Early neutrophil sequestration after injury: a pathogenic mechanism for multiple organ failure

Polymorphonuclear neutrophils (PMNs) play a pivotal role in the inflammation that precedes multiple organ failure (MOF). In a rat model of MOF, PMNs become primed for enhanced superoxide anion (O2-) release and CD11b expression, sequester in end organs, and produce organ failure. Therefore, we hypothesized that circulating PMNs harvested in the first 24 hours after injury from trauma patients at risk for MOF would (1) exhibit a primed O2- release, (2) upregulate CD11b expression, and (3) show evidence of sequestration in tissues. Extracellular PMN O2- release and CD11b receptor expression were measured at 3, 6, 12, and 24 hours after injury in 33 torso trauma patients with Injury Severity Scores > 15; eight patients (24%) developed MOF. Healthy adults served as controls. PMNs after injury were primed for enhanced in vitro O2- release at 3, 6, 12, and 24 hours after injury, indicating prior in vivo priming. CD11b expression was also increased at 6, 12, and 24 hours after injury. Circulating PMN numbers increased sharply at 3 hours after injury, before decreasing dramatically at 6 and 12 hours, suggesting end organ sequestration. At 12 hours after injury, declines in circulating PMNs were significantly greater in MOF than in non-MOF patients (p < 0.05). These data indicate that PMNs are quickly mobilized into the circulation after injury and then primed for enhanced O2- release and CD11b expression. PMN priming appears to be a necessary preamble to PMN sequestration in patients with major torso trauma. Upregulation of PMN function, accompanied by subsequent end organ sequestration, may represent an important early event in the pathogenesis of MOF after injury.     

Functional characterization of the rat chemokine KC and its importance in neutrophil recruitment in a rat model of pulmonary inflammation

Expression of mRNA for the neutrophil (PMN) chemokine, KC, in rat models of lung injury suggests a role for this chemokine in pulmonary inflammation. We addressed this hypothesis at the protein level by functionally characterizing recombinant rat KC (rKC) in vitro and in vivo. In vitro, rKC induced PMN chemotaxis and increased the expression of CD11b/CD18 on PMNs. Recombinant KC also induced a respiratory burst (quantitated by flow cytometry) in rat PMNs, similar to that caused by its human structural homologue, gro/melanoma growth-stimulating activity, on human PMNs, but less than that caused by IL-8 on human PMNs. Intratracheal instillation of rKC induced dose-dependent PMN influx into airspaces (average PMNs in bronchoalveolar lavage: vehicle = 1.5%, n = 4; rKC (1 microgram) = 11.5%, n = 2; rKC (10 micrograms) = 77.3%, n = 2). A neutralizing anti-KC Ab reduced the chemotactic activity of rat bronchoalveolar lavage fluid collected after the intratracheal administration of LPS (48.3 +/- 8% of control, n = 4). Anti-KC neutralizing Ab markedly inhibited PMN accumulation (71 +/- 6%) within the lungs in response to an intratracheal challenge of LPS. We conclude that rat KC is a major but not exclusive mediator of PMN activation and recruitment during LPS-induced pulmonary inflammation.     

The role of G-CSF in mature neutrophil function is not related to GM-CSF-type cell priming

Because of uncertainties regarding the comparability of granulocyte-macrophage and granulocyte colony-stimulating factors with regard to their effects on mature neutrophils (PMNs), we compared the actions of the two cytokines on reactive oxidant production and granular secretion by these cells. We found that chemiluminescence (CL) stimulated by formylmethionyl-leucyl-phenylalanine (fMLP) was not influenced by G-CSF (0.1-100 ng/ml), whereas GM-CSF priming (10 ng/ml) caused a nearly twofold increase in this PMN response. Moreover, the reactivity of PMNs treated with GM-CSF and G-CSF in combination was not different from that of PMNs treated with GM-CSF alone. GM-CSF (10 ng/ml) increased the rate of O2- production by 79%, caused a fivefold increase in fMLP-induced myeloperoxidase (MPO) secretion, and strongly enhanced CD11b expression. In contrast, G-CSF (50 ng/ml) only slightly increased O2- production (by 15%), and MPO secretion and CD11b expression remained unchanged. Both cytokines together gave results similar to those obtained with GM-CSF alone. In the presence of platelets (which by themselves enhanced PMN reactivity), the differences in the effects of the two cytokines persisted. We conclude that the priming effect of G-CSF on mature PMNs is negligible compared with that of GM-CSF. Our results are in conflict with previous reports of much more pronounced G-CSF effects but in accord with recent work showing the failure of this cytokine to induce a range of effects produced by GM-CSF. We therefore suggest that the primary role of G-CSF in mature PMN function is still unclear but may be related to the control of PMN distribution in view of the mobilizing and marginating effects of the cytokine in vivo.     

