BACKGROUND

Mechanisms of coronary occlusion in ST-elevation acute coronary syndrome are poorly understood. We have previously reported that neutrophil (polymorphonuclear cells [PMNs]) accumulation in culprit lesion site (CLS) thrombus is a predictor of cardiovascular outcomes.

OBJECTIVE

The goal of this study was to characterize PMN activation at the CLS. We examined the relationships between CLS neutrophil extracellular traps (NETs), bacterial components as triggers of NETosis, activity of endogenous deoxyribonuclease, ST-segment resolution, and infarct size.

RESULTS

We analyzed coronary thrombectomies from 111 patients with ST-elevation acute coronary syndrome undergoing primary percutaneous coronary intervention. Thrombi were characterized by immunostaining, flow cytometry, bacterial profiling, and immunometric and enzymatic assays. Compared with femoral PMNs, CLS PMNs were highly activated and formed aggregates with platelets. Nucleosomes, double-stranded DNA, neutrophil elastase, myeloperoxidase, and myeloid-related protein 8/14 were increased in CLS plasma, and NETs contributed to the scaffolds of particulate coronary thrombi. Copy numbers of Streptococcus species correlated positively with dsDNA. Thrombus NET burden correlated positively with infarct size and negatively with ST-segment resolution, whereas CLS deoxyribonuclease activity correlated negatively with infarct size and positively with ST-segment resolution. Recombinant deoxyribonuclease accelerated the lysis of coronary thrombi ex vivo.

CONCLUSIONS

PMNs are highly activated in ST-elevation acute coronary syndrome and undergo NETosis at the CLS. Coronary NET burden and deoxyribonuclease activity are predictors of ST-segment resolution and myocardial infarct size.

Thiazolidinedione rosiglitazone and 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2), are two peroxisome proliferator-activated receptor (PPAR)-gamma ligands. The aim of this study was to investigate the effect of rosiglitazone and 15d-PGJ2 on the lung injury caused by bleomycin administration. Mice subjected to intratracheal administration of bleomycin developed significant lung injury. An increase in immunoreactivity to nitrotyrosine, poly(ADP ribose) polymerase (PARP) and inducible nitric oxide synthase as well as a significant loss of body weight and mortality was observed in the lung of bleomycin-treated mice. Administration of the two PPAR-gamma agonists rosiglitazone (10 mg x kg(-1) i.p.) and 15d-PGJ2 (30 microg x kg(-1) i.p.) significantly reduced the: 1) loss of body weight, 2) mortality rate, 3) infiltration of the lung with polymorphonuclear neutrophils (myeloperoxidase activity), 4) oedema formation, and 5) histological evidence of lung injury. Administration of rosiglitazone and 15d-PGJ2 also markedly reduced the nitrotyrosine, PARP and inducible nitric oxide synthase formation. In addition, treatment with the PPAR-gamma antagonist bisphenol A diglycidyl ether (1 mg x kg(-1) i.p. 30 min before the rosiglitazone or 15d-PGJ2) significantly antagonised the effect of the two PPAR-gamma agonists. These results demonstrate that the two peroxisome proliferator-activated receptor-gamma agonists, rosiglitazone and 15-deoxy-Delta12,14-prostaglandin J2, significantly reduce lung injury induced by bleomycin in mice.

Left ventricular (LV) remodeling after myocardial infarction (MI) results in LV dilation, a major cause of congestive heart failure and sudden cardiac death. Ischemic injury and the ensuing inflammatory response participate in LV remodeling, leading to myocardial rupture and LV dilation. Myeloperoxidase (MPO), which accumulates in the infarct zone, is released from neutrophils and monocytes leading to the formation of reactive chlorinating species capable of oxidizing proteins and altering biological function. We studied acute myocardial infarction (AMI) in a chronic coronary artery ligation model in MPO null mice (MPO(-/-)). MPO(-/-) demonstrated decreased leukocyte infiltration, significant reduction in LV dilation, and marked preservation of LV function. The mechanism appears to be due to decreased oxidative inactivation of plasminogen activator inhibitor 1 (PAI-1) in the MPO(-/-), leading to decreased tissue plasmin activity. MPO and PAI-1 are shown to have a critical role in the LV response immediately after MI, as demonstrated by markedly delayed myocardial rupture in the MPO(-/-) and accelerated rupture in the PAI-1(-/-). These data offer a mechanistic link between inflammation and LV remodeling by demonstrating a heretofore unrecognized role for MPO and PAI-1 in orchestrating the myocardial response to AMI.

