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PubMed1994

[Pleuropulmonary aspergillosis with defective neutrophil functions--G-CSF effects on neutrophil functions].

A 66-year-old man was admitted to our hospital with productive cough and fever. Chest X-ray films showed pleural thickening of the right apex and calcification of the lung field. Antibiotic therapy was administered but no improvement was obtained. Chest CT scan revealed pleural thickening with effusion in the right apex. Needle aspiration biopsy was performed and Aspergillus fumigatus was isolated from pleural fluid. We began pleural drainage and intrapleural infusion of miconazole, with systemic administration of miconazole, amphotericin B and fluconazole. In spite of normal bone marrow findings, the neutrophil functions, NAP score and phagocytosis, of the patient were found to be subnormal. We commenced to treatment with granulocyte colony stimulating factor and his neutrophil functions improved (NAP score: 156-->309.5, phagocytosis: 64.9-->70.0%). However, his fever persisted and he died following cardiopulmonary arrest. Autopsy revealed Aspergillus mycelia invading the visceral pleura. No intrapulmonary lesions caused by Aspergillus were found.

Aged↗
PubMed1994

Neutrophil-activating peptides NAP-2 and IL-8 bind to the same sites on neutrophils but interact in different ways. Discrepancies in binding affinities, receptor densities, and biologic effects.

IL-8 and the neutrophil-activating peptide 2 (NAP-2) are members of the chemokine family of host defense cytokines. Although IL-8 was shown to interact with two different high affinity receptors on polymorphonuclear neutrophil granulocytes (PMN), direct demonstration of specific binding sites for NAP-2 is difficult, because the NAP-2 molecule lacks iodinable side chains. Here we present a modified labeling procedure for the chemokine that does not affect its biologic activity. The 125I-labeled NAP-2 specifically bound to PMN with two different affinities (KD = 0.65 and 22.4 nM). We observed complete cross-competition of unlabeled IL-8 with 125I-labeled-NAP-2 and of unlabeled NAP-2 with 125I-labeled IL-8, indicating the absence of monospecific binding sites for either chemokine. However, in contrast to former work by others, the total number of accessible sites was considerably lower for NAP-2 (13,000/cell) than for IL-8 (59,000/cell). In addition, PMN prepared from heparinized blood expressed significantly more receptors for NAP-2 than cells prepared from citrated blood, whereas receptor numbers for IL-8 were unchanged. Desensitization experiments suggested a regulatory role for the NAP-2 high affinity site. Short-term priming of PMN with a nonstimulatory dose of NAP-2 (or MGSA) but not with IL-8 led to drastic down-regulation of the subsequent degranulation response, challenged by higher dosages of NAP-2, MGSA, or IL-8. Reduced functional responsiveness of cells correlated with the rapid down-regulation and internalization of NAP-2 and IL-8 high affinity binding sites. Thus, our data indicate that chemokines could mediate by individual modes of interaction with common receptor's different biologic functions.

Binding Sites↗
PubMed1993

Circulating and tissue levels of the neutrophil chemotaxin interleukin-8 are elevated in severe acute alcoholic hepatitis, and tissue levels correlate with neutrophil infiltration.

Acute alcoholic hepatitis is characterized by a unique degree of liver neutrophil infiltration, often accompanied by marked peripheral neutrophilia in the absence of demonstrable bacterial or fungal infection. In this study we assayed plasma and tissue levels of a potent neutrophil activator and chemotaxin, interleukin-8, in patients with a spectrum of alcoholic liver diseases and in normal and diseased control subjects. Levels of circulating interleukin-8 were undetectable in normal subjects but highly elevated in patients with alcoholic hepatitis, particularly in those who died (geometric mean = 600 ng/L; confidence interval = 323 to 1,120 vs. geometric mean = 184 ng/L; confidence interval = 114 to 309 in survivors). Levels correlated with biochemical indicators of severe disease (bilirubin: R = 0.38; international prothrombin ratio: R = 0.28; white blood cell count: R = 0.35; creatinine: R = 0.34) and with tumor necrosis factor-alpha (R = 0.43) and soluble tumor necrosis factor receptors (p55; R = 0.59). In contrast, moderate elevations in the levels of circulating interleukin-8 were seen in alcoholic cirrhosis (geometric mean = 93 ng/L; confidence interval = 40 to 213) and in alcoholic patients undergoing alcohol withdrawal (geometric mean = 137 ng/L; confidence interval = 72 to 259). Levels in nonalcoholic inflammatory liver disease were comparatively low (geometric mean = 17 ng/L; confidence interval = 10 to 29).(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Disease↗
PubMed1993

