Biomedical subjects

L Kobzik

Publications and source records attributed to L Kobzik.

At least 109 records · Page 6Linked to original sources

PubMed1995

Role of nitric oxide in human esophageal circular smooth muscle in vitro.

The role of nitric oxide in human esophageal smooth muscle was examined. Immunostaining for constitutive nitric oxide synthase labeled nerve fibers and bundles within longitudinal and circular smooth muscle layers of resected tissue samples. Strips of circular muscle mounted in organ baths exhibited spontaneous contractions and active tone. When exposed to 5-second trains of electric field stimulation at 20 Hz, most strips exhibited intrastimulus "on" and poststimulus "off" contractions. Exposure to a 0.1 mumol/L (or greater) concentration of atropine converted "on" contractions to "on" relaxations and reduced "off" contractions by 63%. Exposure to NG-nitro-L-arginine resulted in concentration-dependent enhancement of "on" contractions and abolition of "off" contractions. Excess L-arginine enhanced the reversal of these effects. Sodium nitroprusside inhibited both spontaneous and evoked contractions. These results suggest that nitric oxide synthesis is a mediator of neural inhibition of human esophageal circular smooth muscle and is necessary for the occurrence of "off" contractions.

Amino Acid Oxidoreductases↗

PubMed1995

Neutrophil adhesion receptor CD18 mediates remote but not localized acid aspiration injury.

BACKGROUND: Acid aspiration leads to lung polymorphonuclear neutrophil (PMN) sequestration and an associated increase in permeability. Although it is known that the neutrophil adhesion receptor (CD18) plays no role in determining PMN accumulations in the region aspirated, we postulated that this PMN adhesion receptor and its endothelial ligand, intercellular adhesion molecule-1 (ICAM-1), mediate remote neutrophil sequestration. METHODS: Anesthetized rabbits underwent localized aspiration of either 0.1N HCl 0.1 ml/kg (n = 18) or saline solution (n = 18). RESULTS: After 30 minutes leukopenia was noted, 2290 +/- 200 white blood cells/mm3 (p < 0.05). At 3 hours diapedesis occurred in the aspirated segment with accumulations in bronchoalveolar lavage fluid (X10(4)) of 87 +/- 6 PMN/ml versus control of 6 +/- 1 PMN/ml (p < 0.05). Histologic evidence of generalized lung leukosequestration occurred. The wet to dry weight ratio of the nonaspirated lung rose to 5.7 +/- 0.2 versus control of 3.9 +/- 0.1 (p < 0.05). Treatment (n = 18) with the CD18 monoclonal antibody (mAb) (R15.7, 1 mg/kg) had no effect on neutrophil accumulations in the aspirated segment. However, the mAb attenuated the remote inflammatory response: early leukopenia (5790 +/- 400 white blood cells/mm3); lung leukosequestration (24 +/- 4 PMN/10 high-power fields); protein leak in bronchoalveolar lavage fluid (570 +/- 50 micrograms/ml); and edema, wet to dry weight ratio (4.9 +/- 0.1) (all p < 0.05). Treatment with the ICAM-1 mAb (RR1/1, 1 mg/kg) (n = 9) did not reduce neutrophil accumulations in the aspirated segment but limited the remote inflammatory response. CONCLUSIONS: Acid aspiration leads to neutrophil adhesion and edema in regions remote from those aspirated via neutrophil CD18 and endothelial ICAM-1.

Animals↗

PubMed1995

Induction of NO synthase in rat cardiac microvascular endothelial cells by IL-1 beta and IFN-gamma.