Thrombotic microangiopathies (TMAs) are a group of life-threatening disorders characterized by thrombocytopenia, fragmentation of erythrocytes, and ischemic organ damage. Genetic disorders, autoimmune disease, and cancer are risk factors for TMAs, but an additional, unknown trigger is needed to bring about acute disease. Recent studies suggest that DNA and histones are released during inflammation or infection and stimulate coagulation, thrombosis, thrombocytopenia, and organ damage in mice. We show that extracellular DNA and histones as well as markers of neutrophils are present in acute TMAs. Analysis of plasma from TMA patients of different clinical categories revealed elevated levels of DNA-histone complexes and myeloperoxidase (MPO) from neutrophil granules as well as S100A8/A9, a heterocomplex abundant in neutrophil cytosol. During therapy of thrombotic thrombocytopenic purpura, a subtype of TMAs often associated with severe ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motifs, member 13) deficiency, plasma DNA and MPO were inversely correlated with platelet counts, and their levels indicated amelioration or exacerbation of the disease. ADAMTS13 deficiency together with increased levels of plasma DNA and MPO were characteristic for acute thrombotic thrombocytopenic purpura. A minor infection often precedes acute TMA and extracellular DNA and histones released during the inflammatory response could provide the second hit, which precipitates acute TMA in patients with pre-existing risk factors.

Toxic, partially reduced metabolites of oxygen (toxic oxygen radicals) are increasingly implicated in acute leukocyte-mediated tissue injury. To further probe the roles of oxygen radicals in acute lung edema, I studied the effects of a recently described and very potent oxygen radical scavenger, dimethylthiourea (DMTU) (Fox, R. B., R. N. Harada, R. M. Tate, and J. E. Repine, 1983, J. Appl. Physiol., 55:1456-1459) on polymorphonuclear leukocyte (PMN) oxidant function and on two types of lung injury mediated by oxygen radicals and PMN. DMTU (10 mM) blocked 79% of hydroxyl radical (OH) production by PMN in vitro without interfering with other PMN functions, such as O-2 production, myeloperoxidase activity, chemotaxis, degranulation, or aggregation. When isolated rat lung preparations were perfused with PMN activated to produce OH, lung weights were increased from 2.3 +/- 0.2 to 11.2 +/- 0.8 g. DMTU (10 mM) prevented 70% of these increases (lung weights, 5.0 +/- 1.1 g, P less than 0.005). Finally, when intact rats were exposed to 100% O2 for 66 h, lung weight:body weight ratios were increased from 5.78 +/- 0.33 to 8.87 +/- 0.16 g. DMTU (500 mg/kg) prevented 83% of this hyperoxia-induced lung edema in vivo (lung:body weight ratios, 6.05 +/- 0.21, P less than 0.001). Pharmacokinetic studies showed that DMTU diffused effectively into lung interstitial fluids and had a relatively long half-life (25-35 h) in the circulation. Because a variety of oxygen radicals, such as superoxide (O-2), hydrogen peroxide (H2O2), or OH are produced by PMN, there is usually some uncertainty about which one is responsible for injury. However, in these studies, DMTU did not scavenge O-2 and scavenged H2O2 only very slowly while scavenging OH very effectively. Therefore, DMTU may be useful in the investigation of the roles of oxygen radicals, especially OH, in acute granulocyte-mediated tissue injury.

