Injection of chemoattractants into normal cornea: a model of inflammation after alkali injury

PURPOSE: The objective of this study was to establish and characterize the invasion of polymorphonuclear leukocytes (PMNs) into a normal cornea after intrastromal injection of the tripeptide chemoattractants generated from alkali-degraded corneas. METHODS: The following samples were injected into the midstroma of normal rabbit corneas: ultrafiltered tripeptide chemoattractants (N-acetyl-proline-glycine-proline and N-methyl-proline-glycine-proline) generated from alkali-degraded corneas, synthetic N-acetyl-PGP, positive control (leukotriene B4 [LTB4]), or negative control (Hanks' balanced salt solution [HBSS]). Timed responses of PMN infiltration were established for effective concentrations of LTB4 or the ultrafiltered chemoattractants. RESULTS: All intrastromal injections resulted in the immediate development of an edematous disc that was 10 mm in diameter. The lesion essentially had cleared in the HBSS-injected eyes by 8 hours, and histologic sections revealed minimal numbers of PMNs in the cornea or limbal tissue. The injection of LTB4 or the ultrafiltered tripeptide chemoattractants induced peak numbers of PMNs within the stroma at 8 hours, subsiding by 16 hours. Seventy units of ultrafiltered chemoattractants yielded a strong PMN response, similar to 1 X 10(-5) M LTB4. The highest concentration of ultrafiltered chemoattractants (350 U) produced a severe PMN response that was characterized by a solid sheet of neutrophils surrounding the injection site. The injection of synthetic N-acetyl-PGP (2 X 10(-4) M) produced a marked PMN response. CONCLUSIONS: PMN invasion of the normal cornea after the injection of the ultrafiltered tripeptide chemoattractants or the synthetic N-acetyl-PGP mimicked early PMN infiltration in the alkali-injured eye, confirming the importance of this chemoattractant as an inflammatory mediator.     

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.     

Stress protein inductions after brain ischemia

To better understand the mechanisms by which neutrophils migrate into the airways, we constructed a novel in vitro model system with human umbilical vein endothelial cell (HUVE) monolayers grown on top of permeable filters and human lung Type II-like alveolar epithelial cell (A549) monolayers grown on the undersurface of the filters. The sequential migration of human neutrophils through the endothelium (apical to basal movement) and subsequently through the epithelium (basal to apical movement) in response to a stimulus located basally to the epithelium was measured. We found that the neutrophil chemoattractants, formylmethionylleucylphenylalanine (FMLP), leukotriene B4 (LTB4), and interleukin-8 (IL-8), induced dose-responsive migration through the double monolayer-filter complex. The pattern of migration was similar to that observed through either a naked filter or single monolayer-filter complex. Maximal chemotaxis through the double monolayer-filter complex was observed by 3 hours. Thus, we have established an in vitro model system to examine the sequential migration of neutrophils through endothelium and the respiratory epithelium in a manner analogous to that occurring with an in vivo airway stimulus causing neutrophil-rich airway inflammatory responses.     

Neutrophil-mediated changes in vascular permeability are inhibited by topical application of aspirin-triggered 15-epi-lipoxin A4 and novel lipoxin B4 stable analogues

Neutrophil (PMN) activation is critical in inflammation and reperfusion injury, suggesting that PMN-directed therapies may be of clinical use. Here, leukotriene B4 (LTB4)-induced PMN influx in ear skin was equivalent between 5-lipoxygenase knockout and wild-type mice. To explore actions of lipoxin (LX) in PMN-mediated tissue injury, we prepared several novel LX stable analogues, including analogues of LXA4 and aspirin-triggered 15-epi-LXA4 as well as LXB4, and examined their impact in PMN infiltration and vascular permeability. Each applied topically to mouse ears inhibited dramatically PMN-mediated increases in vascular permeability (IC50 range of 13-26 nmol) with a rank order of 15(R/S)-methyl-LXA4 > 16-para-fluoro-phenoxy-LXA4 approximately 5(S)-methyl-LXB4 >/= 16-phenoxy-LXA4 > 5(R)-methyl-LXB4. These LX mimetics were as potent as an LTB4 receptor antagonist, yet results from microphysiometry with mouse leukocytes indicated that they do not act as LTB4 receptor level antagonists. In addition, within 24 h of delivery, > 90% were cleared from ear biopsies. Neither IL-8, FMLP, C5a, LTD4, nor platelet-activating factor act topically to promote PMN influx. When applied with LTB4, PGE2 enhanced sharply both infiltration and vascular permeability, which were inhibited by a fluorinated stable analogue of aspirin-triggered LX. These results indicate that mimetics of LXs and aspirin-triggered 15-epi-LXA4 are topically active in this model and are potent inhibitors of both PMN infiltration and PMN-mediated vascular injury.     

