Neutrophil migration during endotoxemia - CiteSeerX

Neutrophil migration during endotoxemia James G. Wagner and Robert A. Roth Department of Pharmacology and Toxicology, Michigan State University, East Lansing

Abstract: Endotoxemia is marked by a global activation of inflammatory responses, which can lead to shock, multiple organ failure, and the suppression of immune and wound healing processes. Neutrophils (PMNs) play a central role in some of these responses by accumulating in tissues and releasing reactive oxygen species and proteases that injure host structures. This review focuses on altered PMN migratory responses that occur during endotoxemia and their consequences in the development of pulmonary infection. The inflammatory mediators that might be responsible for these altered responses are discussed. The oxidant potential of PMNs is increased after exposure to endotoxin both in vitro and during clinical and experimental endotoxemia. However, other functions such as chemotaxis and phagocytosis are often depressed in these same cells. Endotoxin exposure renders PMNs hyperadhesive to endothelium. The sum of these effects produces activated inflammatory cells that are incapable of leaving the vasculature. As such, the endotoxic PMN is more likely to promote tissue injury from within microvascular beds than to clear pathogens from extravascular sites. Moreover, the functional characteristics of endotoxic PMNs are similar to those observed during trauma, burn injury, sepsis, surgery, and other inflammatory conditions. Accordingly, several clinical conditions might have a common effector in the activated, yet migratorially dysfunctional, PMN. Direct effects of endotoxin on PMNs as well as effects of endogenous mediators released during endotoxemia are discussed. Understanding PMN behavior during endotoxemia may provide basic and critical insights that can be applied to a number of inflammatory scenarios. J. Leukoc. Biol. 66: 10–24; 1999. Key Words: endotoxin · lipopolysaccharide · chemokine · inflammation

INTRODUCTION Polymorphonuclear leukocytes (neutrophils; PMNs) are bloodborne inflammatory cells with potent oxidative and proteolytic potential that are usually the first line of defense against invading pathogens. Their ability to exit blood vessels and migrate rapidly to extravascular sites in tissues is crucial for successful elimination of bacterial, parasitic, and viral infec10

Journal of Leukocyte Biology

Volume 66, July 1999

tions. Indeed, defects in PMN migratory ability are associated with increased incidence of infection [1–3]. In addition to killing pathogens, cytotoxic factors produced by activated PMNs can have deleterious effects on the host. For example, activation of PMNs is frequently implicated in the promulgation of deleterious inflammatory processes, including tissue injury associated with exposure to endotoxin (lipopolysaccharide; LPS). A component of the cell wall of Gram-negative bacteria, LPS initiates a global activation of inflammatory pathways that can lead to tissue damage and death in humans and other animals. Depending on dose and route of exposure, virtually any organ or tissue can be affected, and the hemodynamic and inflammatory responses can result in widespread tissue injury and multiple organ failure. Interventions that block PMNs or PMN-derived products protect from tissue injury and death in animal models of endotoxemia, thus illustrating the critical role that activated PMNs play in host pathogenic responses [4–7]. As discussed in detail below, PMNs exhibit a distinctive functional profile during endotoxemia. For purposes of this review, we define the ‘‘endotoxic PMN’’ as a neutrophil with cellular functions altered either by direct exposure to LPS or by the numerous endogenous mediators present during endotoxemia. Included in this definition are PMNs exposed to LPS either in vitro or in vivo.

Pathophysiological Consequences of the Endotoxic PMN A critical aspect of the endotoxic PMN is an inability to respond and migrate to extravascular inflammatory stimuli in vivo [8, 9] and to chemotactic stimuli in vitro [10, 11]. Migratorially depressed yet oxidatively hyperactive, the endotoxic PMN is positioned and primed to injure endothelial cells and increase vascular permeability and, in some organs, cause parenchymal cell death and organ dysfunction. Another consequence of endotoxemia is increased susceptibility to bacterial infection. The inability of the endotoxic PMN to migrate to extravascular

Abbreviations: PMNs, polymorphonuclear leukocytes; LPS, lipopolysaccharide; CLP, cecal ligation and puncture; LAL, Limulus amebocyte lysate; LTB4, leukotriene B4; PAF, platelet-activating factor; ZAS, zymosan-activated serum; fMLP, N-formyl-methionyl-leucyl-phenylalanine; IL, interleukin; ROS, reactive oxygen species; SIRS, systemic inflammatory response syndrome; LBP, LPS binding protein; TNF-a, tumor necrosis factor a; IMF-1, integrin modulating factor; MIP, macrophage inflammatory protein; CINC, cytokineinduced neutrophil chemoattractants; COX-2, cyclooxygenase-2; MAP, mean arterial pressure; FBP, fibrin degradation products. Correspondence: Dr. Robert A. Roth, Department of Pharmacology and Toxicology, Michigan State University, East Lansing, MI 48824. E-mail: [email protected] Received December 25, 1998; revised April 23, 1999; accepted April 24, 1999.

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sites to fight infection can be as serious to the host as PMN-mediated organ injury. In fact, results from animal studies suggest that deficits in migration can occur after doses of endotoxin too small to cause pronounced tissue damage [12]. As such, the endotoxemic host is likely to be more at risk from infections that require a PMN response than from organ injury mediated by activated PMNs. In humans, impaired PMN chemotaxis after surgery correlates with the development of endotoxemia and pulmonary infection [1, 2, 13]. This phenomenon of endotoxemiaassociated airway infection has been modeled with some success in rats. Emigration of PMNs from pulmonary vasculature into airways during bacterial pneumonia or in response to airway endotoxin is blocked in rats made endotoxemic by systemic LPS administration [8, 9, 14–16]. In addition, PMN migration into the peritoneum in response to glycogen is blocked in endotoxemic rats [J. G. Wagner and R. A. Roth, unpublished observations]. Currently, the mediators and mechanisms responsible for altered PMN migratory behavior during endotoxemia are unknown. Expanded efforts in animal models have the potential to clarify these. Experimental protocols in animal studies typically allow endotoxemia to develop for 2 h before challenging airways with bacteria or endotoxin [8, 9, 16], and several more hours pass before endpoints such as airway neutrophilia are measured. Many inflammatory mediators have been produced and released into the circulation by this time, and one or more may be involved in the inhibition of PMN migration into airways. The fact that PMN function can be modulated by several soluble and cellular mediators makes it difficult to discern to which effector(s) the PMN responds during endotoxemia or, even more daunting, how multiple and sometimes conflicting signals are integrated and processed by the PMN. The discussion that follows will focus on the behavior of PMNs during endotoxemia, a comment on clinical conditions associated with endotoxemia in which PMN functions are altered, a discussion of the direct effects of LPS on PMNs, and a survey of endogenous mediators that are present during endotoxemia and that can affect PMN migratory function. Not discussed in this review are details of PMN functions or the mechanisms of PMN migration, which have been reviewed recently elsewhere [e.g., see refs. 17–20].