There are important phenotypic differences between endothelial cells of large vessels and the microvasculature and among microvascular endothelial cells isolated from different tissues and organs. In contrast to most macrovascular endothelial cells, we demonstrate that cultured cardiac microvascular endothelial cells (CMEC) have no detectable constitutive NO synthase (NOS) activity but have a robust increase in NOS activity in response to specific inflammatory cytokines. To determine the identity of the inducible NOS (iNOS) isoform(s) induced by cytokines, we used reverse-transcription polymerase chain reaction techniques to clone and sequence a 217-bp cDNA fragment from CMEC cultures pretreated with interleukin-1 beta (IL-1 beta) and interferon-gamma (IFN-gamma) that was identical to the corresponding portion of the murine macrophage iNOS cDNA. By use of this CMEC iNOS cDNA as a probe in Northern analyses, IL-1 beta, but not IFN-gamma, increased iNOS mRNA content in CMEC, although IFN-gamma markedly potentiated iNOS induction in these cells. In IL-1 beta- and IFN-gamma-pretreated CMEC, dexamethasone only minimally suppressed the rise in iNOS mRNA, protein abundance, or maximal iNOS enzyme activity in whole cell lysates but suppressed nitrite production by 60% in intact CMEC. Dual labeling of cytokine-pretreated CMEC in primary culture with an anti-iNOS antiserum and a fluorescein-labeled lectin specific for the microvascular endothelium of rat heart (GS-1) confirmed the presence of iNOS expression in these cells. iNOS was also detected in microvascular endothelium in situ in ventricular muscle from lipopolysaccharide-, but not sham-injected, rat hearts.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Oxidoreductases↗

PubMed1995

Effects of sodium concentration on human neutrophil bactericidal functions.

What are the ionic requirements for neutrophil (PMN) function and how might altered electrolyte concentrations contribute to airway disease? The in vitro killing of Pseudomonas aeruginosa by human peripheral white blood cells (WBCs) was progressively compromised Na+ concentration was lowered from 124 to 62 mM; at 62 mM Na+, bactericidal activity was 28.8 +/- 7.4% (SE) of normal. In contrast, Cl- concentration affected killing only when lowered to 8 mM. We examined phagocytosis and oxidative metabolism in response to P. aeruginosa or particles opsonized with either immunoglobulin G (IgG) or complement (C'). Phagocytosis of P. aeruginosa and of IgG-coated particles was Na(+)-dependent (31.2 +/- 3.1 and 58.6 +/- 14.2% of normal, respectively, at 62 mM Na+). However, no effect on uptake of C'-coated particles was observed, and the respiratory burst at 70 mM Na+ was normal regardless of stimuli. Thus low Na+ concentration compromises select PMN functions. These results may help explain why airways of cystic fibrosis (CF) patients become colonized with bacteria such as P. aeruginosa. Perhaps the low concentration of Na+ reported for some CF respiratory secretions inhibits bactericidal functions of PMNs, predisposing these patients to airway infections.

Blood Bactericidal Activity↗

PubMed1995

Substance P-induced histamine release in tracheally perfused guinea pig lungs.

The capacity of substance P (SP) and endogenously released tachykinins to liberate histamine was examined in isolated tracheally perfused guinea pig lungs. Increasing doses of tracheally injected SP were associated with the recovery of increasing amounts of histamine from lung effluent. The mechanism of SP-induced histamine liberation was explored in studies with neurokinin-(NK) receptor agonists and antagonists. Tracheal injection of either the NK1 agonist [Sar9,Met(O2)11]SP or the NK2 agonist [beta-Ala8]-neurokinin A-(4-10) was associated with a significant increase in histamine recovery from lung effluent. In addition, both the NK1 antagonist CP-99994 and the NK2 antagonist SR-48968 significantly inhibited SP-induced histamine release. These findings support the hypothesis that SP can liberate histamine from guinea pigs lungs by a mechanism that depends predominantly on NK1- and NK2-receptor activation. The liberation of endogenous tachykinins by acute tracheal injection of capsaicin was also associated with augmented histamine recovery, which was inhibited by combined NK1- and NK2-receptor blockade. Tracheal injection of SP was associated with an increase in the percentage of airway mast cells exhibiting histological evidence of degranulation. This study demonstrates that exogenous SP, as well as endogenous tachykinins released from capsaicin-sensitive neurons, can liberate histamine, most likely from airway mast cells, by a mechanism that depends predominantly on the activation of NK1 and NK2 receptors.

