Potent Chemotactic Activity For Neutrophils Research Articles

Physiological Role of the Oxidative Stress-Sensitive TRPM2 Ca2+ Channel in Immunocytes

It is known that a large amount of reactive oxygen species (ROS) exist at inflamed sites. ROS induce chemokines responsible for the recruitment of inflammatory cells at inflamed sites. Here, we demonstrate that the plasma membrane Ca2+-permeable channel TRPM2 controls ROS-induced chemokine production in monocytes/macrophages. In monocytes from Trpm2-defecient mice, H2O2-induced Ca2+ influx and production of the macrophage inflammatory protein-2 (CXCL2), which exhibit potent neutrophil chemotactic activity, were impaired. In the inflammation model dextran sulfate sodium-induced colitis, CXCL2 expression was attenuated by Trpm2 disruption. Interestingly, the number of recruited neutrophils was significantly reduced in DSS-treated TRPM2 KO mice, whereas that of DSS-induced macrophages after infiltration into inflamed sites, was indistinguishable in WT and TRPM2 KO mice. Importantly, TRPM2 deficiency failed to impair important aspects of CXCL2-evoked neutrophil chemotaxis, including Ca2+ response, in vitro migration, and in vivo infiltration after DSS administration. Thus, TRPM2-mediated Ca2+ influx induces chemokine production in monocytes that aggravates inflammatory neutrophil infiltration. We propose functional inhibition of TRPM2 channels as a new therapeutic strategy for treating inflammatory diseases.

PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR-γ LIGANDS 15-DEOXY-δ12,14-PROSTAGLANDIN J2 AND PIOGLITAZONE INHIBIT HYDROXYL PEROXIDE-INDUCED TNF-α AND LIPOPOLYSACCHARIDE-INDUCED CXC CHEMOKINE EXPRESSION IN NEONATAL RAT CARDIAC MYOCYTES

Ligands for peroxisome proliferator-activated receptor gamma (PPAR-gamma) such as prostaglandin metabolite 15-deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2) or thiazolidinedione pioglitazone have been identified as a new class of anti-inflammatory compounds with possible clinical applications. Reactive oxygen species play an important role in the generation of cellular damage by induction of proinflammatory cytokines and chemokines during myocardial I/R. These events were preceded by activation of the transcription factors nuclear factor (NF)-kappaB pathway. It has been suggested that myocardium overproduces TNF-alpha after I/R, and locally produced TNF-alpha is sufficient to cause severe impairment of cardiac function. LPS-induced CXC chemokine (LIX) is a rodent chemokine with potent neutrophil-chemotactic activity. Based on this concept, we examined the effects of 15d-PGJ2 and pioglitazone on oxidative stress-induced TNF-alpha and LIX expression in neonatal rat cardiac myocytes. Pretreatment of myocytes with 15d-PGJ2 or pioglitazone decreased hydrogen peroxide-induced TNF-alpha and LIX production (mRNA and protein) in a concentration-dependent manner. The beneficial effects of both ligands were associated with reduction of hydrogen peroxide-induced NF-kappaB activation. Treatment with 15d-PGJ2, but not pioglitazone, caused dose-dependent activation of heat shock factor 1, which could render cells unresponsive to stimulation of NF-kappaB. The cytoprotection afforded by pioglitazone was attenuated by the PPAR-gamma antagonist GW9662, which failed to affect the beneficial effects afforded by 15d-PGJ2. Taken together, these results demonstrate that treatment with these chemically distinct ligands of PPAR-gamma results in diverse anti-inflammatory mechanisms.