THE PMN DURING ENDOTOXEMIA

commercial preparations of LPS that are sold as the same product by the same vendor often have different activities as determined in the Limulus amebocyte lysate (LAL) assay. Unfortunately, the specific activity of commercially available LPS is rarely supplied by vendors and is rarely reported in publications. It is the experience of this laboratory that different lots of the same LPS product can vary in LAL activity by more than 10-fold. This might be largely responsible for the disparate observations reported in studies employing similar doses of LPS. Until standard methodologies and reporting practices are adopted, differences in results among laboratories and even within a laboratory can be expected. Host responses during endotoxemia are dynamic and described by indistinct stages. As such, sampling of cellular and soluble factors at different times after the initiation of endotoxemia can yield different results for the same endpoints. Due to the release of immature PMNs from bone marrow during endotoxemia, the PMN population is itself dynamic and can consist of a mixture of mature and young cells, the composition of which changes with time. In addition, PMN responses to endotoxin in vitro can vary somewhat within the same model. Despite the range of temporal and qualitative changes described for cytokine production, organ dysfunctions, and maturation of PMNs, the overall picture of PMN status is fairly consistent across models. With these caveats in mind, the following description of the endotoxic PMN represents an amalgam of observations of PMN behavior in human and animal studies. The profile is one in which some functions are enhanced, whereas others are inhibited (Fig. 1).

Chemotaxis Results from chemotaxis assays using PMNs isolated from endotoxemic subjects suggest that responses are selectively inhibited depending on the chemotactic stimulus. In rabbit PMNs, responses to leukotriene B4 (LTB4), platelet-activating factor (PAF), and zymosan-activated serum (ZAS, a source of C5a) are inhibited, but chemotaxis toward the bacterial peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) is unaffected [10, 21]. A similar pattern is observed with human PMNs isolated during endotoxemia. That is, chemotactic responses to interleukin-8 (IL-8), ZAS, and LTB4 are depressed yet responses to fMLP are intact [11, 22]. Furthermore, this pattern of inhibition is observed at both early and later stages of endotoxemia. These observations suggest that mobilization of PMNs during infections might be controlled in a manner that limits PMN responses to endogenous mediators and maximizes,

Animal Models and Their Challenges It is important to note that endotoxemia, defined as the presence of endotoxin in the blood, can be produced experimentally by a variety of methods that elicit different kinetics in various animal models. Common approaches include bolus intravenous or intraperitoneal administration, prolonged infusion, and the release of endogenous endotoxin into the peritoneal cavity after procedures of cecal ligation and puncture (CLP). Variability in the response of animals during endotoxemia can arise from the size of the dose. For example, responses of inflammatory cells in the same animal model can be enhanced or inhibited as the dose changes. Furthermore,

Fig. 1. Neutrophil status during endotoxemia. Oxidant production capability and adhesiveness of endotoxic PMNs outweigh locomotory functions such as transendothelial migration and chemotaxis.

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Neutrophils during endotoxemia

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or focuses, PMN responses to bacterial stimuli (e.g., fMLP). This possibility is supported by studies in vitro in which fMLP causes unidirectional desensitization of IL-8 receptors on human PMNs [23, 24].

Vascular Sequestration and Adhesion Early (,1 h) neutropenia and pulmonary and hepatic accumulation of PMNs are well documented during experimental endotoxemia [6, 25–27]. A consistent PMN response in human and animal models of endotoxemia is loss of L-selectin and the expression of CD11b/CD18 integrin (Mac-1) [11, 28–31]. These early changes in PMN adhesive protein expression are associated temporally with vascular sequestration, and antibody studies provide evidence both for and against the involvement of CD18 integrins in pulmonary and hepatic leukostasis [32–34]. Extensive work performed in rabbits suggests that prolonged pulmonary PMN localization during inflammation is dependent on CD18, whereas the early sequestration (1–4 min) may be due to CD18-independent changes in cytosolic calcium, cytoskeletal derangement, and increased PMN stiffness [35, 36]. Whatever mechanisms are invoked, the capillary sequestration, L-selectin-shedding, and CD18 expression on PMNs are consistent and reproducible findings in models of endotoxemia. It is instructive at this point to consider the subpopulation of PMNs newly released from bone marrow that are responsible for neutrophilia observed during later stages of endotoxemia (3–8 h). Immature PMNs appear in the plasma as band cells and sequester in microvessels in the endotoxemic rabbit [37]. In a model of Gram-positive infection, these younger PMNs remain sequestered in the pulmonary microvasculature and are less likely to migrate in response to intrapulmonary stimuli than older, mature PMNs [38]. In addition, studies in vitro suggest that bone marrow PMNs are less responsive to stimuli that elicit actin polymerization, shape change, and adhesion molecule expression [39]. Thus, during endotoxemia both mature and immature PMNs have a proclivity to sequester in microvessels, although it is unclear whether these events occur by the same mechanism.

Oxidant Production Increased oxidant production by PMNs as determined by the detection of superoxide anion, hydrogen peroxide, or chemiluminescence is reported consistently after endotoxin administration to humans and experimental animals [5, 40, 41]. Tissue injury during endotoxemia is often attributed to PMN-derived reactive oxygen species (ROS), and treatment with antioxidants or blocking PMN function can prevent endotoxemia-related pulmonary injuries [42]. Some studies suggest that adhered or tissue-associated PMNs demonstrate even greater oxidant potential than circulating PMNs during endotoxemia [4, 43, 44]. However, instances of depressed or unaltered oxidant production by PMNs during endotoxemia are also reported [45–47]. In these cases, decreased oxidant generation might be due to different LPS doses or to the down-regulation that follows previous activation. Kajdacsy-Balla and co-workers [46] showed that the oxidative response of PMNs in endotoxemic rats is dependent on LPS dose and the stage of endotoxemia. For 12



example, small doses increased superoxide release by twofold after 5 min and 10-fold by 6 h. Conversely, larger doses inhibited responses by 5 min, and oxidant releasing ability returned to normal by 3 h but never increased above control levels. Thus, a meaningful interpretation of PMN oxidative status during endotoxemia should be made with consideration for LPS dose, plasma LPS concentration, and time course.