Animals↗

PubMed1995

Contractile responsiveness of ventricular myocytes to isoproterenol is regulated by induction of nitric oxide synthase activity in cardiac microvascular endothelial cells in heterotypic primary culture.

Unlike large-vessel endothelial cells in cell culture, cardiac microvascular endothelial cells (CMEC) isolated from adult rat ventricular muscle exhibit little detectable constitutive nitric oxide (NO) synthase activity after isolation in vitro but respond to specific combinations of inflammatory mediators with an increase in inducible NO synthase (iNOS; type 2 NO synthase) activity. CMEC iNOS is induced by soluble inflammatory mediators in lipopolysaccharide-activated rat alveolar macrophage-conditioned medium at 24 hours, and this induction can be partially prevented by either interleukin-1 (IL-1) receptor antagonist or a polyclonal anti-rat tumor necrosis factor-alpha (TNF-alpha) antiserum. Interferon-gamma (IFN-gamma), which by itself does not induce iNOS in CMEC, potentiates and accelerates iNOS induction by IL-1 beta. Transforming growth factor-beta (TGF-beta) decreases iNOS activity, protein content, and mRNA abundance in IL-1 beta- and IFN-gamma-pretreated CMEC. To determine whether NO released by CMEC would affect myocyte contractile function in vitro, freshly isolated ARVM were allowed to settle onto confluent, serum-starved CMEC that had been pretreated for 24 hours with IL-1 beta, a cytokine that alone does not affect myocyte contractile function in vitro. Baseline contractile amplitude, at 2 Hz and 37 degrees C, of myocytes in heterotypic culture with IL-1 beta-pretreated CMEC was not different from that of myocytes in control, homotypic myocyte cultures. However, cocultured myocytes exhibited decreased contractile responsiveness to 2 nmol/L isoproterenol compared with control cells, and this could be reversed by the addition of 1 mmol/L NG-monomethyl-L-arginine, an inhibitor of NOS.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Oxidoreductases↗

PubMed1995

Induction of nitric oxide synthase activity by cytokines in ventricular myocytes is necessary but not sufficient to decrease contractile responsiveness to beta-adrenergic agonists.

Recent evidence has documented that increased activity of an inducible nitric oxide synthase (iNOS; type 2 NO synthase) in primary isolates of adult rat ventricular myocytes after exposure to soluble mediators in medium conditioned by lipopolysaccharide-activated macrophages is associated with a decrease in their contractile responsiveness to beta-adrenergic agonists. It remained unclear which specific inflammatory cytokines in this medium contribute to the induction of iNOS activity in myocytes and whether induction of iNOS would result in an obligatory decline in contractile function. Interleukin (IL)-1 beta and tumor necrosis factor-alpha (TNF-alpha) were both present in the lipopolysaccharide-activated macrophage-conditioned medium. However, only IL-1 receptor antagonist and not an anti-rat TNF-alpha antiserum diminished the extent of iNOS induction in myocytes exposed to this medium and prevented a decline in contractile responsiveness to isoproterenol. When recombinant cytokines were used, IL-1 beta, TNF-alpha, and IFN-gamma each induced iNOS activity in cardiac myocytes at 24 hours. However, only the combination of IL-1 beta and IFN-gamma reproducibly caused contractile dysfunction in cardiac myocytes. Among the constituents of the defined medium routinely used for maintenance of adult rat ventricular myocytes in primary culture, it was noted that insulin (10(-7) mol/L) was required for NO production, as detected by nitrite release in cytokine-pretreated myocytes, although insulin had no effect on the extent of induction of iNOS mRNA or maximal enzyme activity in myocyte cell lysates.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic beta-Agonists↗

PubMed1995

Increased airway responsiveness to inhaled methacholine in a rat model of chronic bronchitis.