Contrasting responses to multiple chemotactic stimuli in transendothelial migration: heterologous desensitization in neutrophils and augmentation of migration in eosinophils

At inflammatory sites in vivo, leukocytes may confront multiple, competing chemoattractive signals. We found significant differences between eosinophils and neutrophils in transendothelial chemotaxis to a chemoattractant diffusing from the lower chamber, when a chemoattractant that binds to another receptor is present at uniform concentration. The transendothelial migration of eosinophils to FMLP, C5a, RANTES, or MCP-3 was totally inhibited by the presence of the homologous chemoattractant, and only RANTES and MCP-3 showed mutual inhibition. C5a and to a lesser extent FMLP chemokinetically stimulated migration to RANTES and MCP-3, without stimulating random migration. Results with neutrophils contrasted. The presence of FMLP not only abrogated neutrophil transmigration to FMLP but also strongly decreased chemotaxis to C5a, IL-8, and Gro-alpha. Similarly, C5a inhibited neutrophil chemotaxis to IL-8 and Gro-alpha. IL-8 almost totally abrogated chemotaxis to Gro-alpha, but Gro-alpha only moderately inhibited chemotaxis to IL-8. Neither IL-8 nor Gro-alpha significantly inhibited transmigration to FMLP or C5a. Actin polymerization in eosinophils and neutrophils was desensitized by the same combinations of chemoattractants that desensitized chemotaxis. We conclude that eosinophils have at least three noninterfering receptor-signal transduction pathways for chemotaxis and actin polymerization. In contrast, the signaling pathways for FMLP, C5a, and IL-8/Gro-alpha in neutrophils are heterologously cross-desensitized, with a hierarchy of resistance to competing signals of FMLP > C5a > IL-8 > Gro-alpha, in agreement with previous results in neutrophils on the Ca2+-mobilizing response. These results may have important implications for the behavior of these cell types in inflammatory sites.     

Increased endothelin-1 in the rabbit model of middle cerebral artery occlusion

Human neutrophils (5 x 10(4) incubated on fibronectin precoated wells released 2.83 +/- .25 nmoles of superoxide (0(2)-) (x +/- 1 SEM, n = 15) in response to 5.9 nM (100 ng/ml) Tumor Necrosis Factor Alpha (TNF). On the contrary, the 0(2)- production induced by interleukin-8 (IL-8) (doses ranging from 0.1 nM to 1 microM) was comparable to that of "resting" cells (< .6 nmoles/5 x 10(4) cells). IL-8 (100 nM) did not affect the TNF-dependent 0(2)- production when added with TNF at the beginning of the assay, but reduced it by approximately 80% when added with TNF on neutrophils previously incubated for 1 hour on fibronectin. As compared with IL-8, N-formyl-methionyl-leucyl-phenylalanine (FMLP, 100 nM) failed to suppress the TNF-triggering of the oxidative burst in neutrophils plated on fibronectin. The data suggest that the interaction of neutrophils with fibronectin uncovers the capacity of IL-8 to limit the cell response to TNF, without affecting the response to the combination of FMLP and TNF. Thus, although the chemotactic factors IL-8 and FMLP share the capacity of triggering the oxidative burst of neutrophils incubated in suspension, only IL-8 has the potential to down-regulate the responsiveness of fibronectin-adherent cells to TNF.     