Phagocytosis Enhancement of three critical features of the phagocytic process, i.e., bacterial binding, ingestion, and killing, have been demonstrated in PMNs isolated from endotoxemic subjects. For example, adherence and uptake [30, 48] of C3bcoated zymosan particles by PMNs are increased 30 min after endotoxin administration in pigs. In addition, phagocytic killing of yeast is increased in PMNs from endotoxemic rats [49]. This is in contrast to results in an ovine model in which phagocytosis-associated bactericidal and candidicidal activities are depressed by 15 min after endotoxin administration [50]. Furthermore, studies in endotoxemic rats and humans show no changes in phagocytic function of circulating PMNs [22, 51]. Clearly, the manner of endotoxin intoxication and species differences might contribute to the disparity in these outcomes. It is notable that the phagocytic process involves receptor binding, oxidant production, and cytoskeletal rearrangements, and all of these functions can be modulated by LPS. In the CLP model in pigs, IgG-dependent phagocytic responses initially increase and then decrease with time, whereas complement-dependent phagocytosis is unaffected [52]. In addition, PMNs from endotoxemic subjects can have increased extracellular superoxide release but depressed phagocytosis-related oxidant ability [4, 48]. Thus, phagocytic responses of PMNs vary across models of endotoxemia, and making direct comparisons among them is difficult and perhaps not constructive when extrapolating from one animal to another or from animals to humans. Taken together, the functional profile of the endotoxic PMN is one of an activated inflammatory cell with depressed capacity to migrate. Cytotoxic pathways, namely oxidant-producing ability, are primed and/or activated, yet the PMN is unable to leave the vasculature. As such, adhered PMNs have enhanced potential for causing vascular injury from within tissue capillaries while allowing extravascular infections to proceed unchecked.

CLINICAL CONDITIONS ASSOCIATED WITH ENDOTOXIC PMNs Administration of LPS to laboratory animals has been used to model a variety of clinical inflammatory conditions for which the mechanisms are incompletely understood. Included in these are the systemic inflammatory response syndrome (SIRS) and related septic conditions in humans. SIRS is described clinically as hyper- or hypothermia, tachycardia, tachypnea, and either leukopenia or leukocytosis [53]. SIRS can be complicated by infection and at least one hypoperfused organ (sepsis syndrome) and can progress further to include systemic http://www.jleukbio.org

hypotension (septic shock) [54]. Although these conditions have a variety of causes and initiating events, administration of LPS to animals can effectively duplicate many of their pathophysiological manifestations. Common to these conditions is the activation of PMNs. Studies of PMN behavior during endotoxemia and after LPS treatment in vitro have provided insight into potential mechanisms of injury. The status of PMNs during conditions such as sepsis, in which endotoxemia is obvious, is similar to conditions in which the presence of endotoxemia is less obvious. It has become clear that in many of these latter conditions there may exist ‘‘endogenous endotoxemia’’ originating from the release of gut-derived, enterobacterial endotoxin into the circulation. Among these are burn injury, blunt trauma, major surgery, hemorrhagic shock, and nonseptic SIRS (Table 1). In each case, the potential exists for a sequence of events that includes intestinal hypoperfusion, ischemic injury to sensitive gut mucosa, increased intestinal permeability, and translocation of endotoxin and bacteria from the gut lumen into the mesenteric circulation. That PMN behavior is similar in these clinical situations suggests that a common factor might be at work, namely endotoxin. To be sure, unique inflammatory pathways and mediators are likely activated in each case, but the resulting effect on PMN function supports the hypothesis that endotoxin or mediators released during endotoxemia are responsible for the altered oxidative and locomotory character of PMNs.

Burn Patients Endotoxin is detected in the blood of human burn patients and of laboratory animals subjected to thermal skin injury [55–57]. Therapies that inhibit intestinal permeability can prevent endotoxemia as well the inflammatory, immune, and pathophysiological responses observed in thermally injured rodents [58– 60]. Isolated PMNs have enhanced production of superoxide anion and hydrogen peroxide from 5 h to 7 days after experimental burn injury [61–63]. During thermal injury, increased oxidative ability of PMNs appears to contribute to cell and organ injuries, which are prevented by agents that detoxify superoxide anion [64, 65]. However, as is the case with endotoxemia models, instances of depressed oxidative status of PMNs are also reported [66, 67]. PMNs from burn subjects have altered kinetics of actin polymerization that favor the assembly of filamentous actin, which may render the cell rigid and unable to deform and undergo shape change [68, 69]. These features might contribute to the decreased responses of PMNs in chemotaxis assays in vitro [70–72] and to the accumulation of PMNs in the pulmoTABLE 1.

Conditions Associated With Endotoxemia and Altered PMN Responses

Thermal injury Blunt trauma Surgery Hemorrhagic shock a

Evidence of endotoxemia

Enhanced oxidative ability

[55–57]a [77, 78] [75–77] [93, 94]

[61–63] [79, 80] [84, 90] [95, 96]

Altered adhesion

Trauma Patients The appearance of endotoxin in plasma can occur early after major blunt trauma or multiple injuries [77, 78], and PMNs isolated from these patients are often activated in a manner reminiscent of endotoxic PMNs. Specifically, superoxide production is enhanced [79, 80], chemotactic ability is decreased [1, 81], and CD11b/CD18 expression is elevated at the same times that endotoxin levels peak in plasma [82–84]. Furthermore, a pattern of neutropenia, vascular sequestration of PMNs in organs, and oxidative injury occurs in trauma patients [82, 85].

