Induction of lung eosinophilia and neutrophilia in guinea pigs following injection of Sephadex beads

We have developed a method of induction of airway eosinophilia and neutrophilia in guinea pigs by intravenous injection of various types of Sephadex beads. In the first series of experiments, we have shown that G-50 Sephadex beads (Superfine, 24 mg/kg in conscious animals) induced a large accumulation of inflammatory cells in alveolar walls. The bronchoalveolar lavage (BAL) fluid from animals treated with this dose of Sephadex beads contained about 85 × 10⁶ cells as compared to 20 × 10⁶ cells in control animals. The eosinophils corresponded to 41% of the BAL cell population as assessed with Wright-Giemsa staining. However, in the BAL fluid from these bead-treated animals, a significant increase of monocytes, lymphocytes, and neutrophils was also observed. We have also tested the potency of G-75, G-100, and G-200 Sephadex beads (Superfine) to induce eosinophilia in guinea pig. Nonlethal intravenous doses of G-75 (14.27 mg/kg), G-100 (8.0 mg/kg), and G-200 (10.71 mg/kg) Sephadex beads were selected and induced variable levels of airway eosinophilia and neutrophilia in conscious guinea pigs. The percentage of eosinophil recovered in the BAL fluid corresponded to 35, 61, and 44% of total cells for G-75, G-100, and G-200, respectively. The neutrophils corresponded to 24, 2, and 12% of the total BAL cells for G-75, G-100, and G-200, respectively. Since the size of the beads did not seem to correlate with the intensity of airway eosinophilia and neutrophilia, the effect of lower doses of the G-50 Sephadex beads (9.86–0.43 mg/kg) on the inflammatory cell infiltration was also tested. Results showed that there was a correlation between the neutrophil number and the number of beads (r=0.996), whereas the number of eosinophils was less directly correlated to the bead number (r=0.812). The alveolar eosinophils were purified from BAL fluid by centrifugation on a continuous Percoll gradient (65%) to separate eosinophils from neutrophils. Normodense eosinophils (density 1.087–1.100 g/ml) obtained from Sephadex-treated animals were found at the bottom of the continuous Percoll gradient (25 x 10⁶; 98% purity). These highly purified eosinophils released thromboxane A₂ (TxA₂) following stimulation with 2M ionophore A23187. The method of accumulation and purification of guinea pig alveolar eosinophils is simple, rapid, and provides a large number of pure normodense cells for further biological studies. The induction of airway eosinophilia and neutrophilia in guinea pigs following injection of various types of Sephadex beads could also provide an interesting model for the study of the mechanisms of eosinophilia and neutrophilia and their relationship to airway hyperresponsiveness.     

Early Bronchial Hyperresponsiveness Following Injection of Sephadex Beads in the Guinea Pig: Involvement of Platelet Activating Factor and Thromboxane A2

The effects of an intravenous injection (i.v.) of Sephadex beads (20 mg kg^–1) were examined on bronchial responsiveness to ACh (1–200 g kg^–1 i.v.) as well as on cell accumulation in guinea-pig lung. Bronchial hyperreactivity to ACh, measured as increase in pulmonary insufflation pressure (PIP), was observed 3 h following the i.v. injection of Sephadex beads. However, no significant increase in bronchial reactivity to ACh was measured at 6 and 12 h following Sephadex injection. A second later increase in bronchial hyperresponsiveness was observed at 24 h. Bronchoalveolar lavage performed at 3 h following Sephadex treatment showed that there was no significant increase in total or differential cell number. At 6 h and 12 h, a significant increase in total cell counts was observed. At 24 h, a greater than 5-fold increase in cell number was observed and was related to a marked eosinophil, neutrophil and macrophage infiltration. A platelet-activating factor (PAF) antagonist, CV-3988 (10 mg kg^–1 i.v.), and a thromboxane A₂ (TxA₂) antagonist, L655,240 (10 mg kg^–1 i.v.), significantly attenuated the Sephadex-induced bronchial hyperresponsiveness to ACh observed at 3 h. The results show that an i.v. injection of Sephadex beads in guinea pigs can induce an early bronchial hyperresponsiveness to ACh that is mediated by the release of both PAF and TxA₂ and is independent of airway cell infiltration.     