Role of P-selectin and leukocyte activation in polymorphonuclear cell adhesion to surface adherent activated platelets under physiologic shear conditions (an injury vessel wall model)

Carbohydrate moieties on leukocytes adhere to activated platelets via P-selectin under static binding condition studies. We characterize polymorphonuclear cell (PMN) surface interactions with surface adherent platelets and the PMNs response, under physiologic flow conditions corresponding to a shear of 100 s-1, in an in vitro flow chamber. Fluorescent labeled PMNs with red blood cells were drawn through a transparent flow channel and visually quantitated over 30 minutes, interacting with a confluent monolayer of activated, shear-spread platelets expressing P-selectin. PMN adhesion was saturable (2,250 +/- 350/mm2), and time and cation (Ca2+, Mg2+) dependent, and PMNs did not bind to the experimental surface in the absence of a platelet monolayer. P-selectin antibodies completely abolished PMN adhesion in a concentration-dependent manner with half inhibition at 70 micrograms/mL. Antibodies to a putative P-selectin receptor CD15 (80H5 and MMA) maximally inhibited PMN adhesion by 73% and 10%, respectively. Adherent PMNs appeared morphologically activated and flow cytometric analysis of adherent PMNs confirmed activation because CD11b and CD18 surface expression was upregulated (100% and 27%, respectively), whereas L-selectin was downregulated (55%) compared with control nonadherent PMNs. In the presence of the metabolic inhibitor sodium azide (0.02% and 0.1%) there was a 23% +/- 9% and 51% +/- 3% decrease, respectively, in PMN adhesion at 100 s-1. Thus, P-selectin is required for PMN adhesion to a pathophysiologic surface of activated adherent platelets at physiologic shear rates. Furthermore, a secondary step involving PMN activation after platelet binding appears necessary for complete (irreversible) adhesion to occur. This unique flow cell provides a model to explore, under controlled conditions, biologic mechanisms and ligands involved in leukocyte-platelet binding that play important roles in PMN localization at sites of thrombosis and vascular injury.     

Role of interleukin 8 in the genesis of acute respiratory distress syndrome through an effect on neutrophil apoptosis

OBJECTIVE: To evaluate the role of interleukin 8 (IL-8) in the regulation of neutrophil (PMN) apoptosis in normal plasma and plasma from patients with early, fulminant acute respiratory distress syndrome (ARDS). DESIGN: Experimental study using cultured human PMNs. SETTING: University hospital, level I trauma center. PARTICIPANTS: Plasma was obtained from 6 patients with early, fulminant posttraumatic ARDS (mean Injury Severity Score, 26). All samples were drawn within 24 hours after injury. Plasma was also taken from 13 healthy control subjects. These controls were also used as sources of PMNs. MAIN OUTCOME MEASURES: Effect of early, fulminant ARDS and normal plasma on spontaneous apoptosis, CD16, and CD11-b expression in PMNs in vitro; levels of IL-8 in plasma; correlation of extracellular IL-8 concentration with rate of PMN apoptosis; and effect of IL-8 blockade on PMN apoptosis, CD16, and CD11-b expression in ARDS and normal plasma. RESULTS: Plasma from patients with early, fulminant ARDS inhibited spontaneous PMN apoptosis at 24 hours (35%+/-5% vs 54%+/-5%; P=.01). Neither CD16 nor CD1l-b differed significantly between the 2 groups. The mean plasma level of IL-8 in patients with early, fulminant ARDS was 359+/-161 pg/mL vs 3.0+/-0.4 pg/mL in healthy controls (P<.05). Interleukin 8 inhibited apoptosis in plasma-free medium at low doses (1-50 pg/mL) but had no significant effect at higher doses (100-5000 pg/mL) (P<.05). Interleukin 8 blockade with monoclonal antibody suppressed apoptosis in normal plasma (28%+/-5% with monoclonal antibody vs 51%+/-5% without monoclonal antibody; P=.008) but not in plasma from patients with early, fulminant ARDS (29%+/-5% with monoclonal antibody vs 34%+/-6% without monoclonal antibody; P=.67). It had no effect on CD16 or CD11-b expression in either plasma. CONCLUSIONS: Plasma from patients with early, fulminant ARDS contains soluble factors that inhibit PMN apoptosis in vitro. Low levels of IL-8 inhibit PMN apoptosis in normal plasma. Although plasma levels of IL-8 are markedly elevated in early, fulminant ARDS, IL-8 is not directly responsible for the antiapoptotic effect of plasma from patients with early, fulminant ARDS.     

