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Lavage with leukotriene B4 induces lung generation of tumor necrosis factor-α that in turn mediates neutrophil diapedesis
Abstract
In experimental models of acute respiratory failure, leukotriene (LT) B4 is generated in the lungs, followed by a 2- to 3-hour delay before there is substantial neutrophil (PMN) accumulation and increased permeability. This study tests whether lavage with LTB4 induces tumor necrosis factor (TNF) synthesis by the lungs that in turn mediates PMN diapedesis. Anesthetized rats underwent lavage with 0.1 ml LTB4 (10(-6) mol/L) into a lung segment. This led to localized TNF synthesis measured in bronchoalveolar lavage fluid with peak concentrations of 580 pg/ml after 1 1/2 hours and 120 pg/ml after 3 hours. These values were higher than after lavage with 0.1 ml saline solution: 0.7 and 4.3 pg/ml, respectively (both p < 0.05). There was a delay before PMN accumulated in bronchoalveolar lavage fluid (x 10(4)). After 30 minutes, the numbers were 2.2 PMN/ml, whereas at 4 hours there was a rise to 40 PMN/ml and at 5 hours 60 PMN/ml, higher than after saline lavage (all p < 0.05). Pretreatment of rats by lavage into airways with actinomycin D, 12 ng in 0.1 ml, minimized LTB4-induced TNF synthesis after 1 1/2 and 3 hours (38 and 51 pg/ml), as well as the delayed diapedesis after 4 hours (12 PMN/ml) (all p < 0.05). Similarly, pretreatment of other rats by lavage with TNF-alpha antiserum (rabbit antimurine), but not normal serum, limited LTB4-induced diapedesis (13 PMN/ml) (p < 0.05). Interestingly, administration of the protein synthesis inhibitor actinomycin D by lavage 10 minutes after LTB4 did not prevent TNF generation after 1 1/2 or 3 hours (490 and 440 pg/ml). However, this agent did limit PMN diapedesis after 4 hours (14 PMN/ml) (p < 0.05), an event possibly caused by limiting later synthesis of endothelial adhesion proteins, a thesis consistent with the findings that pretreatment of rats by lavage with actinomycin D was without any effect on N-formyl-methionyl-phenylalanine (10(-8) mol/L)-induced diapedesis. This agent is known to induce PMN migration without need for synthesis of endothelial adhesion proteins. The data indicate that lavage with LTB4 induces local TNF-alpha generation that in turn mediates a delayed PMN diapedesis. This event is likely regulated by endothelial synthesis of adhesion proteins.
... The second component was shown in experiments of PMN depletion before aspiration, which reduced lung injury by 66% (14). Injury-derived, locally produced chemoactivators such as eicosanoids (9)(10)(11) and interleukins (5) were thought to recruit and activate the PMNs, leading to their transmigration to alveolar spaces and bronchial walls (4). Experimental inhibition of the abovementioned agents reduced leukosequestration and injury. ...
... Neutropenic mice had a 59% reduction in lung injury after acid aspiration. This confirms similar results reported in other species (9)(10)(11). Previous studies in the rat have shown that locally produced leukotriene B 4 and thromboxane A 2 recruit and activate neutrophils. ...
... The operative adhesion molecule after acid aspiration was speculated to be a selectin because studies have shown that anti- 2 -integrin therapy (immunoneutralization of CD18) did not affect either neutrophil sequestration or lung injury (4,5,(9)(10)(11). We used selectin-deficient mice to determine the role of this adhesion molecule in acid aspiration injury. ...
Acid aspiration may result in the development of the acute respiratory distress syndrome, an event associated with significant morbidity and mortality. Although once attributed to direct distal airway injury, the pulmonary failure after acid aspiration is more complex and involves an inflammatory injury mediated by complement (C) and polymorphonuclear leukocytes. This study examines the injurious inflammatory cascades that are activated after acid aspiration. The role of neutrophils was defined by immunodepletion before aspiration, which reduced injury by 59%. The injury was not modified in either P- or E-selectin-knockout mice, indicating that these adhesion molecules were not operative. C activation after aspiration was documented with immunochemistry by C3 deposition on injured alveolar pneumocytes. Animals in which C activation was inhibited with soluble C receptor type 1 (sCR1) had a 54% reduction in injury, similar to the level of protection seen in C3-knockout mice (58%). However C4-knockout mice were not protected from injury, indicating that C activation is mediated by the alternative pathway. Finally, an additive effect of neutrophils and C was demonstrated whereby neutropenic animals that were treated with sCR1 showed an 85% reduction in injury. Thus acid aspiration injury is mediated by neutrophils and the alternative C pathway.
... Neutropenic wild-type mice had a 58% reduction in lung injury after acid aspiration. This is similar to previous reports including other animal species (5,6,8,14,24). Prior work from this laboratory has examined other potent mediators of PMN chemotaxis in addition to C5a. ...
... Prior work from this laboratory has examined other potent mediators of PMN chemotaxis in addition to C5a. Experimental lavage with leukotriene B 4 into the airways induced the synthesis of local tumor necrosis factor-␣ that, in turn, leads to PMN diapedesis (6). The importance of eicosanoids after aspiration was documented by the observation that acid aspiration induced preliminary synthesis of leukotriene B 4 and thromboxane A 2 , coincident with the recruitment and activation of neutrophils (8). ...
A significant role for the alternative complement pathway in acid aspiration has been demonstrated by the observation that C3 genetic knockout mice are protected from injury. Utilizing C5-deficient mice, we now test the role of the terminal complement components in mediating injury. Lung permeability in C5-deficient mice was 64% less than in wild-type animals and was similar to wild-type mice treated with soluble complement receptor type 1, which gave a 67% protection. Injury was fully restored in C5-deficient mice reconstituted with wild-type serum. The role of neutrophils was established in immunodepleted wild-type animals that showed a 58% protection. Injury was further reduced (90%) with the addition of soluble complement receptor type 1, indicating an additive effect of neutrophils and complement. Similarly, an additional protection was noted in C5-deficient neutropenic mice, indicating that neutrophil-mediated injury does not require C5a. Thus acid aspiration injury is mediated by the membrane attack complex and neutrophils. Neutrophil activity is independent of C5a.
... There is an early and direct chemical airway injury characterized by copious, protein-rich bronchial secretions, resulting in neutralization of the acid (1). This is followed by a delayed humoral (complement, cytokine, eicosanoid) and cellular (neutrophil, macrophage) response, leading to distal alveolar injury and increased capillary leak (4,6,7,13,26). Both the complement system and the polymorphonuclear leukocyte (PMN) have been shown to be important mediators of acid aspiration injury (13,26). ...