Polymeric Osteopontin Employs Integrin α9β1 as a Receptor and Attracts Neutrophils by Presenting a de Novo Binding Site

Osteopontin (OPN) is a cytokine and ligand for multiple members of the integrin family. OPN undergoes the in vivo polymerization catalyzed by cross-linking enzyme transglutaminase 2, which consequently increases the bioactivity through enhanced interaction with integrins. The integrin alpha9beta1, highly expressed on neutrophils, binds to the sequence SVVYGLR only after intact OPN is cleaved by thrombin. The SVVYGLR sequence appears to be cryptic in intact OPN because alpha9beta1 does not recognize intact OPN. Because transglutaminase 2-catalyzed polymers change their physical and chemical properties, we hypothesized that the SVVYGLR site might also be exposed on polymeric OPN. As expected, alpha9beta1 turned into a receptor for polymeric OPN, a result obtained by cell adhesion and migration assays with alpha9-transfected cells and by detection of direct binding of recombinant soluble alpha9beta1 with colorimetry and surface plasmon resonance analysis. Because the N-terminal fragment of thrombin-cleaved OPN, a ligand for alpha9beta1, has been reported to attract neutrophils, we next examined migration of neutrophils to polymeric OPN using time-lapse microscopy. Polymeric OPN showed potent neutrophil chemotactic activity, which was clearly inhibited by anti-alpha9beta1 antibody. Unexpectedly, mutagenesis studies showed that alpha9beta1 bound to polymeric OPN independently of the SVVYGLR sequence, and further, SVVYGLR sequence of polymeric OPN was cryptic because SVVYGLR-specific antibody did not recognize polymeric OPN. These results demonstrate that polymerization of OPN generates a novel alpha9beta1-binding site and that the interaction of this site with the alpha9beta1 integrin is critical to the neutrophil chemotaxis induced by polymeric OPN.

Mediators of neutrophil recruitment in human abdominal aortic aneurysms

AimsNeutrophils/platelet interactions are involved in abdominal aortic aneurysm (AAA). The intraluminal thrombus (ILT) is a human model of platelet/neutrophil interactions. The present study focused on mediators involved in neutrophil recruitment in AAA.Methods and resultsConditioned media from luminal, intermediate, and abluminal layers of 29 human ILTs were analysed for neutrophil markers [elastase/α1-antitrypsin and MMP9/NGAL complexes, myeloperoxidase (MPO), and α-defensin peptides], RANTES, platelet factor 4 (PF4), and interleukin-8 (IL-8). Their time-dependent release into serum from clots generated in vitro and their plasma concentrations in AAA patients and controls were determined. Immunohistochemistry for neutrophils, platelets, IL-8, PF4, and RANTES on AAA sections was performed; and molecules involved in ILT neutrophil chemotactic function were analysed in vitro. Neutrophils and platelets colocalized in the luminal layer of the thrombus. Consistently, neutrophil markers and platelet-derived RANTES and PF4 were released predominantly by the luminal thrombus pole, where their concentrations were significantly correlated. The luminal ILT layer was also the main source of IL-8, whose immunostaining colocalized with neutrophils. All were also released time dependently from clots and were increased in plasma of AAA patients. Luminal ILT layers displayed potent neutrophil chemotactic activity in vitro, which was inhibited by RANTES- and IL-8-blocking antibodies as well as by reparixin, an antagonist of the IL-8 receptors CXCR1 and CXCR2.ConclusionTaken together, these results suggest that platelet-derived RANTES and neutrophil-derived IL-8 are involved in attracting neutrophils to the luminal layer of AAA ILT.

Serum level of growth-related oncogene alpha during abdominal aortic aneurysm repair in humans

High postoperative mortality in patients after open elective aortic abdominal aneurysm (AAA) repair is thought to be the effect of ischemia-reperfusion organ injury followed by Multiple Organ Dysfunction Score. Neutrophils appear to be predominant leukocytes that are important in mediating ischemia-reperfusion injury and organ damage. Growth-related oncogene alpha (GROα) (CXCL1) is the chemokine with potent chemotactic activity for neutrophils. The aim of this study was to determine changes in serum GROα concentrations in the course of ischemia-reperfusion during AAA repair.

Chemokines accumulate in the lungs of rats with severe pulmonary embolism induced by polystyrene microspheres.

