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Effect of cytokines on polymorphonuclear neutrophil infiltration in the mouse. Prostaglandin- and leukotriene-independent induction of infiltration by IL-1 and tumor necrosis factor
Abstract
The i.p. injection of mice with highly purified recombinant human rIL-1 alpha or beta resulted in the rapid influx of a large number of polymorphonuclear neutrophils (PMN) into the peritoneal cavity. Significant increases in the number of PMN were induced by doses of IL-1 which ranged from 0.005 to 5 ng/injection. Interestingly the dose response for PMN influx was bell-shaped because 50 ng of IL-1 did not result in a significant increase in peritoneal PMN. IL-1 induced PMN infiltration was detectable by 1 h with peak levels of PMN obtained by about 2 h, followed by a subsequent decline by 24 h. Other cytokines, IL-2, IFN-gamma, IFN alpha beta, granulocyte-CSF, granulocyte-macrophage-CSF, IL-3, TNF-alpha, and TNF-beta were compared to IL-1 for their ability to induce a PMN influx into the peritoneum. Only TNF-alpha or TNF-beta (lymphotoxin) were able to induce a significant influx of PMN within 2 h. However, based on total protein administered, about 100 times more TNF than IL-1 was required to produce a comparable PMN infiltration. Intraperitoneal injection of inhibitors of the cyclooxygenase or lipoxygenase pathways did not inhibit the IL-1-induced influx of PMN. Also, neither IL-1 nor TNF triggered an increase in PG or leukotriene release from peritoneal cells in vitro. Furthermore, direct peritoneal injection of leukotriene B4, a potent PMN chemoattractant in vitro, did not induce any significant increase in PMN in the peritoneal cavity indicating that chemotactic activity alone is insufficient for inducing peritoneal infiltration. These results suggest that the local production of very low levels of IL-1 in vivo would be sufficient to initiate a sequence of events that results in a rapid accumulation of PMN. Because IL-1 was not chemotactic for PMN in vitro, our data suggest that IL-1 induces production of factors that are chemotactic for PMN. Alternatively, IL-1 may act on other stages of the complex sequence of events that regulates the emigration of PMN into tissue sites in vivo. The synergy apparent in PMN influx when suboptimal concentrations of IL-1 and TNF were injected suggests that the local production of very low concentrations of these cytokines in situ could play a critical role in the emigration of PMN during infection.
... Almost immediately following an injury (as soon as 1 hr), neutrophils form the predominant immune cell infiltrate. Neutrophils migrate into the injured area through areas of intact capillary endothelium and across layers of basement membrane and sarcolemma (Menger and Vollmar 1996 Another potential candidate is IL-1, which has been shown to be involved in the regulation of neutrophil adhesion and migration in vivo and in vitro in several species (Bevilacqaa et al., 1985;Sayer et al., 1988;Moser et al., 1989). In one of the in vivo studies, direct injection of 0.005-5 ng/ml of either IL-1α or β resulted in increased neutrophil chemotaxis into the peritoneal cavity (Sayer et al., 1988). ...
... Neutrophils migrate into the injured area through areas of intact capillary endothelium and across layers of basement membrane and sarcolemma (Menger and Vollmar 1996 Another potential candidate is IL-1, which has been shown to be involved in the regulation of neutrophil adhesion and migration in vivo and in vitro in several species (Bevilacqaa et al., 1985;Sayer et al., 1988;Moser et al., 1989). In one of the in vivo studies, direct injection of 0.005-5 ng/ml of either IL-1α or β resulted in increased neutrophil chemotaxis into the peritoneal cavity (Sayer et al., 1988). However, the role of IL-1 in stimulating neutrophils to migrate across cellular barriers is a bit more complex in in vitro conditions. ...
ENGLISH ABSTRACT: Muscle injuries are associated with changes in skeletal muscle as well as the immune system. All studies investigating possible treatment modalities have found both positive and negative effects on muscle recovery. Since no universally accepted treatment modality exists, this thesis aims to determine whether a plant-derived antioxidant, proanthocyanidolic oligomer (PCO), might prove beneficial as treatment for sports injuries in order for athletes to return to the sports field quicker. The difference in recovery of muscle following both chronic (supplementation started 14 days prior to injury and continued thereafter) and acute supplementation (supplementation started two hours after injury) were also investigated. Both chronic and acute PCO supplementation in a rat hindlimb contusion injury model resulted in earlier muscle recovery, verified by an earlier satellite cell response compared to the placebo group. This effect was most prominent already at the four hour time point following injury, compared to day seven and three after chronic and acute placebo treatment respectively. PCO supplementation also resulted in quicker foetal myosin heavy chain (MHCf) expression compared to placebo treatment. Chronic supplementation specifically resulted in a blunted circulatory pro-inflammatory cytokine response, whilst allowing for a significant increase in IL-10, an anti-inflammatory cytokine, on day three (in the PCO group only). At tissue level, the response of the muscle pro-inflammatory cytokines, TNF- and IL- 6, coincided with the satellite cell response. Macrophage infiltration into the injured muscle also followed a similar pattern to that seen for the pro-inflammatory cytokines. Macrophages invaded the injured area quicker when supplemented with PCO chronically, however, macrophage infiltration could not explain the cytokine response seen with acute supplementation. Both chronic and acute supplementation with PCO was responsible for a severely blunted neutrophil response, a novel finding of this particular antioxidant. The main findings of the in vivo rodent study were that PCO was able to blunt the neutrophil response, whilst allowing for earlier macrophage infiltration. To establish possible mechanisms by which PCO might exert these beneficial effects, further analysis included determining macrophage phenotypes and neutrophil numbers in circulation. An in vitro neutrophil migration assay was also employed to further elucidate PCO’s ability to blunt neutrophil infiltration into the injured area. For this study, conditioned plasma were harvested from experimental animals and added together with neutrophils from control rats and granulocyte colony stimulating factor (G-CSF) to the insert of the migration chamber. A chemotactic factor, N-formyl methionine-leucine-phenylalanine (fMLP), was added to the bottom well and neutrophils were allowed to migrate for two hours. Results from this study indicated that neutrophil migration was attenuated in vitro in the presence of conditioned plasma from PCO supplemented rats only. The studies in this thesis on the effect of PCO on parameters of muscle and the immune system led to the following main conclusions: a) PCO supplementation resulted in earlier muscle recovery as a result of earlier satellite cell activation and MHCf synthesis; b) PCO favours an anti-inflammatory cytokine reaction, whilst blunting the pro-inflammatory cytokine response; and c) PCO blunted the neutrophil response whilst facilitating earlier macrophage infiltration into the injured area. The specific mechanism of action of PCO to blunt the neutrophil response specifically, possibly includes the ability to suppress adhesion molecule expression on the neutrophils themselves. However, this warrants further investigation. AFRIKAANSE OPSOMMING: Spier beserings word geassosiëer met veranderinge in skeletspier sowel as die immuunstelsel. Meeste studies wat moontlike behandelingsopsies ondersoek, het beide positiewe en negatiewe spierherstel gerapporteer. Omrede daar geen universele behandelingsmoontlikheid bestaan nie, is die doel van hierdie tesis om die effek van ‘n plantgebaseerde anti-oksidant, pro-antosianiedoliese oligomeer (PSO), as ‘n voordelige behandelingstrategie vir sportbeserings te toets. Die verskil in spierherstel na beide kroniese (supplementering wat 14 dae voor besering begin is, en volgehou is daarna) en akute supplementering (supplementering het twee uur na besering begin), is ook ondersoek. Beide kroniese en akute PSO supplementering, in ‘n rot agterbeen-kneusbeseringmodel, het gelei tot vroeë spierherstel. Die bevindinge is geverifiëer deur ‘n vroeë satelietselrespons in vergelyking met die plasebo groep. Hierdie effek was reeds opvallend vier uur na besering, in vergelyking met die dag sewe en dag drie tydpunt tydens kroniese en acute plasebo behandeling onderskeidelik. In vergelyking met die kontrole groep, het PSO supplementering ook gelei to vininger uitdrukking van miosienswaarketting (MHCf). Kroniese supplementering het spesifiek gelei to ‘n onderdrukte sirkulatoriese pro-inflammatoriese sitokien response, terwyl ‘n betekenisvolle toename in IL-10 op dag drie (in die PSO groep alleenlik) waargeneem is. Op weefselvlak, het die pro-inflammatoriese sitokiene, IL-6 en TNF- , dieselfde patron gevolg as die van satelietselle. Makrofaaginfiltrasie binne die beseerde spier het ook ‘n soorgelyke patroon gevolg. Makrofage het die beseerde area vinniger geïnfiltreer in die kronies PSO-gesupplementeerde groep, maar kon nie die sitokienrespons, wat waargeneem is met akute supplementasie, verklaar nie. Beide kroniese en akute PSO supplementering was verantwoordelik vir ‘n onderdrukte neutrofiel respons, wat ‘n nuwe bevinding is vir hierdie spesifieke anti-oksidant. Die hoof bevindinge in die in vivo rotstudies, is dat PSO instaat is om die neutrofielrespons te onderdruk, en sodoende vroeë makrofaaginfiltrasie teweeg te bring. Om meganismes waarby PSO hierdie voordelige effekte veroorsaak te ondersoek, is verdere analises gedoen om makrofaagfenotipe en neutrofielgetalle in die sirkulasie te bepaal. ‘n In vitro neutrofielmigrasie studie is ook aangewend om PSO se vermoë om neutrofielinfiltrasie in die beseerde area te onderdruk, te ondersoek. Neutrofiele van kontrole rotte, tesame met gekondisioneerde plasma van eksperimentele diere en granulosiet-kolonie stimulerende faktor (G-KSF), is toegelaat om vir twee ure in die teenwoordigheid van ‘n chemotaktiese faktor, N-formiel metionien-leusien-fenielalanien (fMLP) te migreer. Resultate van hierdie studie het aangetoon dat neutrofielmigrasie, in vitro, alleenlik onderdruk word in die teenwoordigheid van gekondisioneerde plasma van PSO-gesupplementeerde rotte. Die studies in hierdie tesis oor die effek van PSO op parameters van spier en die immuunsisteem, het tot die volgende hoofgevolgtrekkings gelei: a) PSO supplementering het vroeë spierherstel, as gevolge van vroeë satelietselaktivering en MHCf sintese, teweeg gebring; b) PSO verkies ‘n anti-inflammatoriese sitokien reaksie, terwyl dit die proinflammatoriese sitokienrespons onderdruk; en c) PSO onderdruk die neutrofielrespons, terwyl vroeë makrofaaginfiltrasie in die beseerde area gefasiliteer word. Die spesifieke meganisme van aksie van PSO, om die neutrofielrespons te onderdruk, kan moontlik die vermoë van neutrofiele om adhesie molekule uit te druk, insluit. Hierdie aanname moet egter verder ondersoek word. Thesis (PhD (Physiological Sciences))--University of Stellenbosch, 2011. Includes bibliography.