Surgical Patients Endotoxemia is common during and after numerous types of surgery and is especially associated with cardiopulmonary and thoracoabdominal procedures [86–88]. Even general anesthesia has been reported to induce endotoxemia [89], and interrupting the aortic blood supply to the gut is linked to increased intestinal permeability and the appearance of endotoxin in plasma [86]. Activation of isolated PMNs in postsurgical patients is characterized by increased superoxide production and CD11b/CD18 expression [84, 90, 91]. Furthermore, CD11b/ CD18 expression is directly related to plasma endotoxin levels after surgery [88]. Finally, isolated PMNs display depressed chemotactic responses in migration assays in vitro [13, 92].

Hemorrhagic Shock Translocation of bacteria and the appearance of endotoxin in the systemic circulation occur by 30–90 min in animal models of hemorrhagic shock [93, 94]. PMNs isolated during shock display increased oxidant release [95, 96] and decreased motility that is coupled with increased adherence [97]. Impaired migration of PMNs is also evident in vivo [98]. In addition, pulmonary vascular sequestration of activated PMNs is a common phenomenon in animals after hemorrhagic shock [99, 100]. Taken together, isolated PMNs from shock, surgery, trauma, and burn patients resemble the activation profile of PMNs during endotoxemia. Specifically, this profile includes (1) enhanced oxidant production, (2) altered adhesion molecule expression, (3) sequestration in microvasculature, and (4) depressed chemotactic responses. Other changes that are not as widely reported include altered phagocytic responses and actin assembly kinetics. Finally, that endotoxin is often present in each of these conditions suggests that it may be the proximal mediator for these altered PMN functions.

Impaired mobility

[73–76] [64–66] [82–84] [1, 75] [84, 88, 91] [13, 86] [99, 100] [91, 92]

Numbers in brackets refer to relevant reference citations.

nary vasculature in vivo after thermal injury [73, 74]. Increased expression of CD11b/CD18 occurs early (1–2 days) in burn patients [75], and PMNs become unresponsive to endotoxinstimulated CD11b/CD18 expression by 1 week after burn injury [76]. Thus, accumulated evidence indicates that endotoxemia occurs after thermal injury and that PMNs of burn victims have characteristics of endotoxic PMNs.

DIRECT AND INDIRECT EFFECTS OF LPS ON PMNs Two general mechanisms for inhibition of PMN migration are tenable. First, LPS may directly affect PMN migratory function, Wagner and Roth


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and second, one or more of the many inflammatory mediators present during endotoxemia may be responsible for the depressed responses. Accumulated evidence supports both of these possibilities.

Direct Effects CD14 receptor binding

LPS exposure to isolated PMNs engenders a broad array of immediate and delayed, direct (Fig. 2) and indirect effects. Most of these responses are believed to be mediated through interactions of the lipid A portion of the LPS molecule with CD14, a glycosylphosphatidylinositol-linked (GPI) glycoprotein that is expressed on tissue macrophages, monocytes, and PMNs [reviewed in 101–103]. Although the majority of leukocyte work has been performed in monocytes and macrophages, human PMNs behave similarly with respect to mechanisms of activation by LPS. CD14 has been identified on human PMNs but has not been reported to be expressed on rat PMNs. CD14 functions as a receptor that can bind to and promote cell activation by several bacterial cell wall components of both Gram-negative and Gram-positive bacteria. In addition, CD14 might act as a receptor for membrane moieties expressed on cells early during the apoptotic process [104, 105]. Activation of CD14-bearing cells by LPS involves the mobilization of phospholipases, tyrosine kinases, and nuclear factor-kB [106–108], but the transduction molecule responsible for linking CD14 to these pathways is unknown. Studies with human neutrophils and monocytes suggest that a membrane accessory protein is involved, and recent evidence points to a Toll-like-receptor that is known to activate IL-1 receptorassociated kinases [109, 110]. CD18 integrins also associate with CD14, but a functional consequence has not been determined [111]. Binding of LPS to CD14 on cell membranes (mCD14) is greatly enhanced by at least two factors. LPS binding protein (LBP) is a liver-derived plasma glycoprotein, concentrations of which increase in the circulation during infection [89, 112]. The other factor is a soluble form of CD14 (sCD14), which is present in high concentrations in plasma. Each of these plasma proteins serves to shuttle and exchange LPS and phospholipids between themselves and mCD14 [113, 114] and to activate cells that do not express mCD14. However, adhesive pathways in human leukocytes can be activated by LPS/sCD14 in a manner that requires the internalization of LPS and sCD14

[115]. This form of activation might be important in rats because mCD14 has not been reported on rat PMNs, and rat PMNs in vitro are not as sensitive to LPS as are PMNs from humans and other animals. Azurophilic and secretory granules in human PMNs store mCD14 and are mobilized to the cell membrane by activators such as fMLP and tumor necrosis factor a (TNF-a) as well as by LPS [76, 116, 117]. Similar responses in rodent PMNs have not been reported. Compared to humans and rabbits, mice and rats are less sensitive to the pathophysiological effects of endotoxin, a difference reflected in the binding characteristics of LPS to CD14 in each species [118]. Other membrane proteins that bind LPS and activate cells are the b2 integrins (CD18), which include CD11b/CD18 (Mac-1) and CD11c/CD18 (p150,95) [119, 120]. In addition, the adhesive glycoprotein L-selectin binds to LPS and promotes superoxide release in human PMNs [121]. Thus, activation of PMNs by LPS might be accomplished by multiple mechanisms that depend on the presence of receptors, plasma cofactors, and inflammatory mediators. Cytoskeletal changes

Exposure of PMNs to LPS in vitro causes assembly of filamentous actin and reorganization of cytoskeleton in the cell cortex such that cells are resistant to deformation [122, 123]. Endotoxin-treated PMNs become stiff and can lose their ability to deform and pass through vessels of small diameter [123]. Similar responses might contribute to microcapillary localization in vivo. For example, endotoxin-treated PMNs or monocytes that are infused into animals preferentially accumulate in lung microvasculature [26, 33]. The coordinated assembly and disassembly of actin within the cell cortex is essential for normal PMN responses. For example, exposure of PMNs to chemokines causes them to flatten and migrate in vitro, processes that require simultaneous and coordinated actin polymerization and depolymerization [124, 125]. Reorganization of actin filaments is also necessary for phagocytosis, oxidative burst, and degranulation [126, 127]. Altered distribution of microfilaments and microtubules correlates with the inhibition of phagocytosis in macrophages isolated from LPS-treated mice and in macrophages from naive animals treated in vitro with LPS [128]. Conversely, LPS enhances the effectiveness of PMN activators for superoxide production and vesicular mobilization. Accordingly, modulation of PMN actin by LPS exposure may inhibit some functions but enhance others. Adhesion

Fig. 2. Direct effects of LPS on human neutrophils.