Neutrophil transfusions: kinetics and functions of neutrophils mobilized with granulocyte-colony-stimulating factor and dexamethasone

BACKGROUND: The collection of adequate numbers of neutrophils (polymorphonuclear leukocytes, PMNs) from normal donors has long hampered the development of neutrophil transfusion therapy. The stimulation of donors with granulocyte-colony-stimulating factor (G-CSF) plus dexamethasone is a promising way of improving PMN collections. STUDY DESIGN AND METHODS: Sixteen normal subjects received G-CSF (600 micrograms subcutaneously) and dexamethasone (8 mg by mouth) 12 hours before leukapheresis. Measurements included PMN morphology, immunophenotype analysis, chemiluminescence, bactericidal activity, in vivo kinetics, and adverse effects. RESULTS: A mean of 77.4 +/- 6.4 x 10(9) PMNs was collected with each leukapheresis; 14 percent were bands. PMNs had increased surface expression of CD11b, CD18, CD14, CD32, and CD64. Bactericidal capacity against Staphylococcus aureus was normal. Inducible respiratory burst was maintained, although the responses to some agonists were diminished. Returned leukapheresis cells labeled with 3H-diisopropylfluorophosphate had a modestly decreased percentage of recovery and circulated with a prolonged half-life. Migration of these cells to skin chambers was approximately equal to that of the subjects' own blood PMNs. Adverse effects included transient bone pain, headache, hunger, and insomnia. CONCLUSIONS: Precollection treatment of leukapheresis donors with G-CSF plus dexamethasone is an effective way to enhance the collection of PMNs with normal or near-normal functional properties for PMN transfusion therapy.     

Human polymorphonuclear leukocytes adhere to complement factor H through an interaction that involves alphaMbeta2 (CD11b/CD18)

The work presented here demonstrates that human complement factor H is an adhesion ligand for human neutrophils but not for eosinophils. The adherence of polymorphonuclear leukocytes (PMNs) to plastic wells coated with factor H depended on divalent metal ions and was augmented by C5a and TNF-alpha. PMN adhesion to factor H in the presence or absence of C5a was blocked specifically by mAbs against CD11b or CD18. Affinity purification using factor H Sepharose followed by immunoprecipitation using mAbs to various integrin chains identified Mac-1 (CD11b/CD18) as a factor H binding receptor. The presence of surface bound factor H enhanced neutrophil activation resulting in a two- to fivefold increase in the generation of hydrogen peroxide by PMNs stimulated by C5a or TNF-alpha. When factor H was mixed with PMNs, 1.4 to 3.8-fold more cells adhered to immobilized heparin or chondroitin A. In addition, augmented adhesion of PMNs was measured when factor H, but not HSA or C9, was absorbed to wells that were first coated with heparin or chondroitin A. The adhesion of PMNs to glycosaminoglycan-factor H was blocked by mAbs to CD11b and CD18. These studies demonstrate that factor H is an adhesion molecule for human neutrophils and suggest that the interaction of factor H with glycosaminoglycans may facilitate the tethering of this protein in tissues allowing factor H to serve as a neutrophil adhesion ligand in vivo.     