... Having confirmed that UCB-9260 was selective for TNF over other superfamily members, it was tested for activity in vivo using a TNF-dependent mouse model. One such model measures the ability of exogenous TNF injected into the mouse peritoneal cavity to induce neutrophil recruitment 27 . Either human or murine TNF can be tested because both signal through murine TNFR1 28 . ...
Tumour necrosis factor (TNF) is a cytokine belonging to a family of trimeric proteins; it has been shown to be a key mediator in autoimmune diseases such as rheumatoid arthritis and Crohn’s disease. While TNF is the target of several successful biologic drugs, attempts to design small molecule therapies directed to this cytokine have not led to approved products. Here we report the discovery of potent small molecule inhibitors of TNF that stabilise an asymmetrical form of the soluble TNF trimer, compromising signalling and inhibiting the functions of TNF in vitro and in vivo. This discovery paves the way for a class of small molecule drugs capable of modulating TNF function by stabilising a naturally sampled, receptor-incompetent conformation of TNF. Furthermore, this approach may prove to be a more general mechanism for inhibiting protein–protein interactions. While biologics have been successfully applied in TNF antagonist treatments, there are no clinically approved small molecules that target TNF. Here, the authors discover potent small molecule inhibitors of TNF, elucidate their molecular mechanism, and demonstrate TNF inhibition in vitro and in vivo.
... Leukotrienes (LTB 4 and cysteinyl leukotrienes or cysLTs) seem to have an overall proinflammatory effect on innate immune cells as they have been shown to promote bacterial phagocytosis and killing in both AMs and PMNs (70)(71)(72)(73). Similarly, cysLTs have been shown to be involved in the induction of TNFα production by both AMs and recruited PMNs (74,75). Studies on the effects of prostaglandins, particularly PGE 2 , however, suggest they may negatively regulate innate immune responses (76). ...
Infectious pulmonary complications limit the success of hematopoietic stem cell transplant (HSCT) therapy in both autologous and allogeneic patients. Susceptibility to pathogens, like Pseudomonas aeruginosa and Staphylococcus aureus, persists despite successful immune reconstitution. Despite high incidence of infectious pulmonary complications following HSCT, relatively little is known about the mechanisms promoting enhanced susceptibility. Alveolar macrophages (AMs) are the sentinel phagocytes in the lung and following infection polymorphonuclear neutrophils (PMNs) assist in bacterial clearance. Previous human studies implicate AM and PMN impairment post-HSCT. Using a murine syngeneic or allogeneic bone marrow transplant (BMT) model, our studies explore the mechanisms involved in promoting HSCT AM and PMN defects. We show that syngeneic BMT mice display increased susceptibility to both P. aeruginosa and S. aureus, which correlated with impaired AM function. Altered class A scavenger receptors impaired AM uptake of P. aeruginosa but not S. aureus, while defective bacterial killing conferred overall susceptibility to these pathogens. Syngeneic BMT AM susceptibility is promoted by upregulation of prostaglandin E2 (PGE2) and its rate-limiting enzyme, cyclooxygenase-2 (COX)-2. Studies exploring the etiology of enhanced COX-2 expression revealed a loss in DNA methylation of the COX-2 promoter mediated by transforming growth factor (TGF)-??-induced miRNA-29b, resulting in elevated COX-2. Previous data show COX-2/PGE2 impaired PMN bacterial killing but had no effect on phagocytosis post-syngeneic BMT. Here we show that upregulation of COX-2/ PGE2 inhibits PMN extracellular trap (NET) formation post-syngeneic and allogeneic BMT, a novel finding that identifies PGE2 as a physiologically relevant inhibitor of NETosis. Together, these findings highlight the importance of epigenetic changes (DNA methylation and miRNA) in BMT AMs, as they directly and indirectly result in increased COX-2 expression and PGE2 production. Upregulation of this pathway establishes an immunosuppressive environment in the lung through the inhibition of AM and PMN functions. Moreover, these findings identify potential therapeutic avenues to further explore in HSCT patients.
... and leukotrien-B4(LTB4). a potent chemoattraetant ;>; t(7ra. isa poor ehemoattractant in mice ini it o [34]. ...
Intra-articular injections of murine recombinant IL-1 (mrIL-1) during the chronic phase of antigen-induced arthritis (AIA) induced a flare-up of the smouldering inflammation. The exacerbation was characterized by acute and transient joint swelling and this coincided with the extravascular accumulation of neutrophils. IL-1 injected into arthritic joints of neutropenic mice demonstrated that joint swelling was independent of the neutrophil influx into the joint. Both phenomena were absent when IL-1 was injected into a naive joint. The IL-1-induced flare-up was not T cell mediated as in the antigen-induced flare-up, and suggestive evidence is presented that IL-1 sensitivity depended on the resident macrophage population. This explained why the hypersensitivity is not restricted to the immunologically mediated arthritis but reflects a more general hypersensitivily of previously injured joints, e.g. zymosan-induced arthritis and IL-l-affected joints. In addition, IL-1 could also potentiate the antigen-specific flare-up of chronic AIA and prolongs the duration of the exacerbation. Our data indicate that joints bearing a chronic infiltrate are at risk from exacerbations in two ways: a T cell mediated rechallenge with antigen, and a non-specific reactivation by systemic and local IL-1 generation.
... At 6 h postinjection, IL-1␣ elicited the recruitment of neutrophils (ϳ 3.5 ϫ 10 5 /ml) (Fig. 4D and supplemental figure 5). This is in line with previous studies (20). Importantly, the recruitment of neutrophils triggered by IL-1␣ was greatly impaired in mice lacking CXCR2, the receptor for the chemokine CXCL1 (21). ...
... Therefore it is possible that not only IL-1β but also IL-1α mediates the FasL-induced neutrophil infiltration. The intraperitoneal injection of mice with IL-1 results in neutrophil infiltration into the peritoneal cavity 22 . Thus, our results indicate that neutrophil infiltration into FasL-expressing tumor cells proceeds as follows: FasL induces apoptosis in vanguard neutrophils, but they simultaneously release active IL-1β, which then induces massive neutrophil infiltration. ...
Fas ligand is a well-characterized apoptosis inducer. Here we demonstrate that Fas ligand induces the processing and secretion of interleukin-1β (IL-1β) in peritoneal exudate cells. This IL-1β secretion is independent of IL-1β converting enzyme (caspase 1), yet it is inhibited by caspase inhibitors, indicating that a caspase(s) in addition to IL-1β converting enzyme can process IL-1β. Inoculation of tumor cells expressing Fas ligand into wild-type mice induces a massive neutrophil infiltration that is, in contrast, suppressed in IL-1α/β knockout mice. These results demonstrate a newly discovered role for Fas ligand in inflammation, and challenge the dogma that apoptosis does not induce inflammation.
... Leukotrienes (LTB4 and cysteinyl leukotrienes or cys LTs) seem to have an overall pro-inflammatory effect on innate immune cells as they have been shown to promote bacterial phagocytosis and killing in both AMs and PMNs (Bailie, 1996; Mancuso et al., 1998; Mancuso, 2001; Serezani et al., 2005). Similarly, cys LTs have been shown to be involved in the induction of TNFα production by both AMs and recruited PMNs (Sayers, 1988; Ménard, 2000). Studies on the effects of prostaglandins, particularly prostaglandin E2 (PGE2), however, suggest they may negatively regulate innate immune responses (Aronoff et al., 2004). ...
Infectious pulmonary complications limit the success of hematopoietic stem cell transplantation (HSCT) as a therapy for malignant and non-malignant disorders. Susceptibility to pathogens in both autologous and allogeneic HSCT recipients persists despite successful immune reconstitution. As studying the causal effects of these immune defects in the human population can be limiting, a bone marrow transplant (BMT) mouse model can be used to understand the defect in mounting a productive innate immune response post-transplantation. When syngeneic BMT is performed, this system allows the study of BMT-induced alterations in innate immune cell function that are independent of the confounding effects of immunosuppressive therapy and graft-versus-host disease. Studies from several laboratories, including our own show that pulmonary susceptibility to bacterial infections post-BMT are largely due to alterations in the lung alveolar macrophages. Changes in these cells post-BMT include cytokine and eicosanoid dysregulations, scavenger receptor alterations, changes in micro RNA profiles, and alterations in intracellular signaling molecules that limit bacterial phagocytosis and killing. The changes that occur highlight mechanisms that promote susceptibility to infections commonly afflicting HSCT recipients and provide insight into therapeutic targets that may improve patient outcomes post-HSCT.