... Previous experimental work in acid aspiration has shown that anti- 2 -integrin therapy in the form of immunoneutralization of CD18 did not affect either PMN sequestration or lung injury (6,7). We therefore speculated that the endothelial selectins are the operative adhesion molecules after acid aspiration. ...
The potentially enhanced anti-inflammatory effects of the sialyl Lewis(x) (sLe(x))-decorated version of soluble complement receptor type 1 (sCR1) in moderating acid aspiration injury are examined. HCl was instilled in tracheostomy tubes placed in mice, and extravasation of (125)I-labeled albumin in bronchoalveolar lavage (BAL) fluid was used to calculate the vascular permeability index (PI). Neutrophil counts in BAL fluid and immunohistochemistry were performed. PI was moderated by 82% after treatment with sCR1sLe(x) compared with 54% in sCR1-untreated mice (P < 0.05). Respective reductions in PI in mice treated 0.5 and 1 h after acid aspiration with sCR1sLe(x) of 70 and 57% were greater than the decreases in PI of 45 and 38% observed in respective sCR1-treated groups (P < 0.05). BAL fluid neutrophil counts in sCR1sLe(x)-treated mice were significantly less than those in sCR1-treated animals, which were similar to those in untreated mice. Immunohistochemistry stained for sCR1 only on the pulmonary vascular endothelium of sCR1sLe(x)- but not sCR1-treated mice. In conclusion, sCR1sLe(x) moderates permeability by antagonizing complement activation and neutrophil adhesion. Delayed complement and neutrophil antagonism significantly reduces injury.
... Paradoxically, LTB 4 plays a critical role in bacterial clearance but also contributes to long-term inflammatory disorders. Aberrant leukotriene production is hypothesized to alter immune cell function, which results in the hyperproductivity of inflammatory cytokines such as TNF-a, IL-8, and Il-6 during chronic inflammation (117)(118)(119). A genetic screen discovered that mutants of the Ita4h locus in zebrafish resulted in altered LT4AH expression, which caused increased susceptibility to Mycobacterium marinum infection (120). ...
Eicosanoids are lipid-based signaling molecules that play a unique role in innate immune responses. The multiple types of eicosanoids, such as prostaglandins (PGs) and leukotrienes (LTs), allow the innate immune cells to respond rapidly to bacterial invaders. Bacterial pathogens alter cyclooxygenase (COX)-derived prostaglandins (PG) in macrophages, such as PGE2 15d-PGJ 2 , lipoxygenase (LOX)-derived leukotriene LTB 4, which has chemotactic functions. The PG synthesis and secretion are regulated by substrate availability of arachidonic acid and by the COX-2 enzyme, and the expression of this protein is regulated at multiple levels, both transcriptionally and post-transcriptionally. Bacterial pathogens use virulence strategies such as type three secretion systems (T3SSs) to deliver virulence factors altering the expression of eicosanoid-specific biosynthetic enzymes, thereby modulating the host response to bacterial lipopolysaccharides (LPS). Recent advances have identified a novel role of eicosanoids in inflammasome activation during intracellular infection with bacterial pathogens. Specifically, PGE 2 was found to enhance inflammasome activation, driving the formation of pore-induced intracellular traps (PITs), thus trapping bacteria from escaping the dying cell. Finally, eicosanoids and IL-1β released from macrophages are implicated in the efferocytosis of neighboring neutrophils. Neutrophils play an essential role in phagocytosing and degrading PITs and associated bacteria to restore homeostasis. This review focuses on the novel functions of host-derived eicosanoids in the host-pathogen interactions.
... LTB4 can be produced by activated leukocytes, working as a potent chemoattractant for neutrophils and stimulating the production of pro-inflammatory cytokines. [16][17][18] The role of LTB4 in lymphedema has been explored by a recent seminal work showing that LTB4 antagonism reversed edema and improved lymphatic function in a murine model of lymphedema. 19 In this work, using specific LTB4 antagonist, Tian et al. found improved lymphatic clearance, diminished tissue inflammation, and improved tail anatomy. ...
Background:
Early lymphedema detection may reduce the symptoms and improve clinical outcomes. However, the lack of reliable serum biomarkers capable of predicting lymphedema development is a current medical problem. In this study, we investigated if serum levels of hyaluronic acid (HA) and leukotriene B4 (LTB4), two molecules involved in lymphedema development, may work as predictors of this condition.
Methods and Results:
A mouse model of acquired lymphedema was generated through ablation of tail dermal lymphatic network. Tail diameter was measured daily, and HA and LTB4 serum levels were analyzed before and during the development of lymphedema. We found increased serum levels of HA and reduced levels of LTB4 at early days before the appearance of lymphedema signs. Similar results were observed in the lymphedema tissue. Increased local and systemic inflammation was also detected at early time points. Moreover, the ratio LTB4/HA arises as the strongest predictor for lymphedema development. In fact, we found an inverse correlation in our model, where reduced LTB4/HA levels showed increased lymphedema signs.
Conclusions:
These findings suggest that serum ratio of LTB4/HA may be a useful biomarker to predict acquired lymphedema development, with potential to be used in clinical conditions such as breast cancer patients.
... LTB4 enhances phagocytosis [9,15,16] and nitric oxide production [17] in macrophages, activates NADPH oxidase [18], and increases the production of antimicrobials [19,20]. LTB4 also stimulates the production of cytokines such as TNFα [12,21], IL-8 [22] and IL-6 [23] to further augment pro-inflammatory responses. Thus, LTB4 is a key mediator of the host responses to inflammatory stimuli. ...