Adhesion-dependent protein tyrosine phosphorylation in neutrophils treated with tumor necrosis factor

Human neutrophils (PMN) respond to tumor necrosis factor (TNF) by releasing their granules, reorganizing their cytoskeleton, and massively secreting hydrogen peroxide. This response is dependent on adhesion to extracellular matrix proteins and expression of CD11b/CD18 integrins (Nathan, C., S. Srimal, C. Farber, E. Sanchez, L. Kabbash, A. Asch, J. Gailit, and S. D. Wright. 1989. J. Cell Biol. 109:1341-1349). We investigated the role of tyrosine phosphorylation in the response of PMN to TNF. PMN adherent to protein-coated surfaces but not suspended PMN showed tyrosine phosphorylation of several proteins (approximately 150, approximately 115, approximately 75, and approximately 65 kD) in response to TNF. Tyrosine phosphorylation was evident 5 min after addition of TNF and lasted at least 2 h. The tyrosine kinase inhibitors K252a, genistein and ST638 suppressed tyrosine phosphorylation and blocked hydrogen peroxide production in a reversible manner at low concentrations. Tyrosine kinase inhibitors also blocked the spreading of PMN in response to TNF. Dihydrocytochalasin B did not inhibit tyrosine phosphorylation, but in its presence phosphorylation was rapidly reversed. By immunocytochemistry, the majority of tyrosine phosphoproteins were localized to focal adhesions. Thus TNF-induced tyrosine phosphorylation depends on adhesion of PMN to extracellular matrix proteins, and participates in the transduction of the signals that direct the cells to spread on a biological surface and undergo a respiratory burst.     

Regulation of neutrophil-derived IL-8: the role of prostaglandin E2, dexamethasone, and IL-4

Historically, the neutrophil has been perceived as a terminally differentiated leukocyte with limited ability to produce de novo proteins. Furthermore, in the context of acute inflammation the activated neutrophil has been appreciated only for its ability to release various proteases, reactive oxygen, and arachidonic acid metabolites. Recently, the neutrophil has been shown to have the capacity to produce a number of cytokines that may be instrumental in orchestrating the progression of acute inflammation to a more chronic and specific immune response. These cytokines include IFN-alpha, M-CSF, G-CSF, TNF, IL-1, and IL-6. Our laboratory and others have shown that neutrophils produce IL-8 in response to LPS or a phagocytic challenge. Although these studies have shown the induction of IL-8 from polymorphonuclear neutrophils (PMN), relatively little is known regarding the regulation of PMN-derived IL-8. Because PMN and monocytes share the same stem cell, and monocyte-derived IL-8 is regulated by prostaglandin E2 (PGE2), glucocorticoids (dexamethasone; DEX) and the T-Lymphocyte-derived IL-4, we postulated that PMN-derived IL-8 production may be regulated in a similar manner. To test this hypothesis, PMN were isolated (> 99% pure) from peripheral blood and cultured in media with 5% FCS in the presence or absence of LPS (10 ng/ml; a concentration of LPS that induced the half-maximal production of PMN-derived IL-8) and in the presence or absence of DEX (10(-6) M to 10(-10) M), PGE2 (10(-6) M to 10(-10) M), or IL-4 (100 ng/ml to 100 pg/ml). PMN-derived IL-8 was measured using a specific sandwich ELISA. DEX and IL-4 in the presence of LPS were found to inhibit PMN-derived IL-8 in both a dose- and time-dependent fashion. DEX and IL-4 in concentrations of 10(-6) M and 10 ng/ml resulted in maximal inhibition of LPS-induced PMN-derived IL-8, respectively. Moreover, both DEX and IL-4 administration could be delayed 4 hr post-stimulation with LPS and result in significant suppression of PMN-derived IL-8. Interestingly, in contrast to the regulation of monocyte-derived IL-8 by PGE2, PGE2 treatment of PMN failed to inhibit the generation of LPS-induced IL-8. Northern blot analysis of steady-state IL-8 mRNA demonstrated that both DEX and IL-4 treatment of PMN resulted in a 40 and 52% reduction in LPS-stimulated PMN-derived IL-8 mRNA, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Survival and water-balance characteristics of unfed adult Amblyomma cajennense (Acari: Ixodidae)