Chronic exposure of rats to high concentrations of SO2 gas causes pathologic changes in airway similar to those seen in human chronic bronchitis. The purpose of this study was to examine the pulmonary mechanical correlates of these changes and to quantify the extent of mucous hypersecretion by measuring changes in mucous glycoproteins. Female Sprague-Dawley rats were exposed to 250 ppm SO2 gas, 5 h/d, 5 d/wk, for a period of 4 wk. Control rats were exposed to air only. On the day after the last SO2 exposure, rats were anesthetized, instrumented for the measurement of pulmonary resistance (RL) and dynamic compliance (Cdyn), and ventilated. Chronic SO2 exposure caused a small but significant increase in RL and decrease in Cdyn. Airway responsiveness to inhaled aerosolized methacholine was increased in SO2-exposed rats, as indicated by approximately 6.6- and 4.6-fold decreases respectively, in the doses of inhaled methacholine required to double RL or decrease Cdyn to 50% of baseline. SO2 exposure had no effect on the contractile response of the trachea measured in vitro. Tracheae and lungs from SO2-exposed animals exhibited 140 and 535% increases in measured neutral mucous glycoproteins, respectively, and 33 and 37% increases in acid glycoproteins. Our results indicate that this animal model of chronic bronchitis mimics the mucous hypersecretion, airway obstruction, and increased airway responsiveness observed in human bronchitis and may allow us to begin to probe their mechanistic basis.

Animals↗

PubMed1995

Airway neutrophilia and chemokine mRNA expression in sulfur dioxide-induced bronchitis.

Airway inflammation in acute and chronic bronchitis includes a prominent neutrophil influx. Using a rat model of sulfur dioxide (SO2)-induced bronchitis, we investigated the role of the polymorphonuclear leukocyte (PMN) chemokines macrophage inflammatory protein-2 (MIP-2) and KC. Adult female rats were exposed to 230 ppm SO2 for 5 h/day for periods of 1 day to 5 wk. Immunohistochemical identification of rat PMNs in trachea cryostat sections allowed quantitation of a marked neutrophil influx into airways of bronchitic rats (PMNs/trachea ring = 55 +/- 26.2 [1 day SO2] versus 3.6 +/- 2.7 [air]; n = 5, P < or = 0.05). Northern analysis of trachea homogenates demonstrated induction of KC and MIP-2 mRNA expression after 1 day of SO2 and persistence of increased expression after longer exposure periods examined. Pretreatment of rats with dexamethasone (0.5 mg/kg) prior to a 1-day acute SO2 exposure prevented induction of chemokine mRNA and abrogated neutrophil influx completely (PMNs/trachea ring = 6.6 +/- 8.8 versus air controls; n = 5, P = 0.96). To determine if chemokine inhibition by dexamethasone could be further studied in vitro, the rat alveolar macrophage cell line NR8383 was treated with dexamethasone (10(-7) M) before stimulation with lipopolysaccharide (10 micrograms/ml). Pretreatment with dexamethasone substantially decreased induction of both MIP-2 and KC mRNA in response to lipopolysaccharide, indicating the potential utility of in vitro systems to identify additional anti-inflammatory agents. These studies support the hypothesis that the chemokines MIP-2 and KC mediate airway neutrophil influx in both acute and chronic SO2-induced bronchitis in the rat.

Acute Disease↗

PubMed1995

Expression of membrane glycoconjugates on sheep lung endothelium.

BACKGROUND: Carbohydrates play an important role in both the regulation and expression of endothelial cell surface molecules. EXPERIMENTAL DESIGN: To investigate the molecular phenotype of endothelial cells in the sheep lung, we used a panel of 39 lectins and carefully titrated lectin histochemistry to identify small vessel glycoconjugates. Expression of glycoconjugates on isolated cells was studied by flow cytometry and lectin Western blotting. RESULTS: Eight lectins stained small vessel endothelium with minimal background staining. To confirm cell surface binding, endothelial cells from the peripheral lung were isolated and tested for lectin recognition by flow cytometry. Three lectins (Datura stramonium, Griffonia simplicifolia-1, Lycopersicon esculentum) also stained isolated lung cells by flow cytometry. A lectin Western transfer technique demonstrated common binding to a high molecular weight (100 to 130 kD) band. CONCLUSIONS: The use of lectins as probes of cell surface carbohydrate expression supports the possibility of selective glycoconjugate expression on sheep endothelium.