Immune cells sense and react to a multitude of factors including both host and microbederived signals. Understanding how cells translate these cues into particular cellular behaviors is a complex yet critical area of study. We have previously shown that both neutrophils and macrophages are important for controlling the fish pathogen Streptococcus iniae. Here, we report both host and bacterial determinants leading to the formation of organized macrophage aggregates as part of the host inflammatory response in a subset of infected larvae. Streptococcal capsule was a required signal for aggregate formation. Macrophage aggregation coincided with NFκB activity, and the formation of these aggregates is mediated by leukotriene B4 (LTB4) produced by neutrophils. Depletion, inhibition, or genetic deletion of leukotriene A4 hydrolase (Lta4h), which catalyzes the last step in LTB4 synthesis, resulted in the absence of macrophage aggregation. Larvae with impaired neutrophil function also had impaired macrophage aggregation; however, aggregate formation was partially rescued with the addition of exogenous LTB4. Neutrophil-specific expression of lta4h was sufficient to rescue macrophage aggregation in Lta4h-deficient larvae and increased host survival following infection. In summary, our findings highlight a novel innate immune response to infection in which specific bacterial products drive neutrophils that modulate macrophage behavior through eicosanoid signaling. © 2017 Vincent et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
... LTB 4 induces antimicrobial peptide release from neutrophils in vivo, in some cases inhibiting viral replication757677. Lung generation of the proinflammatory cytokine TNF-α is enhanced by LTB 4 [78]. A number of studies have reported that LTB 4 acts synergistically with IL-4 to induce activation, proliferation, and differentiation of human B lymphocytes798081, although a separate study reported that 5-LO inhibitors actually enhanced B lymphocyte proliferation [82]. ...
Viruses are frequent causes of respiratory infection, and viral respiratory infections are significant causes of hospitalization, morbidity, and sometimes mortality in a variety of patient populations. Lung inflammation induced by infection with common respiratory pathogens such as influenza and respiratory syncytial virus is accompanied by increased lung production of prostaglandins and leukotrienes, lipid mediators with a wide range of effects on host immune function. Deficiency or pharmacologic inhibition of prostaglandin and leukotriene production often results in a dampened inflammatory response to acute infection with a respiratory virus. These mediators may, therefore, serve as appealing therapeutic targets for disease caused by respiratory viral infection.
... We established the specificity of the effects of both the MO and RNA by showing that lta4h sense but not antisense RNA rescued the increased bacterial burden of the lta4h morphants ( Figure S2). The LTA4H-high animals displayed evidence of increased LTB 4 activity, both chemoattractant and proinflammatory ( Figure 1A) (Goldman et al., 1993;Haeggstrom, 2004;Samuelsson et al., 1987;Tobin et al., 2010). More phagocytes were recruited to M. marinum infection of their hindbrain ventricle (1.7 fold increase over wildtype siblings, P=0.01) and expressed 1.5 fold more TNF mRNA than wildtype (P=0.02) ...
Susceptibility to tuberculosis is historically ascribed to an inadequate immune response that fails to control infecting mycobacteria. In zebrafish, we find that susceptibility to Mycobacterium marinum can result from either inadequate or excessive acute inflammation. Modulation of the leukotriene A(4) hydrolase (LTA4H) locus, which controls the balance of pro- and anti-inflammatory eicosanoids, reveals two distinct molecular routes to mycobacterial susceptibility converging on dysregulated TNF levels: inadequate inflammation caused by excess lipoxins and hyperinflammation driven by excess leukotriene B(4). We identify therapies that specifically target each of these extremes. In humans, we identify a single nucleotide polymorphism in the LTA4H promoter that regulates its transcriptional activity. In tuberculous meningitis, the polymorphism is associated with inflammatory cell recruitment, patient survival and response to adjunctive anti-inflammatory therapy. Together, our findings suggest that host-directed therapies tailored to patient LTA4H genotypes may counter detrimental effects of either extreme of inflammation.
... Leukotriene A 4 hydrolase (LTA 4 H) is an enzyme that converts leukotriene A 4 (LTA 4 ) into the proinflammatory leukotriene B 4 (LTB 4 ). LTB 4 serves as a potent leukocyte chemoattractant and promotes the production of TNF (100,111), a cytokine critical to M. tuberculosis control. LTA 4 pro-duction is closely tied to the activation of the eicosanoid pathway, whereas LTA 4 H expression is fairly ubiquitous (reviewed in reference 259). ...
Despite the availability of effective treatment for several decades, leprosy remains an important medical problem in many regions of the world. Infection with Mycobacterium leprae can produce paucibacillary disease, characterized by well-formed granulomas and a Th1 T-cell response, or multibacillary disease, characterized by poorly organized cellular infiltrates and Th2 cytokines. These diametric immune responses confer states of relative resistance or susceptibility to leprosy, respectively, and have well-defined clinical manifestations. As a result, leprosy provides a unique opportunity to dissect the genetic basis of human in vivo immunity. A series of studies over the past 40 years suggests that host genes influence the risk of leprosy acquisition and the predilection for different clinical forms of the disease. However, a comprehensive, cellular, and molecular view of the genes and variants involved is still being assembled. In this article, we review several decades of human genetic studies of leprosy, including a number of recent investigations. We emphasize genetic analyses that are validated by the replication of the same phenotype in independent studies or supported by functional experiments demonstrating biological mechanisms of action for specific polymorphisms. Identifying and functionally exploring the genetic and immunological factors that underlie human susceptibility to leprosy have yielded important insights into M. leprae pathogenesis and are likely to advance our understanding of the immune response to other pathogenic mycobacteria. This knowledge may inform new treatment or vaccine strategies for leprosy or tuberculosis.
... We next assessed if the lta4h mutation impacts the TNF pathway, critically important for resistance to mycobacteria in humans, mice and zebrafish (Clay et al., 2008; Flynn et al., 1995; Keane et al., 2001). We confirmed that LTB 4 induces TNF expression in WT zebrafish as in mammals (Goldman et al., 1993). Injection of ~1.5×10 −14 mol LTB 4 into the caudal vein of uninfected larvae induced tnf mRNA expression in WT embryos 3.6 ± 0.7 (SEM)-fold over mock at 2.5 hours. ...
Exposure to Mycobacterium tuberculosis produces varied early outcomes, ranging from resistance to infection to progressive disease. Here we report results from a forward genetic screen in zebrafish larvae that identify multiple mutant classes with distinct patterns of innate susceptibility to Mycobacterium marinum. A hypersusceptible mutant maps to the lta4h locus encoding leukotriene A(4) hydrolase, which catalyzes the final step in the synthesis of leukotriene B(4) (LTB(4)), a potent chemoattractant and proinflammatory eicosanoid. lta4h mutations confer hypersusceptibility independent of LTB(4) reduction, by redirecting eicosanoid substrates to anti-inflammatory lipoxins. The resultant anti-inflammatory state permits increased mycobacterial proliferation by limiting production of tumor necrosis factor. In humans, we find that protection from both tuberculosis and multibacillary leprosy is associated with heterozygosity for LTA4H polymorphisms that have previously been correlated with differential LTB(4) production. Our results suggest conserved roles for balanced eicosanoid production in vertebrate resistance to mycobacterial infection.