Selective Inflammatory Response Induced by Intratracheal and Intravenous Administration of Poly-L-Arginine in Guinea Pig Lungs

Major basic protein (MBP) is a cationic protein found in eosinophil granules that was postulated to participate in the pathogenesis of bronchial asthma. Recently, it has been demonstrated that MBP level in serum or bronchoalveolar lavage (BAL) fluid was correlated with bronchial hyperresponsiveness (BHR) in asthmatics. A number a studies have established that MBP actions could be mimicked by synthetic polycations as poly-L-arginine. In this study, we investigated the effects of intratracheal and intravenous administration of poly-L-arginine on lung inflammatory response development. The intratracheal injection of poly-L-arginine at the doses of 1, 10, 100 nmol/animal increased the number of eosinophils (up to 3.2 fold) and neutrophils (up to 12 fold) in BAL fluid. Eosinophil and neutrophil infiltration was reversed by 88% and 67% respectively following low molecular weight heparin treatment (500 g/animal). The intravenous injection of increasing doses of poly-L-arginine (1, 10, 100, 500 nmol/animal) increased the number of eosinophils (up to 2.7 fold) but not neutrophil infiltration in guinea pig lungs. Eosinophil infiltration was reversed by 87% following low molecular weight heparin treatment (1.5 mg/animal). Intratracheal treatment with poly-L-arginine (100 nmol/animal) produced an important increase of -glucuronidase, histamine, eosinophil peroxidase (EPO) and albumin levels in BAL fluid, whereas the intravenous treatment (500 nmol/animal) did not. These results show that the route of administration of poly-L-arginine greatly influences its effect on inflammatory cell recruitment since both administration routes elicited eosinophil migration but only the intratracheal route stimulated the migration of neutrophils. Moreover, poly-L-arginine appeared to induce other inflammatory responses since it increased -glucuronidase, histamine, EPO and albumin levels in BAL fluid following intratracheal treatment. These results also showed that low molecular weight heparin significantly blocks the inflammatory responses elicited by poly-L-arginine.     

Comparative Study of Trans‐Oral and Trans‐Tracheal Intratracheal Instillations in a Murine Model of Acute Lung Injury

Animal model is of importance to further elucidate the pathogenesis of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). We envisioned a possibility that there might be the differences in lipopolysaccharide (LPS)‐induced acute lung inflammation by the trans‐oral and trans‐tracheal intratracheal instillations. We compared the LPS‐induced early inflammatory responses by these two methods. The evaluative system included bronchoalveolar lavage (BAL) fluid biochemical analysis and differential cell counting, lung wet/dry weight ratio and lung histology. In vitro studies were performed on human bronchial epithelial cell line NCI‐H292 and alveolar Type II epithelial cell line A549 stimulated with LPS. Both interleukin (IL)‐8 release in the BAL fluid and IL‐8 secretions from NCI‐H292 and A549 cells were measured. We found that the trans‐tracheal intratracheal instillation promoted the LPS‐induced cell injury, neutrophil infiltration, and pulmonary edema compared to the trans‐oral one. The LPS‐induced pathological changes by the trans‐oral intratracheal instillation were characterized by pulmonary interstitial edema, but the trans‐tracheal intratracheal instillation was exudative pulmonary edema. More IL‐8 is produced from A549 cells than from NCI‐H292 cells under the treatment of LPS. The increased IL‐8 release in the BAL fluid and enhanced inflammatory responses caused by LPS may be due to more LPS delivered into the alveolar spaces by the trans‐tracheal intratracheal instillation compared to the trans‐oral one. The trans‐tracheal intratracheal instillation is proved to be more suitable to establish the murine model of ALI than the trans‐oral one and helpful to further elucidate the pathogenesis of ALI/ARDS. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.     