The cDNA of the neutrophil antibiotic Bac5 predicts a pro-sequence homologous to a cysteine proteinase inhibitor that is common to other neutrophil antibiotics.

Bac5 is a 5-kDa proline- and arginine-rich antibiotic, stored as inactive precursor (proBac5) in the large granules of bovine neutrophils. A full-length cDNA encoding the precursor form of Bac5 has been cloned. The encoded protein (pre-proBac5) has a calculated mass of 20,031 Da and a pI of 9.21. This comprises a putative signal peptide of 29 amino acid residues and a 101-residue pro-sequence that precede the mature antibiotic. The pro-sequence is acidic and may neutralize the highly cationic Bac5, thus accounting for the inactivation of the antibiotic activity observed in in vitro experiments. The structure of mature Bac5 agrees closely with the amino acid sequence previously determined, with an additional tripeptide tail predicting carboxyl-terminal amidation. A valyl residue is deduced at the cleavage site for the proteolytic maturation of proBac5, consistent with a previous observation showing elastase as the enzyme involved in this processing step. The region upstream of Bac5 reveals high identity to corresponding regions of two neutrophil antimicrobial polypeptides, CAP18 from rabbit and bovine indolicidin. The COOH-terminal sequences of these antibiotics are completely unrelated. The proregion also exhibits remarkable similarity to pig cathelin, an inhibitor of cathepsin L, indicating a common evolutionary origin.

Amino Acid Sequence↗
PubMed1977

A neutrophil-dependent pathway for the generation of a neutral peptide mediator. II. Subcellular localization of the neutrophil protease.

The human neutrophil neutral peptide-generating protease was associated with the plasma membrane marker 5'-nucleotidase on sucrose density gradient centrifugation of sonicates of granule-free fractions following homogenization and velocity sedimentation. The two activities were also associated on sucrose density gradient fractionation of plasma membranes obtained by hypotonic lysis in EDTA containing buffers, a technique which minimizes aggregation. Treatment of fractions containing these enzymatic activities with 1-0 M NaCl separated the neutral peptide-generating proteasein to the eluate while leaving the 5'-nucleotidase in the pellet. Gel filtration of the solubilized neutral peptide-generating protease through Sephadex G-100 in 1-0 M NaCl demonstrated that the protease had an approximate mol. wt of 20,000 while filtration in physiological salt concentrations yielded activity only in the excluded volume. In both cases, there was complete recovery of neutral peptide-generating activity suggesting that the filtration characteristics of the protease were determined by the salt concentration. The solubilized purified protease, the whole cell sonicates, and the intact cells interacted with heat-inactivated plasma to yield the same product, a neutral peptide with a 1000 molecular weight and an isoelectric point of 7-2-7-6. The neutral peptide-generating protease in each instance was inhibited in dose-response fashion by alpha-1-antitrypsin, LBTI, and DFP. Only 30-60% of the protease sites were functional on intact cells as revealed by substrate cleavage or were available to inhibitors. The neutrophil protease which generates neutral peptide is an extrinsic plasma membrane protein with an approximate mol. wt of 20,000 which functions as an ectoenzyme.

Cell Fractionation↗
PubMed1995

Activation of p58c-fgr and p53/56lyn in adherent human neutrophils: evidence for a role of divalent cations in regulating neutrophil adhesion and protein tyrosine kinase activities.