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Exposure of PMNs to LPS in vitro causes both shedding of L-selectin and a transient increase in expression of membrane CD11b/CD18 [129]. This phasic transition is hypothesized to be a necessary sequence for firm adhesion of PMNs to physiological surfaces. The change in PMN adhesive profile from selectin to integrin expression is normally thought to occur at the vessel wall in response to endothelium-derived signals, yet circulating PMNs are identified with this profile during endotoxemia [9, 13, 90]. If the sequence of cell activation in PMNs is critical for transendothelial migration, an ‘‘untimely’’ mobilization of adhesion molecules on circulating PMNs in http://www.jleukbio.org

response to plasma LPS may affect their ability to respond to migratory signals in the microenvironment of the vessel wall. Cleavage of L-selectin is believed to be via the action of a metalloprotease [130, 131]. Although LPS can up-regulate peptidase activity on the PMN surface, similar up-regulation of metalloprotease activity has not been reported [132]. LPS-exposed PMNs demonstrate increased adhesion to fibrinogen and complement (C3b)-coated particles, which are ligands for CD11b/CD18 [133, 134]. In these cases, adhesion may be enhanced by both the increase in the number of membrane-expressed CD11b/CD18 receptors [129] and their functional activation by integrin modulating factor (IMF-1). This lipid enhances CD11b/CD18 binding, and its production is enhanced by LPS [133]. However, LPS also can induce the retraction of CD11b/CD18 from the PMN membrane surface into azurophilic granules in vitro [135]. Clearly, LPS modulation of CD11b/CD18 can promote adhesion in vitro, but because LPS can also bind to CD11b/CD18 and activate intracellular pathways [119] the possibility exists that it might negatively affect adhesive and migratory functions.

of the IL-8 receptor [144]. These peptidases may thus be critical modulators of PMN responses during inflammation. Indeed, exposure of PMNs to LPS can inhibit the expression of IL-8 receptors on human PMNs [145]. Chemotaxis

Pretreatment of PMNs with LPS can selectively inhibit PMN chemotaxis in response to IL-8 but not to fMLP [146]. In a separate study, however, inhibition to both fMLP and C5a chemotactic stimuli was demonstrated with doses of LPS that correlate with the magnitude of inducible superoxide production by PMNs [147]. The possibility that oxidant status might affect chemotactic processes is supported by the up-regulation of IL-8 receptors after PMN treatment with antioxidants [148]. As mentioned above, LPS can regulate chemokine receptor expression [145, 149]. In addition, altered actin assembly dynamics and the modulation of adhesion molecules might also contribute to the depressed locomotor responses observed in these systems.

Cytokine Production

PMN priming

TNF-a and IL-1 are two important, proximal inflammatory mediators produced by macrophages during endotoxemia in humans and animals. Modest amounts of both cytokines can be elicited from PMNs after incubation with LPS [136–138]. Because activated macrophages can synthesize 50–100 times more IL-1 or TNF-a than similarly stimulated PMNs, the physiological importance of synthesis of these cytokines by PMNs is not clear. LPS exposure of PMNs can also cause production of PMN chemoattractants such as PAF and IL-8 [139, 140]. Recently, induction of cyclooxygenase-2 (COX-2) and the production of prostaglandin E2 and thromboxane B2 were demonstrated in human PMNs after LPS exposure in vitro [141]. Thus, it appears that PMNs activated by LPS are capable of producing several inflammatory mediators usually associated with activated mononuclear cells.

PMN responses to activators and secretagogues can be enhanced by a prior exposure to LPS. Probably the most often cited example is augmentation of superoxide production elicited by phorbol esters or fMLP [150–153]. Exposure of human PMNs to LPS causes the membrane localization of cytochrome b558, a subunit of the superoxide-generating enzyme, NADPHoxidase [154]. Cytochrome b558 is localized with CD11b/CD18 in secretory vesicles [142]. In this light, adhesion-dependent enhancement of oxidative burst induced by LPS, TNF, fMLP, or phorbol esters may be due to the mobilization to the membrane of both CD11b/CD18 (Mac-1) and cytochrome b558 [155–158]. Indeed, this form of activation may explain both vascular sequestration and the increased oxidant capacity of PMNs during endotoxemia. Evidence from studies in vitro suggests that an increased oxidant status of PMNs favors adhesion over locomotion. Specifically, oxidant production, filamentous actin formation, mobilization of CD11b/CD18, and adhesion frequently happen simultaneously in activated PMNs [126, 159, 160]. Moreover, agents that activate oxidative and adhesive pathways concomitantly inhibit chemotactic responses of PMNs in vitro [161– 163]. Assembly and activation of NADPH oxidase in PMNs alters intracellular redox status [164], and endogenous oxidants produced in activated PMNs can act as signal transduction factors that modulate kinase activity [148, 165] and gene expression. Thus, it is tempting to conclude that chemotaxis and oxidant-associated functions in PMN are mutually exclusive and determined by the redox state of the cell. In this regard, the disposition of the endotoxic PMN as hyperadhesive, hyperoxidative, and migratorially deficient can be explained solely by direct effects of LPS on PMN functions. However attractive this might be, it ignores the numerous cytokines, cells, chemokines, and other inflammatory and down-regulating factors encountered by PMNs during endotoxemia. As described in the next section, many of these factors are capable of modulating PMN migratory function and may contribute to the behavior of PMNs during endotoxemia.

