PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINE-3 return on or before date due. DATE DUE MTE DUE DATE DUE 1M WHO/0.0.969.“ CHARACTERIZATION OF IMPAIRED PULMONARY NEUTROPHIL TRAFFICKING IN THE ENDOTOXEMIC RAT By James Gerard Wagner A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pharmacology and Toxicology 1 998 ABSTRACT CHARACTERIZATION OF IMPAIRED PULMONARY NEUTROPHIL TRAFFICKING IN THE ENDOTOXEMIC RAT BY James Gerard Wagner Neutrophils (polymorphonuclear leukocytes; PMNs) migrate from the blood to extravascular sites such as lung airspaces by a complex, multi-step process. Migratory functions of PMNs are altered by unknown mechanisms when endotoxin (lipopolysaccharide; LPS) is present in the blood. Endotoxemia involves a global activation of inflammatory responses and there are ample points where numerous endotoxemic mediators might interrupt the precise step-wise process of transendothelial migration. As such, identification of the mechanism of inhibition is difficult and cursory studies using animal models of endotoxemia have yielded little insight. The research described herein was designed to characterize the effects of endotoxemia on pulmonary PMN recruitment and evaluate the role of potential mediators and mechanisms of inhibition. Recruitment of PMNs into lung airspaces of Sprague-Dawley rats was stimulated within 2 hours after intratracheal instillation of endotoxin. Endotoxemia was induced by injecting LPS (2 mg/kg) into the tail vein. My results show that inhibition of pulmonary PMN recruitment occurs within 30 minutes after endotoxemia and is correlated with sequestration of PMNs in the pulmonary microvasculature. Because of the temporal dependence of inhibition, I tested the role of mediators that are present early during endotoxemia. Neither the depletion of complement, platelets nor TNF affected the ability of endotoxemia to cause inhibition of pulmonary PMN recruitment and vascular Ieukostasis. Inhibition of pulmonary PMN trafficking during endotoxemia is dependent on the stimulus for migration. Migration in response to intrapulmonary LPS, interleukin-1, zymosan-activated serum and lipoteichoic acid was inhibited by 100%, 100%, 40%, and 58%, respectively, by endotoxemia. Migration was unaffected by endotoxemia when HCl was instilled in lungs. The pattern of inhibition of migration suggests that endotoxemia selectively inhibits migration in response to stimuli for which migration is dependent on CD18 adhesion molecules. Furthermore, inhibition was related to production in airways by each stimulus of tumor necrosis factor (TNF) and macrophage inflammatory protein —2 (MIP-2), two mediators involved in CD18-dependent migration. In summary, endotoxemia inhibits pulmonary PMN trafficking and causes pulmonary leukostasis within 30 minutes. In addition, the ability of endotoxemia to inhibit pulmonary PMN migration was dependent on the intrapulmonary stimulus and was related to the differential production in ainNays of inflammatory mediators. Taken together, the data suggest that endotoxin may be acting directly on PMNs to inhibit migration, and that the mechanism of inhibition involves CD18 adhesion molecules on PMNs. This dissertation is dedicated to two Pattis, Patricia Joan Wagner and Patricia Kay Tithof, who believed in me. iv ACKNOWLEDGEMENTS First and foremost I would like to thank Dr. Robert A. Roth for his help and guidance in the years both before and during my doctoral research. Without his financial, logistical and grammatical support this dissertation would not be possible. My search for a project was marked by frustrating fits and starts, and it was not until Dr. Jack R. Harkema provided the ainNay endotoxin model did I begin to make progress. For this, and his invaluable advice and honest assessment of my work, I am grateful. Two members of my committee in whom I might have confided more these last few years are Drs. Gregory D. Fink and Norb Kaminski. As a friend and lab-neighbor, Greg has always offered sound advice on both personal and statistical issues. Although Norb was never called on to provide the molecular and immunologic expertise I had hoped my thesis work would require, he always seemed to provide the insight or important question that the rest of us had overlooked. My committee provided me not only with guidance on my research, but also role models and exemplary research styles, elements of which I plan on emulating as a mentor and researcher in the near future. I would also like to acknowledge the many scientists-in-training who have passed through this lab and helped shape both my research experience and the focus of this lab: Drs. Susan White and Jim Reindel were two of my first co-workers and mentors in lung and cell culture work, Dr. Lonnie Dahm showed me it was possible to drink beer and dose rats at the same time, Dr. Cindy Hoom helped push the lab toward more molecular pursuits, Dr. Eric Schultze with whom I worked closely and was gracious enough to include me on most of his publications, and Dr. Jim Hewett, who was possibly the hardest working and most conscientious student to come through the lab. His ideas, initiative and early work with endotoxin has led to two NIH grants, and endotoxin is now the primary focus of not only this laboratory, but of several researchers in the Food Safety and Toxicology Center at MSU. Over the years I was fortunate to have the friendship and technical assistance of Eric Shobe, Kerry Ross, Maria Colligan and John Buchweitz, the expert administrative help of Micki Vanderlip, Diane Hummel, and Nelda Carpenter, the opportunity to teach under Drs. Braselton, Rech and Thomberg, the advice and support of fellow graduate students Drs. Marc Baile, Pat Lappin, Mike Denny, Annette Fleckenstein, Rob Durham and Mike Olszewski, and the genuine concern for my research by Drs. Galligan, Lookingland, Cobbett and Killam. In addition, the members of Dr. Harkema’s lab, Dr. Jon Hotchkiss, Dr. Michelle Fannuci, and Katie Bennett, have been especially helpful and supportive during the last two years. My experience here was not easy. Without those outside the lab who provided the balance, purpose and reality to my life, this dissertation would not be possible. In this light, I have had the inestimable benefit of creature comforts from Amber, Ziggy, Sonny, Willy, Dude, Jane, Arnold, O’Hara, Rawan, Phoebe and Dallas. More importantly I have relied heavily, and perhaps unfairly, on the empathy, patience and unconditional support from Patti, Jeffrey, Kristy and Taylor. vi They made the burdens more tolerable, the good times more precious, and showed me it is perhaps more important to feed my heart than it is to feed my head. I can't imagine doing this without them. It is their trust and support I hope to return someday, to let them know what it feels like to be a hero, if at least for a little while. vii TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS CHAPTER 1 - INTRODUCTION I. Bacterial Pneumonia A. Host Defenses B. Animal Models of Airway Infection C. Bacterial Products in Pneumonia Models 1. Factors from Gram-positive organisms 2. LPS from Gram-negative organisms a. Mediator Production b. Chemokine Production c. Negative Modulators d. Extrapulmonary Effects of IT LPS D. Summary ll. Nosocomial Pneumonia A. Risk Factors B. Animal Models of Nosocomial Pneumonia C. Summary Ill. Polymorphonuclear Leukocytes viii xiii xiv xvii 10 12 13 13 15 16 17 19 20 Distribution Mobilization Killing Summary DOW? IV. Mechanisms of PMN Migration A. Capture and Rolling 1. L-selectin 2. P-selectin 3. E-selectin B. Firm Adhesion 1. lntegrins 2. Intracellular Adhesion Molecule-1 C. Transmigration D. Mediators of Migration Cytokines Chemoattractants Chemokines Rat Chemokines Cytokine/Chemoattractant Interactions During Migration 9'?pr E. Summary V. PMN Migration in the Lungs A. Margination B. Site of Migration C. Adhesion Molecule Requirements 1. lntegrins 2. Selectins D. Models of Pulmonary PMN Migration 1. Bacterial and bacterial products ix 21 22 23 24 25 26 26 29 31 32 32 35 37 39 40 42 44 45 46 49 51 51 53 54 54 57 59 59 2. Acid aspiration 3. Interleukin-1 4. Complement Protein C5a 5. Immune-complex deposition E. Summary VI. Animal Models of Endotoxemia A. Rat Responses During Endotoxemia 1. Liver 2. Lung B. Summary VII. Effects of LPS on PMNs A. Direct LPS-induced PMN Activation Cytoskeletal Adhesion Cytokine Production Vesicle Mobilization Chemotactic Responses Priming Effect of LPS on PMNs P’P‘PP’N.