... Overexpression of VEGF in murine skin via the K14-promoter (K14-VEGF) has been reported to upregulate both angiogenesis and lymphangiogenesis (Hong et al., 2004; Nagy et al., 2002; Xia et al., 2003). TNF-α has been shown to be a potent chemoattractant for neutrophils (Lukacs et al., 1995; Sayers et al., 1988; Widegren et al., 2008). We evaluated VEGF and TNF-α expression in both unwounded and wounded TG (high expressor) and WTLM skin by Western blot (Figure 6a). ...
Chronic, nonhealing wounds and inadequate tissue repair characterized by excessive fibrosis continue to have a considerable negative effect on health and quality of life. Understanding the molecular events required for adequate healing, including the transcriptional control of wound repair, will be important for the development of future therapies. We previously showed that loss of Hoxb13 from murine skin results in enhanced cutaneous wound healing, suggesting that Hoxb13 has a negative effect on wound repair. To test this, we generated skin-specific Hoxb13 transgenic (TG) mice that overexpress Hoxb13 in the basal layer of the epidermis by the human keratin 14 promoter. Using these mice, we evaluated the effects of Hoxb13 overexpression on cutaneous wound healing. Transgenic wounds were characterized by persistence of the fibrin clot and prolonged inflammation. Notably, neutrophils, which had cleared from wild-type wounds, were still pronounced in TG wounds. Marked epidermal hyperplasia was observed at TG wound edges, and dermal vessels were grossly abnormal compared with wild-type mice. Both vascular endothelial growth factor and tumor necrosis factor-alpha were upregulated in Hoxb13 TG skin. Together, our results identify Hoxb13 as a potential important clinical target in wound healing and other pathologies characterized by abnormal or excessive inflammation, angiogenesis, or epidermal proliferation.
We have previously described the cloning of a group of novel cellular immediate-early response genes whose expression in human umbilical vein endothelial cells is induced by tumor necrosis factor alpha in the presence of cycloheximide. These genes are likely to participate in mediating the response of the vascular endothelium to proinflammatory cytokines. In this study, we further characterized one of these novel gene products named B61. Sequence analysis of cDNA clones encoding B61 revealed that its protein product has no significant homology to previously described proteins. Southern analysis suggested that B61 is an evolutionarily conserved single-copy gene. B61 is primarily a hydrophilic molecule but contains both a hydrophobic N-terminal and a hydrophobic C-terminal region. The N-terminal region is typical of a signal peptide, which is consistent with the secreted nature of the protein. The mature form of the predicted protein consists of 187 amino acid residues and has a molecular weight of 22,000. Immunoprecipitation of metabolically labeled human umbilical vein endothelial cell preparations revealed that B61 is a 25-kilodalton secreted protein which is markedly induced by tumor necrosis factor.
Ischaemia is a common clinical event leading to both local and remote tissue injury. Evidence suggests that the injury results mainly from subsequent reperfusion which causes activated neutrophils to migrate into the reperfused tissue and sequester in the lungs, inducing permeability and oedema. This thesis examines the mechanisms by which neutrophils are recruited into reperfused tissue and accumulate in the lungs, and the means by which they then induce injury. Rabbits or rats were subjected to 3 or 4 hours of bilateral hindlimb tourniquet ischaemia. When the tourniquets were released there was peripheral neutropenia, due to the microvascular adhesion of neutrophils. A chemotactic factor was generated in plasma which was identified as leukotriene (LT) B4. The plasma was capable of inducing neutrophil diapedesis which, like the neutropenia, was dependent upon the CD 18 complex of neutrophil adhesive glycoproteins. The mechanism of both the neutropenia and the diapedesis was probably a change in the conformation of existing cell surface CD 18, because there was no increase in its quantitative cell surface expression. Following reperfusion, neutrophils were sequestered locally in skeletal muscle and also in the lungs, where they induced permeability and oedema. Moreover, LTB4 was also generated in bronchoalveolar lavage fluid. Neutrophils induced lung injury via a CD 18-dependent mechanism involving elastase and reactive oxygen metabolites. Since the time course of sequestration of neutrophils in the lungs suggested that endothelium was activated directly by tumour necrosis factor-α (TNF), evidence for involvement of this cytokine was sought. TNF was identified in plasma in some, but not all, ischaemic and reperfused rats. Use of polyclonal anti-TNF anti-serum reduced the lung injury. Infusion of TNF induces a similar lung injury, and this suggests a mechanism whereby the lung is a target for reperfusion injury following hindlimb ischaemia.
Control of granulocyte production can no longer be considered in isolation from control of red blood cell, platelet, or lymphocyte production. This is because there is extensive networking with regard to regulation of these different systems, i.e., regulators and regulator cells which have effects on multiple lineages or which interact to augment effects on different hneages. The classic granulocyte-macrophage (GM) regulators, granulocyte-macrophage or granulocyte-colony stimulating factor (CSF), have major effects on in vitro red blood cell (RBC) and platelet (megakaryocyte) producdon (Metcalf et al. 1986b; Sieff et al. 1985; Robinson et al. 1987; Quesenberry et al. 1985) and probably on lymphocyte generation, while interleukin-3 (IL-3) affects megakaryocyte, neutrophil, mast cell, eosinophil, RBC, and monocyte production (Ihle 1983; Prystowsky et al. 1983). Thus, this chapter, while focusing on GM production, will of necessity touch on and describe elements of all the cell production systems housed in the marrow cavity.
The cytokine interleukin 1 β is an important mediator of inflammatory processes capable of inducing eicosanoid production, T-cell activation, and increased vascular permeability. In this study, in situ hybridization techniques were used to delineate the kinetics and cellular source of induced interleukin 1β in acute experimental colitis. The induction of interleukin 1β messenger RNA was an early phenomenon and occurred predominantly in undifferentiated cells located in the basal part of the mucosal crypts but not in differentiated enterocytes. The undifferentiated enterocytes retained the messenger RNA during differentiation and migration to more apical parts of the crypts. These results suggest that induction of interleukin 1β messenger RNA in enterocytes is causally related to the subsequent inflammatory changes seen in acute experimental colitis.
During both the estrous cycle and pregnancy, the uterus undergoes significant changes that have many similarities to inflammatory processes. These changes include edema, prostaglandin and leukotriene production, neovascularization, and infiltration of blood-borne cells including macrophages (1), lymphocytes (2), neutrophils (3, 4) and eosinophils (5); each of these responses occurs as part of the normal physiology of this organ. Leukocyte influx in the rodent uterus occurs in response to estrogen in ovariectomized adult and in immature animals (5), and this infiltration resembles one of the major events in a classical inflammatory response (6). Classical inflammatory processes entail complex interactions between many cell types and involve not only the infiltration, but also the activation of leukocytes. The concomitant production of various soluble mediators, including macrophage-derived cytokines (7, 8), is central to the inflammatory response. Although the presence of macrophages and other inflammatory cells within the endometrial stroma has been detected by morphological and histochemical means, their activation in the uterus during early pregnancy has not been examined.
The body responds to injury in an orderly and orchestrated fashion to arrest the process of injury, provide protection for the rest of the organism against further injury and begin the repair process aimed at returning the body to homeostasis. These responses are both local and systemic. Immediate local responses include alterations in vascular permeability, and the release of active components from cells, including lysosomal enzymes, vasoactive amines and arachidonic acid metabolites. Systemic responses involve the activation of phagocytic cells, further generation of arachidonate metabolites, including prostaglandins and leukotrienes, and the release of polypeptide hormones or cytokines from various cells. The systemic reaction includes fever, an increase in the circulating granulocyte pool, changes in the metabolic activity of the liver and other tissues, with the induction of synthesis and secretion by the liver of a number of plasma proteins termed the “acute phase proteins” or “acute phase reactants” (APR).
Objective To assess the effects of tumor necrosis factor- α (TNF- α ) on adhesion of endometrial stromal cells to peritoneal mesothelial cells, a possible step leading to endometriosis. Design Analysis of cell adhesion in vitro. Setting University research laboratory. Patients Peritoneal fluid and biopsies were obtained from volunteers with regular cycles without endometriosis or endometrial pathology. Interventions Tumor necrosis factor- α (0.1 to 1,000 U/mL) was added to nutrient media with the following supplements: 10% fetal calf serum, epidermal growth factor 20 ng/mL, and gentamicin 10 ng/mL. Main Outcome Measures Radioactivity of chromium-51 was used as a reflection of adherent stromal cells. Results The adherence of endometrial stromal cells to mesothelial cells was significantly increased by pretreatment of mesothelial cells with TNF- α . Conclusions Tumor necrosis factor- α may play a facilitory role in the development of endometriosis.
Cytokines are ubiquitous messenger molecules, produced not only by inflammatory cells but also by a wide variety of different cell types throughout the body. Cytokines have been shown to have effects on most in vitro functions of both neutrophils and monocytes-macrophages. The effector functions of phagocytic cells require stimuli that activate these cells. This chapter discusses information known about the effects of cytokines on phagocytic cellular functions associated with activation. Cytokines represent a more physiological stimulus for phagocytic cell activation compared to the biochemical stimuli used in vitro to define phagocyte activation states. They have been shown to both stimulate and inhibit the activation of phagocytes. The timing, dose, or combinations of cytokines to which phagocytic cells are exposed, markedly influence the type, duration, and degree of phagocytic cell effector response. Cytokines represent an important part of the language of the inflammatory response, and additional studies should more clearly define the precise role cytokines play in controlling in vivo phagocytic cell function.
Growth factors are proteins that work as paracrine or au-tocrine modulators of cell proliferation. Many growth factors have been isolated that were originally purified on the basis of a specific biological effect and subsequently named for this effect. Once the factor was isolated to homogeneity, the amino acid sequence was determined and recombinant proteins developed. Upon testing of recombinant proteins in a variety of systems it was found that many factors not only have overlapping functions but also act on a wide range of cell types to produce a variety of actions. In addition to the effects of individual factors on cells involved in the wound healing process, many of these factors interact with other substances to antagonize or synergize their effects, perhaps through the use of similar second-messenger systems or modulation of receptor-ligand affinity. The susceptibility of tissues to modulation by growth factors may change as wound healing progresses.