Tumor necrosis factor (TNF) stimulates generation of reactive oxygen intermediates, secretion of granule constituents, and rearrangement of the cytoskeleton in neutrophils (PMN); this response requires that PMN be adherent to plasma or extracellular matrix proteins, and is dependent on beta 2 integrins. Tyrosine phosphorylation of distinct proteins [Fuortes et al., J Cell Biol 120:777-784, 1993] and activation of the protein tyrosine kinase p58c-fgr [Berton et al., J Cell Biol 126:1111-1121, 1994] were recently recognized as signals involved in beta 2 integrin-dependent responses of TNF-treated PMN. As the integrin capability to bind their ligands is regulated by divalent cations we investigated whether modulation of PMN adhesion to fibrinogen by divalent cations also affected activation of protein tyrosine kinases. In the absence of divalent cations or in the presence of Ca2+ alone, PMN did not adhere to fibrinogen in response to TNF. However, Mg2+, either alone or together with Ca2+, promoted stimulated adhesion to fibrinogen. We also found that Mn2+ promoted PMN adhesion to fibrinogen without additional stimuli. Analysis of the activity of two src family tyrosine kinases, p58c-fgr and p53/56lyn, showed that their autophosphorylating kinase activity strictly correlated with adhesion. In fact, only in the presence of Mg2+, but not in the absence of divalent cations or in the presence of Ca2+ alone, TNF increased p58c-fgr and p53/56lyn kinase activities; and this was prevented by anti-CD18 antibodies. In addition, Mn2+ strongly promoted activation of p58c-fgr and p53/56lyn without additional stimuli. Analysis of tyrosine phosphorylated proteins with anti-phosphotyrosine immunoblots showed that divalent cations regulated adhesion and protein tyrosine phosphorylation in the same fashion. Detergent extraction of proteins showed that the Mg(2+)-dependent, TNF-stimulated adhesion redistributed p58c-fgr and p53/56lyn to a Triton-insoluble fraction. In addition, analysis of p58c-fgr activity allowed us to demonstrate that the fraction of p58c-fgr which became Triton-insoluble displayed a higher kinase activity. These findings establish that PMN adhesion signals for activation of two different src family tyrosine kinases. The evidence that Mn2+, a strong promoter of integrin function, induces adhesion and activation of tyrosine kinases without additional stimuli suggest the existence of a direct link between beta 2 integrins binding to fibrinogen and activation of tyrosine kinases in neutrophils.

Antibodies↗
PubMed1990

Amino acid sequence of CAP37, a human neutrophil granule-derived antibacterial and monocyte-specific chemotactic glycoprotein structurally similar to neutrophil elastase.

We report the amino acid sequence of CAP37, a human neutrophil granule protein with antibacterial and monocyte-specific chemotactic activity. CAP37 is a single-chain protein consisting of 222 amino acid residues. It has three N-glycosylation sites, at Asn residues 100, 114 and 145. Some species of CAP37 are glycosylated at all three sites; some at Asn-114 alone, others at Asn-114 and Asn-110 or Asn-145. CAP37 has 45% sequence identity to human neutrophil elastase, and 30-37% identity to several other granule serine proteinases. Despite these similarities, CAP37 is not a serine proteinase because the active site residues serine and histidine are replaced.

Amino Acid Sequence↗
PubMed2004

Identification and overexpression of human neutrophil alpha-defensins (human neutrophil peptides 1, 2 and 3) in squamous cell carcinomas of the human tongue.

To date, little attention has been paid to the possible role of alpha-defensins (human neutrophil peptides 1-3), HNP-1, HNP-2 and HNP-3 in innate host defence against tumour invasion. In the current study, using a single-dimensional high pressure liquid chromatography (HPLC) method for peptide separation, followed by mass spectrometry and amino acid sequencing for identification and quantitation, we report the overexpression of HNP-1, HNP-2 and HNP-3 in squamous cell carcinomas of the human tongue compared with autogenous non-tumour tissue. Using a specific antibody we show that the defensins are abundant in neutrophils infiltrating human oral squamous cell carcinoma tissue. In the context of their previously reported oncolytic activity, our results may imply a role for alpha-defensins in host defence against oral squamous cell carcinoma.

Adult↗
PubMed1988

Synthesis and expression in Escherichia coli of the gene encoding monocyte-derived neutrophil-activating factor: biological equivalence between natural and recombinant neutrophil-activating factor.