Peroxidase-catalyzed halogenation reactions have been established as being important in the biosynthesis of the hormone thyroxine and in biological defense mechanisms. Recently these reactions have been recognized as valuable tools for the study of proteins as well as their arrangement in macromolecular structures. The pathways of peroxidase catalyses can be accommodated within the framework of the classical Chance-George mechanism. This implies that the initial steps of the reaction invariably involve oxidation of peroxidases by peroxides--and that the resulting derivative, compound I, is the oxidant of the halide ions. Such reactions may result either in the formation of hypohalous acids, or in halogenation of the enzyme apoprotein, followed by transhalogenation to substrate for halogenation. Chloro- and myeloperoxidases catalyze oxidation of all halide ions, except F-; oxidation of bromide and iodide is mediated by lactoperoxidase, but horseradish peroxidase only oxidizes iodide. All of the above enzymes except horseradish will oxidize the pseudo halide thiocyanate. The origins of this differentiation remain to be defined, but they presumably reflect significant variation in oxidation potential of different peroxidase-peroxide derivatives, rather than constraints on the peroxidase-donor interactions. As pointed out above, halogenation of the amino acids tyrosine and histidine or these residues in proteins can take place on the enzyme. This makes lactoperoxidase-catalyzed iodination selective. The amino acid residues in proteins that are iodinated depend not only on reactivity of the amino acid residue but also on its geometric location. Thus lactoperoxidase-catalyzed iodination can be a useful tool in the study of protein structure and function. It is also useful in establishing the geometric position of proteins within macromolecular structures. Thyroid peroxidase catalyzes iodination of thyroglobulin and is involved in a second important step, the coupling of the iodotyrosines to form thyroxine or triiodothyronine. A proposed mechanism for this reaction suggests that the oxidation is mediated by the iodoenzyme derivative mentioned above followed by a prototropic rearrangement and scission to form the ether bound of thyronine and a serine residue on thyroglobulin.

Fractionation of rabbit heterophil leukocyte homogenates by isopycnic centrifugation as well as by zonal sedimentation has helped to characterize further the particulate components of these cells. Four classes have been identified: (A) Large (0.5-0.8 microm) and dense (1.26) azurophil or primary granules, containing all the myeloperoxidase, one-third of the lysozyme, and a major proportion of the lysosomal acid hydrolase activities of the cells. (B) Smaller (0.25-0.40 microm) and less dense (1.23) specific or secondary granules, containing 90% of the alkaline phosphatase and the remainder of the lysozyme activities, but very little if any acid hydrolases. (C) Particles of low density (1.20), containing the remainder of the lysosomal acid hydrolases. This fraction was heterogeneous, but showed abundant small rod- or dumbbell-shaped particles of moderate electron opacity, surrounded by a single membrane (tertiary granules?). The possible origin of these lysosomes from contaminating macrophages could not be ruled out but appeared unlikely. (D) Slowly sedimenting material of very low density (1.14), made up of large, empty vesicular membrane structures, and containing 10% of the alkaline phosphatase, and all of a thiol-dependent acid p-nitrophenyl phosphatase, an enzyme clearly different from the lysosomal acid phosphatase.

Peroxisome proliferator-activated receptor (PPAR) beta-null mice exhibit exacerbated epithelial cell proliferation and enhanced sensitivity to skin carcinogenesis, suggesting that ligand activation of PPARbeta will inhibit keratinocyte proliferation. By using of a highly specific ligand (GW0742) and the PPARbeta-null mouse model, activation of PPARbeta was found to selectively induce keratinocyte terminal differentiation and inhibit keratinocyte proliferation. Additionally, GW0742 was found to be anti-inflammatory due to inhibition of myeloperoxidase activity, independent of PPARbeta. These data suggest that ligand activation of PPARbeta could be a novel approach to selectively induce differentiation and inhibit cell proliferation, thus representing a new molecular target for the treatment of skin disorders resulting from altered cell proliferation such as psoriasis and cancer.