Pulmonary thromboembolism (PEm) is a serious and life threatening disease and the most common cause of acute pulmonary vascular occlusion. Even following successful treatment of PEm, many patients experience long-term disability due to diminished heart and lung function. Considerable damage to the lungs presumably occurs due to reperfusion injury following anti-occlusive treatments for PEm and the resulting chronic inflammatory state in the lung vasculature. We have used a rat model of irreversible PEm to ask whether pulmonary vascular occlusion in the absence of reperfusion is itself sufficient to induce an inflammatory response in lungs. By adjusting the severity of the vascular occlusion, we were able to generate hypertensive and nonhypertensive PEm, and then examine lung tissue for expression of CXC and C-C chemokine genes and bronchoalveolar lavage (BAL) fluid for the presence of chemokine proteins. Hypertensive and nonhypertensive PEm resulted in increased expression of both CXC and C-C chemokines genes in lung tissues. Hypertensive PEm was also associated with a 50-100-fold increase in protein content in lung BAL fluid, which included the CXC chemokines cytokine-induced neutrophil chemoattractant and macrophage-inflammatory protein 2. The presence of chemokines in BALs was reflected by a potent neutrophil chemotactic activity in in vitro chemotaxis assays. Abs to cytokine-induced neutrophil chemoattractant blocked the in vitro neutrophil chemotactic activity of BAL by 44%. Our results indicate that the ischemia and hypertension associated with PEm are sufficient to induce expression of proinflammatory mediators such as chemokines, and establish a proinflammatory environment in the ischemic lung even before reperfusion.

Ischemia-reperfusion of rat myocardium activates nuclear factor-KappaB and induces neutrophil infiltration via lipopolysaccharide-induced CXC chemokine.

Mechanisms by which neutrophils are attracted to the myocardium in ischemia/reperfusion are not fully defined. Lipopolysaccharide-induced CXC chemokine (LIX), cytokine-induced neutrophil chemoattractant (KC), and macrophage inflammatory protein-2 (MIP-2) are rodent chemokines with potent neutrophil-chemotactic activity. The goals of the present study were to evaluate the roles of these chemokines in a rat model of ischemia/reperfusion and to examine the mechanisms of chemokine induction by oxidative stress and cytokines in cultured cardiomyocytes. Male Wistar-Kyoto rats underwent 45 minutes of ligation of the left anterior descending coronary artery, followed by reperfusion for various periods. Compared with sham-operated controls, myocardium from reperfused animals had higher levels of free radicals, increased neutrophil infiltration evidenced histologically and by elevated myeloperoxidase activity, and increased nuclear factor (NF)-kappaB DNA binding activity. Ischemia-reperfusion also induced the expression of interleukin-1beta, tumor necrosis factor (TNF)-alpha, LIX, KC, and MIP-2 mRNA and protein. LIX expression was localized to resident myocardial cells, whereas KC and MIP-2 were expressed only in infiltrating inflammatory cells. Neutralization of LIX inhibited 79% of neutrophil infiltration into previously ischemic myocardium. In contrast, neutralization of KC and MIP-2 reduced neutrophil infiltration by only 28% and 37%, respectively. In cultured cardiomyocytes, LIX expression was induced by oxidative stress or TNF-alpha and was blocked by the NF-kappaB inhibitor pyrrolidinedithiocarbamate. LIX is expressed by resident myocardial cells during ischemia-reperfusion and is induced in cultured cardiomyocytes by oxidative stress or TNF-alpha via NF-kappaB activation. Although KC and MIP-2 are expressed by inflammatory cells infiltrating the myocardium during reperfusion after ischemia, neutrophil recruitment to reperfused rat myocardium is mainly due to cardiomyocyte expression of LIX.

The Immediate Early Gene 1 Product of Human Cytomegalovirus Is Sufficient for Up-Regulation of Interleukin-8 Gene Expression