‘ B. Summary VIII. Effectors of PMN Function During Endotoxemia A. Early Effectors (0-1 hours) 1. Platelets 2. Complement Activation Product(s) 3. Tumor Necrosis Factor B. Intermediate Effectors (1-3 hours) 1. IL-1 2. IL-6 3. PAF 60 61 62 63 64 65 65 66 67 69 7O 72 74 75 76 76 77 78 78 80 80 80 82 83 83 84 84 85 4. IL-8/CINCs 5. Colony Stimulating Factors 6. IL-10 C. Late Effectors 1.PGE2 2. Nitric Oxide (NO) 3. Coagulation/Fibrinolytic Factors D. Summary IX. Research Goals Chapter 2 - PULMONARY LEUKOSTASIS AND THE INHIBITION OF AIRWAY NEUTROPHIL RECRUITMENT ARE EARLY EVENTS IN THE ENDOTOXEMIC RAT Summary Introduction Materials and Methods Results Discussion Chapter 3 - AN EVALUATION OF EARLY MEDIATORS OF ENDOTOXEMIA ON PULMONARY NEUTROPHIL MIGRATION DYSFUNCTION Summary Introduction Materials and Methods Results Discussion Chapter 4 - INHIBITION OF PULMONARY NEUTROPHIL TRAFFICKING DURING ENDOTOXEMIA IS DEPENDENT ON THE STIMULUS FOR MIGRATION xi 85 86 87 87 87 88 89 90 91 94 95 95 96 102 1 16 120 121 122 122 126 134 138 Summary Introduction Materials and Methods Results Discussion SUMMARY AND CONCLUSIONS BIBLIOGRAPHY xii 139 140 142 144 156 167 175 TABLE 1: TABLE 2 : TABLE 3: TABLE 4: TABLE 5: LIST OF TABLES Selected Mediators of Migration and their Effects Adhesion Molecule Requirements of PMN-Eliciting Airway Stimuli Effectors of PMN Function During Endotoxemia Lavage Fluid Protein 3 hours after IT and IV administrations Cell and Cytokine Content of Bronchoalveolar Lavage Fluid 3 hours after IT instillations. xiii 41 56 81 151 153 LIST OF FIGURES FIGURE 1: Neutrophil Transendothelial Migration 27 FIGURE 2: Neutrophil and Endothelial Cell Adhesion Molecules 30 FIGURE 3: Direct Effects of LPS on Neutrophils 73 FIGURE 4: Summary of MIT LPS Treatment Protocols 99 FIGURE 5: Development of airway PMN accumulation after IT 101 LPS administration. FIGURE 6: Development of neutropenia after IV LPS 104 administration. FIGURE 7: Time-dependent pulmonary Ieukostasis in rats after 105 IV LPS administration. FIGURE 8: Inhibition of airway PMN accumulation by treating with 107 IV LPS at times before IT LPS instillation. FIGURE 9: Pulmonary PMN location after treatments with IT saline 110 and IV saline or LPS. FIGURE 10: Pulmonary PMN location after treatments with IT LPS 112 and IV saline or LPS. FIGURE 11: Inhibition of airway PMN accumulation by treating with 115 IV LPS at various times after IT LPS instillation. FIGURE 12: Summary of protocols for mediator inhibition studies. 124 xiv FIGURE 13: FIGURE 14: FIGURE 15: FIGURE 16: FIGURE 17: FIGURE 18: FIGURE 19: FIGURE 20: FIGURE 21: FIGURE 22: FIGURE 23: FIGURE 24: FIGURE 25: FIGURE 26: Effects of anti-platelet serum on circulating platelet numbers after IV and IT LPS administrations. Effects of platelet depletion on endotoxemia-associated inhibition of airway PMN migration. Effect of CVF treatment on serum complement activity. Effect of complement depletion on endotoxemia- associated nhibition of ainNay PMN migration. Effect of treatment with antibody to TNF on endotoxemia-associated TNF concentration in plasma. Effect of treatment with antibody to TNF on endotoxemia-associated inhibition of airway PMN migration. Inhibition by IV LPS of ainNay PMN accumulation elicited by ainlvay LPS administration. Inhibition by IV LPS of ainNay PMN accumulation elicited by airway lL-1 administration. Inhibition by IV LPS of ainlvay PMN accumulation elicited by airway LTA administration. Inhibition by IV LPS of airway PMN accumulation elicited by ainNay HCI administration. Inhibition by IV LPS of ainlvay PMN accumulation elicited by ainlvay ZAS administration. Effects of IV LPS on airway production of MIP-2 induced by intrapulmonary HCI, ZAS, and LTA. Involvement of 0018 during pulmonary PMN emigration. Pulmonary PMN migration during endotoxemia. XV 127 128 129 130 132 133 145 146 147 149 150 155 162 165 AM ARDS BALF BSA C3i C5a CINC COX-2 CR1 EC EGF E-selectin FDP FMLP HCI HDL G-CSF ICAM-1 ICAM-2 ICU IFN-ry lgG IL-1 lL-1 ra IL—6 IL-8 lL-1 0 IMF-1 IP IT IV LFA-1 LBP LIST OF ABBREVIATIONS alveolar macrophage adult respiratory distress syndrome bronchoalveolar lavage fluid bovine serum albumin complement protein 3, immobilized complement protein 5, fragment a cytokine-inducible neutrophil chemoattractant cyclooxygenase -2 complement receptor -1 endothelial cell epidermal growth factor endothelial selectin; CD62 fibrin degradation products formyl-methione—leucine-phenylalanine hydrochloric acid high-density lipoprotein granulocyte-colony stimulating factor intercellular adhesion molecule -1 intercellular adhesion molecule - 2 intensive care unit gamma- interferon immunoglobulin G interleukin-1 interleukin -1 receptor antagonist interleukin-6 interleukin-8 interleukin-10 integrin modulating factor -1 intraperitoneal intratracheal intravenous lymphocyte function associated antigen-1 lipopolysaccharide binding protein xvi LDL LIF L-NAME LPS L-selectin LTA LTB4 Mac-1 MAP MAP-kinase mCD14 MIP-2 mRNA NADPH NF-xB NO NOS PAF PECAM-1 PGE2 PKC PLA2 PMA PMN P-selectin PSGL-1 PTK RBC sCDI4 SD SEA SIRS sTNFr TNF ZAS low-density lipoprotein leukemia-inhibitory factor N-w -nitro-L-arginine methyl ester lipopolysaccharide leukocyte-selectin; CD62L lipoteichoic acid leukotriene B4 macrophage antigen -1 mean arterial blood pressure mitogen-activated protein -kinase membrane CDI4 macrophage inflammatory protein -2 messenger ribonucleic acid nicotinamide-adenine dinucleotide phosphate nuclear factor - KB nitric oxide nitric oxide synthase platelet-activating factor platelet-endothelial cell adhesion molecule -1 prostaglandin E2 protein kinase C phospholipase A2 phorbol myristate acetate polymorphonuclear leukocyte; neutrophil platelet-selectin; CD62P; granule membrane protein- 1402GMP-140 platelet selectin glycoprotein ligand -1; C0162 protein tyrosine kinase red blood cells; erythrocytes soluble CD14 Sprague-Dawley staphylococal enterotoxin -A systemic inflammatory response syndrome soluble tumor necrosis factor receptor tumor necrosis factor zymosan-activated serum xvii Chapter 1 INTRODUCTION AND REVIEW OF THE LITERATURE place” Summem Polymorphonuclear cells (PMNs; neutrophils) are inflammatory cells which migrate from blood into tissues in response to infection or injury. Recruitment of PMNs into lungs is critical for the killing and removal of bacteria during bacterial pneumonia. During conditions in which endotoxin (lipopolysaccharide; LPS) is present in the blood (i.e., endotoxemia), inflammatory responses, including those of PMNs, are altered. Indeed, in human patients there is a correlation between endotoxemia, altered PMN adhesive function and the risk for developing bacterial pneumonia during hospitalization. In this dissertation, I present and discuss data generated in a rat model of endotoxemia-associated inhibition of pulmonary PMN migration. The process of PMN emigration has multiple steps and can be regulated at many levels by a number of endogenous and exogenous mediators of inflammation. The goal of this literature review is to introduce briefly animal models of neutrophilic inflammation of ainlvays and to discuss how PMN migration is altered by systemic inflammatory conditions, namely endotoxemia. This chapter begins with a discussion of bacterial pneumonia in humans and reviews the use of rodent models to characterize the inflammatory responses associated with bacterial airway infection (Section I). The special case of hospital-acquired pneumonia and its association with endotoxemia is then introduced (Section II). The next three sections (III-V) describe and compare mechanisms of systemic and pulmonary PMN migration and review models of pulmonary inflammation that require PMNs. Section VI is a brief discussion of rat models of endotoxemia, and this is followed by a more detailed overview of the direct effects of LPS on PMNs (section VII) and indirect effects by mediators present during endotoxemia, 3 particularly with respect to how these mediators may affect PMN migratory responses (section VIII). This chapter concludes with a statement of rationale, hypothesis, and research goals that have been designed to evaluate, in a rodent model, the mechanism of endotoxemia-associated inhibition of pulmonary PMN migration. I. Bacterial Pneumonia Pneumonia is inflammation of the lungs and can be caused by bacterial, viral or parasitic infections, aspiration, or inhalation of chemicals