Reactive oxygen species, particularly hydrogen peroxide (H2O2), participate in neutrophil-mediated glomerulonephritis. However, the mechanism of H2O2 neptrotoxicity is unknown. Myeloperoxidase (MPO), a neutrophil cationic enzyme that localizes in glomeruli, can react with H2O2 and halides to form highly reactive products. We tested the hypothesis that the MPO-H2O2-halide system may induce glomerular injury by infusing MPO followed by H2O2 in a chloride-containing solution into the renal artery of rats. Controls received MPO or H2O2 alone. MPO-H2O2-perfused rats developed significant proteinuria, endothelial cell swelling, and epithelial cell foot process effacement, whereas control kidneys were normal. In the presence of free 125I, MPO-H2O2-perfused rats incorporated large amounts of 125I, localized to the glomerular basement membrane and mesangium by autoradiography, into glomeruli. Glomerular iodination was greatly decreased or absent in controls. The MPO-H2O2-halide system causes glomerular injury and may be important in neutrophil-mediated glomerulonephritis.

Neutrophils recruited to the airways in chronic obstructive pulmonary disease (COPD) are thought to mediate tissue destruction. Neutrophil recruitment is increased during bacterial exacerbations. The inflammatory process was studied in patients with an acute exacerbation of COPD in order to ascertain the role of leukotriene B4 (LTB4). The sputum of eight subjects with a bacterial exacerbation of COPD was analysed for neutrophil products (myeloperoxidase, elastase) and chemoattractants (interleukin-8 (IL-8) and LTB4). The contribution of LTB4 to the chemotactic activity of the sputum sol phase was determined using the LTB4 receptor antagonist LY293111. The concentrations of the serum acute phase proteins alpha1-proteinase inhibitor, alpha1-antichymotrypsin and C-reactive protein were measured. All patients received appropriate broad-spectrum antibiotic treatment for 7-14 days. Initially, the sputum myeloperoxidase activity was high, indicating neutrophil influx; this was associated with high levels of IL-8 and LTB4. All these concentrations fell with treatment (p<0.01). The chemotactic activity of the sputum was raised on presentation and fell with treatment (p<0.01). LTB4 contributed approximately 30% of the total chemotactic activity on presentation; this diminished with therapy. All acute phase proteins were raised on presentation and fell with therapy (p<0.01). These findings suggest that leukotriene B4 contributes to neutrophil influx into the airway in chronic obstructive pulmonary disease and may influence disease progression.

A group of 100 totally or subtotally myeloperoxidase (MPO)-deficient individuals was compared to a reference population of 118 probands selected at random. Data for a protective effect of the deficiency against cardiovascular damage are presented. On the other hand, a significantly higher occurrence of severe infections and chronic inflammatory processes was noted among the deficient patients. An increased incidence of cancer among the MPO-deficient individuals was not demonstrated.

OBJECTIVE

R-spondin 1 (Rspo1) is a novel epithelial mitogen that stimulates the growth of mucosa in both the small and large intestine.

METHODS

We investigated the therapeutic potential of Rspo1 in ameliorating experimental colitis induced by dextran sulfate sodium (DSS) or trinitrobenzene sulfonic acid (TNBS) as well as nonsteroidal anti-inflammatory drug-induced colitis in interleukin (IL)-10-deficient mice.

RESULTS

Therapeutic administration of recombinant Rspo1 protein reduced the loss of body weight, diarrhea, and rectal bleeding in a mouse model of acute or chronic DSS-induced colitis. Histologic evaluation revealed that Rspo1 improved mucosal integrity in both villus and/or crypt compartments in the small intestine and colon by stimulating crypt cell growth and mucosal regeneration in DSS-treated mice. Moreover, Rspo1 significantly reduced DSS-induced myeloperoxidase activity and inhibited the overproduction of proinflammatory cytokines, including tumor necrosis factor-alpha, IL-1alpha, IL-6, interferon-gamma, and granulocyte-macrophage colony-stimulating factor, in mouse intestinal tissue, indicating that Rspo1 may reduce DSS-induced inflammation by preserving the mucosal barrier function. Likewise, Rspo1 therapy also alleviated TNBS-induced interstitial inflammation and mucosal erosion in the mouse colon. Furthermore, Rspo1 substantially decreased the histopathologic severity of chronic enterocolitis by repairing crypt epithelium and simultaneously suppressing inflammatory infiltration in piroxicam-exposed IL-10(-/-) mice. Endogenous Rspo1 protein was localized to villus epithelium and crypt Paneth cells in mouse small intestine.