... The participation of TNF-␣ in pulmonary pathophysiology is well recognized, with specific activation of vascular cellular components resulting in endothelial cell barrier dysfunction (3,10,22,49), transendothelial leukocyte diapedesis into tissues (23,63), and marked increases in endothelial cell apoptosis (30, 52-54, 56, 58). In this report, we have addressed potential endothelial cell cytoskeleton-dependent mechanisms that may underlie the effects of TNF-␣ on vascular barrier dysfunction and endothelial cell apoptosis. ...
Tumor necrosis factor (TNF)-alpha is released in acute inflammatory lung syndromes linked to the extensive vascular dysfunction associated with increased permeability and endothelial cell apoptosis. TNF-alpha induced significant decreases in transcellular electrical resistance across pulmonary endothelial cell monolayers, reflecting vascular barrier dysfunction (beginning at 4 h and persisting for 48 h). TNF-alpha also triggered endothelial cell apoptosis beginning at 4 h, which was attenuated by the caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone. Exploring the involvement of the actomyosin cytoskeleton in these important endothelial cell responses, we determined that TNF-alpha significantly increased myosin light chain (MLC) phosphorylation, with prominent stress fiber and paracellular gap formation, which paralleled the onset of decreases in transcellular electrical resistance and enhanced apoptosis. Reductions in MLC phosphorylation by the inhibition of either MLC kinase (ML-7, cholera toxin) or Rho kinase (Y-27632) dramatically attenuated TNF-alpha-induced stress fiber formation, indexes of apoptosis, and caspase-8 activity but not TNF-alpha-induced barrier dysfunction. These studies indicate a central role for the endothelial cell cytoskeleton in TNF-alpha-mediated apoptosis, whereas TNF-alpha-induced vascular permeability appears to evolve independently of contractile tension generation.
... Acid aspiration-induced injury. A volume of 40 l of 0.1 M HCl was administered per mouse as adapted from Goldman and coworkers (21). ...
Platelet endothelial cell adhesion molecule-1 (PECAM-1) (CD31) is an adhesion molecule believed to mediate transendothelial migration of neutrophils and other leukocytes after CD11/CD18-mediated adhesion. Our study evaluated the role of PECAM-1 in neutrophil emigration across the pulmonary capillaries and the bronchial microvasculature using blocking anti-PECAM-1 antibodies in mice and rats. Neutrophil emigration was induced by Escherichia coli, a stimulus eliciting CD11/CD18-dependent emigration, or Streptococcus pneumoniae, a stimulus inducing CD11/CD18-independent emigration. Although anti-PECAM-1 antibodies partially inhibited glycogen-induced neutrophil emigration into the peritoneum, neutrophil emigration across either the pulmonary capillaries or the bronchial microvasculature in response to either E. coli or S. pneumoniae was not prevented when the function of PECAM-1 was inhibited in either mice or rats. There was also no increase in the number of intravascular neutrophils within the bronchial vessels after treatment with anti-PECAM-1 antibody. These studies indicate that either CD11/CD18-dependent or -independent adhesion pathways may lead to PECAM-1-independent transendothelial migration through the pulmonary or the bronchial endothelium.
... Acid aspiration-induced injury. A volume of 40 l of 0.1 M HCl was administered per mouse as adapted from Goldman and coworkers (21). ...
Platelet-endothelial cell adhesion molecule-1 (PECAM-1) (CD31), a 130-kD transmembrane glycoprotein that functions in adhesion and signaling, is thought to play a role in some forms of leukocyte transmigration. In the lung, PECAM-1 is highly expressed, yet there have been few studies examining its role in pulmonary pathology. We therefore examined the inflammatory response (measured by bronchoalveolar lavage cell counts and protein content) after several types of lung injury in wild-type and PECAM-1 knockout mice. Consistent with studies in other organs, instillation of an endothelial stimulant (interleukin-1) was PECAM-1-dependent. In contrast, we noted that three other forms of acute lung injury (acid aspiration, adenoviral instillation, and tumor necrosis factor instillation) were completely PECAM-1-independent. Interestingly, in situ immune complex deposition injury, another complex lung disease, was also PECAM-1-dependent. This surprising finding was investigated in more detail and found to be due to a defect in macrophage activation, and not to a blockade of leukocyte transmigration. Experiments in bone marrow chimeric mice as well as ex vivo data demonstrated that Fcgamma receptor-dependent phagocytosis and tumor necrosis factor release were significantly reduced in macrophages derived from PECAM-1 knockout mice. Although PECAM-1 may not be required for transmigration of leukocytes into the alveolar space in many forms of complex lung inflammation, it is important in the function of Fcgamma receptors on alveolar macrophages.
... Although IL-8 has been reported to induce activation of NF-B (59), a transcription factor that mediates TNF-␣ gene expression (63,64), there is no evidence to date indicating that IL-8 induces the production of TNF-␣. Induction of TNF-␣ expression by IL-8 through mediator cascade(s) may occur, since LTB 4 (inducible by IL-8) has been reported to induce TNF-␣ expression (65,66). Thus, the rationale exists for a direct inhibitory effect of the CXCR2 antagonist on mediator production, although the relative contributions of inhibition of mediator expression and inhibition of leukocyte chemotaxis remain to be determined. ...
Much evidence implicates IL-8 as a major mediator of inflammation and joint destruction in rheumatoid arthritis. The effects of IL-8 and its related ligands are mediated via two receptors, CXCR1 and CXCR2. In the present study, we demonstrate that a potent and selective nonpeptide antagonist of human CXCR2 potently inhibits (125)I-labeled human IL-8 binding to, and human IL-8-induced calcium mobilization mediated by, rabbit CXCR2 (IC(50) = 40.5 and 7.7 nM, respectively), but not rabbit CXCR1 (IC(50) = >1000 and 2200 nM, respectively). These data suggest that the rabbit is an appropriate species in which to examine the anti-inflammatory effects of a human CXCR2-selective antagonist. In two acute models of arthritis in the rabbit induced by knee joint injection of human IL-8 or LPS, and a chronic Ag (OVA)-induced arthritis model, administration of the antagonist at 25 mg/kg by mouth twice a day significantly reduced synovial fluid neutrophils, monocytes, and lymphocytes. In addition, in the more robust LPS- and OVA-induced arthritis models, which were characterized by increased levels of proinflammatory mediators in the synovial fluid, TNF-alpha, IL-8, PGE(2), leukotriene B(4), and leukotriene C(4) levels were significantly reduced, as was erythrocyte sedimentation rate, possibly as a result of the observed decreases in serum TNF-alpha and IL-8 levels. In vitro, the antagonist potently inhibited human IL-8-induced chemotaxis of rabbit neutrophils (IC(50) = 0.75 nM), suggesting that inhibition of leukocyte migration into the knee joint is a likely mechanism by which the CXCR2 antagonist modulates disease.