Prothymosin alpha 1 effects in vitro on chemotaxis, cytotoxicity and oxidative response of neutrophils from melanoma, colorectal and breast tumor patients

Immunoregulatory effects of thymic peptides on functions of polymorphonuclear leukocytes (PMNs) are poorly investigated. We studied the effects of prothymosin alpha 1 (Pro alpha 1) on PMNs from patients with colorectal tumors, breast tumors and melanoma (total n = 37) in comparison with healthy donors (n = 18), with respect to chemotaxis, cytotoxicity against HCT-116 colon tumor cells, oxidative response (chemiluminescence reaction) as well as expression of surface marker molecules. We found that Pro alpha 1 was equally effective in stimulating the chemotactic activity of PMNs from tumor patients and healthy donors (43% increase). PMNs from tumor patients, especially with breast tumor, showed a significant enhancement of cytotoxicity against the tumor target cells in comparison with healthy donors. With respect to the PMNs cytotoxicity, only about 50% of the colorectal tumor patients and healthy donors responded to Pro alpha 1 and FMLP. As to the oxidative response of PMNs, elevated levels were found only among colorectal tumor patients. Pro alpha 1 significantly increased the oxidative response in breast and colorectal tumor patients by 55% and 25%, respectively. Pro alpha 1 decreased the expression of CD16 on PMNs of healthy donors, but not that of CD11a, CD11b, CD11c, CD13, CD14, CD15 and CD32. Therefore, we suggest, that Pro alpha 1 may improve some PMN functions of tumor patients, associated with the proposed role in host-tumor interaction.     

Comparative clinical study of cefonicid, chloramphenicol, and penicillin in community-acquired pneumonia

Telomere length in human somatic cells gradually decreases with the number of cell divisions and is regarded as a marker of somatic cell turnover. Mucosal cells of the affected colon show rapid turnover in individuals with active ulcerative colitis (UC). Telomere length was determined by Southern blot analysis of terminal restriction fragments (TRFs) from the colonic mucosa of 17 patients with UC in remission, two of whom showed dysplasia, and 17 control subjects without colitis. For each individual, mean TRF length was compared between rectal mucosa and unaffected cecal mucosa. The mean TRF length of the rectal mucosa was significantly less than that of cecal mucosa in UC patients (7.87 +/- 0.36kb versus 8.77 +/- 0.21 kb; P = 0.0015, Wilcoxon signed rank test), whereas no significant difference was detected in the control subjects. The extent of telomere shortening was 10.6 +/- 3.35% in UC patients, compared with 0.8 +/- 0.64% in noncolitis controls (P = 0.0024, Mann-Whitney U-test). Four UC patients, two of whom had dysplasia, showed telomere shortening of more than 20% in the rectal mucosa. These observations suggest that telomere shortening in the colonic mucosa of individuals with UC may represent the history of mucosal inflammation during disease of long duration, and that it may contribute to aneuploidy in UC.     

Substantiation of MPEL of acoustic exposure in accordance with its informational value]

Our previous studies have proved that a great number of polymorphonuclear neutrophils (PMN) adhered to endothelial lining and induced endothelial cell (EC) injury. This study was attempted to investigate the change in adhesive force between PMN and EC in response to burn patients' serum within 24 hours and effects of antibody against CD11/CD18 on PMN-EC adhesive force. Burn serum was isolated from 4 burn patients (III degrees 20%-50% TBSA) within 48 hours after burn injury. PMNs were isolated from 8 volunteers. Adhesive force between a PMN and a HUVEC was calculated by means of micropipette technique at 1 h, 3 h, 6 h, 12 h, 24 h after PMN or HUVEC incubated with burn serum or normal serum. Five individual PMN-HUVEC pairs were measured for each sample. All data were analyzed statistically using the t test. RESULTS: 1. The adhesive force between a single PMN-HUVEC pair increased sharply after PMN incubated with burn serum, which reached the maximun level at 1 h after incubation and remained at this level in the following 23 hours. However, the adhesive force was reduced by 78.5%, 72.5%, 75.2% at 1 h, 6 h, 24 h, respectively after the PMNs were pretreated with CD11/CD18 mAb; 2. The adhesive force between a single burn serum stimulated-HUVEC and PMN pair increased gradually, reached peak at 12 h and remained at this level in the following 12 hours. CONCLUSIONS: Burn serum could induce PMN-EC adhesion, with related to PMN aggregation in internal organs and EC damage after burn injury. Antibodies against CD11/CD18 could partially block PMN-EC adhesion and might reduce EC injury induced by activated PMNs.     