Evidence for the activation of blood complement in Sephadex beads-induced lung inflammation in guinea pigs

OBJECTIVE AND DESIGN: This study evaluated the complement activation in guinea pigs that were given an intravenous injection of Sephadex and its correlation with markers of the development of inflammation. MATERIALS AND METHODS: Dunkin Hartley guinea pigs (250-300 g) were used. Whole blood was collected by heart puncture in a sodium citrate solution (0.315 g/ml) for complement measurements. Complement activation was measured using a colorimetric haemolytic assay. Bronchoalveolar lavages (BAL) were performed to monitor cell infiltration and inflammation was monitored by measurements of eosinophil peroxidase (EPO), histamine, beta-glucuronidase, albumin and total proteins in the BAL fluid. TREATMENT: Guinea pigs were pre-treated with aprotinin (40000 IKU/kg) 30 min before they were given an intravenous injection of Sephadex beads (24 mg/kg). Carboxymethyl (CM)-Sephadex (24 mg/kg) was administered alone. RESULTS: Sephadex beads activated the complement system in vitro (14.12+/-2.29 U/ml) and in vivo (9.95+/-0.08 U/ml) reaching a peak 6 h after the injection. This activation was accompanied by other characteristic features of inflammation such as leukocyte infiltration and activation. Both CM-Sephadex and aprotinin reduced the blood complement activation and eosinophil infiltration/activation observed. CONCLUSIONS: Our results strongly suggest that complement is involved in the cascade of events leading to the inflammatory state observed in guinea pig following the intravenous injection of Sephadex beads.     

Evidence that arachidonic acid derived from neutrophils and prostaglandin E<Subscript>2</Subscript> are associated with the induction of acute lung inflammation by lipopolysaccharide of <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objective: The involvement of arachidonic acid (AA) and PGE₂ during the E. coli lipopolysaccharide (LPS)-induced acute lung injury was investigated.Material: Adult male Wistar rats were used. For in vitro studies, rat neutrophils, bronchoalveolar lavage (BAL) fluid, and lug vascular endothelium were used, as described below.Treatment: Rats were given an intratracheal injection of LPS (750 g).Methods: Total and differential cell counts in BAL fluid; enzyme-linked immunoassay (ELISA) analyses of TNF-, IL-1, LTB₄ and PGE₂ in BAL, and immunohistochemical detection of ICAM-1 on lung vascular endothelium were performed six h after LPS challenge. Fatty acid composition of blood neutrophils and plasma was analyzed by HPLC.Results: Rats instilled with LPS presented a sixty three-fold increase in the number of neutrophils in BAL (from 0.5 × 10⁶ to 31.5 × 10⁶ cells), accompanied by increased levels of TNF- and IL-1 (p < 0.001), and a three-fold increase in ICAM-1 expression on vascular endothelium. The content of AA in blood neutrophils was reduced by 50%, whereas the level of PGE₂ in BAL was increased by 3.5 fold, without changes in the levels of LTB₄.Conclusions: These findings suggest that AA and PGE₂ are associated with LPS challenge.Received 19 October 2003; returned for revision 30 November 2003; accepted by N. Boughton-Smith 15 July 2004     

Rapamycin inhibits airway leukocyte infiltration and hyperreactivity in guinea pigs

The effect of rapamycin on cell infiltration to the lung and on bronchial hyperreactivity induced by an intravenous injection of sephadex beads to guinea pigs was investigated. One day following the injection of sephadex the total cell number in bronchoalveolar lavage (BAL) fluid was significantly increased from 24.77 to 83.45×10⁶ cells. This was reflected in an increase in eosinophils, neutrophils, macrophages and lymphocytes. In addition, there was an increase in the reactivity of isolated bronchial strips to histamine. Rapamycin (5 mg/kg), administered two hours before the injection of sephadex, reduced the eosinophil, neutrophil, lymphocyte and macrophage number by 64%, 55%, 50% and 19%, respectively, and also inhibited the increased reactivity of isolated bronchial strips to histamine. These results suggest that rapamycin may reduce bronchial reactivity by the inhibition of leukocyte migration into the airways.