... inhibitors of leukotriene synthesis and cytokine directed antibodies (French and Galicich, 1964;Limet and Lecomte, 1968;Goldman et al., 1993;Canetti et al., 2001). ...
The anti-inflammatory activities of some medicinal plants are attributed to their contents of sesquiterpene lactones. In the present study, the anti-inflammatory and anti-nociceptive activity of a sesquiterpene lactone isolated from Viguiera robusta, budlein A in mice was investigated. The treatment with budlein A dose--(1.0-10.0 mg/kg, p.o., respectively) dependently inhibited the carrageenan-induced: i. neutrophil migration to the peritoneal cavity (2-52%), ii. neutrophil migration to the paw skin tissue (32-74%), iii. paw oedema (13-74%) and iv. mechanical hypernociception (2-58%) as well as the acetic acid-induced writhings (0-66%). Additionally, budlein A (10.0 mg/kg) treatment inhibited the mechanical hypernociception-induced by tumour necrosis factor (TNF-alpha, 36%), Keratinocyte-derived chemokine (KC, 37%) and Interleukin-1beta (IL-1beta, 28%), but not of prostaglandin E(2) or dopamine. Budlein A also inhibited the carrageenan-induced release of TNF-alpha (52%), KC (70%) and IL-1beta (59%). Furthermore, an 8 days treatment with budlein A inhibited Complete Freund's adjuvant (10 microl/paw)-induced hypernociception, paw oedema and paw skin myeloperoxidase activity increase while not affecting the motor performance or myeloperoxidase activity in the stomach. Concluding, the present data suggest that budlein A presents anti-inflammatory and antinociceptive property in mice by a mechanism dependent on inhibition of cytokines production. It supports the potential beneficial effect of orally administered budlein A in inflammatory diseases involving cytokine-mediated nociception, oedema and neutrophil migration.
... The leukotrienes represent major mediators of inflammation with LTB 4 recognized as a remarkably potent effector of PMN chemotaxis and aggregation as well as enhanced adhesion to the endothelium. 8,22 Targeted disruption of LTB 4 synthesis or activity confers a distinct survival advantage in response to PLA 2-derived lipids. 1,12 Additionally, LTB 4 is a consistent mediator of ALI in animal models 7 and pretreatment with the PLA 2 inhibitor quinacrine attenuates lung injury in a rodent model of splanchnic I/R. 10 The effect of this inhibition alters the PSML lipid profile as observed by normal phase HPLC, 10 implying that regulation of the lipid component(s) within PSML may be an important means of mediating gut-derived cytotoxicity following T/HS. ...
Mesenteric lymph is the mechanistic link between splanchnic hypoperfusion and acute lung injury (ALI), but the culprit mediator(s) remains elusive. Previous work has shown that administration of a phospholipase A(2) (PLA(2)) inhibitor attenuated postshock ALI and also identified a non-ionic lipid within the postshock mesenteric lymph (PSML) responsible for polymorphonuclear neutrophil (PMN) priming. Consequently, we hypothesized that gut-derived leukotriene B(4) (LTB(4)) is a key mediator in the pathogenesis of ALI. Trauma/hemorrhagic shock (T/HS) was induced in male Sprague-Dawley rats and the mesenteric duct cannulated for lymph collection/diversion. PSML, arachidonic acid (AA), and a LTB(4) receptor antagonist were added to PMNs in vitro. LC/MS/MS was employed to identify bioactive lipids in PSML and the lungs. T/HS increased AA in PSML and increased LTB(4) and PMNs in the lung. Lymph diversion decreased lung LTB(4) by 75% and PMNs by 40%. PSML stimulated PMN priming (11.56 +/- 1.25 vs. 3.95 +/- 0.29 nmol O(2)(-)/min; 3.75 x 10(5) cells/ml; P < 0.01) that was attenuated by LTB(4) receptor blockade (2.64 +/- 0.58; P < 0.01). AA stimulated PMNs to produce LTB(4), and AA-induced PMN priming was attenuated by LTB(4) receptor antagonism. Collectively, these data indicate that splanchnic ischemia/reperfusion activates gut PLA(2)-mediated release of AA into the lymph where it is delivered to the lungs, provoking LTB(4) production and subsequent PMN-mediated lung injury.
Introduction
Danggui Buxue Decoction (DBD) is a clinically proven, effective, classical traditional Chinese medicine (TCM) formula for treating blood deficiency syndrome (BDS). However, its effects and effective constituents in the treatment of BDS remain unclear, limiting precise clinical therapy and quality control. This study aimed to accurately evaluate the effects of DBD and identify its effective constituents and quality markers.
Methods
BDS was induced in rats by a combined injection of acetylphenylhydrazine and cyclophosphamide, and the efficacy of DBD against BDS was evaluated based on body weight, body temperature, energy metabolism, general status, visceral indices, histopathology, biochemical markers, and metabolomics. The effects of DBD on urinary and serum biomarkers of BDS were investigated, and the associated metabolic pathways were analyzed via metabolomics. Guided by Chinmedomics, the effective constituents and quality markers of DBD were identified by analyzing the dynamic links between metabolic biomarkers and effective constituents in vivo .
Results
DBD improved energy metabolism, restored peripheral blood and serum biochemical indices, and meliorated tissue damage in rats with BDS. Correlation analyses between biochemical indices and biomarkers showed that 15(S)-HPETE, LTB4, and taurine were core biomakers and that arachidonic acid, taurine, and hypotaurine metabolism were core metabolic pathways regulated by DBD. Calycosin-7-glucoside, coumarin, ferulic acid sulfate, cycloastragenol, (Z)-ligustilide + O, astragaloside IV, acetylastragaloside I, and linoleic acid were identified as effective constituents improving the hematopoietic function of the rats in the BDS model. Additionally, calycosin-7-glucoside, ferulic acid, ligustilide, and astragaloside IV were identified as quality markers of DBD.