We previously observed that human cytomegalovirus (CMV) infection induced a massive production of a chemokine with potent neutrophil chemotactic activity, interleukin-8 (IL-8). Hence, we examined the effect of CMV immediate early (IE) gene products on IL-8 production by the human astrocytoma cell line, U373MG. Transient or stable transfection with a CMV IE1 gene expression vector, but not with a IE2 gene expression vector, significantly augmented IL-8 protein secretion and IL-8 mRNA expression. Luciferase activity was enhanced in U373MG cells when the cells were cotransfected with CMV IE1 and chimeric firefly luciferase reporter genes driven by the transcriptional regulatory region of the human IL-8 gene. Moreover, IE1 gene-mediated enhancement of luciferase activity was abolished by the introduction of mutations into the AP-1 or NF-κB factor binding elements in the regulatory region of the IL-8 promoter. Furthermore, electrophoretic mobility shift assays demonstrated that CMV IE1 gene products induced the formation of NF-κB or AP-1 complexes. Finally, Western blotting analysis demonstrated that the CMV IE1 gene product increased the amount of NF-κB complexes translocated into the nucleus. Collectively, CMV IE1 gene expression may be sufficient to activate AP-1 and NF-κB, resulting in IL-8 gene expression.

The Biphasic mRNA Expression Pattern of Bovine Interleukin-8 inPasteurella haemolyticaLipopolysaccharide-Stimulated Alveolar Macrophages Is Primarily due to Tumor Necrosis Factor Alpha

Pasteurella haemolytica serotype 1 is the bacterial agent responsible for the pathophysiological events associated with bovine pneumonic pasteurellosis. Our previous studies support a role for the lipopolysaccharide (LPS) from P. haemolytica in the induction of proinflammatory cytokines. One of the pathological hallmarks of bovine pneumonic pasteurellosis is an influx of neutrophils into the alveolar spaces. This pronounced influx suggests the local production of a chemotactic factor(s) such as interleukin-8 (IL-8). In the context of the lung, the alveolar macrophage appears to be the major producer of IL-8, a proinflammatory cytokine with potent neutrophil chemotactic activity. By using Northern blot analysis, we have examined the kinetics of IL-8 mRNA expression in P. haemolytica LPS-stimulated bovine alveolar macrophages and found that 1 ng of LPS per ml induces maximal expression of IL-8 mRNA. The results also indicate a biphasic time course expression pattern in which IL-8 mRNA levels peak between 1 and 2 h in the first phase and between 16 and 24 h in the second phase (P < 0.01). In addition, monospecific polyclonal antibodies were used to demonstrate the role of tumor necrosis factor alpha (TNF-alpha) in the second phase of IL-8 mRNA expression. Our findings support a role for P. haemolytica LPS and TNF-alpha in the induction of IL-8 from bovine alveolar macrophages.

Synthesis, biological effects and pathophysiological implications of the novel arachidonic acid metabolite 5-oxo-eicosatetraenoic acid (Review).

Arachidonic acid is rapidly metabolized by several distinct enzymes including the 5-lipoxygenase generating leukotrienes and 5-hydroxyeicosatetraenoic acid (5-HETE). These well studied metabolites cause a variety of physiological and pathophysiological effects in different tissues. Recently, oxidation of 5-HETE to 5-oxo-eicosatetraenoic acid (5-oxo-ETE) by an NADP+-dependent dehydrogenase has been demonstrated. Calcium ionophors and protein kinase C activators stimulate the synthesis of 5-oxo-ETE in neutrophils, eosinophils and monocytes. This novel arachidonic acid metabolite has a potent chemotactic activity for neutrophils and eosinophils. It stimulates adhesion of neutrophils and induces reactive oxygen metabolites in eosinophils. There is evidence that 5-oxo-ETE and 5-HETE interact with a specific G-protein coupled receptor. Since in contrast to 5-oxo-ETE much higher concentrations of 5-HETE are needed to provoke cell responses, 5-oxo-ETE might be the physiological relevant ligand for this putative receptor. Further downstream signalling pathways of this ligand include calcium transients, actin polymerization, activation of phosphatidylinositol-3-kinase and MAP-kinase. 5-oxo-ETE has been extracted from scales of psoriatic patients and injection of 5-oxo-ETE into rabbit subcutis causes a severe edema with an inflammatory cell infiltrate resembling an urticarial lesion. These findings indicate, that 5-oxo-ETE might play a role in different cutaneous inflammatory diseases.