Role of lipopolysaccharide in signaling to subepithelial polymorphonuclear leukocytes

Polymorphonuclear leukocyte (PMN) infiltration and migration across colonic intestinal epithelia is a hallmark of inflammation in Shigella flexneri-mediated dysentery. To identify bacterial signals associated with this process, potential stimulatory factors mediating initial PMN association with the epithelium and subsequent transepithelial migration were examined in an in vitro model system. Quantitative analyses revealed that purified S. flexneri lipopolysaccharide (LPS) deposited at the apical surface of polarized intestinal epithelial cells transcytosed to the basolateral pole, a process dependent on the stage of epithelial cell differentiation. Transcytosed LPS in the presence of normal human serum (NHS), a source of LPS binding protein and soluble CD14, mediated both interleukin-8 secretion at the basolateral pole and enhanced PMN adherence. In addition, LPS stimulated a significant degree of directed transepithelial migration of PMNs, an event that was further enhanced in the presence of NHS. These results implicate LPS in signaling subepithelial PMN emigration and enhancing PMN-epithelium interactions prior to and during subsequent Shigella-induced transepithelial migration.     

Functional activity of blood polymorphonuclear leukocytes as an oxidative stress biomarker in human subjects

In the present work, we studied the role of polymorphonuclear leukocytes (PMN) in aged individuals and coronary heart disease (CHD)-bearing patients, two physiopathological processes associated with overproduction of reactive oxygen species (ROS). The effects of antioxidant supplementation on the functional activity of PMN from CHD patients were also determined. The function of PMNs was evaluated by measuring of phagocytosis, killing activity, and ROS production. Luminol amplified chemiluminescence (CL) was used to estimate ROS production by stimulated PMNs. Total cholesterol and the LDL-cholesterol fraction from CHD patients were found to be higher than those recommended, returning to normal levels after antioxidant therapy. PMN CL of CHD patients was found to be higher than the associated control groups. Antioxidant therapy administrated to CHD patients lead to an increase in the killing activity accompanied by a decrease in PMN CL of these subjects. The study also showed that killing activity of PMN from human subjects over 60 years was significantly lower than the activity measured in younger subjects. PMN CL produced after stimulation was found to be positively correlated with the increasing age of human subjects (r=.946, p < .01).     

Beta2 integrins (CD11/CD18) promote apoptosis of human neutrophils

Apoptosis of human polymorphonuclear neutrophils (PMN) is thought to be critical for the control of the inflammatory process, but the mechanisms underlying its regulation in physiological settings are still incompletely understood. This study was undertaken to test the hypothesis that the beta2 integrin (CD11/CD18) family of leukocyte adhesion molecules contributes to the control of activated PMN by up-regulating apoptosis. Apoptosis of isolated human PMN was investigated by 1) analysis of DNA content, 2) detection of DNA degradation, 3) morphological studies, and 4) measurement of CD16 expression on the cell surface. We found that beta2 integrins potentiated the tumor necrosis factor alpha (TNF-alpha) -induced apoptosis within 4 and 8 h after stimulation. The effect required aggregation of the beta2 integrin Mac-1 (CD11b/CD18), which was induced by antibody cross-linking, and was independent of Fc receptors. An enhancement of apoptosis was also observed after migration of PMN through an endothelial cell monolayer. TNF-alpha-induced apoptosis as well as potentiation by beta2 integrins was prevented by inhibition of tyrosine kinases with herbimycin A or genistein. The present study provides a new model for the regulation of PMN apoptosis by a functional cross-talk between beta2 integrins and TNF-alpha with a promoting role for the beta2 integrins. This mechanism, which allows enhanced elimination of previously emigrated PMN, may be critical to abate local inflammatory processes in vivo.     