Vesicle mobilization

Within the PMN cytoplasm are granules and secretory vesicles. Azurophilic, specific, and gelatinase-containing granules possess a host of cytocidal enzymes and inflammation-mediating proteins. LPS has not been reported to induce degranulation directly, however, it can cause the mobilization of secretory vesicles to the plasma membrane [142]. Secretory vesicles do not contain the roster of inflammatory enzymes that granules do, but they express on their membranes important receptors including CD14, CD11b/CD18, complement receptor (CR1), and fMLP receptors [116]. This mobilization clearly makes the PMN competent to react to bacterial products (through CD14 or via formyl peptides) and pathogens opsonized with complement [through CD11b/CD18 (Mac-1) and CR1]. Vesicles also contain alkaline phosphatase, an enzyme capable of dephosphorylating critical phosphate groups on LPS, thereby diminishing its activity [143]. Along with CD14, this enzyme may assist in the removal and detoxification of LPS. Two peptidases, neutral endopeptidase (CD10) and aminopeptidase (CD13), are coexpressed in the secretory vesicle. CD10 hydrolyzes fMLP [132] and has been implicated in the cleavage

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Indirect Effects: Effectors of Endotoxemia Endotoxemia is characterized by a sequential appearance of mediators that is fairly consistent across various animals and humans (Table 2). An understanding of the kinetics and effects of these mediators can provide the rationale for protocol design and the interpretation of experimental results in models of endotoxemia. For example, in existing models of endotoxemiaassociated inhibition of pulmonary PMN emigration, the timing of endotoxemia might be manipulated such that different factors predominate during the various phases of transendothelial migration [8, 9, 12, 16]. In this manner, the onset of inhibition might coincide with a specific phase of endotoxemia, and hence specific mediators could be associated with the inhibitory effect. The following is a brief survey of soluble and cellular agents that are present during endotoxemia and that might be responsible for PMN migratory dysfunction observed in humans and animals. It should be kept in mind that a complex and intertwined cascade of inflammatory pathways typifies endotoxemia. Although several factors are discussed below individually, they must be considered together in an appropriate temporal context to understand their complicity in the inhibition of PMN migration. Early Effectors (0–1 h)

Complement activation product(s). Activation of the complement cascade in blood leads to the production of anaphylatoxins (complement proteins C3a and C5a) and the assembly of the membrane attack complex (proteins C5b–C9) on cell membranes of pathogens and susceptible host cells. The appearance of C5a in blood occurs as soon as 5 min after intravenous LPS administration in rats and reaches a plateau by 30 min [166]. This early increase in C5a matches temporally the rapid up-regulation of PMN adhesion molecules after exposure of rats to LPS [167]. Infusion of rabbits with ZAS, a source of C5a, causes pulmonary leukostasis in a manner similar to that induced by intravenous LPS in rats [36]. Activation of PMNs by C5a can lead to desensitization to other stimuli. C5a-treated PMNs demonstrate decreased chemotactic responses in vitro [24], and activation of the C5a-receptor TABLE 2.

Endogenous Factors that Alter PMN Function During Endotoxemia

Stage of endotoxemiaa

Early (0–1 h) Intermediate (1–3 h)

Late (3–6 h)

a

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Factor

Complement activation products Platelets Tumor necrosis factor Interleukin-1 Interleukin-6 Platelet activating factor Interleukin-8 CINCs, MIPs Colony-stimulating factors Interleukin-10 Prostaglandin E2 Nitric oxide Coagulation factors Fibrinolytic factors

Based on results of animal and human studies cited in text.



on isolated PMNs can induce desensitization of receptors for chemoattractants and other inflammatory mediators [23, 168]. C5a that is produced early in endotoxemia may bind to PMN receptors and render circulating PMNs insensitive to chemotactic signals that appear later on the pulmonary endothelial surface.

Platelets. Within 30 min after administration of LPS to rats, platelets colocalize with PMNs in pulmonary and hepatic capillary networks [169]. Platelets can bind to both endothelial cells and PMNs and modulate adhesion and transendothelial migration [170]. In addition to these physical interactions, activated platelets secrete a variety of products that directly affect PMNs. Within a-granules of platelets are PMN activators such as platelet factor 4, the chemokine IL-8, and ligands for CD11b/CD18 such as fibrinogen and fibronectin [171–173]. Accordingly, release of granular contents from platelets could affect PMNs colocalized within the microvasculature. Activated platelets also release adenine nucleotides that, in addition to promoting platelet aggregation, inhibit PMN adhesive interactions with endothelial cells [174]. Thus, the platelet is (1) capable by a number of mechanisms of inhibiting PMN migratory responses and (2) anatomically positioned to exert such an effect. TNF-a. TNF-a appears in plasma by 30 min and peaks 1.5 h after LPS is administered to rats or humans [6, 31, 175]. Administration of TNF-a doses much larger than those that occur in plasma during endotoxemia can reproduce the inhibition of PMN migration caused by intravenous LPS [16]. Although this supports a role for TNF-a in modulating pulmonary PMN trafficking, it leaves unconfirmed that smaller, endotoxemia-associated TNF-a concentrations could impart similar effects. For example, studies in which TNF-a production is blocked or its activity neutralized have not been reported. Studies in vitro show that the TNF-a receptor is linked functionally to chemotactic receptors in PMNs [176] and that TNF-pretreated PMNs have depressed migratory responses both ex vivo and in vivo [177]. Thus, circulating TNF-a might modulate chemotaxin receptor activity on PMNs and lead to altered PMN responses to intrapulmonary stimuli during endotoxemia. Furthermore, TNF-a can prime and activate PMNs for superoxide burst [162, 178]. Intermediate effectors (1–3 h)

Plasma concentrations of TNF-a during endotoxemia peak at 90 min in most mammals. By 1–2 h, the synthesis of secondary mediators elicited by TNF-a and LPS begin to appear in plasma and include pro-inflammatory interleukins (IL-1, IL-6) and chemokines such as IL-8 in humans [179] and cytokineinduced neutrophil chemoattractants (CINCs) and macrophage inflammatory proteins(MIPs) in rats [180]. In addition, antiinflammatory factors such as IL-10 and PGE2 begin to appear by 2–3 h [181, 182]. This stage is also characterized by neutropenia, activation of coagulation pathways, the beginning of PMN release from bone marrow, and the production of nitric oxide by macrophages, endothelial, and smooth muscle cells. Thus, factors are present that might act in synergy or in opposition to modulate the responses of PMNs during this time. http://www.jleukbio.org