The specific type of phospholipase A2 (PLA2) involved in formation of leukotriene B4 (LTB4) and platelet activating factor (PAF) in inflammatory cells has been controversial. In a recent report we characterized activation of the 'cytosolic' form of PLA2 (cPLA2) in human neutrophils (PMN) permeabilized with Staphylococcus aureus alpha-toxin under conditions where the secretory form of PLA2 (sPLA2) was inactive. In the current study, generation of both LTB4 and PAF in porated PMN are demonstrated. PMN, prelabeled with [3H]arachidonic acid (3H-AA, to assess AA release and LTB4 production) or with 1-O-[9',10'-3H]hexadecyl-2-lyso-glycero-3-phosphocholine (3H-lyso-PAF, for determination of lyso-PAF and PAF formation), were permeabilized with alpha-toxin in a 'cytoplasmic' buffer supplemented with acetyl CoA. Maximum production of both PAF and LTB4 required addition of 500 nM Ca2+, G-protein activation induced with 10 microM GTP gamma S, and stimulation with the chemotactic peptide, N-formyl-Met-Leu-Phe (FMLP, 1 microM); LTB4 production was confirmed by radioimmunoassay. Removal of acetyl CoA from the system had little effect on LTB4 generation but blocked PAF production with a concomitant increase in lyso-PAF formation LTB4 and PAF production occurred in parallel over time and at differing ATP and Ca2+ concentrations. Further work demonstrated that: (i) maximum production of both inflammatory mediators required a hydrolyzable form of ATP; (ii) blocking phosphorylation with staurosporin inhibited production of both; (iii) the reducing agent, dithiotreitol, had little affect on LTB4 formation but slightly enhanced PAF generation. This study clearly shows that cPLA2 activation can provide precursors for both LTB4 and PAF, that maximum PAF and LTB4 formation occur under conditions that induced optimal cPLA2 activation, that a close coupling between LTB4 and PAF formation exists, and that, after substrate generation, no additional requirements are necessary for LTB4 and PAF generation in the permeabilized PMN system.     

Diltiazem compared with placebo in the prevention of myocardial ischemia during non-cardiac surgery

OBJECTIVE: Because eosinophils likely play important roles in the pathophysiology of allergic diseases, specific inhibitors of eosinophils may be desirable to treat such diseases. To evaluate the capacity of a novel compound, sulochrin, as an inhibitor of eosinophilic inflammation, we examined the effects of this compound on various effector functions of eosinophils. MATERIALS AND METHODS: We examined the effects of sulochrin on degranulation of human eosinophils stimulated with platelet-activating factor (PAF) or Sepharose 4B beads coated with secretory IgA (sIgA) or IgG. The effects of sulochrin on other effector functions of human eosinophils, including superoxide anion (O2-) production, leukotriene (LT) C4 release, and interleukin (IL)-8 production induced by sIgA-beads were also studied. Finally, using PAF and LTB4 as chemoattractants, we evaluated the potency of sulochrin to inhibit eosinophil migration in vitro and in vivo. RESULTS: Sulochrin inhibited EDN release by eosinophils stimulated with sIgA-beads. IgG-beads and PAF in a concentration-dependent manner; IC50 values were 0.75 microM, 0.30 microM and 0.03 microM. Eosinophil O2- production, LTC4 release, and IL-8 production were also inhibited by sulochrin. Furthermore, PAF-induced chemotaxis of human eosinophils and LTB4-induced chemotaxis of guinea pig eosinophils were abolished by 1 microM of sulochrin. Finally, sulochrin potently inhibited LTB4-induced infiltration of eosinophils into the skin of guinea-pig in vivo. CONCLUSIONS: These results suggest that sulochrin is a potent inhibitor of various effector functions of eosinophils. Sulochrin and its derivatives may be useful in the development of therapeutic approaches for patients with allergic diseases.     

Expression and function of the chemokine receptors CXCR1 and CXCR2 in sepsis

Neutrophils (polymorphonuclear neutrophils; PMN) and a redundant system of chemotactic cytokines (chemokines) have been implicated in the pathogenesis of the acute respiratory distress syndrome in patients with sepsis. PMN express two cell surface receptors for the CXC chemokines, CXCR1 and CXCR2. We investigated the expression and function of these receptors in patients with severe sepsis. Compared with normal donors, CXCR2 surface expression was down-regulated by 50% on PMN from septic patients (p < 0.005), while CXCR1 expression persisted. In vitro migratory responses to the CXCR1 ligand, IL-8, were similar in PMN from septic patients and normal donors. By contrast, the migratory response to the CXCR2 ligands, epithelial cell-derived neutrophil activator (ENA-78) and the growth-related oncogene proteins, was markedly suppressed in PMN from septic patients (p < 0.05). Ab specific for CXCR1 blocked in vitro migration of PMN from septic patients to IL-8 (p < 0.05), but not to FMLP. Thus, functionally significant down-regulation of CXCR2 occurs on PMN in septic patients. We conclude that in a complex milieu of multiple CXC chemokines, CXCR1 functions as the single dominant CXC chemokine receptor in patients with sepsis. These observations offer a potential strategy for attenuating adverse inflammation in sepsis while preserving host defenses mediated by bacteria-derived peptides such as FMLP.     