and air pollutants. Community and hospital-acquired bacterial pneumonias kill up to 250,000 annually in the United States (Campbell, 1994; Dal Nogare, 1994). Despite new antimicrobials and sophisticated detection procedures, pneumonia-related mortality has not improved in 25 years and has actually increased slightly in the last decade (Campbell, 1994). Respiratory airways make up the largest epithelial surface exposed to the environment. Considering the constant exposure with each breath to microbes and the nightly auto-inoculation from bacteria-laden oropharyngeal secretions during sleep, it is remarkable that respiratory bacterial infections are not more common. A. Host Defenses The first line of defense against inhaled bacteria in upper airways is the mucociliary clearance apparatus which also effectively removes other particles, including non-bacterial pathogens (Murray, 1986). In addition, soluble antimicrobial factors are secreted from pneumocytes into fluids lining alveolar surfaces. Not all of these factors have been characterized, but they include Iysozyme, Iactoferrin, interferon, and defensins, to name 4 only a few (Widdecombe, 1991 ). Specific clearance of inhaled or aspirated bacteria is accomplished primarily by the phagocytic alveolar macrophage (AM). As the resident inflammatory cell of the lung, AMs constantly patrol the airway lining and are capable of eliminating invading pathogens without initiating a widespread inflammatory response (Kobzik and Schoen, 1994). A large infiltration of some bacterial organisms however, can cause proliferation of macrophages and elicit the mobilization and differentiation of monocytes from blood, resulting in an increase in pulmonary tissue macrophages 5- to 10-fold in animal models of pneumonia (Toews, 1986). It is only in the last 15-20 years that the contribution of infiltrating PMNs to the pulmonary phagocytic response been recognized (Vial et al., 1984; Toews et al., 1979; Pierce et al., 1977). B. Animal Models of Airway Infection ln rodent models of bacterial pneumonia, the differing involvements of PMNs and AMs have been distinguished on the basis of bacteria type and inoculum size. For example, exposure to aerosolized Gram-negative bacteria induces airway PMN infiltration in mice, whereas a similar exposure to Gram-positive organisms produce little PMN recruitment but a large AM response (Pierce, et al., 1977; Toews, 1986). With larger doses of some Gram-positive organisms, however, PMN migration can be elicited in a dose-dependent manner (Vial et al., 1984). When PMN responses are compared between similar doses of bacteria, the recruitment elicited by Gram-positive organisms is about 20-25% of that caused by Gram- negative inoculums (T oews et al., 1979). The inflammatory responses to different types of bacteria are due in a part to their outer structures and secreted products (Tuomanen et al., 5 1995). The capsule surrounding Gram-positive organisms does not elicit an inflammatory response per se and can actually inhibit phagocytosis by macrophages and PMNs (Xu et al., 1992). Accessible through the capsule however, is the highly inflammagenic cell wall, components of which can recruit and stimulate inflammatory cells, fix complement, and induce permeability of pulmonary epithelia (Tuomanen et al., 1987, 1995). The reactivity of the cell wall of Gram-positive organisms is correlated with the content of lipoteichoic acid (Tuomanen et al., 1987; RiesenfeId-Orn et al., 1989). Conversely, Gram-negative bacteria lack outer capsules and related components but instead possess highly Inflammagenic LPS structures in their outer membrane. Recognition of LPS by host cells can engender similar ainlvay responses as Gram-positive cell wall components: activation of inflammatory cells (Wizeman and Laskin, 1994); permeability changes (Wheeldon et al., 1992); and epithelial cell injury (Domenici-Lombardi et al., 1995). Release of teichoic acids, LPS, and other bacterial products into lung airspaces inevitably occurs during cell division and as debris resulting from cytocidal activities of inflammatory cells, antibiotics or factors present in surfactant and alveolar fluid. Because the AM is the first inflammatory cell to encounter ainlvay bacteria, its response to the different stimuli can often determine the nature of inflammation during infection. Interaction of LPS with CDI4, a high affinity LPS receptor on AM membranes, elicits responses for phagocytosis, adhesion and production of secondary cytokines and Chemokines (Sweet and Hume, 1996). The mechanism for activation of AMs by pneumococcal cell wall components is less clear. Both CD14 and the receptor for platelet-activating factor (PAF) have been proposed as the 6 conduit for teichoic acid and cell wall fragment-induced activation of AMs (Cabellos et al., 1992; Cauwels, et al., 1997). Production of secondary mediators by AMs may play a critical role in the differential responses to bacteria, especially with respect to tumor necrosis factor (TNF). TNF is a powerful pleiotropic factor at the top of an inflammatory cascade which includes the production of PAF, interleukins and numerous other cytokines and inflammatory mediators. LPS induces release of both TNF and IL-1 from AMs (Sweet and Hume, 1996; Takai et al., 1997), whereas Gram-positive pneumococcal cell wall components elicit only modest IL-1 production and no TNF release (Riesenfeld-Orn et al., 1989). Both LPS and lipoteichoic acid can induce nitric oxide (NO) production in macrophages (Kengatharan et al., 1996). However, whereas either lipoteichoic acid or LPS infusion in rat can precipitate NO-associated circulatory shock, only LPS causes TNF production and multiple organ injury (de Kimpe et al., 1995). Thus, although lipoteichoic acid and LPS can elicit some common responses in vivo and in vitro, TNF produced by Gram-negative stimuli may represent the distinguishing effector for different inflammatory responses elicited by each bacterial type in animal models. C. Bacterial Products in Pneumonia Models Recently, bacterial products have been effectively substituted for live bacteria in animal models of pneumonia. Isolation of individual factors has helped to develop an understanding of the mechanisms of invasion, colonization, cytotoxity and host defense responses. Results from studies using bacterial components have provided the basis for vaccines, antibodies and antiinflammatory compounds in therapeutic strategies to combat pneumonia and respiratory infections. 7 1. Factors from Gram-positive Organisms Pneumolysin is found in the cytoplasm of Gram positive pneumococci. It is released on autolysis and cell replication and can injure cultured airway epithelium by its insertion into the cell membrane and formation of pores (Rubins et al., 1993). lnstillation of pneumolysin into ainNays induces an alveolar neutrophilia in rats similar to that seen with live Gram-positive bacteria (Feldman et al., 1991). Pneumolysin also acts to thwart host defense responses by inhibiting the oxidative burst and bactericidal activities of PMNs and AMs (Rubins and Janoff, 1998). A related bacterial product is autolysin, which degrades bacterial cell wall components during mitosis but also can be toxic to ainNay epithelial cells. The outer capsule of Gram-positive organisms is made of polysaccharides that are less inflammagenic than the cell wall components it conceals. Both capsular and cell wall fragments can elicit recruitment of inflammatory cells into airways (Tuomanen et al., 1987). A variety of cell wall components, muramyI-peptides, peptidoglycans, and lipids can elicit host defense responses to various degrees. The chemical and structural determinants in capsules and cell wall fragments are not clearly defined, but there is an association between the severity of inflammatory responses and the presence of teichoic acid in cell wall fragments. Another gram-positive inflammagen is staphylococcal enterotoxin-A (SEA), which is secreted from S. aureus. SEA can be a potent ainNay inflammagen and has been implicated as a mediator in food poisoning (Miller et al., 1996). SEA can directly stimulate the production of cytokines and chemoattractants from macrophages. 