The Biphasic mRNA Expression Pattern of Bovine Interleukin-8 inPasteurella haemolytica Lipopolysaccharide-Stimulated Alveolar Macrophages Is Primarily due to Tumor Necrosis Factor Alpha

Pasteurella haemolytica serotype 1 is the bacterial agent responsible for the pathophysiological events associated with bovine pneumonic pasteurellosis. Our previous studies support a role for the lipopolysaccharide (LPS) from P. haemolytica in the induction of proinflammatory cytokines. One of the pathological hallmarks of bovine pneumonic pasteurellosis is an influx of neutrophils into the alveolar spaces. This pronounced influx suggests the local production of a chemotactic factor(s) such as interleukin-8 (IL-8). In the context of the lung, the alveolar macrophage appears to be the major producer of IL-8, a proinflammatory cytokine with potent neutrophil chemotactic activity. By using Northern blot analysis, we have examined the kinetics of IL-8 mRNA expression in P. haemolytica LPS-stimulated bovine alveolar macrophages and found that 1 ng of LPS per ml induces maximal expression of IL-8 mRNA. The results also indicate a biphasic time course expression pattern in which IL-8 mRNA levels peak between 1 and 2 h in the first phase and between 16 and 24 h in the second phase (P < 0.01). In addition, monospecific polyclonal antibodies were used to demonstrate the role of tumor necrosis factor alpha (TNF-α) in the second phase of IL-8 mRNA expression. Our findings support a role for P. haemolytica LPS and TNF-α in the induction of IL-8 from bovine alveolar macrophages.

Requirement for C-X-C chemokines (macrophage inflammatory protein-2 and cytokine-induced neutrophil chemoattractant) in IgG immune complex-induced lung injury.

The C-X-C chemokines of the IL-8 family possess potent chemotactic activity for neutrophils, but their in vivo role in inflammatory responses is not well understood. In the IgG immune complex-induced model of acute lung inflammatory injury in the rat we have evaluated the roles of two rat chemokines, macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (CINC). Both mRNA and protein for MIP-2 and CINC appeared in a time-dependent manner after initiation of IgG immune complex deposition in lung. There exists a 69% homology between the amino acid sequences for these proteins, and we found cross-reactivity between polyclonal Abs raised to these chemokines. By purifying the blocking Abs using double affinity methods (with Ag-immobilized beads), this cross-reactivity was removed. Individually, anti-MIP-2 and anti-CINC Ab significantly reduced lung injury (as measured by 125I-labeled albumin leakage from the pulmonary vasculature) and reduced neutrophil accumulation in the lung (as determined by myeloperoxidase (MPO content) and neutrophil counts in bronchoalveolar lavage (BAL) fluids); however, no change in TNF-alpha levels in BAL fluids was found. Chemotactic activity in BAL fluids collected 2 h after injury from animals undergoing immune complex deposition could be shown to be chiefly due to the combined contributions of MIP-2 (39%), CINC (28%), and C5a (21%). When either MIP-2 or CINC was blocked in vivo, up-regulation of Mac-1 expression on neutrophils obtained from BAL fluids was significantly reduced. These data suggest that, in the model studied, both MIP-2 and CINC contribute significantly to the influx of neutrophils and their activation.

S100 protein CP-10 stimulates myeloid cell chemotaxis without activation.

Leukocyte recruitment to inflammatory foci is generally associated with cellular activation. Recent evidence suggests that chemotactic agents can be divided into two classes, "classical chemoattractants" such as FMLP, C5a, and IL-8, which stimulate directed migration and activation events and "pure chemoattractants" such as TGF-beta 1 which influence actin polymerisation and movement but not oxidative burst and associated granular enzyme release. The studies reported here demonstrate that the murine S100 chemoattractant protein, CP-10, belongs to the "non-classical" group. Despite its potent chemotactic activity for neutrophils and monocytes/macrophages, CP-10 failed to increase [Ca2+]i in human or mouse PMN, although chemotaxis was inhibited by pertussis toxin, confirming the suggestion of a novel Ca(2+)-independent G-protein-coupled pathway for post-receptor signal transduction triggered by "pure chemoattractants." The co-ordinated up-regulation of Mac-1 and down-regulation of L-selectin induced by FMLP on human PMN in vitro was not observed with CP-10. Quantitative changes in immediate (30 s) actin polymerisation occurred with FMLP and CP-10-treated human PMN. The relative F-actin increases induced in WEHI 265 monocytoid cells by FMLP and CP-10 was optimal at 60 s and declined over 120 s. F-actin changes reflected the concentration and potencies of the agonists required to provoke chemotaxis. After 90 min, CP-10 profoundly altered cell shape and increased both cell size and F-actin within pseudopodia. These changes are typical of those mediating leukocyte deformability, and CP-10 may mediate leukocyte retention within microcapillaries and thereby contribute to the initiation of inflammation in vascular beds.