Conclusion
The hematopoietic function of DBD was confirmed through analysis of energy metabolism, biochemical markers, histopathology, and metabolomics. Moreover, by elucidating effective constituents of DBD in BDS treatment, quality markers were confirmed using a Chinmedomics strategy. These results strengthen the quality management of DBD and will facilitate drug innovation.
AIM: To investigate production of TNFα from murine peritoneal macrophages stimulated with inflammatory mediator, such as IL-1, IL-6, IL-8, LTB4, LTC4 and LTD4. METHODS: L929 cytotoxicity was used to show the level of released and cell-associated TNFα in murine peritoneal macrophages measured by means of crystal violet staining assay. RESULTS: rhIL-8(40 ∼ 4 000 U·mL-1) was shown to induce dose-dependent increase of released TNFα from murine macrophages. rmIL-1β(0.001 ∼ 1 μg·mL-1) was found to increase the level of released TNFα dose-independently, whereas rhIL-6(10 ∼ 10 000 U·mL-1), LTB4 (5.64 × 10-9 ∼ 5.64 × 10-7 mol·L-1), LTC4(1.73 × 10-9 ∼ 1.73 × 10-7 mol·L-1), LTD4(4.03 × 10-9 ∼ 4.03 × 10-7 mol·L-1) did not induce macrophages to produce TNFα. They all can not elevate cell-associated TNFα. CONCLUSION: The results suggest that rhIL-8 and rmIL-1 can induce production of TNFα from murine peritoneal macrophages, while rhIL-6, LTB4, LTC4 and LTD4 can not induce production of TNFα from murine peritoneal macrophages.
Postinjury multiple organ failure (MOF) is the net result of a dysfunctional immune response to injury characterized by a hyperactive innate system and a suppressed adaptive system. Acute lung injury (ALI) is the first clinical manifestation of organ failure, followed by renal and hepatic dysfunction. Circulatory shock is integral in the early pathogenesis of MOF, and the gut has been invoked as the motor of MOF. Mesenteric lymph is recognized as the mechanistic link between splanchnic ischemia/reperfusion and distant organ dysfunction, but the specific mediators remain to be defined. Current evidence suggests the lipid fraction of postshock mesenteric lymph is central in the etiology of ALI. Specifically, our recent work suggests that intestinal phospholipase A2 generated arachidonic acid and its subsequent 5-lipoxygenase products are essential in the pathogenesis of ALI. Proteins conveyed via postshock mesenteric lymph also may have an important role. Elucidating these mediators and the timing of their participation in pulmonary inflammation is critical in translating our current knowledge to new therapeutic strategies at the bedside.
PUFA and their eicosanoid metabolites are potent biological modifiers. They have beneficial effects in a number of diseases, which may result in part from their direct actions on neutrophils as well as from their ability to modulate eicosanoid biosynthesis. A consideration of their interactions with other cell types, e.g. lymphocytes and macrophages, is beyond the scope of this review. Small alterations in structure can result in large changes in the neutrophil response. This will have important implications for the further development and use of fatty acids for therapeutic purposes.
Lipid fractions obtained from Mycobacterium avium serovar 8 were assessed for the ability to affect various immune functions of human peripheral blood mononuclear cells (PBM). Lipids included a total lipid fraction and fractions eluted from silicic acid column separation of that total lipid fraction, using chloroform and chloroform-methanol combinations. Lipid fractions were assayed for total carbohydrate and total 6-deoxyhexose content and were assessed for the ability to influence human macrophage function and the capacity to induce secretion of prostaglandin E2 (PGE2) and tumor necrosis factor alpha in PBM. The total lipid and serovar-specific glycopeptidolipid (GPL) fractions both induced significant levels of tumor necrosis factor alpha, as well as PGE2, in PBM exposed to a sublethal concentration of 100 micrograms lipid per 2 x 10(6) cells. In addition, the same concentrations of the 5 to 7% and GPL fractions induced significant levels of leukotriene B4 in PBM. Comparison of carbohydrate and 6-deoxyhexose contents of each fraction suggested a relationship to carbohydrate content and ability of fractions to induce immune modulator secretion. Analysis of GPL fractions from M. avium serovars 4 and 20 revealed that those GPL lacked the ability to induce PGE2. These results are explained by considering the difference in the carbohydrate residues of the oligosaccharide moieties.
This study was undertaken to identify those events of bacteremic shock that pathophysiologic levels of leukotriene C4 (LTC4) alone were sufficient to cause. Sixteen adult swine were studied for 4 h in three groups: ANES (n = 6) received anesthesia only; Septic (n = 6) received Aeromonas hydrophila, 10(9)/mL, intravenously, increased incrementally from .2 to 4.0 mL/kg/h; LTC4 (n = 4) received LTC4 infused intravenously, at rates that approximated LTC4 levels of Septic animals. Measurements included mean arterial pressure and arterial PO2, mmHg, pulmonary and systemic (SVRI) vascular resistance indexes, cardiac index (CI), oxygen extraction ratio, hematocrit; thromboxane B2 (TxB2), prostaglandin 6 keto F1 alpha (6 keto), leukotrienes B4 and C4D4E4, and tumor necrosis factor were measured in pg/mL by ELISA. Statistical analysis was performed by ANOVA and general linear model). Mean arterial pressure increased from 100 +/- 5 to 141 +/- 9 in the LTC4 group, but decreased in the Septic group from 90 +/- 7 at baseline to 62 +/- 6 at 3 h. In the LTC4 group, SVRI did not differ from ANES, and pulmonary vascular resistance, PO2, and CI did not change from baseline. In the LTC4 group, TxB2 and 6 keto levels decreased from 149 +/- 26 to 87 +/- 18 and 58 +/- 10 to 44 +/- 12, respectively; in the Septic group, TxB2 increased 140-fold and 6 keto increased 60-fold. Pathophysiologic LTC4 is not sufficient alone to cause the derangements in CI and SVRI, and tissue metabolism induced by graded bacteremia. Significantly increased systemic blood pressure suggests that endogenous pathophysiologic LTC4 may be involved. LTC4 does not increase plasma eicosanoids and tumor necrosis factor, but may down-regulate prostaglandin and leukotriene release.
The purpose of the study was to identify the changes in plasma prostaglandin, leukotriene, and cytokine levels during clinical severe sepsis for which interleukin-1 was necessary.