CONCLUSIONS

Our results show that Rspo1 may be clinically useful in the therapeutic treatment of inflammatory bowel disease by stimulating crypt cell growth, accelerating mucosal regeneration, and restoring intestinal architecture.

The endocannabinoid (EC) system mediates protection against intestinal inflammation. In this study, we investigated the effects of blocking EC degradation or cellular reuptake in experimental colitis in mice. Mice were treated with trinitrobenzene-sulfonic acid in presence and absence of the fatty acid amide hydrolase (FAAH) blocker URB597, the EC membrane transport inhibitor VDM11, and combinations of both. Inflammation was significantly reduced in the presence of URB597, VDM11, or both as evaluated by macroscopic damage score, myeloperoxidase levels, and colon length. These effects were abolished in CB(1)- and CB(2)-receptor-gene-deficient mice. Quantitative reverse transcription polymerase chain reaction after induction of experimental colitis by different pathways showed that expression of FAAH messenger RNA (mRNA) is significantly reduced in different models of inflammation early in the expression of colitis, and these return to control levels as the disease progresses. Genomic DNA from 202 patients with Crohn's disease (CD) and 206 healthy controls was analyzed for the C385A polymorphism in the FAAH gene to address a possible role in humans. In our groups, the C385A polymorphism was equally distributed in patients with CD and healthy controls. In conclusion, drugs targeting EC degradation offer therapeutic potential in the treatment of inflammatory bowel diseases. Furthermore, reduction of FAAH mRNA expression is involved in the pathophysiological response to colitis.

Survival and proliferation of skeletal myoblasts within the cardiac environment are crucial to the therapeutic efficacy of myoblast transplantation to the heart. We have analyzed the early dynamics of myoblasts implanted into the myocardium and investigated the mechanisms underlying graft attrition. At 10 min after implantation of [14C]thymidine-labeled male myoblasts into female mice hearts, 14C measurement showed that 39.2 +/- 3.0% of the grafted cells survived, and this steadily decreased to 16.0 +/- 1.7% by 24 h and to 7.4 +/- 0.9% by 72 h. PCR of male-specific Smcy gene calculated that the total (surviving plus proliferated) number of donor-derived cells was 18.3 +/- 1.6 and 23.3 +/- 1.3% at 24 and 72 h, respectively, indicating that proliferation of the surviving cells began after 24 h. Acute inflammation became prominent by 24 h and was reduced by 72 h as indicated by myeloperoxidase activity and histological findings. Multiplex RT-PCR revealed corresponding changes in IL-1beta, TGF-beta, IL-6, and TNF-alpha expression. Treatment with CuZn-superoxide dismutase attenuated the initial rapid death and resulted in enhanced cell numbers afterward, giving a twofold increased total number at 72 h compared with the nontreatment. This effect was associated with reduced inflammatory response, suggesting a causative role for superoxide in the initial rapid graft death and subsequent inflammation. These data describe the early dynamics of myoblasts implanted into the myocardium and suggest that initial oxidative stress and following inflammatory response may be important mechanisms contributing to acute graft attrition, both of which could be potential therapeutic targets to improve the efficiency of cell transplantation to the heart.