TNFα and increased chemokine expression in rat lung after particle exposure

Macrophage inflammatory protein 2 (MIP-2) and CINC (Cytokine-Induced-Neutrophil-Chemoattractant) are members of the chemokine family of inflammatory and immunoregulatory cytokines. MIP-2 and CINC exhibit potent neutrophil chemotactic activity and are thought to be key mediators of inflammatory cell recruitment in response to tissue injury and infection. In the present studies, we examined the potential involvement of MIP-2 and CINC in particle-elicited inflammation in the rat lung and the role of TNFα in particle-induced chemokine expression. Acute intratracheal instillation exposure of F344 rats to α quartz or titanium dioxide was shown to markedly increase steady-state levels of MIP-2 and CINC mRNA in lung tissue; a response which was associated with a significant increase in neutrophils in the bronchoalveolar lavage fluid. Additional studies demonstrated that acute inhalation of crocidolite fibers by rats also induced increased MIP-2 and CINC expression. Since previous studies had demonstrated that TNFα stimulates MIP-2 and CINC expression in vitro and that particle exposure induces TNFα production in rat lung we examined the role of TNFα in a quartz-induced MIP-2 gene expression. We demonstrated that passive immunization of mice against TNFα markedly attenuated the increased lung MIP-2 mRNA seen in response to a quartz inhalation. Collectively, these findings suggest that the chemokines MIP-2 and CINC play a role in neutrophil recruitment to the rat lung after particle exposure and indicate that particle-induced expression of these chemokines is mediated, at least in part, by production of TNFα.

Expression of cytokine-induced neutrophil chemoattractant in rat cardiac myocytes

Accumulation and adhesion of leukocytes to cardiac myocytes play important roles in the pathogenesis of inflammation-mediated myocardial injury such as ischaemia/reperfusion and myocarditis. The involvement of leukocyte chemotactic factors has been speculated in these processes. We investigated the expression of cytokine-induced neutrophil chemoattractant (CINC) in rat cardiac myocytes. CINC is a rat equivalent of human interleukin-8. On exposure to interleukin-1α (IL-1α), cultured neonatal rat cardiac myocytes released appreciable levels of CINC both dose-and time-dependently. Tumor necrosis factor-α and lipopolysaccharide also significantly increased CINC accumulation in the culture supernatant. CINC mRNA expression was not observed in unstimulated myocytes, however, the expression was markedly induced by exposure to IL- 1α with a peak elevation at 3 h. Potent chemotactic activity for neutrophils was detected in the supernatant of cultured rat cardiac myocytes by stimulation with IL-1α. This IL-1α-induced chemotactic activity was significantly inhibited by polyclonal anti-CINC antiserum. Addition of dexamethasone, genistein, actinomycin D or cycloheximide significantly suppressed the IL-1α-induced CINC accumulation. Under hypoxia (95%N 2+5%CO 2), CINC accumulation was increased in a time-dependent manner, and reoxygenation after hypoxia further intensified CINC accumulation. This hypoxia reoxygenation-induced CINC expression was significantly inhibited by pretreatment with dexamethasone. In conclusion, inflammatory stimuli induce the expression of CINC in rat cardiac myocytes, which may lead to myocardial injury via accumulation and activation of neutrophils.

Proinflammatory properties of molluscum bodies.