The neutrophil-activating factor (NAF) purified from the conditioned medium of lipopolysaccharide-stimulated human monocytes was sequenced and found to consist of 72 amino acids: SAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRA ENS. Purified preparations of natural NAF contained, in addition to this main form, minor amounts of three amino-terminal variants with 77 (+AVLPR), 70, and 69 residues. A gene coding for the 72-amino acid NAF was synthesized, cloned, and expressed in Escherichia coli. Western (immunologic) blot analysis of crude bacterial extracts, with an antiserum raised against natural NAF, revealed a single band that comigrated with natural NAF. Recombinant NAF purified to homogeneity had identical amino- and carboxyl-terminal sequences to the 72-amino acid natural NAF. Recombinant NAF was tested on human neutrophils and had the same activity and potency as natural NAF in inducing chemotaxis, rapidly increasing cytosolic free Ca2+, activating the respiratory burst, and releasing specific and azurophilic granular contents.

Amino Acid Sequence↗
PubMed2002

Neutrophil elastase and acute lung injury: prospects for sivelestat and other neutrophil elastase inhibitors as therapeutics.

OBJECTIVES: To review the evidence and rationale that suggest that neutrophil elastase (NE) may contribute to the development of acute lung injury (ALI) and the acute respiratory distress syndrome. To review selected preliminary data regarding the effectiveness of NE inhibition in animals, in in vitro models, and in patients with ALI. DATA SOURCES: The published literature and observations provided by Ono Pharmaceutical and Eli Lilly investigators and their colleagues. DATA SUMMARY: Taken en toto, the data suggest that NE could contribute to ALI and endothelial cell injury that is relevant to ALI. Moreover, the toxic effects of NE are greatly enhanced by increased oxidative stress, which commonly occurs in patients with ALI. In addition to neutrophils, xanthine oxidase, a constituent of endothelial cells, is a potential source of oxidative stress in ALI; xanthine oxidase-derived oxidants enhance NE toxicity in in vivo, isolated lung, and in vitro endothelial cell test systems. Not surprisingly, endogenous nonoxidatively sensitive NE inhibitors (e.g., eglin C) are more effective in combating the detrimental effects of NE than oxidatively sensitive NE inhibitors (e.g., alpha-1-proteinase inhibitor). In addition, a synthetic NE inhibitor, sivelestat (ONO-5046 and LY544349), is effective in reducing measures of inflammation and injury in multiple animal models of ALI. In a trial of ALI patients with systemic inflammatory response syndrome, conducted in Japan by Ono Pharmaceutical scientists, sivelestat treatment improved the investigator assessment of global improvement and the percentages of patients who were removed from ventilators and transferred out of the intensive care unit. CONCLUSIONS: Further study of the role of NE inhibition as a treatment for ALI is warranted. Additional clinical and preclinical studies with sivelestat and various other NE inhibitors should not only clarify the clinical potential of this intervention strategy, but also better define the activities of NE in inflammatory disorders such as ALI and multiple organ failure.

Animals↗
PubMed1995

Proteolysis of classic anti-neutrophil cytoplasmic autoantibodies (C-ANCA) by neutrophil proteinase 3.

C-ANCA, which are directed against neutrophil proteinase 3 (PR3), are specific markers for the diagnosis of active Wegener's granulomatosis (WG). The correlation between C-ANCA titre and WG disease activity suggests that these autoantibodies are involved in the development of WG. We have previously observed that C-ANCA interfere with PR3 proteolytic activity and with complexation of PR3 with its major physiologic inhibitor alpha 1-antitrypsin (alpha 1-AT). The possible pathogenic importance of C-ANCA may be related to the stability of C-ANCA IgG-PR3 complexes. In the present study we tested proteolysis by PR3 of human IgG and proteolysis of C-ANCA IgG complexed to the enzyme. All human IgG subclass proteins were cleaved by PR3. Digestion products were compared with those obtained by human neutrophil elastase (HNE)-mediated proteolysis of human IgG subclass proteins. Although cleavage products of similar size could be identified, the proteolytic activity of both enzymes towards human IgG differed. Furthermore, inhibiting C-ANCA IgG were cleaved into small peptides when complexed to PR3. The possible pathogenic consequences of these findings will be discussed.