The effects of human recombinant interleukin-1 receptor antagonist (IL-1ra) on inflammation, tissue damage, and production of inflammatory mediators in rabbit formalin-immune complex colitis were examined. Treatment of rabbits with intravenous administration of IL-1ra before and periodically throughout the first 43 hours after the induction of colitis (total 7 doses) suppressed inflammation and tissue damage in a dose-related manner. Maximum inhibition of inflammatory index (from 3.0 +/- 0.3 to 0.8 +/- 0.3, P less than 0.001), edema (from 2.3 +/- 0.2 to 0.6 +/- 0.2, P less than 0.001), percent of mucosal necrosis (from 44% +/- 7% to 7% +/- 3%, P less than 0.02) and myeloperoxidase (MPO) activity (from 4.9 +/- 1.0 U/g to 1.0 +/- 0.3 U/g, P less than 0.001) occurred with the dose of 5 mg/kg. Colonic prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) production, measured in rectal dialysates by specific radioimmunoassays, was also dose dependently suppressed (from 1124 +/- 319 pg/mL to 190 +/- 75 pg/mL, P less than 0.001 and 568 +/- 192 pg/mL to 92 +/- 51 pg/mL, P less than 0.001, respectively, at 5 mg/kg). In contrast, colonic IL-1 alpha tissue levels measured by a specific radioimmunoassay after tissue extraction were similar in all groups. When only two doses of IL-1ra, 10 minutes before and 3 hours after the induction of colitis were given, there was no longer an inhibitory effect on inflammation or production of inflammatory mediators. However, delaying the initial IL-1ra treatment 3 hours after the induction of colitis (total 6 doses) was effective in reducing inflammatory index (by 60%), MPO activity (by 79%), PGE2 (by 62%), and LTB4 (by 72%) whereas colonic IL-1 alpha levels were unchanged compared with vehicle-treated animals. These studies show the ability of human recombinant IL-1 receptor antagonist to suppress the proinflammatory activity of IL-1 produced in the colon during the induction and progression of rabbit immune complex colitis.

Two different techniques were utilized to identify the infiltration of polymorphonuclear leukocytes (PMN) into cerebral tissue following focal ischemia: histologic analysis and a modified myeloperoxidase (MPO) activity assay. Twenty-four hours after producing permanent cortical ischemia by occluding and severing the middle cerebral artery of male spontaneously hypertensive rats, contralateral hemiparalysis and sensory-motor deficits were observed due to cerebral infarction of the frontal and parietal cortex. In hematoxylin-and-eosin-stained histologic sections, PMN, predominantly neutrophils, were identified at various stages of diapedesis from deep cerebral and meningeal vessels at the periphery of the infarct, into brain parenchyma. When MPO activity in normal brain tissue was studied initially, it could not be demonstrated in normal tissues extracted from non-washed homogenates. However, if tissue was homogenized in phosphate buffer (i.e., washed), MPO activity was expressed upon extraction. Utilizing this modified assay, MPO activity was significantly increased only in the infarcted cortex compared to other normal areas of the brain. This was observed in non-perfused animals and after perfusion with isotonic saline to remove blood constituents from the vasculature prior to brain removal. The increased PMN infiltration and MPO activity were not observed in forebrain tissue of sham-operated control rats. Also, MPO activity was not increased in the ischemic cortex of MCAO rats perfused immediately after middle cerebral artery occlusion, indicating that blood was not trapped in the ischemic area. By using a leukocyte histochemical staining assay, activity of peroxidases was identified within vascular-adhering/infiltrating PMN in the infarcted cortex 24 hr after focal ischemia. An evaluation of several blood components indicated that increased MPO activity was selective for PMN. The observed increase of approximately 0.3 U MPO/g wet weight ischemic tissue vs. nonischemic cerebral tissues probably reflects the increased vascular adherance/infiltration of approximately 600,000 PMN/g wet weight infarcted cortex 24 hr after focal ischemia. This combined biochemical and histological study strongly suggests that PMN adhere within blood vessels and infiltrate into brain tissue injured by focal ischemia and that the associated inflammatory response might contribute to delayed progressive tissue damage in focal stroke. This modified MPO assay is a useful, quantitative index of PMN that can be utilized to elucidate the potential deleterious consequences of neutrophils infiltrating into the central nervous system after cerebral ischemia, trauma, or other pro-inflammatory stimuli.

