于其他三组。对照组CD4＋水平高于其他三组（P〈0.05）,对照组CD8＋计数明显高于第3天时的水平。对照组的CD4＋/CD8＋明显低于其他三组,混合用药组CD4＋/CD8＋低于骨化三醇组和环孢菌素组（P〈0.05）;术后第30天：混合用药组的阻塞率、上皮缺失率明显低于其他三组（P,0.05）。对照组CD4＋、CD8＋水平与第10天相比明显降低（P〈0.05）。环孢菌素组、骨化三醇组的CD8＋与10天相比明显增加（P〈0.05）。混合组CD4＋、CD8＋、CD4＋/CD8＋没有明显变化。结论骨化三醇对小鼠胸腺内CD4＋、CD8＋水平的表达有抑制作用。骨化三醇与环孢菌素A表现出免疫协同性。     

Evaluation of rabbit tracheal inflammation using optical coherence tomography

BACKGROUND: Optical coherence tomography (OCT) is an evolving technology that is capable of delivering real-time, high-resolution images of tissues. The purpose of this study was to evaluate the feasibility of using OCT for detecting airway pathology in a septic animal model. METHODS: The tracheas of New Zealand white rabbits were inoculated endobronchially with various concentrations of live Streptococcus pneumoniae bacteria. After the development of pneumonia/sepsis, the animals were killed. OCT tracheal images and corresponding histologic specimens from these experimental animals were compared to control rabbit tracheas for morphologic features and quantitative tracheal mucosal thickness measurements. RESULTS: The results revealed significant airway mucosal thickening in the experimental group that was consistent with tracheal edema. Morphologic changes, including epithelial denuding and mucosal sloughing, were evident in regions of the experimental tracheas. CONCLUSION: This study suggests that OCT is a potentially valuable imaging modality that is capable of evaluating superficial airway pathology with high-resolution in vivo images. Numerous applications of OCT can be envisioned in the realm of pulmonary medicine and thoracic surgery that may substantially increase the precision and accuracy of current bronchoscopic diagnostic and surgical techniques.     

Evidence against a major role for angiotensin converting enzyme-related carboxypeptidase (ACE2) in angiotensin peptide metabolism in the human coronary circulation

OBJECTIVE: To investigate the role of angiotensin-converting enzyme-related carboxypeptidase (ACE2) in angiotensin peptide metabolism in the human coronary circulation. METHODS: Angiotensin I and angiotensin II, and their respective carboxypeptidase metabolites, angiotensin-(1-9) and angiotensin-(1-7), were measured in arterial and coronary sinus blood of heart failure subjects receiving angiotensin-converting enzyme (ACE) inhibitor therapy and in normal subjects not receiving ACE inhibitor therapy. In addition, angiotensin I, angiotensin II and angiotensin-(1-7) were measured in arterial and coronary sinus blood of subjects with coronary artery disease before, and at 2, 5 and 10 min after, intravenous administration of ACE inhibitor. RESULTS: In comparison with normal subjects, heart failure subjects receiving ACE inhibitor therapy had a greater than 40-fold increase in angiotensin I levels, but angiotensin-(1-9) levels were low (1-2 fmol/ml), and similar to those of normal subjects. Moreover, angiotensin-(1-7) levels increased in parallel with angiotensin I levels and the angiotensin-(1-7)/angiotensin II ratio increased by 7.5-fold in coronary sinus blood. Intravenous administration of ACE inhibitor to subjects with coronary artery disease rapidly decreased angiotensin II levels by 54-58% and increased angiotensin I levels by 2.4- to 2.8-fold, but did not alter angiotensin-(1-7) levels or net angiotensin-(1-7) production across the myocardial vascular bed. CONCLUSIONS: The failure of angiotensin-(1-9) levels to increase in response to increased angiotensin I levels indicated little role for ACE2 in angiotensin I metabolism. Additionally, the levels of angiotensin-(1-7) were more linked to those of angiotensin I than angiotensin II, consistent with its formation by endopeptidase-mediated metabolism of angiotensin I, rather than by ACE2-mediated metabolism of angiotensin II.     