8 _2._L_E§ from gram-negative Organisms Although Gram-negative bacteria make a number of toxic and inflammagenic products, LPS is probably the most important component from these organisms in precipitating host defense responses. LPS has been used as an effective tool for studying pneumonias caused by Gram- negative organisms. lntratracheal (IT) administration of LPS into rat airways produces robust PMN migration into pulmonary airways (Ulich et al., 1991a; Frevert et al., 1995b), and this migration has a similar time course as that elicited by live bacteria (Harris et al., 1988; Nelson et al., 1990). Furthermore, PMNs collected from ainNays of rats instilled with bacteria or LPS have similar profiles for oxidant production and protease secretion (Delclaux et al., 1997). Models using IT LPS have also been used to examine effects of environmental endotoxin which might be inhaled in urban and occupational settings (Rylander and Vesterlund, 1982; Harrison et al., 1992; Dong et al., 1996). Endotoxin contamination has been proposed as a contributing factor in the inflammatory response to inhaled organic grain dusts (Jagielo et al., 1996), diesel exhaust and aerosolized machining fluids (Gordon and Harkema, 1995). Furthermore, the comparison of experimental and clinical data reveals that the responses of rodents to IT LPS treatment is similar to those seen in humans with ainNay endotoxin (Burrell et al., 1988; Burrell and Ye, 1990; Michel et al., 1995). Thus, instillation of LPS in rat ainNays provides models not only for bacterial pneumonias but also for environmental exposures to LPS in the absence of infection. Neutrophil accumulation in respiratory airways is comparable whether LPS is administered by aerolization or direct instillation, and it is similar when the application is intranasal or intratracheal (Wheeldon et al., 9 1992; Szarka et al., 1997). A bolus airway administration of microgram quantities of endotoxin elicits a transient and reproducible accumulation of large numbers of PMNs which is accompanied by increased permeability and long term morphologic changes in lung epithelium (Gordon and Harkema, 1994; Domenici-Lombardo et al., 1995; Wagner et al.,1996). In most models, accumulation of airway PMNs begins within 2 hours after LPS, peaks in 4-6 hours, and wanes to pretreatment levels by 48 hours after dosing (Garat et al., 1995; Hirano, 1997; van Helden et al., 1997). An accompanying monocytic and lymphocytic infiltration persists for 2-3 days (Ulich et al., 1991a). Larger doses (5—20 mglkg) of IT LPS resembles the condition of Adult Respiratory Distress Syndrome (ARDS) in humans (Hyers, 1991; Wheeldon, 1992; van Helden et al., 1997). Lethality is high in these models, as LPS administration leads to ARDS-like characteristics such as functional alterations, epithelial cell injury, and severe lung vascular leak. a. Mediator Production The result of lower dosages of LPS (10-500 pg) in lung airspaces initiates the production of a similar roster of cytokines and inflammagens to that seen in the circulation during endotoxemia. TNF and IL-1 are two of the earliest mediators to appear in the lungs after as little as 10 pg LPS is instilled in the rat (Ulich et al., 1991a). Detection of messenger ribonucleic acid (mRNA) for TNF and IL-1 occurs by 1 hour after LPS treatment, a time at which airway PMNs are just beginning to appear. Substitution of lL-1 for LPS in airways can induce PMN emigration similar to IT LPS, but PMN accumulation after IT lL-1 reaches a maximum sooner than the LPS-induced response. lntratracheal instillation of TNF 10 results in a delayed and much weaker PMN response relative to LPS or IL- 1, suggesting that lL-1 is the more potent mediator in initiating neutrophilia after IT LPS. Studies with receptor antagonists, however, suggest the opposite. Cotreatment of IT LPS with lL-1 receptor antagonist (lL-1ra) results in 46% inhibition of BALF PMNs whereas cotreatment with soluble TNF receptor (sTNFr) causes a slightly greater inhibition (63%). Treatment with both ILI- ra and sTNFr results in 68% inhibition. Taken together, these observations suggest that TNF is the more important mediator of the two, though it is not sufficient for the full PMN response (Ulich et al.,1992, 1993). As seen with airway LPS administration, TNF plays a critical role in initiating the inflammatory response after IT administration of Gram-negative bacteria (Rezaiguia et al., 1997), although again, its presence is necessary but insufficient for full development of ainNay neutrophilia. Ainlvay TNF and IL-1 production after LPS exposure is largely from AMs. However, mRNA for TNF has been detected in PMNs which have migrated into airspaces (Xing et al., 1994), and TNF production still occurs in rat airways depleted of AMs (Broug-Holub et al., 1997). b. Chemokine Production Although LPS, IL-1 and TNF are not directly Chemotactic for PMNs, they can induce production of chemoattractant cytokines, or chemokines, from macrophages and endothelial and epithelial cells. Interleukin-8 (IL-8) is an important PMN chemokine in rabbits and humans (Furie and Randolph, 1995). An analogue to IL-8, cytokine-inducible neutrophil chemoattractant (CINC) is found in rats (Watanabe et al., 1993; Nakagawa et al., 1994). Another critical rat PMN chemokine is 11 macrophage inflammatory protein -2 (MlP-2). Chemokines will be discussed in detail later in this chapter (Section IV). Strieter and coworkers (1990) stimulated human AMs with IL-1, TNF or LPS to produce IL-8. Similar responses have been demonstrated in rat macrophages, except that CINC and MIP-2 are produced instead of IL-8 (Huang et al., 1992; Nakagawa et al., 1996). Both CINC and MlP-2 have been characterized as important mediators of neutrophil infiltration in the inflammatory response to airway LPS. Expression of mRNA for both chemokines precedes the appearance of airway PMNs (Huang et al. 1992), and co-instillation of antibodies to either CINC or MlP-2 inhibits LPS-induced PMN migration by 70% (Ulich et al., 1995; Frevert et al., 1995a, 1995b). Besides rat macrophages, expression of CINC and MIP-2 has been demonstrated in pulmonary Type II epithelial cells and in activated PMNs (Xing et al. 1994; Crippen et al., 1995; Furie and Randolph, 1995). In addition, cultured Type II cells grown on filter supports preferentially secrete MlP-2 into medium of the basal chamber rather than apically, thus suggesting a preference in vivo for secretion toward the vasculature, where it can affect circulating PMNs (Crippen et al., 1995). Production of MlP-2 after IT LPS is dependent on pulmonary TNF production (Tang et al., 1995). Macrophages collected in bronchoalveolar lavage fluid (BALF) after IT LPS administration exhibit production of MlP-2 mRNA as early as 30 minutes after treatment in vivo (Xing et al., 1994). Interestingly, after 6 hours, PMNs recovered in BALF showed mRNA expression for both TNF and MlP-2. This observation suggests that migrated PMNs fuel the inflammatory process by providing additional Chemotactic mediators over and above those produced by pulmonary cells. This contrasts with some 12 IT LPS studies in which pulmonary PMN infiltration was inhibited yet TNF and chemotactic activities were greater than that seen in PMN—containing BALF from uninhibited rats (Frevert et al., 1994; Ulich et al., 1995). This result suggests that emigrated PMNs promote the down-regulation of mediator production by macrophages and that PMN expression of mRNA for mediators may not translate into their secretion. The central role for the AM in the development of ainlvay neutrophilia after IT LPS is consistent with observations in rat models of pneumonia in which depletion of macrophages decreases PMN responses (Hashimoto et al., 1996). c. Negative Mogglators PMN accumulation after lT LPS is limited by the action of anti-inflammatory mediators produced in the lungs. Interleukin-6 (IL-6) and leukemia-inhibitory factor (LIF) are commonly produced during endotoxemia in rats and are capable of down-regulating TNF production from macrophages in vitro (Schindler et al., 1990; Waring et al., 1995). Detection of mRNA for lL-1ra, IL-6, and LIF in lung tissue of rats treated with IT LPS peaks at 4-6 hours, and this coincides with the peak in PMN accumulation in airways (Ulich et al., 1991a; 1991b;1994a). Co-instillation of LPS with either lL-6 or LIF results in a 50% reduction in PMN infiltration. Production of another cytokine, interleukin-10 (IL-10), has been implicated in the regulation of the inflammatory response to IT bacteria in mice (Standiford, et al., 1996). Furthermore, production of TNF and chemokines from PMNs or AMs stimulated with LPS can be inhibited by IL- 10 (Cassatella et al., 1993; Armstrong et al., 1996). Thus, treatment with IT LPS initiates a cascade of pro- and anti-inflammatory events that produce a self-limiting airway neutrophilia with TNF and AMs as central players. 