IL-1. IL-1 has effects similar to TNF-a on endothelial cells, PMNs, and macrophage/monocytes. Unlike TNF-a, however, IL-1 has not been reported to desensitize PMNs to chemotactic stimuli. IL-1 can enhance the response of PMNs to TNF-a and other stimuli present during endotoxemia [183]. In this regard, IL-1 may act as a cofactor that contributes to the modulation of PMN/EC interactions. IL-6. IL-6 in plasma is associated with severity of injury and mortality during endotoxemia and has a broad array of proinflammatory effects on PMNs and macrophages in vitro. For example, exposure of PMNs to IL-6 enhances phagocytic and oxidant production capabilities [184]. In addition, IL-6 causes PMNs to produce PAF [185]. Although there is little evidence that IL-6 directly affects the adhesion and migratory functions of PMNs, it may promote the activity and production of other important PMN modulators [186]. PAF. PAF receptor antagonists can block mortality and morbidity in a variety of endotoxemia models [187]. PAF promotes the activation of PMNs within pulmonary vascular beds and contributes to lung injury [188]. Exposure of PMNs to PAF causes loss of L-selectin and CD11b/CD18 up-regulation [189], but it does not desensitize PMNs to other chemoattractants [190]. Furthermore, PAF is not involved in pulmonary PMN sequestration that occurs early during endotoxemia [191,192]. Thus, by virtue of its chemotactic ability and effect on PMN adhesion molecule expression, PAF might influence PMN responses during endotoxemia. Chemokines. Concentrations of the primary PMN chemokines, IL-8 (humans, primates, rabbits, and sheep) and CINCs (rat), peak in blood by 2–3 h after LPS administration [175, 180]. Sources of plasma CINCs in rat are not known, but these chemokines are produced in both the liver and lungs during endotoxemia [193–196]. Injection of IL-8 into rabbits blocks PMN emigration into extravascular sites of inflammation [197]. Similarly, treatment of isolated PMNs with IL-8 inhibits their migration through endothelial monolayers [190]. That IL-8 causes both the expression of chemoattractant receptors and CD18 integrins on PMNs in vitro [198] suggests that activation of these membrane proteins on circulating PMNs may be related to the inhibition of transendothelial migration in vivo. In this regard, plasma IL-8, TNF-a, and C5a may occupy chemotactic receptors on PMNs and thereby negate the effects of a chemotactic gradient that might otherwise be sensed by marginated PMNs. Colony-Stimulating Factors. Rebound neutrophilia that occurs by 4–6 h of endotoxemia is mediated in part by the production and release from activated macrophages of granulocyte-colony stimulating factor (G-CSF), which peaks in plasma at about 2 h after LPS exposure [29]. Treatment of isolated PMNs with G-CSF mobilizes CD11b/CD18, enhances transendothelial migration, and has no effect on adhesion [199]. In addition G-CSF can enhance the function of IL-8 receptors on human PMNs [149]. Cotreatment of humans with endotoxin and G-CSF produces different results depending on the time of dosing. When G-CSF is given 2 h before LPS, it enhances production of TNF-a, IL-6, and IL-8, whereas a 24-h pretreatment generally inhibits LPS

responses, including pulmonary PMN sequestration [31]. The latter effect is associated with an increase in anti-inflammatory mediators. Thus, endogenous G-CSF production that occurs during endotoxemia may have biphasic effects on PMNs.

IL-10. IL-10 appears in plasma by 1 h after endotoxemia and peaks by 3 h. Both IL-10 and PGE2 work in part through cAMP pathways to turn off the production of inflammatory mediators in macrophages and PMNs [200–201]. Increases in intracellular cAMP in PMNs is associated with decreased adhesion to epithelial cells [202]. That IL-10 can increase intracellular cAMP in PMNs and reverse agonist-induced up-regulation of CD11b/CD18 suggests that it might modulate the adhesive and migratory capabilities of PMNs during endotoxemia [203]. Late Effectors (3–6 h)

Production of mediators such as IL-10 and PGE2 actually occurs during the first 3 h of endotoxemia but peaks by 3 h or later. Thus, they may have effects on PMNs that are not clearly demarcated by the time frames used in this review. In addition, the importance of these factors might be viewed in the context of what other mediators are concurrently produced. For example, PMNs may be more responsive to PGE2 in the absence of TNF-a or IL-8, and thus PGE2 may exert its effects more strongly by 3–6 h when these early mediators are gone. PMNs that have been primed or desensitized by factors present early during endotoxemia may therefore respond differently to subsequent exposure to other factors. Moreover, newly released PMNs from bone marrow may respond differently than older leukocytes.

PGE2. PGE2 is an anti-inflammatory prostaglandin. It is produced by cyclooxygenase 2 (COX-2) that is up-regulated during endotoxemia and initiates the conversion of arachidonate to PGH2, which undergoes further cell-specific metabolism to PGE2 and other prostanoid products [204–205]. PGE2 appears in plasma about 2–4 h after intravenous LPS administration and inhibits the production of inflammatory cytokines by negative regulation of NF-kB pathways [181]. Studies in vitro show that PGE2 can inhibit PMN chemotaxis [206] and aggregation [207]. Nitric Oxide (NO). One of the more life-threatening complications of endotoxemia is hypotensive shock, which has been linked to the induction of nitric oxide synthase (iNOS) in rat macrophages, neutrophils, endothelial cells, and smooth muscle cells among others [208, 209]. The hypotensive course during endotoxemia in rats is marked by an initial (10–20 min) fall in mean arterial pressure (MAP) from 120 to 40–50 mmHg [210, 211]. By 2 h MAP recovers to 100 mmHg, only to decrease gradually to 60 mmHg by 6 h. That nitro-arginine-L-methyl ester (L-NAME, effective against constitutive NOS) blocks the initial drop in MAP, whereas the iNOS inhibitor aminoguanidine prevents the later hypotension suggests that NO mediates both events. Plasma concentrations of nitrite and nitrate, metabolites of NO, are not evident for at least 1 h after endotoxemia in rats, after which they increase 10-fold by 5–6 h [210, 212–214]. This suggests that NO release by endothelial cells early during endotoxemia is localized to the vascular wall or is sufficient to promote vasodilation without producing detectable nitrite and nitrate in plasma. NO inhibits CD18-dependent PMN adhesion Wagner and Roth


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to endothelial cells in a dose-dependent manner [215, 216]. Therefore, NO might have a phasic inhibitory effect on PMNs in vivo in parallel to its hypotensive effects. Induction of iNOS in rat lungs occurs by 2 h of endotoxemia, and by 3 h the levels of nitrite and nitrate in lungs are greater than in other organs [211]. In a model of IL-8- and TNF-ainduced leukosequestration, inhibition of NOS leads to increased PMN migration [213]. Furthermore, PMN rolling in systemic venules and sequestration in lungs is significantly greater in endotoxemic, iNOS-deficient mice compared with normal mice. Taken together, these results suggest that NO production during endotoxemia probably has effects on PMN responses, but its role in pulmonary airway recruitment of PMNs is unknown.