Circulating prostaglandins, leukotrienes, and cytokines have been implicated as causative agents of systemic inflammation due to sepsis. However, interactions between interleukin-1 and the other cytokine and eicosanoid mediators of severe sepsis are not well-defined.
As part of two sequential multisite, prospective, randomized, double-blind, placebo-controlled clinical trials, 37 patients with severe sepsis received interleukin-1 receptor antagonist (IL-1ra) 100-mg bolus followed by 2 mg/kg per hour intravenously for 72 hours (n = 20) or placebo (n = 17). Plasma thromboxane B2 (TxB2), prostaglandin 6-keto-F1alpha (PGI), leukotriene B4 (LTB4), leukotriene C4D4E4 (LTC4D4E4), interleukin-1 beta (IL-1), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-alpha) were measured by enzyme-linked immunosorbent assay before study drug infusion (baseline) and at 24, 48, and 72 hours after the beginning of the study drug infusion.
Differences between placebo and IL-1ra for plasma LTB4 were not significant, but only IL-1ra LTB4 increased from baseline. Plasma TxB2, PGI, LTC4D4E4, TNF, and IL-6, expressed as % baseline, decreased significantly in patients receiving IL-1ra compared with the placebo group (p < 0.05), whereas plasma IL-1 increased significantly.
Interleukin-1 may be a necessary mediator of increased circulating PGI, TxB2, LTC4D4E4, TNF, and IL-6 levels in patients with severe sepsis. Plasma IL-1 and LTB4 are increased with infusion of IL-1 receptor antagonist. The clinical significance of IL-1 in modifying circulating eicosanoid and cytokine concentrations in clinical sepsis is not clear from the data.
Transforming growth factor-beta (TGF-beta), interleukin-8 (IL-8), and leukotrienes are potent neutrophil chemoattractants that are released in several lung diseases. There is limited information about the release of TGF-beta in bronchoalveolar lavage fluid (BALF) of patients with pneumonia. Furthermore, it is not clear if TGF-beta is differentially expressed in different lung diseases. The aim of our study was to compare the concentrations of TGF-beta1 and TGF-beta2 in the BALF of patients with pneumonia and other lung diseases. Furthermore, correlation of the TGF-beta levels with the concentration of chemoattractant mediators as well as with indicators of macrophage and granulocyte activation should be investigated. Patients with pneumonia, interstitial lung disease (ILD), or chronic obstructive pulmonary diseases (COPD) were included. Patients with ischemic heart disease without pulmonary involvement served as controls. The concentrations of TGF-beta1 and TGF-beta2, of the chemoattractant cytokine IL-8, of leukotriene B4, and of the leukotrienes C4, D4, and E4 were measured. Neutrophil elastase and granulocyte content (PMN) were used as markers for granulocyte activation, and neopterin was used as a marker for the activation of macrophages. Significantly elevated levels of TGF-beta1 (mean = 0.216 ng/ml, p < 0.01) were found in patients with microbiologically positive pneumonia but not in patients with ILD or COPD. A significant (p < 0.001) correlation was found between the TGF-beta1 concentrations and the IL-8 levels and the percentage of granulocytes (r = 0.76, and r = 0.44, respectively). Elevated TGF-beta2 concentrations were measured in the BALF of patients with pneumonia (mean = 1.4 ng/ml, p < 0.01) and with ILD. Pneumonia was also associated with increased concentrations of leukotrienes C4, D4, and E4 (mean = 91.61 pg/ml, p < 0.05) and leukotriene B4 (mean = 203.9 pg/ml, p < 0.01), significantly elevated levels of PMN elastase (mean = 2958.26 ng/ml, p < 0.01), and neopterin (mean = 0.42 nmol/L). Our results strongly suggest that different lung diseases do differ with regard to the released cytokines. TGF-beta1 probably plays a key role in regulation of pulmonary inflammation, particularly in pneumonia.
A central role for the polymorphonuclear leukocyte (PMN) in experimental acid aspiration has been demonstrated by the observation that PMN depletion reduced pulmonary vascular permeability. This study investigates the role of recombinant soluble P-selectin glycoprotein ligand-immunoglobulin fusion protein (rPSGL-Ig), a P- and E-selectin antagonist in moderating acid aspiration lung injury.
Tracheostomy tubes were placed in male C57BL/6 mice and 0.1 N HCl was instilled into the trachea at 2 mL/kg after intravenous injection of (125)I-albumin. After 4 hours the lung vascular permeability index (PI) and PMN accumulation in the bronchoalveolar lavage fluid were assessed.
PI in neutropenic mice was 63% reduced compared with the untreated group and similar to the PI of mice treated with 1 mg/kg rPSGL-Ig before acid aspiration. PMN count of 19 +/- 5 in the bronchoalveolar lavage fluid in rPSGL-Ig treated mice was significantly less than the untreated group PMN count of 586 +/- 72. The respective PI in mice treated with rPSGL-Ig (1/2) hour and 1 hour after acid aspiration was 45% and 39% reduced compared with the untreated group.
Endothelial selectin blockade is as effective as PMN depletion in moderating acid aspiration induced lung permeability. Delayed antiselectin therapy can decrease lung injury.
Leukotrienes are bronchoconstrictor and vasoactive lipid mediators that are targets in the treatment of asthma. Although they are increasingly recognized to exert broad proinflammatory effects, their role in innate immune responses is less well appreciated. These molecules are indeed synthesized by resident and recruited leukocytes during infection. Acting via cell surface G protein-coupled receptors and subsequent intracellular signaling events, they enhance leukocyte accumulation, phagocyte capacity for microbial ingestion and killing, and generation of other proinflammatory mediators. Interestingly, a variety of acquired states of immunodeficiency, such as HIV infection and malnutrition, are characterized by a relative deficiency of leukotriene synthesis. The data reviewed herein point to leukotrienes as underappreciated yet highly relevant mediators of innate immunity.