13 d. Extrapulmonary Effects of IT LPS No endotoxin and only minor amounts of alveolar TNF spill into the vascular compartment of rats after IT LPS doses which initiate PMN migration (Ghofrani et al., 1996). Predictably, there is little effect on blood pressure, heart rate or vascular functions which are normally associated with intravascular LPS (deBoisblanc et al., 1996). There is, however, a decrease in circulating leukocytes which occurs in parallel with the increase in ainlvay PMNs. Larger doses of LPS in excess of those required for PMN emigration can cause the appearance of TNF in the circulation three hours after administration (Turner et al., 1993). It is uncertain if this is due to LPS or TNF leaking into the vascular compartment, or to TNF production at extrapulmonary sites. Activation of adhesion molecules is evident in rat pulmonary microvascular endothelial cells as early as one hour (Tang et al., 1995; Beck-Schimmer et al., 1997). This upregulation precedes significant PMN accumulation by 1 hour. Changes in adhesion molecule expression are dependent on TNF generation within the pulmonary compartment (Tang et al., 1995; Beck-Schimmer et al., 1997). Q SJmeailt Animal models of bacterial pneumonia have revealed distinct host defense responses which are dependent on the form of infection, i.e, the dose of inoculum and Gram-type of organism. Bacterial products can effectively substitute for live bacteria and reproduce some of the features of host responses, including the recruitment of PMNs from the circulation. Multiple bacterial products are responsible for inflammatory responses to 14 Gram-positive bacteria. For Gram-negative bacteria, LPS is the most effective inflammagen derived from these organisms. IT LPS initiates a progressive inflammatory response which is characterized by the production of both pro- and anti-inflammatory mediators. Recruitment of PMNs into airspaces, ostensibly to combat bacteria, also contributes to long term decrements in lung morphology and function. Thus, the IT LPS model in rat is useful for examining the mechanics of PMN trafficking in response to bacterial infection and for understanding the progression of pathologies which are associated with endotoxin inhalation. 15 II. Nosocomial Pneumonia Nosocomial pneumonia is defined as pneumonia that is acquired after at least 48 hours of hospitalization and excludes infections incubating at the time of admission. This is in contrast to community-acquired pneumonia which has a different etiology, risk factors, target populations, and bacterial strain profile (Campbell, 1994). Each year, about 400,000 people acquire pneumonia while hospitalized, or between 5 to 10 cases per 100,000 admissions. Pneumonia is currently the second most common nosocomial infection, but has the highest morbidity and mortality and can increase hospital stays 7-9 days (Craven et al., 1991; 1993). Between 30- 40% mortality is associated with nosocomial pneumonia, and it is as high as 70% when accompanied with high risk factors including age, mechanical ventilation, and major surgery (Leu, et al., 1989; Loumann-Nielsen et al., 1992; Craven, et. al., 1993). Estimates for the added hospital costs due to these nosocomial infections approach $5 billion (Wenzel, 1989; Singh-Naz etaL,1996) Nosocomial bacterial pneumonias are often due to several organisms rather than a single pathogen. A common “core” of bacteria include Gram-negative organisms such as Enterobacter spp., Escherichia coli, Klebsiellas spp., Proteus spp, and Gram positive organisms such as S. aureus and S. pneumonia (Laforce, et al., 1992; Craven et al., 1993; Campbell et al., 1995). Several of these are frequently found together in cultures from nosocomial pneumonia patients. In many cases, highly resistant Gram-negative organisms like Pseudomonas aemginosa and Acinetobacters spp. are the predominant pathogens. As much as 40% of nosocomial pneumonias are of the Entembacten’aceae family, which are normal colonic flora but are often found to colonize the trachea of hospital 16 patients (Dal Nogare et al., 1994). The growth of P. aemginosa is common in the aqueous hospital environments, while S. aureus colonizes the nares of healthy hospital workers and has been detected along with other Gram- negative organisms on the hands of critical care personnel (Dal Nogare et al., 1994). Thus, sources of infection appear to be both from the hospital environment and from opportunistic colonization by endogenous bacteria from the patient. A. Risk Factors Attempts at identifying risk factors have provided both common and disparate findings. Prospective and retrospective studies have identified at least 30 different descriptors of patients who eventually contract pneumonia. The most cited factors include nasogastric or tracheal intubation, mechanical ventilation, trauma, thoracolabdominal surgery, prolonged hospitalization, and age (Leu et al. 1989; Celis et al., 1988; Joshi et al., 1992; Dal Nogare et al., 1994; Campbell et al., 1995; Singh-Naz et al., 1996). Other factors cited less frequently include depressed consciousness, severity of underlying disease, large volume aspiration, hypotension, diabetes, inappropriate use of corticosteroids and antibiotics, cerebrovascular accidents and neoplasms, among others. As much as 70% of cases occur in intensive care units (ICU). In fact, 20—40% of mechanically ventilated patients in the ICU develop pneumonia with an accompanying mortality rate of up to 60% (Langer, et al. 1989; Wenzel, 1989). Thus, intubation devices have been proposed as a primary route for tracheal colonization and subsequent infection of the lower lung (Kollef and Schuster, 1994). That nosocomial infection is less frequent in patients who undergo bowel decontamination prior to surgery suggests that 17 gut flora are the major source of hospital-acquired infections (Kollef, 1994). However, a study of mechanically ventilated patients showed that tracheal colonization was from both gastric and nongastric flora (de Latorre et al., 1995). Recently, associations have emerged between the incidence of hospital-acquired pneumonia and extrapulmonary sepsis or endotoxemia. Patients suffering multiple trauma or undergoing thoracic or abdominal surgery represent high risk groups for developing nosocomial pneumonia (Celis, et al., 1988; Leu et al., 1989: Joshi et al., 1992; Campbell et al., 1995). Endotoxemia or altered neutrophil function in these patients correlates with increased mortality and incidence of nosocomial infections when compared with patients having normal PMN function and no endotoxemia (Buffone et al., 1984; Buttenschoen et al., 1996; Brinkmann et al., 1996; Berger et al., 1997; Simms and D’Amico, 1997). Because PMNs demonstrate an altered adhesion molecule profile when exposed to endotoxin in vitro (Lynman et al., 1994; Witthaut et al., 1994) or when isolated from endotoxemic patients (Duigan et al., 1986; Wakefield et al., 1993; Solomkin et al., 1994), impaired PMN inflammatory and migratory responses may predispose individuals with endotoxemiat to the development of bacterial pneumonia. Modified PMN functions have been demonstrated in septic, post-surgical patients (Cheadle et al., 1996; Egger et al., 1996; Foulds et al. 1997), as well as in animal models of ischemia or hypoxia of the gut (Kadesky et al., 1995) and liver (Colleti et al., 1995), conditions which may prevail in surgical and ICU patients at risk for developing nosocomial pneumonia. Thus, PMN dysfunction and endotoxemia are linked to nosocomial infections, especially pneumonia, in humans. An experimental model that addresses these associations has 18 been recently introduced in the rat, however, intensive efforts using mechanistic approaches have not been undertaken. Mimgl Mod_els of Nosocomial Pneumonia Alterations in pulmonary host defense responses during endotoxemia were first shown in rats and mice inoculated with bacteria into airways (White et al., 1986; Nelson et al., 1990). Intravenous administration (IV) of endotoxin blocks PMN emigration into airspaces, and this allows the growth of bacteria go unchecked. Recent studies have shown that LPS administered IT could be substituted for bacteria and that endotoxemia from either IV or intraperitoneal (IP) routes could block PMN migratory responses (Frevert et al., 1994; Hirano, 1996; Mason et al., 1997). Impairment of PMN trafficking in lV/IT LPS models is due neither to insufficient PMN numbers in the pulmonary vascular bed nor to decreased chemotactic activity within the airways (Frevert et al., 1994; Hirano, 1996). Consistent with similar findings during early endotoxemia in humans, isolated rat PMNs have altered expression of adhesion molecules (Frevert et al., 1994; Solomkin et al., 1994; Wakefield et al., 1997). In addition, inhibition of PMN trafficking into ainrvays is associated with systemic TNF production (Nelson et al., 1990) and complement activation (White et al., 1986), two common systemic responses during endotoxemia which will be discussed further in section VIII. In fact, the inhibitory effect can be duplicated by substituting non-physiological doses of TNF in place of systemic LPS administration (Mason et al., 1997). Since concentrations of TNF used in this study were 50-fold greater than those measured in plasma during endotoxemia, it is difficult to conclude whether or not TNF plays a causal role in the inhibition of PMN migration in clinical situations. 