Animals↗

PubMed1994

Nitric oxide in skeletal muscle.

Reactive oxygen intermediates modulate skeletal muscle contraction, but little is known about the role of nitric oxide (NO). Here we show that rat skeletal muscle expresses neuronal-type NO synthase and that activity varies among several respiratory and limb muscles. Immunohistochemistry showed prominent staining of type II (fast) fibre cell membranes with antibodies against neuronal-type NO synthase. NO synthase activity in muscles correlated with type II fibre density. Resting diaphragm muscle produced detectable NO chi, but no reactive oxygen intermediates. In contrast, actively contracting muscle generated increased levels of reactive oxygen intermediates. Contractile function was augmented by blockers of NO synthase, extracellular NO chelation, and guanylyl cyclase inhibition; it was depressed by NO donors and by increased levels of cyclic GMP. Force-frequency plots of different muscles showed an inverse correlation between NO synthase activity and force development. Our results support two physiological functions of NO in skeletal muscle. The first is to promote relaxation through the cGMP pathway. The second is to modulate increases in contraction that are dependent on reactive oxygen intermediates and which are thought to occur through reactions with regulatory thiols on the sarcoplasmic reticulum.

Amino Acid Oxidoreductases↗

PubMed1994

Defective pulmonary recruitment of neutrophils in a rat model of endotoxemia.

We have characterized a defect in the pulmonary recruitment of neutrophils (PMNs) in rats with experimental endotoxemia. Rats pretreated with intravenous (IV) 0.9% saline (NaCl) showed abundant PMNs in bronchoalveolar lavage (BAL) fluid after intratracheal (IT) lipopolysaccharide (LPS) (5 mg/kg) (54.27 +/- 9.80 x 10(6), n = 7, versus IT saline, 0.73 +/- 0.62 x 10(6), n = 4). In contrast, endotoxemic rats (IV LPS 1.0 mg/kg) failed to show PMN influx after IT LPS (0.40 +/- 0.13 x 10(6) PMNs in BAL fluid, n = 7). Four hours after the IT administration of LPS, the chemotactic activity of BAL fluid from endotoxemic rats (87 +/- 9.92% of maximal chemotaxis toward zymosan-activated serum [ZAS], n = 4) was not significantly different (P > 0.05), from rats pretreated with IV NaCl (61.09 +/- 6.17% of maximal chemotaxis toward ZAS, n = 4). Endotoxemic and control rats showed similar chemotactic gradients in determinations of the BAL/plasma chemotactic activity ratio (BAL/plasma ratio: 2.16 +/- 0.14, n = 4, IV NaCl versus 2.98 +/- 0.14, n = 4, IV LPS, P > 0.05). Serum from untreated rats, rats pretreated with IV NaCl, and endotoxemic rats caused minimal effects on rat PMN chemotaxis in vitro (78.17 +/- 8.16%, 79.29 +/- 7.09%, and 69.28 +/- 9.04% of maximal chemotaxis toward ZAS, respectively, n = 4/group, P > 0.05). Quantitation of PMN adhesion molecules revealed a loss of L-selectin (8 +/- 5% of control group, n = 3), an increase in Mac-1 (776 +/- 82.60% of control group, n = 3), and no change in LFA-1 when normal PMNs were incubated with plasma from rats pretreated with IV LPS (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Effects of chronic airway inflammation on the activity and enzymatic inactivation of neuropeptides in guinea pig lungs.