     

Cytokine-Induced Neutrophil Chemoattractant in a Rat Model of Lipopolysaccharide-Induced Acute Lung Injury

To elucidate the role of major chemotactic factors, cytokine-induced neutrophil chemoattractant (CINC), leukotriene B₄ (LTB₄) and C5a in lipopolysaccharide (LPS)-induced acute lung injury in rat, we employed three reagents: anti-CINC-1 antibody, an LTB₄ receptor antagonist (ONO-4057) and an anti-complementary agent (K-76COONa). Rats were divided into five groups: (1) control group; (2) LPS group, which received intratracheal instillation of LPS (100 g/kg); (3) Anti-CINC group, which received intratracheal coinstillation of LPS with anti-CINC-1 antibody (1 mg/kg); (4) LTB₄-Ra group, which received intravenous ONO-4057 (10 mg/kg) prior to intratracheal LPS; (5) Anti-C5a group, which received intravenous K-76COONa (100 mg/kg) prior to intratracheal LPS. The number of neutrophils in bronchoalveolar lavage (BAL) fluids 6 h after LPS instillation was significantly reduced in the Anti-CINC group, however, no reduction was found in either the LTB₄-Ra group or Anti-C5a group. The levels of CINC-1, CINC-2 and CINC-3 in BAL fluids were significantly higher in the LPS group than in the saline-instilled control group. In vitro, the production of CINC-1 and CINC-3 from LPS-stimulated macrophages was significantly elevated compared to unstimulated macrophages 6 h later. The increase in CINC-2 production was markedly less than that of CINC-1 or CINC-3. These results indicate that CINCs, especially CINC-1 and CINC-3 play an important role in the recruitment of neutrophils to the lung in LPS-induced acute lung injury.     

Glycosidase activities in alveolar macrophages from guinea pigs stimulated with a glyco-proteic complex extracted fromKlebsiella pneumoniae

An increased resistance of laboratory animals to pulmonary infections followingper os administration of a glyco-proteic complex extracted fromKlebsiella pneumoniae has been reported. This was associated with an increased phagocytic capacity of alveolar macrophages (AM). In this report, the effect of treating guinea pigs with this extract on the alveolar macrophage (AM) glycosidase machinery has been studied. AM were collected by bronchoalveolar lavage, the cells were pelleted by centrifugation and AM were purified by adherence on plastic dishes. Sialidase, -galactosidase, -glucuronidase and N-acetyl--d glucosaminidase activities were measured in the AM homogenate. In order to evaluate an extracellular release of these enzymes, they were also assayed in the cell free lavage fluid. Lactic dehydrogenase (LDH) activity was assayed as a control for cell lysis.In treated animals, the total number of cells as well as the number of AM increased by 25% (ns). The protein concentration was slightly reduced in the cell homogenate and unchanged in the lavage fluid. The only significant change was a decreased sialidase activity, in AM homogenate (p0.01) and in lavage fluids (ns). The LDH activity was not increased in the lavage fluids.This work was presented in part at the 1988 Meeting of the American Thoracic Society.     

Eotaxin expression in Sephadex-induced lung injury in rats

The CC chemokine eotaxin is a potent and specific eosinophil chemoattractant. Eosinophil-dependent tissue injury has been shown to contribute to airway inflammation such as that in asthma. In the present study, We investigated eotaxin expression in a rat model of pulmonary inflammation (featuring accumulation of eosinophils) induced by intratracheal instillation of cross-linked dextran beads (Sephadex G200). Intratracheal instillation of 5 mg/kg Sephadex caused a time-dependent eosinophil infiltration into the lung, reaching a peak at 24 hours. Eotaxin mRNA in the lung paralleled the eosinophil influx. Eotaxin protein in bronchoalveolar (BAL) fluids and lung homogenates was shown by Western blot and immunostaining to be maximally expressed by 24 hours. Sephadex-induced lung injury, as measured by (125)I-labeled albumin leakage from the pulmonary vasculature, developed in a time-dependent manner. Intravenous injection of blocking antibody to eotaxin significantly decreased eosinophil infiltration and lung permeability. These data suggest that, in the Sephadex model of lung inflammation, eotaxin up-regulation mediates intrapulmonary accumulation of eosinophils and the development of lung injury.     