Flaxseed (FS) is a nutritional supplement with high concentrations of (n-3) fatty acids and lignans that have anti-inflammatory and antioxidant properties. The use of FS in the prevention or treatment of acute lung disease is unknown. In this study, we evaluated diets with high FS content in experimental murine models of acute lung injury and inflammation. The kinetics of lignan accumulation in blood, following 10% FS supplementation, was determined using liquid chromatography tandem mass spectrometry. Mice were fed isocaloric control and 10% FS-supplemented diets for at least 3 wk and challenged by hyperoxia (80% oxygen), intratracheal instillation of lipopolysaccharide, or acid aspiration. Bronchoalveolar lavage was evaluated for white blood cells, neutrophils, and proteins after a 24 h postintratracheal challenge of hydrochloric acid or lipopolysaccharide, or after 6 d of hyperoxia. Lung lipid peroxidation was assessed by tissue malondialdehyde concentrations. The plasma concentrations of the FS lignans, enterodiol and enterolactone, were stable after mice had eaten the diets for 2 wk. Following hyperoxia and acid aspiration, bronchoalveolar lavage neutrophils decreased in FS-supplemented mice (P = 0.012 and P = 0.027, respectively), whereas overall alveolar white blood cell influx tended to be lower (P = 0.11). In contrast, neither lung injury nor inflammation was ameliorated by FS following lipopolysaccharide instillation. Lung malondialdehyde levels were lower in hyperoxic mice than in unchallenged mice (P = 0.0001), and decreased with FS treatment following acid aspiration (P = 0.011). Dietary FS decreased lung inflammation and lipid peroxidation, suggesting a protective role against pro-oxidant-induced tissue damage in vivo.
Acid aspiration leads to pulmonary endothelial and epithelial cell (EC/EpC) injury characterized by increased permeability and polymorphonuclear (PMN) leukocyte diapedesis. Actin in the EC/EpC cytoskeleton has been shown to play a significant role in maintenance of the microvascular junction barrier. This study tests indirectly whether the development of permeability and diapedesis following acid aspiration is via disruption of the pulmonary cytoskeleton. Manipulation was achieved by the actin microfilament assembler phalloidin. Anesthetized rats (n = 88) underwent segmental lung installation of 0.1 ml saline or phalloidin (2 x 10(-6) M). Twenty minutes later 0.1 N HCl, saline, or phalloidin was introduced. After 3 h there was an increase in wet-to-dry weight (W/D) ratio of 6.6 and 5.1 in the HCl-injected and noninjected sides, protein concentration, 3,970 and 2,530 micrograms/ml, and accumulation of 93 PMN/ml (x 10(4] in bronchoalveolar lavage (BAL) of the HCl-injected lung. These values were higher than control animals. Local pretreatment with phalloidin attenuated acid-induced localized but not generalized permeability with reduction in W/D ratio, BAL protein concentration, and diapedesis (P less than 0.05). Acid injection into airways also led to elevated thromboxane B2 and leukotriene B4 levels in plasma and BAL (P less than 0.05) and generalized lung leukosequestration, events not affected by phalloidin. Taken together, these data suggest that acid aspiration lung injury is determined largely by loss of integrity of the pulmonary EC/EpC cytoskeleton with resultant loss of barrier function.
Although several types of acute lung microvascular injury require active participation of circulating leukocytes (presumably neutrophils), the relationship between neutrophils and microvascular injury and edema is controversial. We attempted to answer the question, Does neutrophil migration in response to a chemical signal alter endothelial permeability? If so, is this to be regarded as a significant control mechanism for microvascular protein permeability? Because of our interest in pulmonary injury, we attempted to induce neutrophil chemotaxis into the lungs of anesthetized sheep by using leukotriene B4 (LTB4) (2 nmol/ml, 50 ml total) placed in the alveoli of one caudal lung lobe through a fiberoptic bronchoscope. Measurements of pulmonary hemodynamics, lymph dynamics, and alveolar liquid dynamics were made. Compared to chemotaxis into skin, neutrophil migration into alveoli was slow. LTB4 caused a small increase in lung lymph and protein flow, which was slightly more than that in saline controls. Likewise, excess plasma protein entering the alveolar liquid was slightly increased over saline controls. Both results indicate a small increase in liquid and protein flow across the endothelial and alveolar epithelial barriers in response either to the LTB4 or to the neutrophil chemotaxis. Using a worst case analysis, the slight increase in liquid and protein flow across the endothelium and epithelium may be related to quantal leakage of plasma during neutrophil transit, but there is no evidence for any persistent change in microvascular or epithelial permeability.
The present study investigated the in vitro effect of four different chemotherapeutic agents, namely, cyclophosphamide (CTX), vincristine (VCR), Adriamycin (Adria Laboratories, Columbus, Ohio) (ADR), and actinomycin D (ACT-D) on human polymorphonuclear leukocyte (PMN) function. Human PMNs suspended in phosphate-buffered saline (PBS) at 1 X 10(7) cells/mL were incubated with increasing concentrations of CTX (0, 10(-5), 10(-4), 10(-3) mol/L) or VCR (0, 10(-7), 10(-6), 10(-5), 10(-4) mol/L), ADR (0, 10(-6), 10(-5), 10(-4), 10(-3) mol/L), or ACT-D (0, 5 X 10(-8), 1 X 10(-7), 5 X 10(-7), and 10(-6) mol/L). The cells were then tested for bacterial killing against Staphylococcus aureus, chemotaxis activity stimulated by Escherichia coli endotoxin, N-formyl-methionyl-leucyl-phenylalanine (FMLP)-stimulated aggregation, and cytochalasin B (Cyto B)/FMLP-stimulated superoxide production and enzyme degranulation. High concentration of CTX, an alkylating agent, showed a significant depression of PMN superoxide production, (124 +/- 13 v 161 +/- 15 nmol/10(7) cells, 5 minutes, P less than or equal to .025). ADR, an intercalating agent and membrane inhibitor, showed a significant depression of PMN degranulation and lysozyme release at 10(-4) and 10(-3) mol/L (15.3% +/- 1.7% v 24% +/- 7%, P less than .01; and 15.0% +/- 2.5% v 24% +/- 7%, P less than or equal to .025). VCR, a microtubule inhibitor, showed a significant depression of PMN aggregation at 10(-6), 10(-5), and 10(-4) mol/L (P less than .05), lysozyme release at 10(-4) mol/L (P less than .004), and beta-glucuronidase release at 10(-4) mol/L (P less than .004). In addition, chemotaxis was inhibited by VCR in a dose-dependent manner at all concentrations (10(-7) mol/L, P less than .02; 10(-6) mol/L, P less than .007; 10(-5) mol/L, P less than .006, and 10(-4) mol/L, P less than .003). ACT-D showed no significant effect on the PMN functions tested. These studies conclude that chemotherapeutic agents have modulating in vitro effects on PMN function. Further in vivo studies are therefore needed to assess PMN abnormalities in patients receiving cancer chemotherapy to determine their role in infectious complications.