The effects of airway inflammation induced by chronic antigen exposure on substance P (SP)-induced increases and vasoactive intestinal peptide (VIP)-induced decreases in airway opening pressure (Pao), and the recovery of intact and hydrolyzed radiopeptide were studied in tracheally perfused guinea pig lungs. SP (10(-6) mol/kg) induced a significantly greater increase in Pao in lungs from antigen-exposed (30 +/- 5 cm H2O) than saline-exposed animals (15 +/- 1 cm H2O, P < 0.05). Significantly more intact 3H-SP and significantly less 3H-SP 1-7, a neutral endopeptidase (NEP) hydrolysis product, were recovered from the lung effluent of antigen-exposed than saline-exposed animals (P < 0.05). Injection of VIP (10(-9) mol/kg) induced significantly more pulmonary relaxation in saline-exposed compared with antigen-exposed lungs (62 +/- 4%, P < 0.001). In contrast to effluent from saline-exposed animals, lung effluent from antigen-exposed lungs contained less intact VIP, increased amounts of a tryptic hydrolysis product, and no products consistent with the degradation of VIP by NEP. These data indicate that inflamed lungs are more sensitive to the contractile effects of SP because it is less efficiently degraded by NEP and are less sensitive to the relaxant effects of VIP because it is more efficiently degraded by a tryptic enzyme. Changes in airway protease activity occur with allergic inflammation and may contribute to airway hyperresponsiveness.

Animals↗

PubMed1994

Inhibition of sphincter of Oddi function by the nitric oxide carrier S-nitroso-N-acetylcysteine in rabbits and humans.

Nitric oxide (NO) is an inhibitor of gastrointestinal smooth muscle. Model systems of the gut predict the NO will complex with biological thiol (SH) groups, yielding S-nitrosothiols (RS-NO), which may limit the propensity to form mutagenic nitrosamines. The inhibitory effects of NO and its biologically relevant adducts on sphincter of Oddi (SO) motility have been inferred from animal studies; however, their importance in regulating human SO is not known. The objectives of this study were to (a) provide histologic confirmation of nitric oxide synthase (NOS) in human SO; (b) characterize the pharmacology of S-nitroso-N-acetylcysteine (SNAC), an exemplary S-nitrosothiol, on SO motility in a rabbit model; and (c) study the effects of topical SNAC on SO motility in humans. Immunocytochemical and histochemical identification of NOS was performed in human SO. The pharmacologic response of SNAC was defined in isolated rabbit SO using a standard bioassay. Topical SNAC was then applied to the duodenal papilla in patients undergoing endoscopic retrograde cholangiopancreatography (ERCP) and biliary manometry. NOS was localized to nerve fibers and bundles of the SO in rabbits and humans. SNAC inhibited spontaneous motility (frequency and amplitude) as well as acetylcholine-induced elevations in SO basal pressure in the rabbit model. In patients undergoing ERCP and biliary manometry, topical SNAC inhibited SO contraction freqency, basal pressure, and duodenal motility. NOS is localized to neural elements in human SO, implicating a role for NO in regulating SO function. Supporting this concept, SNAC is an inhibitor of SO and duodenal motility when applied topically to humans during ERCP. Our data suggest a novel clinical approach using local NO donors to control gastrointestinal motility and regulate sphincteric function.

Acetylcysteine↗

PubMed1994

Membrane glycoconjugates expressed on sheep airway epithelium.

In mammalian lung, selective airway inflammatory reactions have been associated with viral infections, transplant rejection, and autoimmune diseases. Although the molecular basis for this selective reactivity is unknown, the importance of carbohydrates in immunologic processes suggests a potential role for membrane glycoconjugates in tissue-specific inflammatory reactions. In the present work we examined a panel of 39 lectins for their pattern of reactivity in the peripheral airways of the sheep lung. The size of the panel facilitated a comprehensive description of the glycoconjugate localization on the airway epithelium. Four lectins (agglutinins for Helix aspersa, Psophocarpus tetragonolobus, Trichosanthes kirilowii, and Griffonia simplifolia II) revealed selective reactivity with the small airway epithelium. On lectin Western blotting, these four lectins demonstrated a common low molecular weight banding profile that was distinct from control lectins. The histochemical staining patterns and Western blotting profiles provided evidence for the selective expression of membrane glycoconjugates in the peripheral airways of the sheep lung.