Direct cell/cell contact with stimulated T lymphocytes induces the expression of cell adhesion molecules and cytokines by human brain microvascular endothelial cells

Upon inflammation, stimulated, but not resting T lymphocytes cross the blood-brain barrier and migrate into the central nervous system. This study shows that direct contact between stimulated T lymphocytes and human brain microvascular endothelial cells (HB-MVEC) induces phenotypic and functional changes on the latter cells. Plasma membranes isolated from stimulated T lymphocytes (S-PM) up-regulated the expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin on isolated HB-MVEC. In addition, HB-MVEC activated by S-PM secreted interleukin (IL)-6 and IL-8. The levels of ICAM-1, E-selectin, IL-6, and IL-8 expressed in S-PM-activated HB-MVEC were similar to those observed with 1000 U/ml tumor necrosis factor (TNF). In contrast, VCAM-1 expression was 15% of that induced by TNF. Inhibitors of TNF diminished (< or = 45%), but did not abolish the expression of cell adhesion molecules and IL-6 induced by S-PM, IL-8 production being insignificantly affected (< or = 10%). This suggests that membrane-associated TNF was partially involved in HB-MVEC activation. The present study demonstrates that stimulated T lymphocytes are able to activate HB-MVEC upon direct cell contact. This novel mechanism of inducing the expression of cell adhesion molecules may prompt the initial adhesion of stimulated T lymphocytes to brain endothelium.     

Thyroid function in a population with an extra iodine intake]

Polymorphonuclear leukocyte (PMN) superoxide (.O2-) production has been implicated in the pathogenesis of cardiopulmonary bypass (CPB)-related end organ injury. PMN "priming" has been described as an event which enhances the release of .O2- following a second, activating insult. We hypothesized that PMN priming occurs during CBP and is temporally related to the plasma level of complement (C3a), interleukin (IL)-6, and IL-8. PMNs were isolated from 10 CPB patients pre-bypass (preCPB), 5 min after protamine administration (PROT), and at 6 and 24 h post-CPB. PMN .O2- production was measured by a cytochrome c reduction assay in the presence or absence of either phorbol 12-myristate-13-acetate (PMA, 0.4 microgram/ml) or N-formyl-methionyl-leucyl-phenylalanine (FMLP, 1 microM) and also after priming with 2000 nM platelet-activating factor (PAF) followed by activation with either PMA or FMLP. Plasma levels of C3a, IL-6, and IL-8 were determined by enzyme-linked immunosorbent assay. PMA-activated PMN .O2- production was significantly elevated at 6 h post-CPB compared to pre-CPB levels (11.04 +/- 0.9 vs 7.62 +/- 0.57, P = 0.009), indicating that CPB is associated with in vivo PMN priming. When PMNs were primed in vitro with PAF and then activated with PMA or FMLP, .O2- release at 6 h post-CPB was also significantly greater than pre-CPB levels (16.04 +/- 0.74 vs 12.2 +/- 0.92, P = 0.038; and 17.33 +/- 1.38 vs 13.33 +/- 1.35, P < 0.05), indicating that CPB acts synergistically with PAF to prime PMNs. Levels of C3a rose significantly over pre-CPB levels at PROT (P = 0.001), and IL-6 and IL-8 rose over pre-CPB levels at 6 h post-CPB (P = 0.01 and P = 0.006, respectively). These findings demonstrate that CPB not only directly primes PMNs, but also potentiates priming of PMNs by PAF. This "primed" PMN state, which coincided with the increased plasma levels of inflammatory mediators, may suggest a mechanism of predisposition to organ dysfunction following CPB.     