It is general experience that some patients receiving long-term peritoneal dialysis suffer from repeated episodes of peritonitis while others remain relatively free of this complication. It has been suggested that differences in the ability of individual subjects to resist infection, i.e., their host defence, are at least partly responsible for this clinical observation. This hypothesis was strengthened by the report by Verbrugh et al. on the potential efficacy of peritoneal macrophages and opsonins in the antibacterial defence of such patients [1]. In this chapter we review the available evidence on the various components of host defences of peritoneal dialysis patients including the effect of the dialysate.
The cytokines IL-1 and TNF cannot by themselves account for the pathology of rheumatoid arthritis (RA) or osteoarthritis. Nonetheless, their actions mimic in detail many processes observed in arthritic disease, and circumstances that could lead to continuous synthesis and release of IL-1 or TNF would be expected to lead to a chronic arthritis. Thus, both IL-1 and TNF could play a significant role in the pathogenesis of arthritis. Moreover, the ability of the cytokines to cause a flare or increase the severity of an arthritis suggest that they would play a role even in arthritis lacking a cytokine etiology if they were induced by concurrent infection or other processes. These models of cytokine-induced arthritis then have value for studies into possible mechanisms of arthritic disease and as tools for design and development of cytokine-directed therapy. Transgenic animals may prove to be a useful tool in understanding the pathogenesis of arthritic diseases by demonstrating the role of cytokines and growth factors. In vitro studies often fail to mimic the full range of physiological and pathological effects that these products have in the whole animal.
The importance of locally produced prostaglandins (PGs) in the inflammatory response has been well recognized since the observation by Vane and colleagues that aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) inhibit PG synthesis1–3. More recently, it has been suggested that other products of arachidonic acid metabolism, such as leukotrienes, are also mediators of allergic and inflammatory reactions. Inhibitors of leukotriene synthesis and antagonists of their action have recently been developed and are presently undergoing clinical evaluation; the exact role and importance of these mediators in human disease will be clarified only when the outcome of these clinical studies is known. In this review we will describe the pharmacological effects of selective lipoxygenase inhibitors in tests conducted in animals, and will discuss the possible relevance to humans.
Adequate biological defences against infection by foreign organisms are essential to the survival of the host. A phylogenetically relatively primitive, but nevertheless essential, aspect of these defences is the inflammatory response. In contrast to the immune responses involving antibodies and effector T lymphocytes, inflammation is relatively non-specific, but it does provide a quick and usually effective anti-invasive response. The inflammatory response is mediated by host cells and their soluble products, the cytokines, and in general is beneficial to the host, limiting potentially lethal infections. However, inflammation may also be accompanied by local pathological effects leading to host tissue damage or to systemic deleterious effects such as shock and catabolic states. These inflammatory changes are orchestrated and mediated by cytokines. Tumour necrosis factor (TNF) is one of these soluble mediators and it appears to have a central role in the initiation, development and augmentation of inflammation, as well as in the tissue remodelling and healing events occurring after the response.
It is now known that there are two classes of IL-1 molecules known as IL-1α and IL-1β, each encoded by a single gene. The IL-1 literature prior to 1984, when the first IL-1 cDNAs were cloned (Lomedico et al. 1984; Auron et al. 1984), has been comprehensively reviewed (Dinarello 1984). This review will therefore largely focus on the literature from 1984 onward.
It is now known that there are two classes of IL-1 molecules known as IL-α and IL-1β, each encoded by a single gene. The IL-1 literature prior to 1984, when the first IL-1 cDNAs were cloned (Lomedico et al. 1984; Auron et al. 1984), has been comprehensively reviewed (Dinarello 1984). This review will therefore largely focus on the literature from 1984 onward.
Cows (48) tested positive for endometritis on the basis of appearance of cervical mucus and white side test were randomly divided into 4 groups with 12 cows in each group. In group A, 100 μg of Escherichia coli LPS once, in group B, 500 mg OG once, in group C, enrofloxacin was infused intrauterine for 3 consecutive days and in group D, 20 ml PBS was infused once which served as control group. Endometrial biopsy was collected at 0 h and 72 h from all the cows for histopathological study. Uterine flushing was collected at estrus prior to infusion of drug (0 h) and at 24, 48 and 72 h post treatment for evaluation of changes in total protein concentration, TLC, PMNs%, and the total immunoglobulin concentration. The E. coli LPS and OG treatment significantly increased (P<0.05 or P<0.01) the total protein concentration, TLC, PMN s% and the concentration of total immunoglobulin at 24, 48 and 72 h post treatment. However, intrauterine enrofloxacin therapy reduced (P<0.01) the total protein concentration and PMN s% at 72 h post treatment than pretreatment levels. It is concluded that E. coli LPS and OG may show better therapeutic efficacy to stimulate non-specific uterine defense mechanisms in crossbred cows suffering from endometritis. Further, E. coli LPS and OG may show better therapeutic efficacy than intrauterine enrofloxacin in curing the endometritis in crossbred cows.
It is well known that recombinant human granulocyte increases dramatically the number of granulocytes in peripheral blood. It is interesting to see if G-C SF enhances the functions of neutrophil since the neutrophil is one of the most important factors of host defence mechanism against infections. In this study, we have demonstrated that G-CSF induced the polarization of neutrophil while it could not be chemoattractant for the cell.Polarization is considered to be the initial reaction of chemotaxis. Therefore, G-CSF might enhance the defence effect of the neurophil to infections in this point.
The movement of leukocytes from blood into the tissues in response to inflammatory stimuli was observed and described as early as 1891 by Metchnikoff1 and 1888 by Leber2; however, only within the past few decades has some light been shed on the cellular and molecular mechanisms involved in the process of leukocyte emigration. During inflammation, blood leukocytes:(1) marginate and adhere to the endothelial cells of the postcapillary venules,(2) migrate across the endothelial cell layer and basal membrane, and (3) move in the tissues towards the source of inflammation where they fulfil their function in host defence. Whereas altered parameters of circulation and the expression of recently identified adhesion molecules by the leukocytes and endothelial cells are responsible for margination and adhesion, the chemotaxis of leukocytes (their capacity of directed movement along a concentration gradient of molecules referred to as chemotaxins or chemoattractants) is considered essential for leukocyte emigration and movement in extravascular tissues.
Recombinant human interleukin 1 receptor antagonist (IL-1ra) and 35F5, a neutralizing monoclonal antibody (mAb) to the type I mouse IL-1 receptor, were examined for their ability to bind to IL-1 receptors (IL-1Rs) on various types of mouse cells and to block immune and inflammatory responses to IL-1 in vitro and in mice. IL-1ra competed for binding of 125I-IL-1 alpha to type I IL-1R present on EL-4 thymoma cells, 3T3 fibroblasts, hepatocytes, and Chinese hamster ovary cells expressing recombinant mouse type I IL-1R. The IC50 values for IL-1ra binding (ranging from 2 to 4 ng/ml) were similar to those of IL-1 alpha. In contrast, IL-1ra bound with very low affinity (IC50 values ranging from 10 to 200 micrograms/ml) to cells expressing type II IL-1R, i.e., 70Z/3 pre-B cell line and polymorphonuclear leukocytes (PMN) derived from bone marrow and acute inflammatory exudates. The mAb 35F5 bound specifically to type I IL-1R; no inhibition of 125I-IL-1 alpha binding to cells having type II IL-1R was observed with very high concentrations of antibody. While neither IL-1ra nor 35F5 had intrinsic activity in bioassays using T helper D10.G4.1 cells and mouse thymocytes, both agents blocked the ability of IL-1 to stimulate proliferation of these cells. The effects of IL-1ra and 35F5 on acute inflammatory responses in mice were also evaluated. IL-1ra and 35F5 blocked the local accumulation of PMN after intraperitoneal injection of rIL-1 alpha. The response to IL-1 was inhibited when IL-1ra or 35F5 was administered simultaneously with or before administration of IL-1. IL-1ra and 35F5 also blocked PMN accumulation after intraperitoneal injection of lipopolysaccharide or proteose peptone, suggesting IL-1 is important in mediating responses to these agents. In addition, IL-1ra and 35F5 significantly blocked the ability of IL-1 to stimulate egress of PMN from bone marrow, to induce a transient neutrophilia, and to elevate serum levels of hepatic acute phase proteins, IL-6, and corticosterone. Thus, IL-1ra and 35F5 competitively inhibit the binding of IL-1 to the IL-1R on certain cell types. These two IL-1 receptor antagonists act to inhibit biological responses induced by IL-1 and other inflammatory agents.
Purpose:
Using an in vitro cell culture model, bovine lactoferrin (BLF) stimulates healing of alkali-induced human corneal epithelial wounds. The present study examined the efficacy of BLF in promoting healing of corneal injury in vivo and explored BLF modulation of interleukin-1 (IL-1) during wound healing.
Methods:
Alkali injury was induced to BALB/c mice by exposure of the mouse cornea to a sodium hydroxide (NaOH)-soaked filter disc for 2 min. The corneal surface was irrigated after the injury with saline. Topical BLF in phosphate buffered saline (PBS) (10 µl, 62.5 μM), bovine serum albumin (BSA) (10 µl, 62.5 μM in PBS) or PBS only (10 µl) were applied three times daily to both the alkali-injured and uninjured eyes for 3 d. Wound healing was assessed using 0.1% fluorescein staining under slit lamp microscope. The corneas at 6 h, 24 h or 3 d post-injury and treatment were excised and examined histologically, homogenized corneal tissue was evaluated for expression of IL-1α and IL-1β.