Antibodies, Antineutrophil Cytoplasmic↗
PubMed2005

Changes in coagulation-fibrinolysis marker and neutrophil elastase following the use of tourniquet during total knee arthroplasty and the influence of neutrophil elastase on thromboembolism.

BACKGROUND: To clarify in detail the mechanism underlying the development and exacerbation of deep venous thrombosis (DVT) and/or pulmonary thromboembolism (PTE), we focused on the following factors: the thrombin-antithrombin III complex (TAT), D-dimer and neutrophil elastase (NE). We basically investigated whether NE played an important role in the development of PTE I a mice model. METHODS: Nineteen rheumatoid arthritis (RA) and six osteoarthritis (OA) patients underwent total knee arthroplasty (TKA) with tourniquet, and 13 RA and 12 OA patients underwent TKA without tourniquet in each group. The blood levels of TAT, D-dimer and NE were measured before surgery, immediately after and during the days following surgery. For the induction of experimental PTE due to coagulation of platelets, adenosine diphosphate (ADP) was administrated, and human NE with ADP was also administrated for the development of DVT and/or PTE. RESULTS: The rates of increase in the mean TAT, D-dimer and NE levels in the group with tourniquet were statistically higher than those in the group without tourniquet after surgery. The mortality of the mouse due to PTE increased from 43 to 67% following ADP and human NE administration compared to a single ADP administration. Histological changes of the lungs in the mice receiving NE and ADP injections were characterized by a diffuse and extensive accumulation of platelets and fibrin in alveolar capillaries and other microvessels. CONCLUSION: We suggest that during TKA, the use of tourniquet induces local release of a large amount of NE from neutrophils, inducing the development of DVT and/or PTE and their exacerbation.

Adenosine Diphosphate↗
PubMed1999

[Clinical reference values for laboratory hematology tests calculated using the iterative truncation method with correction: Part 2, Reference values for white blood cell (WBC) count, WBC differential including segmented neutrophil, band neutrophil, lymphocyte, monocyte, eosinophil, basophil, platelet count and mean platelet volume].

Age and sex dependent differences in the clinical reference values for white blood cell(WBC) count, WBC differential including segmented neutrophils(SEG), band neutrophils(BAND), lymphocytes (LYMPH), monocytes(MONO), eosinophils(EOSINO), basophils(BASO), platelet count(PLT) and mean platelet volume(MPV) were calculated by the iterative truncation method with correction(Usui's method) using the result from tests on 6,300 patient specimens obtained at Kyoto University Hospital. WBC counts exhibited log-normal distribution in all age either in male and female. PLT counts and MPV showed normal or log-normal distribution. WBC differential (SEG, BAND, LYMPH, MONO, EOSINO, BASO) usually showed log-normal distribution. As to the reference value for WBC, high limit values gradually decreased in the age range of 5-14 year old in males and females. After 15 year old, it gradually increased and reached adult's level. Low limit values for WBC gradually decreased with age both in males and females. The reference value for SEG showed similar change to WBC. The reference value for LYMPH gradually decreased age both in males and females. The reference values for BAND, MONO, EOSINO, BASO were generated by the percentile method instead of the iterative truncation method because of the strong deviation in the distribution pattern. BAND, MONO and BASO values were very stable for all ages in males and females. High limit values for EOSINO in the age range of 5-10 year old in males and females remained constant, and gradually decreased in 10-20 year old, and were stable at ages above 20 year old. PLT counts gradually decreased with age, and the MPV remained constant in all age populations both in male and female.

Adolescent↗
PubMed2003

Plasma neutrophil lipocalin, elastase-alpha1-antitrypsin complex and neutrophil protease 4 in preterm infants with respiratory distress syndrome.