Effects of serine protease inhibitor,tame, on IL-1β in LPS-stimulated human monocytes: Relationship between synthesis and release of a 33-kDa precursor and the 17-kDa biologically active species

LPS stimulation of human monocytes in vitro induced release of the 17-kDa mature IL-1 (mIL-1) but did not result in release of precursor IL-1 (pIL-1). In contrast, the presence of a serine protease inhibitor, N-(p-toluene sulfonyl)-L-arginine methyl ester (TAME; 10 mM) for 6 or 18 h was associated with the LPS-stimulated release of the 33-kDa pIL-1 as well. These effects were initially discerned from observations that the fraction of the total IL-1 produced (as detected by ELISA) that was released from monocytes increased in the presence of TAME, and immunoblot assays confirmed that this fraction was predominantly 33-kDa IL-1. A global decrease in monocyte protein synthesis was also observed after prolonged (18-h) exposure to TAME and was associated with a decrease in IL-1 synthesis, predominantly affecting 31-kDa pIL-1, and a dose-dependent inhibition of TNF- production. Parallel examination of lactate dehydrogenase (LDH) release indicated thatpIL-1 release was unrelated to cell lysis. These results demonstrate that TAME-inhibitable serine proteases are probably involved in the production and eventual proteolysis of the 33-kDa pIL-1 in situ but are probably not mechanistically related to either maturation of the IL-1 molecule or signaling of IL-1 release. IL-1 release appears to be dependent on the amount of total IL-1 synthesized. Serine proteolysis may constitute a degradative pathway for excess precursor, which, if interfered with, could result in release of the higher-molecular-weight forms of IL-1.     

Role of Airway Eosinophilia and Eosinophil Activation in Sephadex-induced Inflammation

The effects of an intravenous injection of Sephadex beads on lung eosinophil infiltration and eosinophil peroxydase activity and its relationship to bronchial hyperresponsiveness was examined in guinea pigs. This Sephadex beads injection led to blood, lung and airway eosinophilia in association with bronchial hyperresponsiveness. Histologic examination of the lower bronchus indicated that the eosinophil number increased markedly in the mucosa and submucosa. In addition, the eosinophils surrounding the bronchioles 1 day after the Sephadex injection migrated further in airway submucosa and mucosa 7 and 14 days after. Moreover, the bronchial hyperresponsiveness is observed without histologic evidence of airway epithelium damage. Therefore, the bronchial hyperresponsiveness seems to be more related to the eosinophil infiltration in the airway epithelium and possibly eosinophil activation rather than to the eosinophil number recovered in the BAL fluid. We conclude that the maintenance of hyperresponsiveness state could be associated with the persistence of blood and airway eosinophilia.     

4‐Thiouridine induces dose‐dependent reduction of oedema,leucocyte influx and tumour necrosis factor in lung inflammation

Recent reports demonstrate a role for nucleotides as inflammatory modulators. Uridine, for example, reduces oedema formation and leucocyte infiltration in a Sephadex‐induced lung inflammation model. Tumour necrosis factor (TNF) concentration was also reduced. Previous in vivo observations indicated that 4‐thiouridine might have similar effects on leucocyte infiltration and TNF release. The aim of this study was thus to investigate the effects of 4‐thiouridine in greater detail. We used a Sephadex‐induced acute lung inflammation model in Sprague–Dawley rats. The dextran beads were instilled intratracheally into the lungs, which were excised and examined after 24 h. Sephadex alone led to massive oedema formation and infiltration of macrophages, neutrophils and eosinophils. Microgranulomas with giant cell formations were clearly visible around the partially degraded beads. A significant increase in bronchoalveolar lavage fluid (BALF) content of TNF and leukotrienes was also seen. 4‐Thiouridine co‐administration affected all variables investigated in this model, i.e. oedema, microscopic and macroscopic appearance of lung tissue, total leucocyte and differential leucocyte counts in BALF, TNF and leukotrienes C₄ (LTC₄), LTD₄ and LTE₄ in BALF, indicating a reproducible anti‐inflammatory effect. In conclusion, we have demonstrated that 4‐thiouridine has anti‐inflammatory effects similar to those of uridine. To our knowledge, this is the first demonstration of pharmacological 4‐thiouridine effects in vivo. The results suggest nucleoside/nucleotide involvement in inflammatory processes, warranting further studies on nucleoside analogues as attractive new alternatives in the treatment of inflammatory diseases.     