Molluscum contagiosum, a condition characterized by benign viral tumours, occasionally becomes inflamed and regresses spontaneously, an event probably initiated by a host cell-mediated immune rejection against the lesion, but it inevitably involves the disruption of the epidermal tissue to expose the molluscum bodies to the tissue fluids of the dermis. It has been suggested that the molluscum bodies induce inflammation by a mechanism similar to that involved in ruptured epidermal cysts or in acne. Despite the occasional development of inflammation in molluscum contagiosum, the proinflammatory properties of molluscum bodies have never been studied in vitro. Thus, in the present study we sought to determine whether molluscum bodies exert a proinflammatory effect by inducing neutrophil chemotaxis. When exposed to fresh serum in vitro, water-insoluble components of molluscum bodies activated the alternative complement pathway to produce chemotactic C5a/C5a des Arg. We also found that an aqueous extract of molluscum bodies exerted potent chemotactic activity for neutrophils. Remarkably high amounts of the immunoreactive proinflammatory cytokines IL-8 and GRO alpha were present in the extract even when compared with psoriatic scale extracts. Gel filtration HPLC of the extract demonstrated the presence of neutrophil chemotactic activity over a wide range of molecular mass. These data suggest that disruption of the epidermal wall of molluscum bodies induces acute inflammatory changes by activation of the alternative complement pathway on exposure to the tissue fluids, and that the molluscum bodies themselves release proinflammatory cytokines and other neutrophil chemotactic factors on decomposition.

Interleukin-8 is a chemo-attractant for eosinophils purified from subjects with a blood eosinophilia but not from normal healthy subjects.

Interleukin-8 (IL-8), a pro-inflammatory cytokine with potent neutrophil chemotactic activity, was studied for its effect on eosinophil migration responses, in vitro. Normal density eosinophils were isolated from healthy, non-atopic subjects (<0.35 x 10(9) eosinophils/l) and individuals with various diseases associated with a blood eosinophilia (range 0.56 x 10(9)-12.2 x 10(9) eosinophils/l). IL-8 produced a dose-dependent migrational response for eosinophils from subjects with an eosinophilia, optimal at 10(-8) M (P < 0.01) and the major component of the migrational response was chemokinesis. On a molar basis, IL-8 (EC50 approximately 10(10) M) was 100-fold more potent than platelet activating factor (PAF), although a comparison of the migrational responses showed that at optimal concentrations IL-8 (10(-8) M) produced only 30% maximal responses stimulated by PAF (10(-6) M). In contrast, IL-8 tested over a wide concentration range had a negligible effect on eosinophils from normal subjects. A direct correlation between the total blood eosinophil counts for all subjects and the absolute magnitude of the migrational response to IL-8 (r = 0.727, P < 0.01 at 10(-8) M), PAF (r = 0.551, P < 0.03 at 10(-6) M) and N-formyl-methionyl-leucyl-phenylalanine (fMLP) (r = 0.689, P < 0.02 at 10(-8) M), suggested that heightened eosinophil migrational responses to inflammatory mediators may arise as a consequence of in vivo priming mechanism(s) associated with the development of an eosinophilia. In this regard, eosinophils derived from human cord blood mononuclear cells cultured in the presence of eosinophilopoietic cytokines IL-3 and IL-5, produced migrational responses to IL-8 and PAF, that were comparable with that of eosinophils from eosinophilic subjects. Furthermore, incubation of eosinophils from normal donors with IL-5 (optimal concentration 10(-9) M), significantly enhanced the subsequent migrational responses to both IL-8 (10(-8) M, P < 0.01) and PAF (10(-8) M, P < 0.05). Therefore, the increased responsiveness of eosinophils from eosinophilic subjects may reflect in vivo priming by IL-5 and this phenomenon may contribute partly to the mechanism(s) by which eosinophils preferentially accumulate at sites of allergic inflammation.

Chemoattractants for neutrophils in lipopolysaccharide-induced inflammatory exudate from rats are not interleukin-8 counterparts but gro-gene-product/melanoma-growth-stimulating-activity-related factors.