Results:
After 6 h post-wounding and treatment no significant reduction of wound area was observed between treatments and infiltrating cells or IL-1 expression were not elevated in any group. By 24 h, BLF-treatment resulted in accelerated wound closure (100%) compared to PBS and BSA treatment (70% and 65%, respectively). BLF treatment reduced infiltrating cells compared to controls and no elevation of IL-1, whereas controls displayed elevated infiltrating cells and increased levels of IL-1. After 3 d, mice treated with BLF exhibited complete wound closure while control corneas still exhibited some minor defects. Resolution of inflammation with minimal remaining infiltrating cells was observed in all corneas by day 3, coincident to normal levels of IL-1α and IL-1β.
Conclusion:
BLF accelerated healing of corneal alkali injury in BALB/c mice which was associated with suppression of IL-1 and reduced infiltrating cells.
Mucosal inflammation and damage to the bronchial epithelium are two important pathophysiological components in asthma. The links between these two events remain poorly understood. The recent description of fibrosis lesions under the basal membrane of the bronchial epithelium in this disease led us to envisage such fibrosis as a true attempt at healing of the inflammatory process, itself induced by epithelial aggression. This general review contains thoughts regarding the immunobiochemical mechanisms capable of orchestrating these different stages of inflammation.
Objective: Fas ligand (FasL) is an important mediator of immune function and induces apoptosis by binding to its receptor Fas on sensitized cells. It has recently been shown that malignancies may express FasL and acquire immune privilege by inducing apoptosis of lymphocytes. Acquired resistance to Fas mediated apoptosis is known to be an early event in carcinogenesis. The aim of this study was to determine the extent of FasL expression in patients with colorectal cancer and examine its relationship with several prognostic pathological features and survival. Design and methods: Sixty-eight patients (median age 66 years) with colorectal cancer, whose diagnosis was made between 1988 and 1991 and in whom long-term follow-up was available, were evaluated. The tumours were of varying stages at diagnosis (eight Dukes' A, 28 Dukes' B, 23 Dukes' C and nine Dukes' D). The expression of FasL was detected immunohistochemically with a rabbit polyclonal IgG using the DAKO EnVision+ System. The specificity of FasL binding was confirmed by preincubation of the antibody with the immunizing peptide prior to staining. The relationship with several pathological features was determined using Kendalrs τ-b correlation. Overall survival was estimated using the Kaplan-Meier product limit curves. Differences in observed survival were tested for statistical significance using the Mantel-Haenszel log rank test. Both the extent and intensity of staining were graded by a blinded observer. Results: FasL was predominantly expressed in tumour epithelial cells in 88% of the cases. The positive staining of tumours varied in extent. FasL staining was higher in earlier Dukes' stage turnouts in that the extent of FasL staining negatively correlated with Dukes' stage (Kendall τ-b = -0.22, P = 0.038). Consistent with this, the overall survival was better with a greater extent of FasL expression (log rank χ2 = 5.68, P = 0.017). There was a lower extent of FasL expression in mucinous adenocarcinomas (Kendall τ-b = 0.288, P = 0.01) and in those tumours with neural invasion (Kendall τ-b = -0.26, P = 0.03). No relationship was detected between FasL and turnout site, size, margin, differentiation, vascular invasion, necrosis or Crohn's-like reaction. Conclusions: FasL is widely expressed in colorectal cancers. This finding suggests that the extent of FasL expression in colorectal turnouts is directly related to patients' survival.
In vitro experiments were conducted to determine the effects of recombinant cytokines on phagocytic and oxidative burst activities of bovine neutrophils. Neutrophils were isolated (purity >91%, viability >97%) from EDTA-anticoagulated blood from healthy Holstein-Friesian heifers. Aliquots of neutrophils (10 × 106 cells/ml) were incubated for 1 h at 37 °C with equal volumes of recombinant human cytokines, namely, tumour necrosis factor-alpha (rhTNF-α, 0.5–1000 ng/ml), interleukin-1-alpha (rhIL-1-α, 0.001–10 ng/ml), interferon-gamma (rhIFN-γ, 0.01–100 ng/ml), granulocyte colony-stimulating factor (rhG-CSF, 25 ng/ml), and granulocyte-macrophage colony-stimulating factor (rhGM-CSF, 10 ng/ml). Then, the percentage phagocytosis and average number of intracellular bacteria per cell were evaluated by flow cytometry and/or fluorescent microscopy using FITC-labelled opsonised bacteria (Escherichia coli 0111:B4). Unlabelled opsonised bacteria and dichlorofluorescin diacetate were used to evaluate H2O2 production, a measure of oxidative burst, by flow cytometry. The results showed that all five cytokines significantly (p 2O2 production (31.3–58.2%) when compared to untreated neutrophils. A gradual increase in mean channel fluorescence but not in percentage phagocytosis was consistently seen with increasing concentrations of rhTNF-α, rhIL-1-α, and rhIFN-γ, thereby indicating a concentration-dependent stimulation of phagocytic capacity by these three cytokines.
A recombinant soluble form of human Fas ligand (sFasL) was tested for its chemotactic activity against human and mouse polymorphonuclear neutrophils (PMN) by the Boyden chamber method. sFasL exhibited a potent chemotactic activity against both human and mouse PMN and HL-60 cells when differentiated into neutrophils or monocytes. A neutralizing anti-FasL mAb abolished the chemotactic activity, while control mAb did not. Ligation of Fas by either IgM- or IgG-type anti-Fas mAb also induced PMN migration. PMN derived from lpr mice that express few Fas molecules did not respond to sFasL. In contrast, those derived from lprcg mice that express Fas molecules with a mutated death domain normally responded to sFasL chemotaxis. These results directly indicated a chemotactic activity of sFasL against PMN and suggest a novel signaling function of Fas, which appears to be independent of the death domain-mediated apoptosis.
Accumulation of neutrophils within the epidermis and formation of pustules represents a unique, however, ill-understood phenomenon in human skin. In this overview, we compare localized versus systemic pustular disorders. Localized pustules are mostly infectious and appear to be generated by the interaction of skin cells with Th17 cells and macrophages. IL-1β, IL-17 and TNF-α in concert stimulate IL-8 secretion by keratinocytes. This cutaneous defense circuit creates the ensuing pustular pathology. In systemic pustular disease, dendritic cells (DCs) are activated by TLR-mediated signaling. IL-23 secreted by myeloid DCs feeds the cutaneous defense circuit, which results in neutrophilic infl ammation and pustules, as seen in infectious pustules. The distinction between local and systemic infl ammation, leading to neutrophil migration into the epidermis appears to be helpful in improving understanding of these common disorders of the skin.
Cutaneous exposure to sulfur mustard [bis(2-chloroethyl) sulfide (SM)] produces a delayed inflammatory skin response that is followed by severe dermal injury. Assessment of anti-inflammatory therapies against SM-induced skin injury has mainly relied on qualitative histopathological evaluation. The goal of this study was to identify proinflammatory biomarkers in the hairless mouse vesicant model that could be used as additional indicators of SM-induced skin injury for evaluating anti-inflammatory treatment. SM-induced inflammation was determined at 2, 6, and 24 hr postexposure by changes in edema. Ribonuclease protection assay (RPA) was used to determine changes in gene expression of inflammatory mediators. At 2, 6, and 24 hr postexposure, a time-dependent increase in edema was observed in SM-exposed skin, which was significant at 6 and 24 hr when compared to unexposed controls. Ribonuclease protection assay analysis revealed a two-fold or greater increase in monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-2 (MIP-2), MIP-1α, tumor necrosis factor-α, and interleukin (IL)-1β following exposure to SM when compared to unexposed controls. A significant time-dependent increase was observed in MCP-1, MIP-1α, and IL-1β over the 24 hr time period. At 24 hr postexposure, skin treated with the anti-inflammatory drug olvanil showed a significant decrease in SM-induced edema. Additionally, mRNA levels of MCP-1, MIP-2, and IL-1β were decreased when compared to skin exposed to SM alone. In this study, we identified molecular biomarkers at the mRNA level, up-regulated in skin exposed to SM, which can be partially suppressed by olvanil. Further characterization of the mRNA and protein expression patterns of proinflammatory biomarkers may enable the use of other classes of anti-inflammatory drugs or therapeutic treatments against SM dermal injury.
Treatment of the dorsal epidermis of SENCAR mice with 12-O-tetradecanoylphorbol-13-acetate (TPA) induced a time- and dose-dependent stimulation of interleukin-1α (IL-1α) gene expression. Levels of IL-1α mRNA were elevated as early as 15 min and peaked at 3–4 h after a single application of TPA (2 μg or 10 μg). IL-1α gene expression increased in epidermal tissue isolated from SENCAR mice at 1, 3, 6, 10, 16, and 22 wk after a single treatment with 10 nmol 7,12-dimethylbenz[a]anthracene (DMBA) and subsequent twice-weekly application of 2 μg TPA. IL-1α-immunoreactive protein was specifically localized within suprabasal keratinocytes in cutaneous tissue isolated from mice treated with DMBA-TPA for 1–22 wk and in nonproliferating cells located within papilloma tissue isolated from SENCAR mice at 22 wk after initiation and promotion. Basal cells within hyperplastic epidermis did not produce IL-1α-immunoreactive protein. DMBA treatment alone did not induce IL-1α gene expression. Injection of IL-1α-specific antibodies (50 μg) into SENCAR mice via the tail vein 2 h before treatment with TPA (2 μg or 10 μg) significantly (P < 0.05) inhibited the skin thickening usually observed 24 h after treatment with TPA. Autoradiography studies showed that injection of anti—IL-1α antibodies inhibited incorporation of [methyl-3H]thymidine by keratinocytes within the epidermis and by cells within hair follicles. It also inhibited neutrophil infiltration into the dermis, which usually results from topical application of TPA. These data suggest that IL-1α is a pivotal cytokine produced by specific subpopulations of epidermal keratinocytes and that IL-1α primarily regulates the epidermal proliferative response of a distinctly separate population of keratinocytes after topical exposure of murine epidermis to TPA and secondarily modulates neutrophil migration into the dermis. Consequently, manipulation of IL-1α may be a way to attenuate or abrogate the cutaneous response to TPA by altering keratinocyte proliferation, the resultant hyperplasia, and a portion of the inflammatory response characterized by dermal infiltration of neutrophils.