Animals↗

PubMed1993

Particle opsonization and lung macrophage cytokine response. In vitro and in vivo analysis.

Inhaled inert particles and organisms cause a spectrum of pulmonary responses, ranging from minimal changes to marked acute inflammation. During ingestion and clearance of such particles, alveolar macrophages (AM) can initiate pulmonary inflammation by production of TNF and neutrophil chemoattractant cytokines. We tested the role of opsonization in determining the AM response to ingestion of inert particles in vitro and in vivo. As measures of AM activation after phagocytosis in vitro, we measured release of TNF and mRNA expression for the platelet-factor 4 family neutrophil chemoattractants, KC and macrophage-inflammatory protein-2 (MIP-2). Using albumin-coated fluorescent latex particle as phagocytic targets, we found a marked release of TNF by AM ingesting particles opsonized with antialbumin IgG, although uptake of similar numbers of unopsonized particles caused little or no release (e.g., 4648 +/- 1147 pg/ml, opsonized beads vs 576 +/- 205 unopsonized, 10:1 particle:cell ratio, n = 4, mean +/- SD). Flow cytometry confirmed equal uptake of the two particle types. Northern analysis of AM mRNA showed marked induction of KC and MIP-2 mRNA after ingestion of opsonized particles only. Instillation of opsonized particles into hamster lungs caused a marked neutrophil influx, although unopsonized particles did not. TNF was elevated in lavage fluid after instillation of opsonized particles, but not after unopsonized beads (92.7 +/- 136 pg/ml opsonized, n = 7 vs 1.3 +/- 3.6 unopsonized, n = 6). KC and MIP-2 mRNA were induced in lavaged cells after instillation of opsonized but not after unopsonized particles or vehicle control. The nature of particle interaction with the AM surface during phagocytosis determines the subsequent AM response. Although many unopsonized inert particles are ingested with minimal AM activation, specific opsonization of pathogens or non-specific adsorption of Ig onto other particles may activate AM and lead to pulmonary inflammation.

Animals↗

PubMed1993

Rapamycin treatment depresses intragraft expression of KC/MIP-2, granzyme B, and IFN-gamma in rat recipients of cardiac allografts.

Rapamycin (RPM) treatment prevents accelerated rejection of cardiac allografts in sensitized rats. The prominent feature of this brisk 24-h rejection, which includes a panoply of both cellular and humoral host immune responses, is a massive infiltration of rejecting grafts with neutrophils. In this study we tested the hypothesis that RPM-mediated therapeutic effects on accelerated rejection may be linked to decreased expression of protein encoded by gro/melanoma-growth stimulatory activity gene (KC) and macrophage inflammatory protein-2 (MIP-2) genes, the operational rat homologues of the human intercrine-alpha cytokines with proinflammatory IL-8-like neutrophil activation/chemotactic properties. The induction of these genes was then correlated with mRNA profiles encoding for Th1-selective IFN-gamma and CTL-specific granzyme B proteins. Northern blot analysis of RNA from cardiac allografts of sensitized untreated recipients, revealed maximal levels of KC and MIP-2 mRNA at 3 to 6 h after transplantation. In contrast, IFN-gamma mRNA, which was at most very weakly expressed at 3 h, peaked between 6 to 12 h. As with IFN-gamma, granzyme B transcripts were undetectable at 3 h, but peaked around the time of actual graft rejection at 24 h. RPM therapy abrogated accelerated rejection and prolonged cardiac allograft survival to ca. 46 days. This effect was associated with markedly reduced expression of KC and MIP-2 mRNA in the first 24 h as well as at 7 and 34 days after transplantation. Moreover, RPM completely blocked intragraft appearance of granzyme B and IFN-gamma mRNA in long term cardiac allografts. Immunohistologic analysis has revealed that accelerated rejection was associated with extensive neutrophil infiltration, which peaked at 18 to 24 h. At this time, leukocytes and endothelium were intensely stained for IL-8 and IFN-gamma antibodies. In contrast, the allografts from RPM-treated hosts showed essentially no neutrophil infiltration and minor, focal staining for IL-8 and IFN-gamma. This study demonstrates an association between the early expression of genes for proinflammatory IL-8-dependent neutrophil chemotactic activity, and later expression of genes associated with activation/effector activity of CTL and NK cells. It also documents a novel effect of RPM in vivo, which results in the suppression of intragraft IL-8-like and CTL-dependent mRNA/protein production and diminished neutrophil infiltration; these may contribute to the striking efficacy of RPM therapy in sensitized graft recipients.