Exclusively synthesized by normal neutrophil and monocyte precursor cells, myeloperoxidase (MPO) functions not only in host defense by mediating efficient microbial killing but also can contribute to progressive tissue damage in chronic inflammatory states such as atherosclerosis. The biosynthetic precursor, apoproMPO, is processed slowly in the ER, undergoing cotranslational N-glycosylation, transient interactions with the molecular chaperones calreticulin and calnexin, and heme incorporation to generate enzymatically active proMPO that is competent for export into the Golgi. After exiting the Golgi the propeptide is removed prior to final proteolytic processing in azurophil granules, resulting in formation of a symmetric MPO homodimer linked by a disulfide bond. Some proMPO escapes granule targeting and becomes constitutively secreted to the extracellular environment. Although the precise mechanism is unknown, the pro-segment is required for normal processing and targeting, as propeptide-deleted MPO precursor is either degraded or constitutively secreted. Characterizing the molecular consequences of naturally occurring mutations that cause inherited MPO deficiency provides unique insight into the structural determinants of MPO involved in biosynthesis, processing and targeting.

Myeloperoxidase (MPO), a lysosomal heme protein found exclusively in neutrophils and monocytes, is necessary for efficient oxygen-dependent microbicidal activity. Acquisition of heme by the heme-free MPO precursor apopro-MPO appears to be a prerequisite for its subsequent proteolytic processing and advancement along the biosynthetic pathway to mature MPO. We present data indicating that calreticulin (CRT), a high capacity calcium-binding protein residing in the lumen of the endoplasmic reticulum of a wide variety of cells, interacts specifically with fully glycosylated apopro-MPO. Biosynthetically radiolabeled CRT (60 kDa) and apopro-MPO (90 kDa) were coprecipitated from PLB 985 cells by monospecific antiserum against CRT when the immunoprecipitations were performed either under nondenaturing conditions or following reversible crosslinking. Nonglycosylated MPO precursors synthesized in the presence of tunicamycin did not interact with CRT. The CRT-apopro-MPO interaction was restricted to an early phase of MPO biosynthesis, and CRT did not interact with the later appearing, heme-containing species of MPO, i.e. pro-MPO or the heavy subunit of mature MPO. These data show that CRT participates in the post-translational processing of MPO, perhaps by maintaining apopro-MPO in a conformation competent to accommodate insertion of the heme group. In this general way, CRT shares certain functional properties with the structurally homologous transmembrane calcium-binding endoplasmic reticulum protein calnexin. Both interact with glycosylated biosynthetic precursors of proteins selectively expressed in specialized cells.

Toxin A, a 308,000-Mr enterotoxin from Clostridium difficile, mediates antibiotic-associated diarrhea and colitis in humans. Injection of toxin A into animal intestine triggers an acute inflammatory response characterized by activation of sensory neurons and immune cells of the intestinal lamina propria, including mast cells and macrophages, and migration of circulating neutrophils in the involved intestinal segment. In this study we show that mice genetically deficient in the neurokinin-1 receptor are protected from the secretory and inflammatory changes as well as from epithelial cell damage induced by toxin A. The protective effect of neurokinin-1R deletion correlates with diminished intestinal levels of the cytokine TNF-alpha and its mRNA and the leukocyte enzyme myeloperoxidase. These results demonstrate a major requirement for substance P receptors in the pathogenesis of acute inflammatory diarrhea.

We sought to determine the mechanisms responsible for the reduced renal tissue injury by agonists of A(2A) adenosine receptors (A(2A)-ARs) in models of ischemia-reperfusion (I/R) injury. DWH-146e, a selective A(2A)-AR agonist, was administered subcutaneously to Sprague-Dawley rats and C57BL/6 mice via osmotic minipumps, and animals were subjected to I/R. I/R led to an increase in plasma creatinine and kidney neutrophil infiltration. Infusion of DWH-146e at 10 ng. kg(-1). min(-1) produced a 70% reduction in plasma creatinine as well as a decrease in neutrophil density in outer medulla and cortex and myeloperoxidase activity in the reperfused kidney. Myeloperoxidase activity in kidney correlated with the degree of renal injury. P-selectin and intercellular adhesion molecule 1 (ICAM-1) immunoreactivity were most prominent in endothelial cells of peritubular capillaries and interlobular arteries of cortex and outer and inner medulla of vehicle-treated mice whose kidneys were subjected to I/R. DWH-146e treatment led to a pronounced decrease in P-selectin- and ICAM-1-like immunoreactivity. These data are consistent with our hypothesis that A(2A)-AR agonists limit I/R injury due to an inhibitory effect on neutrophil adhesion.