Azurophilic granules of human neutrophils contain CD14

CD14, the leukocyte receptor for lipopolysaccharide (LPS), is important in the response of human polymorphonuclear leukocytes (PMNs) to infection with gram-negative bacteria. The level of CD14 on the PMN surface increases after exposure to some inflammatory stimuli such as N-formyl-methionyl-leucyl-phenylalanine (fMLP). These newly expressed CD14 molecules probably come from an intracellular pool of preformed receptors. We sought to further characterize PMN CD14 expression, upregulation, and shedding and to define the intracellular location of CD14 molecules. Our results demonstrate that both LPS and fMLP significantly increased CD14 cell surface expression; however, neither phorbol myristate acetate (PMA) or A23187 increased receptor levels on the PMN surface. Neither fMLP, PMA, or A23187 stimulated the release of soluble CD14 from PMNs. Intracellular CD14 was observed in >90% of PMNs examined by flow cytometry and confocal microscopy. Additional analyses using CD14 enzyme-linked immunosorbent assays and electron microscopy studies, examining PMN granules separated by discontinuous sucrose or Percoll gradients, showed that CD14 was present in both the plasma membrane-secretory vesicle fractions and azurophilic granules.     

The sugar permeability test reflects disease activity in children and adolescents with inflammatory bowel disease

OBJECTIVES: To investigate the relationship of intestinal permeability in children and adolescents with inflammatory bowel disease (IBD) to disease activity, disease extent, and response to therapy. Study design: Patients with new and established diagnoses of IBD (12 Crohn's disease [CD] and 18 ulcerative colitis [UC]) were studied. Intestinal permeability was evaluated by measuring with high-performance liquid chromatography 5-hour urinary excretion ratio of lactulose/L-rhamnose (L/Rh). RESULTS: In 8 of 9 patients with active CD, the L/Rh ratio was higher than the reference range (0.006 to 0.074, n = 36). In inactive CD (n = 3) the L/Rh ratio was within the reference range. In 6 of 7 patients with active extensive UC, the L/Rh ratio was elevated. In inactive extensive UC (n = 6) the normal permeability ratio was shown. In both active CD and active extensive UC, the frequency of elevated intestinal permeability was significantly greater than values in both inactive forms. The permeability ratio was normal in 4 of 5 patients with active left-sided colitis. In 5 of 7 patients (3 CD, 4 UC), repeat permeability values entered the reference range after acute phase therapy. Two patients with persistently elevated intestinal permeability (1 CD, 1 UC) had a disease flare-up within 6 months. CONCLUSIONS: Intestinal permeability is a marker of disease activity in CD and extensive UC. Serial permeability test may be useful in monitoring disease activity.     

The role of neutrophils in peritoneal adhesion formation

The most common cause of intraperitoneal adhesions is previous abdominal surgery. Postoperative adhesion formation results from a fibroproliferative inflammatory reaction that begins with an influx of polymorphonuclear leukocytes (PMNs) into the peritoneal cavity. Adherence of the PMNs to the endothelial cells (EC) is necessary for PMN migration into the tissue in response to a stimulus. Several receptor-counterreceptor pairs of ligands such as CD11/CD18 on the PMN and ICAM-1 (CD54) on EC have been identified. Monoclonal antibody against CD11/CD18 (R15.7) inhibits PMN adherence and migration and consequently protects against PMN-induced tissue injuries. We therefore studied the effect of preventing PMN-EC adherence, using anti-CD18 monoclonal antibody, on postoperative adhesion formation in rabbits. Group 1 was a control receiving physiologic saline, and group 2 received anti-CD18 antibody (R15.7, 2 mg/kg). The treatment was administered iv at the end of surgery and repeated on the first and second postoperative days. Peritoneal adhesions were induced at laparotomy by repairing two peritoneal defects, by oversewing the defect (model 1), and by resuturing the removed parietal peritoneum in its place as an ischemic graft (model 2). Adhesions were evaluated blindly at 10 days after operation by measuring the percentage of the suture line covered with adhesions (model 1) or by a scoring system (model 2). All control animals developed intraperitoneal adhesions and the percentage of the suture line covered with adhesions was 25 +/- 5.9% (mean +/- SEM) and the mean score in model 2 was 0.9 +/- 0.2. Anti-CD18 antibody, R15.7, increased the degree of postoperative adhesion formation in both models, but the results were significant only in model 2. Also, anti-CD18 antibody significantly decreased peritoneal neutrophils from 11.1 x 10(7) +/- 1.8 x 10(7) to 2.2 x 10(7) +/- 0.4 x 10(7) (P < 0.001) on the first postoperative day. It is concluded that inhibition of PMN-EC adherence does influence the postoperative adhesion formation. These results might suggest that PMNs have a role in modulating postoperative adhesion formation.     