Nitric oxide and vasoactive intestinal peptide as co-transmitters of airway smooth-muscle relaxation: analysis in neuronal nitric oxide synthase knockout mice

BACKGROUND: Both vasoactive intestinal peptide (VIP) and nitric oxide (NO) relax airway smooth muscle and are potential co-transmitters of neurogenic airway relaxation. The availability of neuronal NO synthase (nNOS) knockout mice (nNOS-/-) provides a unique opportunity for evaluating NO. OBJECTIVE: To evaluate the relative importance of NO, especially that generated by nNOS, and VIP as transmitters of the inhibitory nonadrenergic, noncholinergic (NANC) system. STUDY DESIGN: In this study, we compared the neurogenic (tetrodotoxin-sensitive) NANC relaxation of tracheal segments from nNOS-/- mice and control wild-type mice (nNOS(+/+)), induced by electrical field stimulation (EFS). We also examined the tracheal contractile response to methacholine and its relaxant response to VIP. RESULTS: EFS (at 60 V for 2 ms, at 10, 15, or 20 Hz) dose-dependently reduced tracheal tension, and the relaxations were consistently smaller (approximately 40%) in trachea from nNOS-/- mice than from control wild-type mice (p < 0.001). VIP (10(- 8) to 10(-6) mol/L) induced concentration-dependent relaxations that were approximately 50% smaller in nNOS-/- tracheas than in control tracheas. Methacholine induced concentration-dependent contractions that were consistently higher in the nNOS-/- tracheas relative to wild-type mice tracheas (p > 0.05). CONCLUSION: Our data suggest that, in mouse trachea, NO is probably responsible for mediating a large (approximately 60%) component of neurogenic NANC relaxation, and a similar (approximately 50%) component of the relaxant effect of VIP. The results imply that NO contributes significantly to neurogenic relaxation of mouse airway smooth muscle, whether due to neurogenic stimulation or to the neuropeptide VIP.     

Acute exposure to cigarette smoke induces airway hyperresponsiveness without airway inflammation in guinea pigs. Dose-response characteristics

We examined whether acute exposure to a low dose of cigarette smoke causes an increase in airway responsiveness in guinea pigs and whether the changes in airway responsiveness are accompanied by increased vascular permeability or neutrophil influx in the trachea. Animals were divided into four groups: groups exposed to 5, 10, or 20 puffs of cigarette smoke and a control group. Airway responsiveness was assessed by measuring specific airway resistance (SRaw) as a function of increasing concentration of inhaled methacholine (Mch) aerosol immediately, 5 h, and 24 h after exposure. In parallel studies, tracheal vascular permeability was quantified by measuring the tracheal extravasation of intravenously administered Evans blue dye, and neutrophil influx into the tracheal mucosa was quantified by counting cells within whole mounts of tracheas that were stained with Giemsa. Exposure to 5 puffs of cigarette smoke caused no changes in airway responsiveness. Exposure to 10 puffs induced airway hyperresponsiveness only immediately after exposure. Exposure to 20 puffs induced airway hyperresponsiveness not only immediately but also 5 h after exposure. There was a significant correlation between the dose (puffs) of cigarette smoke and increase in airway responsiveness immediately after exposure (r = 0.77; p less than 0.001). The tracheal extravasation of intravenously administered Evans blue dye and the number of neutrophils in the tracheal mucosa did not differ significantly from the corresponding control values at any time or in any exposed group. Furthermore, none of these changes was observed in the airways distal to the trachea of any animal immediately after exposure to 20 puffs of cigarette smoke.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of the tachykinin NK(1) receptor in mediating contraction to 5-hydroxytryptamine and antigen in the mouse trachea

Neuroimmune interactions are important in airway diseases such as asthma. We evaluated the role of the tachykinin NK(1) receptor in the contractile response of isolated trachea from tachykinin NK(1) receptor wild type (WT) and knockout (KO) mice, to the antigen ovalbumin and the contractile agonist serotonin (5-hydroxytryptamine). One percent ovalbumin induced contractions of tracheas obtained from ovalbumin-immunized and exposed mice. The tracheas from WT animals showed larger contractions compared to the KO mice. Tracheas from sensitized and ovalbumin-exposed animals released 5-hydroxytyptamine upon addition of ovalbumin. No higher levels of 5-hydroxytryptamine were released from tracheas of WT animals. Tracheas of non-sensitized animals did not release 5-hydroxytryptamine upon ovalbumin challenge. Responses to ovalbumin were abrogated by methysergide, a broad 5-hydroxytryptamine receptor antagonist. Exogenous 5-hydroxytryptamine contracted tracheas but WT tracheas responded significantly more. Atropine and tetrodotoxin (TTX) reduced 5-hydroxytryptamine-induced contractions of the WT tracheas, while they did not affect 5-hydroxytryptamine-induced contractions of KO tracheas. 5-Hydroxytryptamine-induced contractions from atropine- or TTX-treated WT tracheas did not differ significantly from the contractions of the KO tracheas. Single tachykinin NK(1) receptor antagonists SR140,333 and RP67,580 had no effect on 5-hydroxytryptamine-induced contractions. In conclusion, the 5-hydroxytryptamine-induced tracheal contraction includes a cholinergic mechanism that requires the presence of the tachykinin NK(1) receptor.     