Regulation of polymorphonuclear cell activation by thrombopoietin

Thrombopoietin (TPO) regulates early and late stages of platelet formation as well as platelet activation. TPO exerts its effects by binding to the receptor, encoded by the protooncogene c-mpl, that is expressed in a large number of cells of hematopoietic origin. In this study, we evaluated the expression of c-Mpl and the effects of TPO on human polymorphonuclear cells (PMN). We demonstrate that PMN express the TPO receptor c-Mpl and that TPO induces STAT1 tyrosine phosphorylation and the formation of a serum inducible element complex containing STAT1. The analysis of biological effects of TPO on PMN demonstrated that TPO, at concentrations of 1-10 ng/ml, primes the response of PMN to n-formyl-met-leu-phe (FMLP) by inducing an early oxidative burst. TPO-induced priming on FMLP-stimulated PMN was also detected on the tyrosine phosphorylation of a protein with a molecular mass of approximately 28 kD. Moreover, we demonstrated that TPO by itself was able to stimulate, at doses ranging from 0.05 to 10 ng/ml, early release and delayed synthesis of interleukin 8 (IL-8). Thus, our data indicate that, in addition to sustaining megakaryocytopoiesis, TPO may have an important role in regulating PMN activation.     

Eosinophil accumulation induced by human interleukin-8 in the guinea-pig in vivo

Interleukin-8 (IL-8) is a neutrophil chemoattractant cytokine. Initially IL-8 appeared to exhibit specificity for neutrophils over other cells of the immune system. However, several recent studies have shown that this mediator can also activate other leucocyte types in vitro. In this study we have used an in vivo model of local [111In]leucocyte accumulation in the guinea-pig and an in vitro assay of leucocyte activation (changes in cytosolic-free Ca2+) to investigate the eosinophil chemoattractant activity of IL-8. The intradermal injection of recombinant human (rh)IL-8 induced a dose-dependent accumulation of intravenously administered [111In]eosinophils into the skin sites over 4 hr. Time-course experiments revealed that this cell infiltration was delayed in onset, occurring between 1 and 2 hr after injection of IL-8. The delay may indicate that IL-8 operates via an indirect mechanism. In contrast, eosinophil accumulation induced by the complement fragment C5a occurred within the first hour following injection. Other human cytokines, IL-1, IL-3, IL-5, tumour necrosis factor (TNF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), were not eosinophil chemoattractants in this in vivo test system. Direct activation of eosinophils by IL-8 was demonstrated in vitro by a transient elevation in cytoplasmic-free Ca2+ levels where it was less potent than either rhC5a or leukotriene B4 (LTB4). Experiments using [111In]neutrophils in vivo indicated that rhIL-8 and rhC5a were similar in potency in inducing local neutrophil infiltration into guinea-pig skin. The demonstration of the eosinophil chemoattractant activity of IL-8 in vivo raises the possibility that this cytokine, or a structurally related molecule, contributes towards eosinophil infiltration in a number of inflammatory conditions such as asthma, helminthic infections and adult respiratory distress syndrome.     

TNF alpha-induced activation of eosinophil oxidative metabolism and morphology--comparison with IL-5

Human dermal mast cells are capable of releasing cytokines, particularly preformed TNF alpha, upon appropriate stimulation. Mast cell activation in vivo was shown to be associated with an influx and activation of inflammatory cells, initially PMN (polymorphonuclear neutrophilic granulocytes) then eosinophils. In order to learn more about the mechanisms by which TNF alpha is capable of activating eosinophils, in the present study the effect of TNF alpha on morphology and function of highly purified normal eosinophils (> or = 95%) was examined. As estimated by transmission and scanning electron microscopy, TNF alpha-stimulated eosinophils appeared to be strictly adherent and flattened exhibiting a characteristic "hemispheric" shape. TNF alpha induced a dose-dependent, long-lasting production of reactive oxygen species as measured by lucigenin-dependent chemiluminescence (CL), even at a concentration of 0.001 U/ml. The maximal response upon stimulation with TNF alpha, however, was significantly lower than optimal effects induced by IL-5. TNF alpha-induced responses were completely inhibited by cytochalasin B and staurosporin, and partially blocked by pertussis toxin. Separation of eosinophils by discontinuous density gradients revealed the existence of at least two hypodense eosinophil populations with a distinct susceptibility to stimulation with TNF alpha. Based on functional assay systems, in contrast to a significant extracellular, only a small intracellular H2O2 production was detected. Accordingly, H2O2 production, detected by an ultrastructural technique, was observed only on the outer surface of the plasma membrane in the contact zones in between adjacent cells. Extracellular as well as intracellular production of H2O2 was completely inhibited by cytochalasin B. TNF alpha-induced activation of eosinophils is most probably mediated by binding to the 55 kD and the 75 kD TNF-receptor since both receptor molecules could be detected by FACS analysis and immune electron microscopy using receptor-specific antibodies. However, in contrast to its effect on eosinophil oxidative response, TNF alpha did not induce the release of significant concentrations of eosinophil cationic protein or eosinophil peroxidase in supernatants of cytokine-stimulated eosinophils, as detected by functional as well as immunological assay systems. These results clearly indicate that TNF alpha represents a potent eosinophil-activating cytokine which may be of relevance in the allergic inflammatory response.     