Release of monokines by pulmonary macrophages following antigen challenge in sensitized guinea pigs

Guinea pigs were passively sensitized with immune serum to ovalbumin (OA), control serum, or saline. Twenty-four hours later, they inhaled aerosols of OA (2% in saline), saline, or lipopolysaccharide (LPS). Following anesthesia, bronchoalveolar lavage (BAL) was performed at 30, 60, 90 and 120 min postinhalation. Alveolar macrophages (AM) were isolated from the BAL fluid and incubated (18 h) in medium alone or with zymosan (1 mg/ml). Supernatants were collected and levels of interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-) determined by bioassays. Unstimulated AM from animals that inhaled OA, saline, or LPS secreted similar amounts of IL-1 at 30, 60, and 90 min postinhalation. Zymosan (1 mg/ml) significantly increased IL-1 secretion by AM collected at 60 and 90 min from OA-sensitized animals that inhaled OA or saline. AM from guinea pigs sensitized to OA that inhaled OA or LPS secreted significantly increased amounts of IL-6 at 30, 60, 90, and 120 min postchallenge compared to saline sensitized controls. In all groups, AM from LPS-treated animals secreted large amounts of TNF- at all sampling times postchallenge; AM from OA-sensitized and challenged animals secreted increasing amounts of TNF- with time postchallenge, spontaneously and in response to zymosan. By contrast, AM from saline sensitized and challenged guinea pigs did not release detectable amounts of TNF- spontaneously and secreted very low amounts in the presence of zymosan. These findings show that antigen challenge results in a rapid activation of AM isolated from BAL and suggest AM may initiate the development of inflammatory processes associated with antigen challenge.     

Lactoferrin,lysozyme, and β2-Microglobulin levels in cerebrospinal fluid

The CSF levels of lactoferrin, lysozyme, and ₂-microglobulin ( ₂ ) were measured in patients with evident, probable, or possible inflammatory CNS reactions and compared to those found in neurologically apparently healthy patients. Patients with viral CNS infections had significantly raised ₂ and lysozyme levels but normal lactoferrin levels, indicating a local activation of lymphocytes and monocytes but not of granulocytes. Patients with bacterial CNS infections had significantly raised levels of all three cell markers, but the increase of lysozyme and lactoferrin was relatively more pronounced than that of ₂ , indicating that the inflammatory response to bacterial agents is dominated by monocytes and granulocytes. Patients with primary or secondary malignant brain tumors were characterized by a moderate increase of ₂ and a considerable increase in both lysozyme and lactoferrin, i.e., the same protein pattern as observed in bacterial CNS infection. The lysozyme levels were moderately increased in half the patients with benign cerebral tumors while the levels of ₂ and lactoferrin were normal, indicating that benign and malignant brain tumors induce different local inflammatory CNS reactions. Half the patients with pituitary gland adenoma had elevated ₂ and lysozyme levels but normal lactoferrin levels, suggesting that immunological mechanisms are associated with the adenoma development. Patients with MS had moderately but significantly raised CSF levels of ₂ and lysozyme and a third of them also had raised levels of lactoferrin, a protein pattern suggesting a low-active inflammatory process in CNS involving mononuclears and granulocytes. A similar protein pattern was found in Guillain-Barré syndrome. In cerebrosarcoidosis we noted considerably increased lysozyme and ₂ but normal lactoferrin levels, consistent with the idea that the sarcoid granuloma mass is dominated by monocytic inflammatory cells. The data obtained indicate a clinical value of lactoferrin, lysozyme, and ₂ as differential indices of inflammatory cell reactions taking place in various CNS processes.     

Direct evidence for granuloma-inducing activity of interleukin-1. Induction of experimental pulmonary granuloma formation in mice by interleukin-1-coupled beads.