Our understanding of the process of peritoneal infection and the key role played by resident cells (mesothelial cells, peritoneal macrophages and lymphocytes) and their secreted mediators (cytokines, chemokines and prostaglandins) in host defence has increased significantly over the past decade. This is in part as a result of the increasing use of peritoneal dialysis (PD) as a form of renal replacement therapy. Peritoneal infection is the single largest reason for treatment failure and infection associated inflammation is believed to be responsible for the observed changes in the peritoneal membrane structure (fibrosis and angiogenesis) that precede treatment failure. As a result of this clinical problem research in the area concentrated initially on understanding inflammation in the dialysed peritoneum. More recently, however, more fundamental questions about the process and stages of the peritoneum''s response to infection have been addressed.
We have investigated the mechanisms of transmembrane signalling implicated in the activation of the respiratory burst of adherent neutrophils by tumor necrosis factor-α/cachectin (TNF). The activation of the respiratory burst by TNF is insensitive to pertussis toxin and weakly sensitive to protein kinase C inhibitors. Cytochalasin B and dibutyryl cyclic AMP have an inhibitory effect. The activation of the respiratory burst by TNF takes place in the absence of formation of 3H-inositol phosphates, 32P-phosphatidic acid, and 3H-arachidonic acid. These results demonstrate that the activation of the respiratory burst by an endogenous, physiologic stimulus can be independent of the formation of messengers derived from hydrolysis of phosphoinositides.
This study investigated the effect of a novel LTD4 receptor antagonist LY203647 on Salmonella enterindis endotoxin-induced shock sequelae in anesthetized rats. LY203647 (30 mg/kg i.v.) or vehicle was given 10 min prior to endotoxin (10 mg/kg i.v.) or its vehicle, and the hematocrit, mean arterial pressure and circulating leukocyte counts were determined. LY203647 significantly inhibited endotoxin-induced hemoconcentration up to 90 min post-endotoxin (46.7 ± 1.3 vs. 51.9 ± 2.4% at 30 min post-endotoxin, 45.9 ± 1.1 vs. 53.1 ± 1.4% at 90 min post-endotoxin, N = 8–9, P < 0.05). The endotoxin-induced decreases in mean arterial pressure were also attenuated by LY203647, −29 ± 5 vs. −56 ± 9 mm Hg at 60 min post-endotoxin and −42 ± 4 vs. −60 ± 9 mm Hg at 90 min post--endotoxin (N = 9–10, P < 0.05). LY203647 also attenuated endotoxin-induced decreases in leukocyte count in arterial blood. A study of differential counts in circulating leukocytes (N = 3) showed that endotoxin induced complete disappearance in circulating neutrophils. The circulating lymphocyte count was decreased by 30 ± 10 and 41 ± 1% at 15 and 30 min post-endotoxin, respectively. LY203647 inhibited endotoxin-induced lymphopenia (P < 0.05) but failed to alter endotoxin-induced neutropenia. These data suggest that LTD4 may play an important role in mediating hemoconcentration, hypotensive and lymphocytopenic sequelae of endotoxin shock.
Eine der bedeutendsten Komplikationen der Abdominalchirurgie ist die Bildung von postoperativen Verwachsungen. Innerhalb der Peritonealhöhle spielt als Ursache einer Adhäsionsbildung ein gestörtes Gleichgewicht antifibrinolytischer und profibrinolytischer Prozesse eine entscheidende Rolle. Eine Exsudation von Fibrin in die Bauchhöhle bedeutet einen Reiz für Fibroblasten, das Exsudat zu organisieren. Es entwickelt sich eine dauerhafte Organverwachsung, deren gefürchtetste Komplikation ein Ileus ist. Humane peritoneale Mesothelzellen (HOMC), die größte Zellpopulation intraabdominal, exprimieren sowohl das fibrinolytische Enzym t-PA (tissue-Plasminaogenaktivator) als auch dessen Inhibitor PAI-1 (Plasminogenaktivatorinhibitor-1). Sie sind an dem fibrinolytischen Gleichgewicht maßgeblich beteiligt. Unsere Untersuchungen an kultivierten humanen Mesothelzellen konnten einen Anstieg von PAI-1 und einen gegenläufigen Abfall von t-PA unter entzündlicher Stimulation zeigen. Die Zellstimulation erfolgte durch TNF-alpha (Tumornekrosefaktor-alpha), TGF-beta (Transforming-growth-Faktor-beta) und IL-1beta (Interleukin-1beta). Dabei erlaubte unser Fibrinclotmodell einen genauen inblick in die zeitlichen Abläufe der Fibrinolyse. Darüber hinaus untersuchten wir den Einfluss von aktivierten Granulozyten auf die fibrinolytische Potenz der Mesothelzellen. Eine Stimulation der HOMC mit Granulozytenüberständen führte zu einer Abnahme der PAI-1 Sekretion und damit zu einer scheinbaren Unterstützung des Lyseprozesses. Dabei blieb unklar, ob dieser Effekt auf einer regulatorischen Funktion der Granulozyten oder auf eine Zellschädigung der Mesothelzellen durch Superoxydionen, Wasserstoffperoxyd und Hydroxidradikale, die von den Granulozyten sezerniert werden, hervorgerufen wird. One of the most important complications of abdominal surgery is the formation of postoperative adhesions. A major factor for adhesion formation is an inbalance of pro- and antifibrinolytic intraabdominal processes. Deposition of a fibrin-rich exsudate leads fibroblasts to organisate the exsudate. A permanent adhesion with the fatal complication of inducing an ileus develops. One of the key players in regulation the pro- and antifibrinolytic process are mesothelial cells, the biggest cell population intraabdominal. They produce the fibrinolytic enzyme t-PA (tissue plasminogen-activator) and its inhibitor PAI-1 (plasminogen-activator-inhibitor-1). Our investigations of cultured human mesothelial cells show an increase of PAI-1 and a decrease of t-PA concentration under inflammatoric stimulation. We used TNF-alpha (tumor-necrosis-factor-alpha), TGF-beta (transforming-growth-factor-beta) und IL-1beta (interleucin-1beta) for stimulation. Our fibrin-clot model allowed an investigation of the time-dependant effects. Moreover we investigated the influence of activated granulocytes on the fibrinolytic potency of the mesiothelial cells. A stimulation of the HOMC with supernatants of granulocytes leaded to a decrease of PAI-1 secretion and therefore to an apparent support of the lytic process. It was not clear if this effect was based on the regulatoric function of the granulocytes or on the cell destruction of the mesothelial cells, which was caused by superoxyd ions, hydrogenperoxyd and hydroxid radicals, produced by the granulocytes.
The 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase inhibitors (statins) are a class of drug used to lower low-density lipoprotein (LDL) levels. However, in recent years, statins have been shown to possess a pleiotropic effect beyond its cholesterol lowering ability, including attenuating the effect of ischaemia reperfusion injury. This review considers the biomolecular processes that may lead to this beneficial effect.
Endogenous danger signals released from necrotic cells are thought to be sensed by phagocytes leading to secretion of IL-1alpha and neutrophilic recruitment. However, the mechanisms for IL-1alpha production and IL-1alpha-mediated sterile inflammation remain poorly understood. We report here that necrotic cell extracts elicited little secretion of CXCL1 and IL-6 from macrophages but robust production in mesothelial cells. The induction of CXCL1 as well as activation of NF-kappaB and MAPKs by cytosolic extracts required the presence of IL-1alpha in the necrotic cell. Conversely, expression of IL-1R and MyD88 but not IL-1alpha, RICK, TLR2, TLR4, TRIF, or inflammasome components in mesothelial cells was critical for the production of CXCL1. Furthermore, IL-1alpha was critical to induce the recruitment of neutrophils in the peritoneal cavity via CXCR2. These studies show that IL-1alpha is a key danger signal released from necrotic cells to trigger CXCL1 secretion and recruitment of neutrophils via IL-1R/MyD88 on neighboring mesothelial cells.
The present study demonstrates that tumour necrosis factor (TNF) and FMLP, but not IL-1 or IL-8, enhanced the adherence of polymorphonuclear neutrophil (PMN) to fibronectin, an extracellular matrix protein. The adherence induced by FMLP was very rapid, within 5 min while the induction of adherence by TNF was much slower, reaching maximum at 60 min. TNF also enhanced an adhesion of PMN to other extracellular matrix proteins, such as laminin, collagen IV and gelatin II, but not to human serum albumin. Anti-CD18 MoAb completely inhibited the binding of TNF-stimulated PMN to fibronectin and partially inhibited the binding to laminin. Further investigation showed that adhesion of TNF-stimulated PMN to fibronectin and laminin was inhibited by anti-CD11b MoAb and to a lesser extent by CD11a MoAb. In contrast to TNF-stimulated PMN the binding of unstimulated PMN to fibronectin and laminin was only inhibited by anti-CD11a MoAb. Anti-CD11c had no effect on PMN adherence. These results suggest that unstimulated PMN adhere to extracellular proteins through the CD11a/18, while TNF-stimulated PMN adhere through the CD11b/18. These results suggest that TNF secreted at the site of inflammation may enhance the interaction of PMN with the extravascular environment through the CD11b/18 complex.