The conversion of iodide to a trichloroacetic acid-precipitable form (iodination) by polymorphonuclear leukocytes (PMN's) has been re-evaluated as a measure of neutrophil function. Optimum conditions are described which result in an iodination value for normal cells during the phagocytosis of zymosan of 64.1 +/- 13.2 (S.D.) nmol. per 10(7) PMN's per hour. Iodination is inhibited by agents which decrease phagocytosis, inhibit myeloperoxidase-catalyzed reactions, or degrade H2O2 and is stimulated by superoxide dismutase, an enzyme which catalyzes the conversion of the superoxide anion to oxygen and H2O2. When patients' cells and normal serum are employed, the iodinating capacity of the patients' cells is evaluated. It is low in patients with myeloperoxidase deficiency and chronic granulomatous disease, and an intermediate value was observed in a carrier of chronic granulomatous disease. When normal cells and patients' serum are employed, the iodination reaction is an indirect measure of the opsonic activity of the patients' serum. The decreased opsonic activity for zymosan of human sera deficient in the fourth or third component of complement was demonstrated in this way. Thus measurement of iodination is a convenient and sensitive screening test for cellular or humoral abnormalities of the phagocytic process.

The oxidation-reduction midpoint potential of the cytochrome b found in the plasma membrane of human neutrophils has been determined at pH 7.0 (Em,7.0) from measurements of absorption spectra at fixed potentials. In both unstimulated and phorbol myristate acetate-stimulated cells Em,7.0 was -245 mV. Changes in pH affected the Em of the cytochrome b, with a slope of approx. 25 mV/pH unit change. The Em,7.0 of the haem group(s) of the membrane-bound myeloperoxidase of human neutrophils was found to be +34 mV. The plasma membranes contained no detectable ubiquinone, and no iron-sulphur compounds were detected by e.p.r. spectroscopy at 5-20 K. No flavins were detected by e.p.r. spectroscopy. The cytochrome b-245 was not reduced by added NADH or NADPH. Dithionite-reduced cytochrome b-245 formed a complex with CO, supplied as a saturated solution, which was dissociated with 26 microseconds illumination from a xenon flash lamp, and the recombination with CO had a half-time of approx. 6 ms. Partly (80%) reduced cytochrome b-245 was oxidized by added air-saturated buffer with a half-time faster than 1 s at 20 degrees C, a resolution limited by mixing time. These results are compatible with cytochrome b-245 acting as an oxidase.

The interaction of Staphylococcus epidermidis slime with human neutrophils (PMN) was examined by using isolated slime and allowing bacteria to elaborate slime and other extracellular products in situ. S. epidermidis slime was found to contain a chemoattractant. Incubation of PMN with 50 micrograms or more of slime per ml inhibited subsequent chemotaxis of the PMN to n-formyl-methionyl-leucyl-phenylalanine by 27% and to zymosan-activated serum by 44 to 67% with increasing slime concentrations. S. epidermidis slime stimulated little degranulation of untreated PMN. After pretreatment of PMN with 5 micrograms of cytochalasin b per ml, slime predominantly induced release of specific granule contents (33.8% lactoferrin release by 250 micrograms of slime per ml versus 10% myeloperoxidase release by 250 micrograms of slime per ml). By a surface phagocytosis assay, PMN uptake of radiolabeled S. epidermidis which were incubated for 18 h on a plastic surface for slime expression was less than that for S. epidermidis adhered to the plastic for 2 h or grown in unsupplemented nutrient broth. These results suggest that S. epidermidis slime interaction with PMN may be potentially detrimental to host defense and may contribute to the ability of this organism to persist on surfaces of foreign bodies in the vascular or central nervous system.