19 Complement activation, the presence of circulating TNF and alteration of PMN adhesive proteins could potentially cause PMN dysfunction, but experiments to test their roles in models of lV/IT endotoxin models have not been performed. Existing animal models of endotoxemia-induced inhibition of pulmonary PMN recruitment typically allow endotoxemia to develop for 2 hours before challenging ainrvays with bacteria or endotoxin (Nelson et al., 1990, Frevert et al., 1994; Mason et al., 1997). Several 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. Furthermore, 3-4 hours pass after lT LPS administration before pulmonary PMNs are collected; thus the time during which circulating PMNs are exposed to multiple stimuli is 5 to 6 hours. The fact that PMN function can be modulated by several soluble and cellular mediators during this time makes it difficult to discern to which effector the PMN responds during endotoxemia, or, even more daunting, how multiple and sometimes conflicting signals are integrated and appropriately processed by the PMN. C. Summary Cotreatment of rats with IV and IT LPS is an attractive animal model with which to study endotoxemia-related pneumonia. To date, the mechanism for migratory dysfunction is unknown, but it may be due to a direct effect of LPS on the PMN or from activity of downstream mediators of endotoxemia. To better understand how PMN migration might be modulated during inflammation, the next few sections give brief descriptions of the PMN and how competent transendothelial processes occur in systemic and pulmonary circulatory systems. 20 Ill. Polymorphonuclear Leukocytes By virtue of their large numbers, rapid response, and relatively simple mission, PMNs represent the infantry in the inflammatory defense response of mammals to invading pathogens. In humans, neutrophils account for 50-65% of circulating white blood cells, and their numbers can swell 2 to 4-fold during conditions of infection or disease (Edwards, 1994). Equally important as their large numbers in the body is their rapid response to inflammatory stimuli. The normal host response to infection, injury, or wounding is usually the recruitment of PMNs within minutes to the site. Once they have arrived and identified the invading pathogen, their role is to kill, and PMNs possess an impressive arsenal of proteases and degradative enzymes stored in their numerous cytoplasmic granules. PMNs can also transform oxygen into several highly toxic metabolites, which, when combined with proteolytic activity from granules, destroy a broad array of infecting organisms (Edwards, 1994). A secondary role of PMNs is to produce and release cytokines that mediate the recruitment and activation of a second wave of cells to the inflammatory focus, namely monocytes, macrophages, and lymphocytes. Macrophages assist in killing highly infectious organisms, but they also are critical to the healing and reorganization of injured tissue (Cotran et al., 1994). Should the invading component be so immunogenic as to elicit antibody production, the presence of lymphocytes ensures a timely response of the appropriate systems. Thus, the PMN is a pivotal cell not only for the initiation of inflammation, but also for proper resolution of damage and modulation of immune responses. 21 A. Distribution Given their short half-life of 6-7 hours in the circulation, large numbers of new PMNs must be constantly produced and released into the blood. In adult humans for instance, 5 X 1010 PMNs are generated daily (Edwards, 1994). Neutrophils arise from precursor myeloblast cells in bone marrow, then divide and differentiate over a two week period before their release into the circulation as mature cells. Blood PMN concentration can increase quickly by the early release of stored, mature neutrophils, and the premature release of underdeveloped cells. The nucleus of an immature neutrophil appears as a band across the cell, distinct in appearance from the segmented, polylobular nucleus and granule-rich cytoplasm of the mature cell (Terashima et al., 1996b; Seebach et al., 1997). Some experimental observations suggest that band cells are less active in the inflammatory response such as migration and oxidant production (van Eeden et al., 1997; Sato et al., 1998). Therefore, the sudden increase in PMN numbers, which include "band" cells may not be matched with an equal increase of inflammatory responsiveness. In contrast, mediators present during persistent inflammation or infection can cause an acceleration of the PMN maturation and release processes in marrow, and thereby prolong neutrophilia and responsiveness during these conditions (Ulich et al., 1989; Selig and Nothdurft, 1995). Thus, differential counting of mature versus banded neutrophils can suggest the mechanism of neutrophilia and perhaps assist in a clinical diagnosis. Enumerating PMNs in a blood sample does not reflect the true number of circulating neutrophils. An estimated 40-60% of vascular PMNs are "marginated", or adhered tenuously to vessel walls, and are inaccessible to counting in a sample of whole blood (Doerschuk et al., 22 1987). Most marginated PMNs are associated with the pulmonary vasculature, and may be due to the longer PMN transit time through the large, microvascular network of the pulmonary circulation. The degree of pulmonary PMN margination is inversely correlated with pulmonary pressure and flow and increased cardiac output by epinephrine (Doerschuk et al., 1988; Lien et al., 1990; Kuhnle et al., 1995). Therefore, pulmonary vasculature may serve as an important storage site from which PMNs can be mobilized in response to acute stimuli. B. Mobilization Migration of the PMN to the locus of inflammation requires its departure from the blood vessels and transit into and through extravascular tissues. It must first escape the shear flow of the blood, usually within capillaries or post-capillary venules, and then proceed to associate with the vascular wall in a delicate sequence of adhesion events which culminate in egress between endothelial cells (Lawrence and Springer, 1991) . Briefly, PMNs are captured via the action of membrane glycoproteins called selectins which slow the passing PMN to a rolling motion outside the shear flow of circulating blood (Bevilacqua and Nelson, 1993; Albelda, 1994). The transition to a firm adhesion and flattening of the PMN is mediated by lntegrins and immunoglobulin (lgG)—like adhesive proteins before the PMN migrates to an endothelial tricellular junction through which it will eventually migrate into the surrounding tissue (Luscinskas and Lawler, 1994; Malik and Lo, 1996; Burns et al., 1997). The events leading to and through the cell-cell associations at the bloodztissue interface represent the crossroads of inflammation, a defining moment when the host response commits the recruitment of cytotoxic cells to (counter)attack foreign insults. 