Pulmonary granulomas were induced in BALB/c mice by the intratracheal injection of Sephadex G-50 and latex beads. Very large granulomas developed around Sephadex G-50 beads. Minimal inflammation was produced in mice given latex beads. Aqueous extracts prepared from pulmonary granuloma lesions induced in mice by Sephadex G-50 beads contained high levels of interleukin-1 (IL-1) activity but not interleukin-2 (IL-2) activity. IL-1 activity in the extracts correlated with granuloma size. In a subsequent step, large granulomas were induced by the intratracheal injection of Sepharose 4B beads coupled to fractions of the extracts containing IL-1 activity (ie, granuloma-derived IL-1) prepared from Sephadex G-50-induced granulomatous lungs. In addition, large granulomas were induced by the intratracheal injection of recombinant IL-1-coated Sepharose 4B beads. In contrast, very small granulomas were seen when IL-2-coated or plain Sepharose 4B beads were injected into mice. These results indicate that IL-1 participates in the induction and/or expression of granulomas.     

Comparison of two models of bleomycin-induced lung fibrosis in mouse on the level of leucocytes and T cell subpopulations in bronchoalveolar lavage

Bleomycin produces pulmonary fibrosis in mice when given as a single intratracheal instillation or as multiple subcutaneous injections. Both models are associated with a significant deposition of collagen in the lungs but differ in the location of the initial lesions. Intratracheal instillation of bleomycin directs lesions to peribronchial or peribronchiolar sites, whereas subcutaneous injection produces lesions in subpleural and perivascular locations. It was therefore of interest to analyse the bronchoalveolar lavage (BAL) fluid for differences in the cellular composition, especially of intraepithelial T lymphocytes characterised by the expression of the integrin E7.Intracheal instillation of bleomycin induced a 5 to 6 times higher number of neutrophils and lymphocytes in BAL fluid than the subcutaneous model. Furthermore, intratracheal instillation of bleomycin induced the infiltration of eosinophilic neutrophils, a peculiar subtype of neutrophils often found in rodents, which were not found in BAL after subcutaneous injection of bleomycin. In both models of bleomycin injection, however, CD4⁺, CD8⁺, NK, and T lymphocytes were detected with dominance of the CD4⁺ T cell population. Analysis of the expression of the integrin E7 revealed similar numbers of E7⁺ cells in the CD4⁺ and T cell populations in both models but significantly lower numbers of E7⁺ T cells were found in the BAL fluid within the CD8⁺ T cell population after subcutaneous injection of bleomycin compared to intratracheal instillation.These results show that a difference in route of bleomycin administration not only causes changes in the localisation of the lesions but that this variation may be reflected in alterations within the BAL leucocyte population especially within the intraepithelial T lymphocytes.     

Functional Characterization of Recombinant Rat Macrophage Inflammatory Protein-1α and mRNA Expression in Pulmonary Inflammation

Chemokines are important inflammatory mediators that function by activating and recruiting leukocytes to an inflamed tissue. We have recently cDNA cloned the rat chemokine macrophage inflammatory protein–1 (MIP-1) (1). In the present study, we characterize the biological function of recombinant MIP-1 protein and describe expression of its mRNA both in vitro and in a rat model of lung inflammation. In vitro rat rMIP-1 protein was chemotactic for both polymorphonuclear leukocytes (PMNs) and macrophages with maximal activity at 50 nM for both cell types. In in vivo studies, we found that intratracheal instillation of 1 and 5 g of rMIP-1 resulted in a significant (P < 0.05) influx of cells, primarily monocytes/macrophages, into the airspace of the lungs after 6 h. Mean numbers of lavagable PMNs were not elevated significantly (P < 0.05) for either dose of MIP-1. As a model of inflammation, rats were intratracheally instilled with 0.1 mg/kg bacterial lipopolysaccharide (LPS). Bronchoalveolar lavage (BAL) was performed 3 h later. Instillation of LPS resulted in an acute neutrophilia, but no significant change in lavagable macrophages. BAL cells from control animals (saline instilled) displayed no basal mRNA expression of either MIP-1 or MIP-2 (positive control). In contrast, both MIP-1 and MIP-2 mRNA levels increased markedly in BAL cells from rats instilled with LPS. The rat alveolar macrophage cell line (NR8383) also showed increased MIP-1 mRNA levels in response to LPS (10 g/ml) with a maximal increase after 6–8 h. The induction of MIP-1 mRNA expression by LPS in NR8383 cells was attenuated by cotreatment with the antioxidants N-acetylcysteine and dimethylsulfoxide, suggesting that the induction of MIP-1 mRNA by LPS is mediated via the generation of reactive oxygen species. We conclude that MIP-1 is a potent chemoattractant for macrophages in vivo, and its mRNA expression in macrophages and BAL cells in response to inflammatory stimuli suggests a fundamental role in acute pulmonary inflammation.     