Respiratory distress syndrome (RDS) and chronic lung disease of prematurity (CLD) are associated with inflammation of the airways and interstitial tissue of the lung. It is hypothesized that RDS severity and the risk of developing CLD may be correlated with neutrophil gelatinase-associated lipocalin (NGAL), a marker of leucocyte activity, human elastase-alpha1-antitrypsin complex (HEAT) or free and complexed neutrophil protease 4 (NP4), markers of proteolytic enzyme secretion from granulocytes. Thirty-three preterm infants with RDS were enrolled in the study and plasma sampled between 3 and 14 days of life. NGAL, HEAT and NP4 concentrations varied widely in infants with RDS. Significant correlations between subsequent development of CLD and plasma concentrations of HEAT and NP4, respectively, were found on days 3-4 of life, p=0.006 and p=0.02, respectively.

Acute-Phase Proteins↗
PubMed1999

alpha9beta1 integrin is expressed on human neutrophils and contributes to neutrophil migration through human lung and synovial fibroblast barriers.

Accumulation of leukocytes in inflamed tissue involves their migration through vascular endothelium and then in the connective tissue. Recently we utilized a barrier of human synovial, dermal, and lung fibroblasts (HSF, HDF, and HLF) grown on polycarbonate filters as a model of human polymorphonuclear leukocyte (PMN) migration through connective tissue. The beta2 integrins (CD 11/ CD18) and alpha4, alpha5, and alpha6beta1 (VLA-4, -5, and -6) integrins each contributed to this PMN migration. Here we report that on human blood leukocytes, alpha9beta1 (VLA-9) is expressed only on PMNs and that it is up-regulated after PMN activation. Based on monoclonal antibody (mAb) blocking studies, alpha9beta1 integrin contributed to C5a-induced PMN migration through fibroblast (HLF and HSF) barriers. This role was apparent only when alternate mechanisms such as CD18, alpha4, alpha5, and alpha6beta1 integrins were blocked and then mAb to alpha9beta1 integrin inhibited the residual PMN migration (by 40-50%) through the HLF or HSF barrier, resulting in > or = 75% inhibition overall. In contrast, PMN migration across interleukin-1-activated endothelium (HUVEC) in response to a C5a gradient, which is partly (30-40%) via CD11/CD18-independent mechanisms, was not inhibited by adhesion blocking by mAbs to alpha4, alpha5, alpha6, and alpha9beta1 even in combination. These results indicate that alpha9beta1 integrin on PMN may have a special role, in conjunction with other beta1 integrins, in mediating PMN migration in the extravascular space, and may contribute to differential neutrophil function within tissues.

Animals↗
PubMed1996

Neutrophil activation and tissue neutrophil sequestration in a rat model of thermal injury.

Neutrophil (PMN) deposition in tissues (leukosequestration) after shock may produce local tissue injury from proteases and oxygen intermediaries which are released from sequestered PMNs. We quantified leukosequestration in tissues in burned rats using two methods of analysis: 1), measurement of lung myeloperoxidase (MPO); 2), measurement of radiolabeled PMNs and erythrocytes deposited in multiple tissues. After tracheostomy and venous cannulation, rats received 17% TBSA full-thickness contact burns and were resuscitated with 20 cc intraperitoneal saline. Lung PMNs were estimated by measuring MPO in lung tissue. PMN influx into lung, liver, spleen, gut, skin, muscle, kidney, and brain was determined by removing (preburn) and differentially radiolabeling PMNs (111In) and erythrocytes (51Cr), reinfusing cells 4.5 hr postburn, and measuring tissue radioactivity 5 hr postburn. Tissue edema was measured by determining extravasation of 125I-labeled albumin in tissues. Peripheral blood PMNs were analyzed for intracellular H2O2 content utilizing a fluorescent dye which reacts with H2O2 coupled with analysis of cell fluorescence by flow cytometry. MPO was elevated in lungs 8 hr postburn (P < 0.05). PMN influx into lung tissues was confirmed by histologic examination. Radioisotope studies demonstrated significant (P < 0.05) leukosequestration into lung, gut, kidney, skin, and brain tissues at 5 hr postburn. Respiratory burst activity of peripheral blood PMNs was increased 5 hr postburn (P < 0.05). Flow cytometric analysis indicated that peripheral blood PMNs were capable of producing markedly increased H2O2 levels 5 hr postburn. Tissue edema, manifested by radiolabeled albumin influx, was not seen in any tissues. Since others have shown that sequestration of metabolically active PMNs may induce remote tissue injury, therapies which block postburn leukosequestration may be able to improve clinical outcomes by limiting remote tissue injury.