23 C. Killing However efficient the PMN is at escaping the vasculature and traversing through interstitial spaces, most of its structural and biochemical components are geared to recognize and kill foreign invaders. Its preferred mode is to first engulf the pathogen and seal it off within an intracellular vacuole called a phagosome. At least three distinct types of granules within the PMN cytoplasm can then fuse with endocytic vesicles and release their cytotoxic contents onto the entrapped pathogen (Sengelov, 1996; Borregaard and Cowlands, 1997). Among the granular products are a variety of proteases, hydrolases and degraditive enzymes that are effective against a broad spectrum of bacteria, fungi, and parasites. In some pathologic and toxicologic scenarios, granule contents are released extracellularly, perhaps to target a pathogen too large to engulf. The PMN component responsible for its oxidant-producing potential is the membrane-bound enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Thelen et al., 1993; Henderson and Chappell, 1996). While it performs the simple transfer of an electron to elemental oxygen, the enzyme requires a complex procedure for activation and consumes large amounts of energy in the process. Before the enzyme can function, cytoplasmic and membrane bound subunits must be sequentially mobilized through a series of signal-transducing mediators. Myeloperoxidase activity within an endocytic vesicles results in highly toxic species such as hypochlorous acid and hydroxyl radical - a free radical against which few cells and organisms have effective defenses. As with granular enzymes, the oxidative burst can be turned to the extracellular environment, the consequences of which expands the killing zone but 24 might also result in injury to host tissue. D. Summary This brief introduction of the neutrophil has omitted several aspects of its inflammatory functions and roles in disease processes. The emphasis presented here is on the PMN’s ability to kill, and kill quickly. The tissue macrophage is larger, more phagocytic, and possesses similar cytotoxic weapons. However they are slower to accumulate at sites of insult, arriving after many hours compared to minutes required for the PMN. Compared to the macrophage's role in inflammation, the inherent strength of the PMN is its ability to mobilize quickly in great numbers where and when needed. Proper trafficking of neutrophils to sites of pathogenic insult is a critical control point for a successful host inflammatory response. 25 IV. Mechanisms of PMN Migration Circulating leukocytes can migrate from vessels into tissues under both normal and pathologic circumstances. Unlike migration by monocytes and lymphocytes, the PMN requires chemotactic stimuli and/or endothelial cell activation to accumulate in tissue in significant numbers. Indeed, rapid influx of large numbers of PMNs into tissues is a hallmark of acute inflammation. In comparison, small numbers of mononuclear leukocytes are already present in healthy tissue and only show significant accumulation in inflamed tissue much later than PMNs (Cotran et al., 1994). It is well accepted that leukocyte migration from the vasculature occurs by a multi-step process, dictated by the sequential activation of adhesive proteins and their ligands on both leukocytes and endothelial cells (von Andrian et al 1991; Lawrence and Springer, 1991; Konstantopoulos and McIntire, 1996). Understanding the mechanisms of leukocyte trafficking and recruitment has implications in diverse conditions from cancer, atherosclerosis, organ transplantation, trauma, and ischemia/reperfusion, to the body’s response to wounding, microbial infection and toxicant exposure. Monocytes, lymphocytes and PMNs all migrate by similar, sequence- dependent mechanisms but differ in their response to chemotactic and inflammatory signals, particularly in their qualitative and quantitative expression of adhesion molecules (Dransfield et al., 1992; Springer, 1994; Li et al., 1996; Ager, 1996). Initiation of migration begins with the “capture” by the vessel wall of PMNs from flowing blood, and is followed by their end-over-end “rolling” along the vessel wall. This process is known as margination, and is believed to be a normal behavior of circulating PMNs. Only after proper stimuli are present do rolling leukocytes become firmly 26 adhered to endothelial cells (ECs) and thus positioned themselves for potential migration from the blood vessel into inflamed tissue. The general steps of transendothelial migration by PMNs and adhesion molecules the functions of which predominate in each step are depicted in Figure 1. A. Capture and Rolling Both the capture, or initial tethering and removal of PMN from the blood flow, and rolling of PMNs along the vessel wall, is due to the reversible binding of transmembrane glycoprotein adhesive molecules called selectins, which are found on both PMNs and endothelial cells (Bevilacqua and Nelson, 1993; Albelda et al., 1994; Luscinskas and Lawler, 1994; Crockett-Torabi and Fantone, 1995; Tedder et al., 1995). Selectins have a calcium-dependent lectin domain on their extracellular N- terminus, which is attached to an epidermal-growth factor (EGF) -like domain, and then to a number of short consensus sequences. A short, intracellular domain is linked to signal transduction proteins (reviewed by Crockett-Torabi and Fantone, 1995; Crockett-Torabi, 1998). Selectin-type adhesive proteins are found on most cell types of hematopoetic origin and on endothelial cells of blood and lymph vessels. 1. L-Selectin lntravital microscopic analysis of the microvascular circulation in normal tissue offers visual evidence of the transient "stick and release" behavior of PMNs rolling along the vessel wall. In non-inflamed tissues, the tenuous association of PMNs within postcapillary venules can be blocked by treatment with antibodies to leukocyte-selectin (L-selectin, Mel— 14, LAM-1, CD62L) on circulating PMNs (Spertini et al., 1991; von Andrian et al., 1991). L—selectin is constitutively expressed on PMNs, and newly 27 60:22.: 3:266:35: E EoEo>_c>:_ 2:022: Exec—=5 use «0339... ._. 959". Z 4.9m: £__._u.:=_ xEC £323. - A— Etc—3-4 ..-.\.- ,l .3. 14¢ .i . 0 l-- .I u . .el . ’ I .5 I O Q. a .I i D. I nu o n . I i ”111/. . . . A u . I ; A... u. .s a .. III. . I. .a i .c . . . t . . .l l ... C l . .. 3.x . 4. \y ' .. V. I p I i n. . n u n u . . I ‘ i I ‘ n u I n ‘I h I ~ m. . I .l I ' I O. ‘ I'l I.“ I I ~ ‘ . 92% 28 released PMNs from bone marrow possess high levels of the L-selectin when compared with older, circulating PMNs (Matsuba et al., 1997). The mechanism of L-selectin loss on PMNs may be due to proteolytic activity which results in rapid shedding from the PMN surface and the appearance of bioactive L-selectin in the blood (Kishimoto et al., 1995). Auto- proteolysis of L-selectin can occur after exposure to various inflammatory mediators such as LPS and TNF, but it may also occur from normal rolling interactions with the vessel wall. For example, metalloprotease inhibitors significantly reduce rolling velocity and cleavage of L-selectin on PMNs in vitro, suggesting that L-selectin is routinely shed during the normal rolling processes (Walchek et al., 1996). Thus, the longer a PMN has been in the circulation and interacting with the vessel wall, the more L-selectin it has lost to transient binding and cleavage. Replacement of L-selectin on circulating PMNs has not been demonstrated, and a low expression level is associated with apoptosis and may be a signal for the removal of the PMN from the circulation (Matsuba et al., 1997). High plasma levels of soluble L-selectin can occur during infection and are believed to inhibit PMN rolling at noninflamed sites. Bioactive soluble L-selectin can bind to endothelial ligands and block their interactions with PMN-borne L-selectin (Schleiffenbaum et al., 1992; McGill et al., 1996; Ohno et al., 1997a). Although not fully characterized, the corresponding endothelial ligand of PMN L-selectin is a member of a group of sialomucin oligosaccharides which share affinity for selectins expressed on platelets, lymphocytes and monocytes (Varki, 1997). Studies in vitro demonstrate an endothelial ligand for PMN L-selectin which is induced by LPS, TNF, or lL-1 exposure to endothelial monolayers (Spertini et al., 1991). In addition, L-selectin dependent rolling of PMNs which occurs in noninflamed tissues suggests 29 the existence of a constitutively expressed endothelial counterpart to L- selectin (von Andrian et al., 1991; Walchek et al., 1996). The best characterized ligand for L-selectin is CD34 (Fig. 2), which is found on high vein endothelial cells and binds selectively to L-selectin of lymphocytes (Tedder et al., 1995; Ager, 1996). CD34 