Treatment of vascular endothelial cells with inflammatory cytokines stimulates surface expression of E-selectin (previously known as endothelial-leukocyte adhesion molecule-1) and promotes the transendothelial migration of neutrophils. To assess participation of E-selectin in cytokine-mediated neutrophil migration, an in vitro model consisting of monolayers of human umbilical vein endothelial cells (HUVEC) grown on amniotic connective tissue was used. When HUVEC-amnion cultures were stimulated for 4 h with relatively low concentrations of IL-1 (0.1 to 0.15 U/ml), mAb BB11 or H18/7 to E-selectin partially inhibited migration of subsequently added neutrophils. However, when the cultures were stimulated with 15 U/ml of IL-1 for 4 or 24 h, little to no inhibition was observed. mAb to E-selectin also failed to inhibit migration of neutrophils across HUVEC-amnion cultures treated with low doses of IL-1 when the leukocytes were additionally stimulated by the chemoattractant leukotriene B4. In contrast, migration of neutrophils across IL-1-treated HUVEC was profoundly inhibited by mAb to CD11/CD18 leukocytic integrins under all conditions tested. Results of these studies suggest that participation of E-selectin is not essential for migration of neutrophils across cytokine-stimulated HUVEC in vitro; rather, E-selectin can be bypassed in favor of CD11/CD18-dependent mechanisms under appropriate circumstances.
The start of the field of cytokine biology has been variously dated to 1951 with the discovery of nerve growth factor, 1954 with the discovery of interferon, the 1960s with the first description of "lymphokines," and 1966 with the discovery of "migration inhibitory factor" (1). These observations were followed by a virtual avalanche of descriptions of bioactivi ties in supernatants from unstimulated and stimulated cell populations. The implicit belief behind these studies was that biologic processes are mediated by mono-functional mole cules that are produced by distinct populations of cells and regulate the function of other well-defined cell populations in the absence of input from the microenvironment. More re cent studies, however, have markedly revised these assump tions. We now know that cytokines are produced by and regulate the function of virtually all nucleated cells. We also know that cytokines, with few exceptions, are multifunc tional molecules that can regulate a wide spectrum of bio logic events relevant to inflammation, metabolism, cell growth and differentiation, morphogenesis, fibrogenesis, and/or homeostasis. Importantly, we also now appreciate that the biologic effects of cytokines are often situation specific since they vary depending on the concentration of the cytokine, the state of activation and/or maturation of the target cell, the composition and state of degradation of the surrounding matrix, and the presence or absence of other cytokines in the local microenvironment. The realization that the bioactivities of most cytokines are so diverse as to seem unrelated to one another and the appreciation of the highly complex processes that modulate cytokine effector function have resulted in the concept of cytokine networking. In this conceptualization, biologic events are believed to be regulated by networks or cascades of interacting cytokines. In these networks, individual cytokines can be best viewed as specialized symbols in a language of intercellular commu nication whose meaning is controlled by context (1, 2). It is only with an appreciation of this context that the biologic role of a cytokine can truly be appreciated.
Conventional cytotoxic chemotherapy fails to cure the majority of patients with advanced-stage ovarian cancer, in spite of encouraging initial antitumor responses. With the emergence of drug resistance in refractory tumors, new biologic and immunologic treatment strategies are needed. Small-volume residual peritoneal disease remains an attractive target for therapeutic trials; however, even in this optimal circumstance, few regimens have yet achieved a high frequency of pathologically confirmed complete remissions. Considerable progress has been made in understanding the impact of growth factors and their receptors on tumor growth regulation and modulation of response to chemotherapy. Better characterization of the antigens recognized by monoclonal antibodies, as well as sequencing of the antibodies themselves, has permitted the rational design of therapeutic reagents that take full advantage of molecular biology techniques for production and conjugation. Important limitations of preclinical models for prediction of host toxicity are recognized, and the reasons for treatment failure in situ, as well as strategies to prevent serious dose-limiting toxicities, are being explored. Further developments in cytokine biology, adoptive cellular therapy, monoclonal antibody conjugation, and molecular biology will continue to provide a growing array of reagents for critical evaluation.
As a putative mediator of inflammation interleukin-1 has been implicated in the recruitment of leukocytes during the early stages of the inflammatory reaction. In the present report we have investigated the release of endogenous IL-1 in the rat zymosan pleurisy and in the mouse zymosan peritonitis. In both cases the release of the cytokine was maximal 4 hours after zymosan injection and appeared to be time-related to neutrophil migration into the inflammatory site. The effect of in vivo treatment with dexamethasone in rat pleurisy and with polyclonal anti-murine IL-1 beta antibody in mouse peritonitis was also assessed. The steroid reduced both cell migration and the release of IL-1-like activity as well as the formation of exudate and the release of eicosanoids. The anti-IL-1 beta serum inhibited selectively the number of neutrophil that migrated to the inflamed site (approximately 40%) and the IL-1 activity recovered in (approximately 70%) the exudate. In vitro incubation of the inflammatory exudate with polyclonal anti-murine IL-1 alpha or anti-murine IL-1 beta sera allowed the identification of the IL-1 species present. In the rat pleurisy IL-1 biological activity was mainly due to the alpha species, whereas IL-1 beta was the only species apparently present in the mouse peritoneal exudate.
Tumor necrosis factor/cachectin (TNF) has been implicated as a mediator of the host response in sepsis and neoplasia. Recent work has shown that TNF can modulate endothelial cell hemostatic properties, suggesting that endothelium is a target tissue for TNF. This led us to examine whether endothelial cells have specific binding sites for TNF and augment the biological response to TNF by elaborating the inflammatory mediator, IL-1. Incubation of 125I-recombinant human TNF with confluent, cultured human umbilical vein endothelial cells resulted in time-dependent, reversible, and saturable binding. Binding was half-maximal at a TNF concentration of 105 +/- 40 pM, and at saturation 1,500 molecules were bound per cell. Heat-treated TNF, which is biologically inactive, did not bind to endothelium. In addition to surface binding, TNF induced the elaboration of IL-1 activity by endothelial cells in a time-dependent manner. Generation of IL-1 activity required protein synthesis and was half-maximal at a TNF concentration of 50 +/- 20 pM. IL-1 activity from TNF-treated endothelium could be adsorbed by an immobilized antibody to IL-1. Heat-treated TNF was ineffective in eliciting endothelial cell IL-1. These data indicate that TNF can bind specifically to endothelium and initiate a cascade of inflammatory and coagulant events on the vessel surface potentially central to the host response to neoplasia and sepsis.
Products of bacteria are potent chemoattractants for mammalian leukocytes. Several reports suggest that these attractants are small peptides. We compared the properties of culture fluids of Staphylococcus aureus with fMet-Leu-Phe, considered a prototype of bacterial attractant. Chemotactic activity for human monocytes of Staph. aureus culture filtrates was determined in multiwell chemotaxis chambers. At optimal concentrations, the filtrate attracted almost twice as many monocytes as fMet-Leu-Phe (53 +/- 5% of input number compared with 30 +/- 3%, in a series of ten experiments). Gel-filtration characteristics and susceptibility to proteolytic digestion suggested that chemotactic activity was due to peptides with a molecular size range of 500-2,000 daltons. Reverse-phase high-pressure liquid chromatography (HPLC) of unfractionated filtrate revealed nine peaks of chemotactic activity, most of which was in five of the peaks. One peak accounted for 40% of total activity. Individual peaks, like the unfractionated material, were capable of attracting about twice as many monocytes as the optimal concentration of fMet-Leu-Phe. Quantitative bioassay of the HPLC peaks showed that only 5% of the total Staph. aureus chemotactic activity eluted in the position of fMet-Leu-Phe. This is in contrast to the report that fMet-Leu-Phe accounted for 70% of neutrophil lysosomal enzyme-releasing activity of Escherichia coli culture fluid. In summary, chemotactic activity for monocytes of Staph. aureus culture fluid is due to peptides other than fMet-Leu-Phe; these peptides recruit a higher percentage of monocytes than fMet-Leu-Phe.
In this study we investigated the role of interleukin 1 (IL 1) in the induction of inflammatory lesions and in the preparation and provocation of the local Shwartzman reaction. Both of these phenomena can be induced with a variety of agents. This suggested to us that a common endogenous mediator may be crucial to the development of these two lesions. When IL 1 was injected intradermally into shaved rabbit backs, 51Cr-labeled neutrophils accumulated at the injection site. Neutrophils began to accumulate less than 1 hr after injection, and the maximum rate of accumulation was observed by 4 hr. This activity was dose dependent. It was calculated that in all animals, 10(-14) mol of IL 1 induced significant neutrophil accumulation, whereas in many animals, as little as 10(-15) mol of IL 1 sufficed. When 4.2 X 10(-9) mol of E. coli 0111:B4 lipopolysaccharide W was injected i.v. 24 hr after an intradermal injection of IL 1 (2.9 X 10(-13) mol), a local Shwartzman reaction was seen 4 hr later at the intradermal injection site. IL 1 injected i.v. 24 hr after an intradermal injection of either IL 1 or lipopolysaccharide also produced a local Shwartzman reaction. These data indicate that IL 1 may be the common endogenous mediator of the inflammatory response, and IL 1 may serve in the same role for the preparation and provocation of the local Shwartzman reaction.
The in vivo efficacy of glycosylated and nonglycosylated recombinant human granulocyte macrophage colony-stimulating factor (rh GM-CSF) expressed in Chinese hamster ovary cells and Escherichia coli respectively was studied in rhesus monkeys following a daily subcutaneous (SC; three times) or intravenous (IV; over six hours) dose for seven consecutive days. The monkeys responded to the rh GM-CSF with a prompt (within 24 hours) rise in circulating white blood cells (WBCs). Thereafter the total cell counts increased steadily in a dose- dependent manner with repeated dosing to numbers six times over the pretreatment levels. Overall, granulocyte counts increased fivefold, lymphocytes twofold to fourfold, and monocytes threefold to fourfold. Platelets and erythrocytes were unaffected. Within 1 week after the end of treatment the leukocytosis had disappeared. Of the two routes of treatment, SC (three times daily)-administered rh GM-CSF was more effective than the same dose given by a six-hour IV infusion. In addition to inducing leukocytosis, parenterally administered rh GM-CSF primed mature circulating granulocytes for enhanced oxidative metabolism and killing of an E coli strain. These results show that exogenously administered glycosylated or nonglycosylated rh GM-CSF is both an effective stimulator of leukocytosis and a potent activator of the phagocytic function of mature granulocytes in monkeys.