Activation of nitric oxide release and oxidative metabolism by leukotrienes B4, C4, and D4 in human polymorphonuclear leukocytes

Because arachidonate metabolites are potent mediators of inflammation, we have studied the effects of leukotriene B4 (LTB4) and the cysteinyl leukotrienes C4 and D4 (LTC4 and LTD4) on the release of nitric oxide (NO), in vitro, by human polymorphonuclear granulocytes (PMN). Two independent and highly sensitive real-time methods were used for these studies, ie, the NO-dependent oxidation of oxyhemoglobin (HbO2) to methemoglobin and a NO-sensitive microelectrode. When activated with LTB4, LTC4, or LTD4, but not with other lipoxygenase products such as 5S-HETE, 5-oxo-ETE or 5S, 12S-diHETE, PMN produced NO in a stimulus- and concentration-dependent manner. The rank order of potency was LTB4 = LTC4 > LTD4, corresponding to 232 +/- 50 pmol of NO/10(6) PMN for 100 nmol/L LTB4 after 30 minutes. The kinetic properties of the responses were similar for all three leukotrienes with a maximum response at 13 +/- 3 minutes. Cysteinyl leukotriene and LTB4 antagonists inhibited the agonist-induced NO production by 70%, and treatment with Bordetella pertussis toxin, or chelation of cytosolic Ca2+, [Ca2+]i, also efficiently inhibited this response. In contrast, treatment of PMN with cytochalasin B (5 microg/mL) enhanced the LTB4-induced NO formation by 86%. Thus, this is the first demonstration that the cysteinyl leukotrienes LTC4 and LTD4, as well as LTB4, activate NO release from human PMN by surface receptor, G-protein and [Ca2+]i-dependent mechanisms. This effect differs from activation of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, for which only LTB4 is an activator.     

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.     

Pig I alpha I appears unmodified in plasma in case of endotoxin-induced disseminated intravascular coagulation

The unrestricted activity of leukocyte proteinases is thought to contribute to the degradation of plasma proteins and thus amplify the coagulation disorders occurring in septic shock. Inter-alpha-inhibitor (I alpha I) is a plasma protein particularly susceptible to their action. Therefore we investigated its behavior in a porcine model of endotoxin shock which reproduces the coagulation changes observed in human sepsis. We did not detect any qualitative or quantitative modification of porcine I alpha I in plasmas collected from pigs after endotoxin infusion. To explain these data, I alpha I was incubated with polymorphonuclear neutrophils (PMN) stimulated by FMLP in the presence of cytochalasin B. We found that, unlike human PMN, porcine cells were unable to proteolyze I alpha I. Moreover, in the incubation medium of pig PMN, triggered either by FMLP or PMA, no measurable elastase activity was evidenced. Therefore, we urge to better take into account species differences in functional responses of PMN, to explain the experimental results obtained in animal models of septic shock.     

CD11b/CD18 mediates the neutrophil chemotactic activity of fibrin degradation product D domain

Coagulation and fibrinolysis universally accompany tissue injury and repair. The accumulation of regionally generated fibrin degradation products (FDP) may modify the local inflammatory response. We have found FDP to be potent neutrophil chemotaxins. We separated plasmin FDP by chromatofocusing and found chemotactic activity limited to fractions containing the fibrinogen D domain (D-D dimer and D monomer). The bioactivity of the D-D dimer did not require an intact cross link site as removal of this sequence with puff adder venom or hypocalcemic plasmic digestion did not decrease chemotaxis. Peptide inhibition studies confirmed that the chemotactic region did not involve terminal gamma chain sequences or alpha chain RGD motifs. The internal gamma chain peptide KYGWTVFQKRLDGSV (P1), known to bind CD11b/CD18, exhibited concentration dependent chemotactic activity. Similarly, monoclonal antibodies directed against CD11b/CD18 blocked PMN migration to FDP without similar inhibition of chemotaxis to IL-8 or LTB4. Thus, neutrophil chemotaxis to FDP is mediated by interactions between the fibrinogen D domain and CD11b/CD18.