Sensitive diagnosis of cardiac allograft rejection by detection of cytokine transcription in situ

BACKGROUND & AIMS: Interleukin 12 (IL-12) is a heterodimeric, macrophage-derived cytokine that is elevated in Crohn's disease (CD). Epstein-Barr virus-induced gene 3 (EBI3) is a recently characterized human glycoprotein that is homologous to the 40-kilodalton chain of IL-12 and forms a heterodimer with the 35-kilodalton chain of IL-12. We investigated the expression of EBI3 in colonic mucosa of normal control subjects, patients with ulcerative colitis (UC), and patients with CD. METHODS: Colonic tissue was analyzed for messenger RNA (mRNA) expression by quantitative polymerase chain reaction and for protein expression by immunohistology and Western blotting. RESULTS: EBI3 mRNA was present in intestinal biopsy specimens from healthy subjects and patients with CD but was elevated only in active UC. EBI3 levels in UC specimens correlated with histological scores of activity and T-cell infiltration. EBI3-positive cells that had a shape consistent with that of macrophages were identified in the lamina propria, and protein was detected by Western blotting. CONCLUSIONS: EBI3 is a novel IL-12-related cytokine that is expressed by macrophage-like cells in normal intestine and CD and has enhanced expression in active UC but not in active CD.     

CD11b/CD18-dependent stimulation of leukotriene B4 synthesis by human neutrophils (PMN) is synergistically enhanced by tumour necrosis factor alpha and low dose diacylglycerol

Unopsonised zymosan particles bind to the CD11b/CD18 integrin on human neutrophils (PMN) and are phagocytosed. Binding stimulates the release of leukotriene (LT) B4. The present study examined the effect on this interaction of two agents that 'prime' PMN for augmented responses to a variety of agonists. The cell permeable diacyl glycerol, 1,2-dioctanoyl-glycerol (DiC8) and TNF alpha each increased CD11b/CD18 expression on PMN [maximal at 10-9 M TNF alpha or 10-8 M DiC8]. There was a decrease, however, in CD11b/CD18 expression above 10-8 M DiC8, which was not observed at high concentrations of TNF alpha. Pre-treatment with either DiC8 or TNF alpha dose-dependently augmented the zymosan-stimulated release of LTB4 from PMN. DiC8 and TNF alpha in combination, however, synergistically increased LTB4 release. In contrast, at concentrations above 10-8 M DiC8, whether in the presence or absence of TNF alpha, LTB4 release was inhibited and this was ameliorated by protein kinase C inhibitors. The response to neither TNF alpha nor DiC8 (below 10-8 M) was kinase inhibitor sensitive. Doses of DAG, which activate protein kinase C, inhibit CD11b/CD18-dependent responses by down-regulating receptor expression. In contrast, the mechanisms of TNF alpha and low dose DAG 'priming' are not clear but are independent of PKC activation. The synergy between these two priming agents, however, suggests independent, complementary signalling pathways that provide a novel, potentially important mechanism for the control of PMN CD11b/CD18 integrin-dependent activation.     