The effects of LPS on macrophages in vitro have been examined. LPS triggers macrophages to produce LAF and PGE2 in vitro. LPS is also cytotoxic for macrophages derived from LPS-sensitive mice and will significantly inhibit their phagocytic ability. Both LAF production and cytotoxicity are due to the direct effects of LPS on the macrophage and do not require the particpation of lymphocytes. Each of these functions is abnormal in C3H/HeJ mice. The nature of the gene(s) controlling these macrophage responses to LPS has been determined. The response of (C3H/HeJ X C3H/HeN)F1 macrophages was intermediate when compared to the parental responses and no sex linkage was found. Backcross linkage analysis suggested that the same autosomal codominant gene controls both macrophage and B lymphocyte-LPS sensitivity.
Docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA) was facilely incorporated into phospholipids of mouse peritoneal macrophages following incubation with pure fatty acids complexed to bovine serum albumin. Following stimulation with calcium ionophore A23187, the DHA-enriched cells synthesized significantly smaller amounts of leukotriene C4 and leukotriene B4 compared to control or EPA-enriched cells. The EPA-enriched cells synthesized lower amounts of leukotriene C4 and leukotriene B4 compared to control cells. The stimulated macrophages utilized endogenously released arachidonic acid for leukotriene B4 and leukotriene C4 synthesis. Exogenous arachidonic acid increased the formation of 12-hydroxyeicosatetraenoic acid (12-HETE) and 15-HETE and macrophages enriched with DHA or EPA produced similar amounts of 12-HETE and 15-HETE compared to control cells. These studies demonstrated that the synthesis of leukotriene C4, leukotriene B4 and HETE in macrophages is differentially affected by DHA and EPA.
The n-3 polyunsaturated fatty acids (PUFA) of fish oils alter arachidonic acid (AA) metabolism in macrophages. The present investigation studied the efficacy of eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA), two n-3 PUFA of fish, to alter lipid composition and specific functions of mouse peritoneal macrophages. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were readily incorporated by macrophages in vitro and replaced 25-50% of AA in cellular lipids. The EPA- or DHA-enriched cells synthesized significantly less (50-65%) prostaglandin E2 (PGE2), thromboxane B2 (TXB2) and 6 keto prostaglandin F1(1) alpha (6 keto PGF1 alpha) when stimulated with opsonized zymosan. The enrichment with EPA or DHA did not affect phagocytosis nor superoxide anion formation in macrophages. These studies demonstrated that EPA or DHA can be used to decrease prostaglandin synthesis selectively without affecting the other physiological functions of macrophages.
A murine monoclonal antibody (H4/18) raised against cultured human endothelial cells (HEC) prestimulated by the monokine interleukin 1 (IL 1) recognizes a cell surface molecule inducible by IL 1 or by the distinct monokine tumor necrosis factor (TNF) in primary or serially passaged HEC. H4/18 binding is not basally expressed or inducible by IL 1 in an SV-40 transformed HEC line, in human dermal fibroblasts, or in blood leukocytes. Expression of this molecule by HEC in response to IL 1 can be blocked by protein and RNA synthesis inhibitors but not by cyclooxygenase inhibitors. In addition, H4/18 can immunoprecipitate two biosynthetically labeled polypeptides (Mr 100,000 and 120,000) from HEC stimulated with IL 1 but not from control HEC. Thus, the H4/18 binding site appears to be an inducible surface protein specific for HEC. The majority of HEC in a culture can be induced to express the H4/18 binding protein, but expression is transient (peak 4 to 6 hr) and over the next 24 hr declines to near basal levels either in the continued presence of or upon removal of IL 1. The magnitude of the peak response depends upon IL 1 concentration (peak 5 to 10 U/ml), and the response is optimized by the continued presence of IL 1 during the initial 4- to 6-hr induction period. The time of peak H4/18 binding does not appear to be a function of IL 1 concentration. The decline of H4/18 binding from peak levels is prevented by cycloheximide, a protein synthesis inhibitor. HEC maintained in the presence of IL 1 for 24 hr become refractory to restimulation by IL 1; however, IL 1-stimulated cells rested in the absence of IL 1 for 20 hr can be stimulated by fresh IL 1. HEC expression of the H4/18 binding protein is not induced by interleukin 2 or by interferon-alpha, -beta, or -gamma. Induction of H4/18 binding by TNF is also concentration dependent, transient, and dependent upon protein and RNA synthesis. Several observations suggest that IL1 and TNF act independently on HEC. Our TNF is a recombinant protein, expressed from a cloned cDNA and thus free of IL 1 contamination; it also has no activity in a highly sensitive IL 1 assay. Our standard IL 1 preparation is affinity purified and lacks TNF activity on L929 cells. Thus, our monokine preparations are not cross-contaminated. Most interestingly, HEC incubated with IL 1 and refractory to IL1 restimulation can be restimulated by TNF to express H4/18 binding and vice versa.(ABSTRACT TRUNCATED AT 400 WORDS)
We have investigated the effect of tumor necrosis factor on the release of interleukin-1 and PGE2 from murine resident peritoneal macrophages. Tumor necrosis factor causes an increase in the production of interleukin-1 and PGE2 with a maximum induction for both noted at 5.9 X 10(-8) M. While indomethacin decreased tumor necrosis factor induced PGE2 production, this cyclooxygenase inhibitor augmented tumor necrosis factor induced interleukin-1 production. Our data suggests that tumor necrosis factor may be an important immunopotentiating agent in addition to its previously described cytolytic and metabolic activities.
Human recombinant tumor necrosis factor (TNF) induced migration across polycarbonate and nitrocellulose filters of human peripheral blood monocytes and polymorphonuclear leukocytes, TNF was active in inducing migration at concentrations less than 1 U/ml, and maximal responses (observed at greater than 100 U/ml) were comparable to those elicited by standard reference chemoattractants (FMLP, 10 nM; activated human serum, 5%). Checkerboard analysis performed by seeding different concentrations of TNF above and below the filter revealed that maximal induction of migration required a positive concentration gradient between the lower and upper compartments and that TNF elicited an actual chemotactic response in phagocytes. An anti-TNF rabbit antiserum and anti-TNF mouse monoclonal antibody abolished the chemotactic activity of TNF. Recombinant lymphotoxin was also chemotactic for phagocytes, and its activity was blocked by an anti-lymphotoxin antiserum. Human umbilical vein endothelial cells and blood large granular lymphocytes did not respond chemotactically to TNF under conditions in which appropriate reference chemoattractants were active. The chemotactic activity of TNF may serve to recruit phagocytic cells from the blood compartment to amplify resistance against noxious agents.
Human epidermal cell thymocyte-activating factor (ETAF) derived from either normal epidermal cells or a squamous cell carcinoma cell line has recently been shown to be a low m.w. protein that is indistinguishable from human macrophage-derived interleukin 1 (IL 1). As with human IL 1, human ETAF elutes from Sephadex S-200 gel in two major peaks at m.w. 70,000 to 40,000 and m.w. 25,000 to 12,000. Rechromatography of the higher m.w. fraction of ETAF yielded some of the lower m.w. activity, and chromatofocusing of high m.w. ETAF yielded the same three isoelectric points as the lower m.w. ETAF. Partially purified human ETAF as well as IL 1 were chemotactic for polymorphonuclear and mononuclear leukocytes. In addition, exposure of PMN to ETAF stimulated increased metabolic activity as demonstrated by greater reduction of intracellular nitroblue tetrazolium. Therefore, this study lends further support for an important role of ETAF in the pathogenesis of inflammatory skin diseases.
Escherichia coli organisms induce polymorphonuclear leukocyte (PMNL) infiltration during clinical infection and also in a rabbit dermal model of inflammation. We investigated the factors which may mediate this host response to E. coli. In vitro incubation of Formalin-killed E. coli in heat-inactivated rabbit plasma or balanced salt solution generated in the supernatant factors which induced in vivo PMNL infiltration upon intradermal injection into rabbits. However, these supernatants, in the presence or absence of plasma, did not induce PMNL migration in vitro. The in vivo activity was stable at 100 degrees C and of high molecular weight (30,000). Antiserum to O antigen or to core glycolipid, but not to K or H antigen, as well as polymyxin B inhibited the release or activity of these E. coli-derived factors. The intradermal injection of 0.02 to 0.2 mug of four different endotoxin preparations or lipid A also induced marked PMNL infiltration in vivo. However, these preparations did not stimulate PMNL migration in vitro and failed to generate chemotactic activity in plasma except at very high concentrations (500 mug/ml). Anti-O serum inhibited PMNL infiltration induced by endotoxins with the corresponding O antigen and anti-core glycolipid serum inhibited all four endotoxins tested, whereas polymyxin B inhibited the activity of the endotoxins as well as that of lipid A. Base hydrolysis of endotoxin abolished PMNL infiltration. It is concluded that (i) endotoxin shed from E. coli (killed or live) may be one factor mediating the PMNL infiltration induced by this organism, (ii) endotoxin probably acts independent of in vivo complement activation, (iii) the activity is dependent on the lipid A moiety, and (iv) antibody binding to O or core glycolipid antigens can modify endotoxin so as to diminish its capacity to induce PMNL infiltration in vivo.
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