Animals↗

PubMed1993

Neutrophil and nonneutrophil-mediated injury in intestinal ischemia-reperfusion.

OBJECTIVE: The role of polymorphonuclear neutrophils (PMN) was examined in local and remote organ injury after intestinal ischemia-reperfusion. SUMMARY BACKGROUND DATA: PMN have been found to mediate the local injury in low flow intestinal ischemia-reperfusion. However, in complete intestinal ischemia-reperfusion, prevention of PMN adhesion by monoclonal antibodies to CD11b and CD18 reduces remote but not local intestinal injury. The role of PMN was further investigated in this setting. METHODS: In a rat model of 1-hour complete intestinal ischemia and 4-hour reperfusion. PMN were manipulated in the following four ways: (1) inhibition of oxygen-free radicals using manganese superoxide dismutase and catalase (SOD/CAT), (2) antagonism of PMN elastase using secretory leukocyte protease inhibitor (SLPI), (3) neutropenia using PMN antisera, and (4) inhibition of activation and adhesion using interleukin-1 receptor antagonist (IL-1ra) and tumor necrosis factor binding protein (TNFbp). Lung injury was quantified by the pulmonary permeability index, which is the ratio of bronchoalveolar lavage to blood concentration of radiolabeled bovine serum albumin, and PMN sequestration by myeloperoxidase (MPO) activity. Liver injury was estimated by PMN counts using quantitative histologic examination and by serum glutamic pyruvic transaminase (SGPT). Local injury was quantified by MPO activity and histologic grading. RESULTS: Neutropenia reduced the pulmonary permeability 80% from 11.0 +/- 0.5 x 10(-3) with saline treatment to 5.50 +/- 0.12 x 10(-3); IL-1ra, to 5.62 +/- 0.44 x 10(-3); and TNFbp, to 4.32 +/- 0.18 x 10(-3) (all p < 0.05). Pulmonary MPO rose from 0.03 +/- 0.01 U/g to 0.25 +/- 0.03 U/g (p < 0.05). This was reduced by neutropenia, 0.01 +/- 0.003 U/g, but not by inhibition of oxygen-free radicals or PMN elastase. IL-1ra inhibited PMN sequestration, reducing MPO to 0.12 +/- 0.01 (p < 0.05). Liver injury was 60% dependent on PMN. Ischemia-reperfusion increased SGPT from 20.3 +/- 0.7 IU/L in the sham-treated animals to 97.0 +/- 3.1 IU/L in the experimental animals. Neutropenia reduced this to 48.1 +/- 3.9 IU/L; IL-1ra, to 44.7 +/- 3.7 IU/L; SOD/CAT, to 64.0 +/- 3.38 IU/L; and SLPI, to 57.1 +/- 3.4 IU/L (p < 0.05 in all cases). Local injury was severe and unaffected by manipulation of the PMN. CONCLUSIONS: These data suggest that PMN and their products mediate most of the lung, part of the liver, and none of the local gut injury after intestinal ischemia-reperfusion.

Animals↗