Angiotensin converting enzyme is the main contributor to angiotensin I-II conversion in the interstitium of the isolated perfused rat heart

OBJECTIVES: Recent studies in homogenized hearts suggest that chymase rather than angiotensin converting enzyme (ACE) is responsible for cardiac angiotensin I to angiotensin II conversion. We investigated in intact rat hearts whether (i) enzymes other than ACE contribute to angiotensin I to angiotensin II conversion and (ii) the localization (endothelial/extra-endothelial) of converting enzymes. DESIGN AND METHODS: We used a modified version of the rat Langendorff heart, allowing separate collection of coronary effluent and interstitial fluid. Hearts were perfused with angiotensin I (arterial concentration 5-10 pmol/ml) under control conditions, in the presence of captopril (1 micromol/l) or after endothelium removal with 0.2% triton X-100. Endothelium removal was verified as the absence of a coronary vasodilator response to 10 nmol bradykinin. Angiotensin I and angiotensin II were measured in coronary effluent and interstitial fluid with sensitive radioimmunoassays. RESULTS: In control hearts, 45% of arterial angiotensin I was metabolized during coronary passage, partly through conversion to angiotensin II. At steady-state, the angiotensin I concentration in interstitial fluid was three to four-fold lower than in coronary effluent, while the angiotensin II concentrations in both fluids were similar. Captopril and endothelium removal did not affect coronary angiotensin I extraction, but increased the interstitial fluid levels of angiotensin I two- and three-fold, respectively, thereby demonstrating that metabolism (by ACE) as well as the physical presence of the endothelium normally prevent arterial angiotensin I from reaching similar levels in coronary effluent and interstitial fluid. Captopril, but not endothelium removal, greatly reduced the angiotensin II levels in coronary effluent and interstitial fluid. With the ACE inhibitor, the angiotensin II/I ratios in coronary effluent and interstitial fluid were 83 and 93% lower, while after endothelium removal, the ratios were 33 and 71% lower. CONCLUSIONS: In the intact rat heart, ACE is the main contributor to angiotensin I to angiotensin II conversion, both in the coronary vascular bed and the interstitium. Cardiac ACE is not limited to the coronary vascular endothelium.     

Effects of platelet-activating factor on vascular permeability and granulocyte recruitment in the rat trachea.

Platelet-activating factor (PAF) is a phospholipid mediator known to produce several features of airway inflammation. We examined the effects of intravenous PAF on vascular permeability and granulocyte recruitment in the rat trachea. To assess vascular permeability, anesthetized rats were given injections of Evans blue dye (30 mg/kg, iv) and PAF (1-10 micrograms/kg, iv), and then their tracheas were removed and assayed spectrophotometrically for dye content. We found that a PAF dosage of 6 micrograms/kg increased the tracheal dye content 7-fold compared to controls. The amount of extravasated dye in the tracheas was significantly increased 1 min after PAF injection, was maximal at 5 min, and had returned to control levels by 10 min. To assess granulocyte recruitment, anesthetized rats were given an injection of PAF (6 micrograms/kg, iv), and then their tracheas were removed and stained to reveal myeloperoxidase-containing neutrophils and eosinophils. We found that the number of neutrophils in the tracheal mucosa was increased 7-fold from controls 5 min after PAF injection, but was not significantly increased 6 h later. The number of eosinophils in the tracheal mucosa was not significantly increased from controls at any time after PAF injection. We conclude that intravenous PAF causes a rapid but transient increase in vascular permeability in the rat trachea, and that intravenous PAF also causes a rapid but transient recruitment of neutrophils into the tracheal mucosa without a similar effect on eosinophils.