Fish oil supplementation with and without added vitamin E differentially modulates plasma antioxidant concentrations in healthy women

5-[4-(2-Carboxyethylcarbamoyl)phenylazo]salicylic acid disodium salt dihydrate (CAS 80573-04-2, BX661A) is developed as a therapeutic agent for ulcerative colitis. To clarify the mechanisms of action of BX661A, the effects of BX661A and its metabolites 5-aminosalicylic acid (5-ASA) and 4-aminobenzoyl-beta-aline (4-ABA) on polymorphonuclear (PMN) leukocyte chemotaxis and production of reactive oxygen species (ROS) from PMN cells were investigated and compared with the effects of 2-hydroxy-5-[[4-[(2-pyridinylamino)sulfonyl]phenyl]azo]-benzoic acid (CAS 599-79-1, SASP) and its metabolite 4-amino-N-2-pyridinyl-benzenesulfonamide (CAS 144-83-2, SP). 1. BX661A, SASP and SP concentration-dependently inhibited guinea pig PMN cell chemotaxis induced by zymosan-activated serum (IC50 = 1.39, 2.17 mmol/l, respectively) and by N-formyl-methionyl-leucyl-phenylalanine (FMLP) with IC50 values of 0.55, 0.06 and 0.66 mmol/l, respectively. 5-ASA and 4-ABA weakly affected the PMN cell chemotaxis induced by zymosan-activated serum (both IC50 values > or = 10 mmol/l) and by FMLP (IC50 > or = 10 and 8.05 mmol/l, respectively). 2. BX661A, SASP and SP concentration-dependently inhibited human PMN cell chemotaxis induced by FMLP with IC50 values of 0.68, 0.05 and 2.68 mmol/l, respectively, but both IC50 values of 5-ASA and 4-ABA were > 10 mmol/l. 3. BX661A, SASP, 5-ASA, 4-ABA and SP inhibited ROS production from rat PMN cells stimulated by FMLP in a concentration-dependent manner (IC50 = 58.4, 27.5, 0.61, 1242 and 13.9 mmol/l, respectively). 4. BX661A, SASP, 5-ASA, 4-ABA and SP inhibited ROS production from human PMN cells stimulated by FMLP in a concentration-dependent manner (IC50 = 67.4, 46.1, 0.69, 748 and 8.31 mumol/l, respectively). These results suggest that BX661A itself has inhibitory effects against PMN cell chemotaxis and ROS production from PMNs and that 5-ASA, which is the active moiety of BX661A, has a potent inhibitory effect against ROS production from PMNs. Therefore, these effects may be partially involved in the therapeutic effects of BX661A on ulcerative colitis.     

Network of inflammatory cytokines and correlation with disease activity in ulcerative colitis

OBJECTIVE: The inflammatory component of most human inflammatory chronic diseases implicates the production of proinflammatory cytokines. Tumor necrosis factor alpha (TNFalpha) and interleukin 1beta (IL1beta) seem to play an important role in ulcerative colitis (UC) in relevant experimental models. Moreover, antiTNF therapy seems promising experimentally and clinically. However, these cytokines, and TNFalpha more particularly, are hardly seen in vivo in such patients. The mediators of choice, correlated with disease activities or drug efficacy, remain unclear. To characterize in vivo the network of colonic cytokines in patients with UC, and the contribution of the various cytokines to disease activity we performed this study, using the colonic perfusion method. METHODS: A 20-cm colon length was perfused. Perfusate samples were collected for cytokine determination by enzyme-linked immnoassays. Nineteen perfusions were performed in mild to moderate UC, including two successive perfusions in four patients. Six healthy control patients and four having Crohn's disease (CD) with rectal involvement were studied. Endoscopic score, leukocyte scintigraphy, and systemic markers of inflammation were simultaneously quantified. RESULTS: Large amounts of IL1beta, TNFalpha, IL6, and IL8 were produced in UC patients with a highly significant correlation between TNFalpha, IL1beta and IL8 two by two. Multivariate factorial analysis indicated that IL1beta showed the best correlation with disease activity. Locally produced IL6 was strongly associated with circulating platelet counts. Moreover, production of inflammatory cytokines was associated with similar variations of disease activity in the four patients with two successive perfusions performed. The level of inflammatory cytokines in CD was lower than in UC; TNFalpha, IL1beta, and IL6 were not found in any control patients. CONCLUSION: UC appears to be a chronic inflammatory disease characterized by high production of all four proinflammatory cytokines (IL1beta, TNFalpha, IL6, and IL8). These results suggest that colonic perfusion may be a suitable method to evaluate the local anticytokine properties of new drugs, in correlation with disease activity and systemic markers of inflammation.