Potent chemotactic activity for neutrophils was detected in rat inflammatory exudate induced by a subcutaneous injection of lipopolysaccharide in a carboxymethyl-cellulose suspension. We purified and characterized chemoattractants from the exudate by the following procedures: carboxymethyl-Sephadex C-25 ion-exchange chromatography; G3000SW gel-filtration chromatography; preparative reverse-phase high-pressure liquid chromatography; rechromatography on reverse-phase HPLC. Two chemotactic factors were purified and their N-terminal amino acid sequences were determined. One factor was a protein in which the first 20 N-terminal amino acids were identical to those of rat cytokine-induced neutrophil chemoattractant (CINC), a counterpart of human gro/melanoma growth-stimulating activity (MGSA). The other factor was highly similar to mouse macrophage inflammatory protein 2 (MIP-2). Mouse MIP-2, a chemotactic factor for neutrophils, is a member of the interleukin-8 family; however the protein we purified had higher similarity to human gro/MGSA than to human interleukin-8. These results indicate that, in rats, chemotactic factors for neutrophils induced by lipopolysaccharide stimulation are not counterparts of interleukin-8, but are gro/CINC-related peptides.

Neutrophilia and high level of interleukin 8 in the bronchoalveolar lavage fluid of diffuse panbronchiolitis.

Diffuse panbronchiolitis (DPB) is a chronic airway disorder exclusively seen in Japan, characterized by chronic inflammation of the respiratory bronchioles leading to air flow obstruction. Although the pathogenesis of the disorder is unknown, current studies based on bronchoalveolar lavage (BAL) have implied an important role of neutrophil-mediated inflammation as a characteristic pathological feature of the disease. Interleukin 8 (IL-8), a cytokine with potent chemotactic activity for neutrophils, has been proven to have close association with many inflammatory disorders. In this study, neutrophil chemotactic activity (NCA) and IL-8 were measured in the BAL fluid in eight patients with DPB to examine the roles of IL-8 in the neutrophil recruitment to the lower respiratory tract in DPB. Significantly higher levels of NCA and IL-8 were demonstrated in the BAL fluid of DPB patients (71.6 +/- 3.6%, 491.9 +/- 48.0 pg/ml, respectively) compared with that of chronic bronchitis (CB) patients (24.8 +/- 2.8%, 54.1 +/- 13.9 pg/ml, p < 0.001) and of healthy control subjects (7.0 +/- 0.9%, 14.2 +/- 3.9 pg/ml, p < 0.001). The IL-8 values were positively correlated with the NCA in the BAL fluid of DPB patients. Treatment with anti-IL-8 could significantly inhibited the NCA. These results suggest that IL-8 plays an important role in the recruitment of neutrophils to the lower respiratory tract in DPB.

Neutrophil-Activating Proteins in Psoriasis

Neutrophil accumulation in the epidermis is a histologic characteristic of psoriasis. We addressed the question: What is the major protein-like chemotactic principle responsible for neutrophil accumulation? Purification of proteinaceous neutrophil chemoattractants from extracts obtained from psoriatic scales by multistep high-performance liquid chromatography (HPLC) yielded three biochemically distinct polypeptides with potent neutrophil chemotactic activity. Aminoterminal amino acid sequence analysis of the quantitatively major neutrophil attractant revealed the sequence ELRXQXIKTYSK, which is identical to that of a 69 residue form of neutrophil-activating peptide-1/interleukin 8 (NAP-1/IL-8). The second major attractant showed the sequence XXVATELRXQXL . . ., which is identical to that of the gene product of the oncogene "gro" as well as "melanoma growth stimulatory activity, MGSA," whereas the third and minor neutrophil chemotaxin has an NH2-terminal sequence identical with NAP-1/IL-8. Estimation of NAP-1/IL-8-related proteins and gro/MGSA by HPLC combined with bioassay revealed a mean of 3.3 +/- 1.7 ng NAP-1/IL-8-related proteins (n = 11) and 3.2 +/- 1.9 ng gro/MGSA (n = 11) per 1 mg psoriatic scales. In normal heel callus (n = 8), these neutrophil attractants were found at concentrations below 0.02 +/- 0.01 ng/mg. The finding of more than 150-times increased amounts of both NAP-1/IL-8 and gro/MGSA in lesional psoriasis material suggest that these mitogenic as well as neutrophil- and lymphocyte-chemotactic compounds may play an important role in the pathogenesis of psoriasis.