Coagulation/Fibrinolytic Factors. Activation of the coagulation process as indicated by the appearance of prothrombin fragments and thrombin-antithrombin complexes in serum begins by 0.5 h but peaks much later at 5–6 h [179]. Despite this, major decreases in plasma fibrinogen do not occur until 2–3 h after LPS administration to rats [217]. The appearance in plasma of plasminogen activators occurs by 1 h and peaks rapidly by 1.5–2 h during endotoxemia, marking the activation of fibrinolytic pathways [182]. Fibrinogen and factor X are two coagulation pathway components that can bind to CD18. These interactions allow for initiation of coagulation on the surface of macrophage/ monocytes that express CD18 integrins during inflammation [218]. Hypothetically, CD11b/CD18 that increases on PMNs during endotoxemia could bind to fibrinogen and factor X, thereby interfering with CD11b/CD18-ICAM-1 interactions and inhibiting PMN migration [219, 220]. That PMNs bind fibrinogen and factor X in vivo to initiate coagulation by a similar CD18-dependent mechanism as macrophages has not been proven. However, both fibrinogen and factor X bind to PMNs in vitro [221, 222]. Furthermore, fibrinogen inhibits PMN chemotaxis in vitro [223], and a peptide derived from factor X can bind to CD11b/CD18 on PMNs and inhibit transendothelial migration [226]. In this scenario, the reduced concentration of fibrinogen in plasma between 2 and 6 h of endotoxemia might free CD11b/CD18 for ICAM-1 binding and thereby promote adhesion and migration. With the decrease in fibrinogen comes an increase in circulating fibrin degradation products (FDPs). These products promote CD18-dependent chemotaxis by PMNs [224]. Therefore, the presence of FDPs in the circulation could modify responses of PMNs to chemotactic factors generated in the extravascular spaces.

SUMMARY Endotoxemia in animals and humans produces PMNs having a profile of altered oxidative burst, hyperadhesion, and migratory deficiency. Similar PMN behavior is noted in a variety of human clinical conditions. As such, endotoxin may ultimately be responsible for the inflammatory responses and altered PMN status that are reported in these individuals. This profile of endotoxic PMNs portends oxidative injury to vascular and parenchymal cells because of enhanced PMN oxidative burst 18



potential and increased risk of infection because of diminished capacity of PMNs to migrate to sites of invaded pathogens. These sequelae are in fact common findings in burn, surgical, shock, trauma, and septic patients. The occurrence of endotoxemia is widespread and more commonplace than the clinical situations detailed above. Elevated plasma endotoxin concentration is associated with various activities such as alcohol consumption and with gastrointestinal distress after strenuous exercise or general anesthesia [89, 225–227]. In otherwise healthy individuals, stress, pharmaceuticals, toxicants, and dietary alterations together or individually can increase plasma endotoxin by enhancing gut permeability and/or by reducing clearance by liver macrophages [reviewed in 228]. It is possible but untested that such individuals have PMNs with reduced migratory capacity and may therefore be more prone to risks (e.g., more severe bacterial infections) associated with this condition.

Knowledge Gaps Still unresolved is the molecular mechanism(s) behind endotoxin-induced PMN chemotactic dysfunction in vitro and whether the same mechanism(s) operates during migratory inhibition in vivo. Available evidence suggests that chemotaxis and adhesion-dependent oxidant production are mutually exclusive activities and might be related to the cell’s oxidant status. The research focus for leukocytes during endotoxemia has been predominantly on monocytic cells. Comprehensive studies that address transendothelial migration of endotoxic PMNs have not appeared. Second, it is not clear from the current animal models whether endotoxin itself or an effector present during endotoxemia is responsible for the inhibition of migration. Detailed characterization of the onset and duration of migratory dysfunction during endotoxemia may suggest which mediators are likely to be involved. In addition, the importance of vascular sequestration to the inhibition of emigration from vessels is not understood. Firm adhesion to endothelium is normally a prerequisite for diapedesis, yet during endotoxemia PMNs are arrested at this step despite the presence of chemokines and chemoattractants that would normally draw them out of the vasculature and into the parenchyma. Finally, a good understanding is needed of the doses of endotoxin required in vivo that elicit various PMN responses: priming for or inhibition of oxidant production, CD18associated adhesion, vascular sequestration, and migratory inhibition. Available evidence suggests that these responses may have different relationships to dose and different kinetics. Furthermore, they might be modified differently by soluble mediators present during endotoxemia. This complexity illustrates the need for standardized studies that examine more closely the plasma levels of endotoxin in relation to cellular and pathophysiological responses. Results in animal studies suggest that small doses of endotoxin that do not cause overt organ injury may be the most effective at inhibiting extravascular recruitment. Risk of severe infection may therefore be increased in people having low, ‘‘non-injurious’’ levels of plasma endotoxin. PMNs represent a potent and rapidly mobilized pool of http://www.jleukbio.org

inflammatory cells, the toxic repertoire of which can be directed at both invading pathogens and host tissues. Their altered behaviors during endotoxemia have been modeled in vitro, but studies are limited in number and scope. A more comprehensive and integrated approach that uses both cellular and whole animal systems is required to (1) understand the molecular, cellular, and physiological mechanisms that underlie the migratory and oxidative responses of endotoxic PMNs, (2) determine the contributions of endotoxin and activated PMNs to the pathophysiological sequelae of patients with endotoxemia, and (3) develop therapies that are based on this new knowledge.

ACKNOWLEDGMENTS This study was supported by NIH Grant ES 02581. The suggestions and advice of Dr. Patricia K. Tithof were critical to the preparation of this review.

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