The guinea-pig step cycle: X-ray cinematographic analysis of the forelimb during pharmacologically induced “stepping automatism”

Summary Stepping automatism has generally been studied in mesencephalic or spinal cats and has been induced electrically with the animal on a treadmill (Shik and Orlovsky 1976). Derivatives of 4-R-2,2,5,5-tetrakis (trifluoromethyl)-imidazoline (SIS = Substances capable of Inducing Stepping) I–IV (Liu 1985, Liu et al. 1984) can induce regular stepping automatism in guinea pigs. The present paper concerns the guinea-pig step cycle of the forelimb during SIS II-induced stepping automatism analysed with the use of X-ray cinematography and electromyography (EMG) studies in suspended animals. Results show that the flexion phase (F E¹) and the extension phase (E² E³) of the SIS-induced step cycle are quite comparable to those of the normal step cycle in other quadrupedal animals walking on the ground. The excursions of elbow, shoulder and scapula joints are all in phase in F and E³, whereas the scapula is largely out of phase with the elbow and shoulder during E¹ and E². It is surprising that during SIS II-induced locomotion in guinea pigs suspended in the air, yield could be seen in both, the elbow and the shoulder joints.     

短期香烟烟雾及脂多糖气道刺激对小鼠气道免疫细胞的影响

目的：探讨香烟烟雾及脂多糖（LPS）短期刺激对小鼠气道免疫细胞的影响。方法：LPS组小鼠气管内（一次性）滴注10 μg LPS，熏烟组小鼠每天熏9支香烟持续熏4 d，观测不同干预措施对小鼠体重的影响；检测支气管肺泡灌洗液(BALF)总细胞数及细胞分类计数，观察细胞形态，并利用PDB（2-丁酸佛波醇酯）诱导BALF中的细胞检测ROS产出率；荧光定量PCR法检测肺组织中GM-CSF和CXCL15 mRNA 表达。结果：与实验前相比，对照组及LPS组小鼠体重未见明显变化（P>0.05），熏烟组小鼠体重减少了189%，其变化差异明显高于对照组和LPS组（P<0.01）。与对照组相比，熏烟组小鼠气道灌洗液细胞总数和各类细胞总数均无明显变化（P>0.05）；LPS组小鼠气道灌洗液细胞总数、巨噬细胞计数和中性粒细胞计数均高于对照组和熏烟组（P<0.01），且LPS组灌洗液巨噬细胞体积较大，形态不规则，中性粒细胞胞核分叶较对照组多。LPS组小鼠气道灌洗液细胞在PDB的诱导下及没有PDB的诱导下均比对照组和熏烟组具有更高ROS产出率（P<0.01）。与对照组相比，LPS组小鼠肺组织中GM-CSF表达显著增高（P<0.01），但CXCL-15和熏烟组小鼠肺组织中GM-CSF及CXCL-15的表达并未发生改变（P>0.05）。结论：短期香烟烟雾刺激明显引起小鼠体重下降，但未能诱发明显气道炎症反应；10 μg LPS气道滴注可通过增加肺组织GM-CSF的表达，增加中性粒细胞的成熟和募集，增加中性粒细胞内氧化应激反应及产生ROS的能力，诱发明显气道炎症反应。