Animals↗
PubMed1997

Regulation of neutrophils in ulcerative colitis by colonic factors: a possible mechanism of neutrophil activation and tissue damage.

The mucosal injury of active ulcerative colitis (UC) could involve enhanced migration and activation of neutrophils (PMNs). Because, in vitro, PMNs from patients with UC appear normal and are not therefore a likely cause for the enhancements, we hypothesized an abnormal colonic milieu. We previously found that factors in the UC colonic milieu markedly increase production of reactive oxygen species (ROS) by control PMNs. We now hypothesize that these factors also regulate PMN surface integrins, that regulation of UC PMNs is different than that of control PMNs, and that the integrin regulation is consistent with the ROS regulation. Using rectal dialysis, we sampled the colonic milieu in patients with active UC, in patients with inactive UC, and in control subjects. We monitored a key PMN adhesion molecule, CD11b. When control PMNs were tested, active UC rectal dialysate was almost as effective (+115%) as N-formyl-methionyl-leucyl-phenylalanine (+132%) in up-regulating CD11b. When inactive UC PMNs were tested, baseline CD11b was 50% higher than that for control PMNs. In contrast, rectal dialysates failed to up-regulate CD11b of inactive UC PMNs and in fact down-regulated CD11b. Preincubating control PMNs with UC rectal dialysates converted their CD11b response to PMN activators from up-regulation to down-regulation, mimicking inactive UC PMNs. Changes in intracellular calcium levels paralleled these changes in CD11b. Rectal dialysate-induced changes in both CD11b and calcium paralleled our previous findings on rectal dialysate-induced changes in ROS production. Thus the net overall effect of factors in the colonic milieu is a consistent and predictable regulation of PMN function--proinflammatory in UC, anti-inflammatory in control subjects. These factors may be a critical part of the pathophysiology of UC.

Adult↗
PubMed1998

Endogenous glucocorticoids modulate neutrophil migration and synovial P-selectin but not neutrophil phagocytic or oxidative function in experimental arthritis.

Pharmacologic glucocorticoids are powerful inhibitors of the inflammatory response at many levels, including leucocyte trafficking and function. The adhesion molecule P-selectin is a key participant in polymorphonuclear neutrophil (PMN) migration to sites of inflammation. The extent to which endogenous glucocorticoids influence PMN migration and activation is not clear. We used the glucocorticoid receptor antagonist RU486 to examine the effect of endogenous glucocorticoid blockade on PMN migration and function in carrageenan monoarthritis in the rat. Arthritis was induced by intraarticular injection of carrageenan and disease severity measured by PMN count in synovial lavage fluid. Decalcified frozen sections of injected joints were analysed for expression of P-selectin by immunohistochemistry. Adrenal glucocorticoid action was blocked in vivo with RU486 20 mg/kg. PMN phagocytosis and reactive oxygen species synthesis were measured by flow cytometry. Carrageenan injection was associated with severe arthritis (synovial lavage PMN 5.9 +/- 0.7 x 10(6), P < 0.01 versus control) which was dose-dependent. P-selectin was not detected in normal joints but was abundant in joints injected with 500 microg carrageenan. RU486 resulted in exacerbation of carrageenan arthritis (9.7 +/- 0.8 x 10(6), P < 0.05). RU486 also altered the threshold for disease induction, in that most RU486-treated animals were susceptible to arthritis at a dose of carrageenan (2.5 microg) which did not induce arthritis in most control-treated animals (P < 0.05), denoting an altered threshold for arthritis induction. RU486 treatment was associated with increased synovial P-selectin expression. Activation status as measured by PMN phagocytic and oxidative function were not influenced by endogenous glucocorticoid blockade. These findings suggest that endogenous glucocorticoids selectively influence PMN migration to inflamed joints via P-selectin expression, but have no effect on PMN activation status.

Animals↗