and related molecules are long protein chains heavily modified with sugar and sialyl groups and have variable binding affinities to all selectins in nonflow systems in vitro. The endothelial ligand for L-selectin is believed to be a fucosylated variant of CD34 (T edder et al., 1995; Krause et al., 1996). Treatment of animals with an antibody to sialyl-Lewisx, an epitope on L- selectin, can block PMN migration in a rat model of hemorrhagic shock and liver failure (Rubio-Avilla et al. 1997). _2_. P-Selectin At least two endothelium-bound selectins, platelet- and endothelial-selectins (P- and E-selectins, respectively) can facilitate PMN- EC adhesions. These selectins are expressed only when appropriate inflammatory stimuli are present. P-selectin (granule membrane protein- 140:GMP-140; CD62P), is stored intracellularly in Weibel-Palade bodies of endothelial cells and in alpha-granules of platelets (Malik and Lo, 1996). Within minutes of exposure of ECs to inflammatory mediators such as complement products, oxygen-derived free radicals or various cytokines, P- selectin is mobilized to the cell surface where it can interact with its PMN counterpart, P-selectin glycoprotein ligand-1 (PSGL-1; 00162). Monocytes and platelets also possess PSGL-I and can bind to P-selectin on activated ECs. PSGL-1 is a sialomucin similar to CD34, but consists of a disulfide bonded homodimer on the PMN surface and is capable of binding two 30 .Aom. 83.0585 5:25 322% 238.52. co =o_u2m_E agape—3:028“ E 60202: 3:89. new 02:020.: 532:3 .N 0.59". ”Sam. 582$-.. .-¢- _1 "\ W t a! u.- '§ 1.’ "d I qr , . ‘ V - 1-."r‘ quurifip‘ ‘ ’ ‘3‘ '..:’T" 4 ~. 7' 7 A . n It ‘ I v .(I ‘ . .I .4; ., . ,. . 31 P-selectin ligands simultaneously (Fig.2) (McEver and Cummings, 1997). PSGL—1 is uniformly distributed on quiescent, rolling PMNs. L- selectin binding occurs first and is more rapid and short lived than P- selectin binding (Ley, 1995). Binding to P-selectin is characterized by longer PMN-EC associations, slower rolling velocities and eventual tethering of PMNs to the vessel surface (Davenpeck et al., 1997; Alon et al., 1997). As with L-selectin binding however, P-selectinzPSGL-1 interaction can be short-lived and reversible if further adhesive events are not invoked (Lawrence and Springer, 1991; Finger et al., 1996; Davenpeck et al., 1997). In the presence of appropriate inflammatory stimuli, however, P-selectin binding is accompanied by a rapid redistribution of PSGL-I to uropods on activated PMN and may signal the transition from capture to rolling (Bruehl et al., 1997). 3. E-Selectin A second endothelial-bome selectin, E-selectin (ELAM- 1,CD62) requires gene transcription for expression. Peak expression and activity in endothelial cells in vitro is 4-6 hours after exposure to inflammatory stimuli such as TNF, LPS or lL-1 (Klein et al., 1995; Scholz et al., 1996). E-selectin can support rolling and tethering of PMNs in a similar fashion as P-selectin (Lawrence and Springer, 1993). Thus, its role in inflammation is probably to maintain PMN rolling after P-selectin has been down-regulated (Malik and Lo, 1996). Characterization of selectin-mediated capture and rolling has been done predominately in systemic vessels (Lawrence and Springer, 1991; von Andrian et al 1991; Ley, 1995; Davenpeck et al., 1997). However, in the pulmonary circulation recent lntravital microscopic studies show 32 different selectin involvement during PMN-EC interactions, and these will be discussed in the next section (Keubler et al., 1997; Yamaguchi et al., 1997). To summarize briefly, L-selectin (capture) and P-selectin (rolling on endothelial cells) work cooperatively to initiate the migration process during inflammation (Ley, 1996; Alon et al., 1997; Davenpeck et al., 1997). Selectins and their PMN ligands are either constitutively expressed or stimulated to mobilize to the cell surface. These reversible selectin-ligand interactions allow time for PMNs to associate with endothelial cells and integrate and respond to stimuli presented on the endothelial surface. B. Firm Adhesion The signal for the next step of firm adhesion is postulated to be either a receptor-mediated event in response to a soluble inflammatory cytokine or an event propagated from signals from activated selectins. Cytoplasmic domains of bound and activated L-selectin and PSGL-1 are linked to signal transduction pathways which appear to lead to integrin activation in PMNs (Simon et al., 1995; Crockett-Torabi and Fantone, 1995; Zimmerman et al., 1996a; McEver and Cummings, 1997). Thus, selectins may function to promote the orderly transition to the adhesion process by invoking integrin pathways in a timely, sequential manner to ensure successful migration (Fig. 1). 1. lntegrins lntegrins are a group of heterodimeric transmembrane glycoproteins found on PMNs and other hematopoietic cells that mediate cell-cell and cell-extracellular matrix adhesions (Hynes, 1992; Luscinskas 33 and Lawler, 1994). All integrins are made of one a and one 3 subunit which together form an extracellular ligand binding site. Cytoplasmic tails of integrins provide phosphorylation sites and linkages to cytoskeletal proteins involved in signal transduction. There are 8 different B-subunit ([313) that associate with one of 16 a—subunits to form 22 known receptors in a variety of cells, including lymphocytes, leukocytes and platelets. lntegrins are capable of mediating cell-cell binding but also are involved in cell interactions with extracellular proteins such as Iaminin, fibronectin, vitronectin and fibrinogen to name a few. PMN binding to activated endothelium is mediated primarily by two integrins which contain [32, or CD18, subunits: macrophage antigen -1 (Mac-I; cam/82 ; CD11b/CD18) and lymphocyte associated function antigen —1 (LFA-1; «L182; CD11a/CD18). LFA-1 is the predominate integrin used for lymphocyte emigration (Li et al., 1996). The low-level of basally expressed LFA-1 on PMNs is unaltered by activators or stimuli. However, both LFA—1 and another CD18 integrin, p150,95 (CD11c/CD18; (Ix/I32). can promote PMN trafficking under certain conditions. Mac-1 has emerged as the more critical CD18 integrin in most models of PMN-dependent inflammatory responses (Luscinskas and Lawler, 1994; Malik and Lo, 1996). Preformed Mac-1 is stored in three separate PMN compartments: secretory vesicles, specific-granules and gelatinase granules (Sengelov, 1996; Borregaard and Cowland, 1997). As such, Mac-1 can be rapidly mobilized to the PMN surface after exposure to degranulation stimuli such as the bacterial peptide n-formyl-methionyl- leucyI-phenylalanine (FMLP), as well as to weaker stimuli which mobilize only the secretory vesicles (Altleri and Edgington, 1988). In human PMNs, these latter stimuli include LPS and TNF, among others. Inflammatory 34 stimuli can also promote transcription and translation of Mac-1 genes, thus prolonging integrin involvement during inflammation. Even when Mac-1 is incorporated into the plasma membrane, only a small percentage (~10%) may be competent for ligand binding (Diamond and Springer, 1993). In addition, small numbers of inactive CD18 molecules, incapable of binding ligand, are constitutively present on the PMN. The consequence of mobilizing inactive Mac-1 to the cell surface of activated PMNs is unclear, but molecular studies have provided some insight into mechanisms which may modulate the activity of membrane- expressed integrins (Amaout et al., 1990). In vitro studies have shown that inactive Mac-1 on PMN membranes becomes competent for ligand-binding in the presence of manganese (Mn"“) and other divalent metals (excluding calcium) (Michishita et al.,1993). Metal binding on the extracellular portion of the a-subunit near the ligand inding site is proposed to cause a conformational shift in the molecule that exposes a requisite epitope for ligand binding. Another site for regulation is the intracellular domain. Sites on the intracelluar portion of (32 are critical for internalization and down-regulation of bound Mac-1 (Rabb et al., 1993). Deletion studies have shown a prolongation of Mac-1 binding to extracellular ligands when critical cytoplasmic residues are missing. In addition, the cytoplasmic tail of the (32 subunit of Mac-1 possesses serine and threonine sites for potential modification by phosphorylation. Granular Mac-1 is unphosphorylated, but it becomes phosphorylated shortly after mobilization into the plasma membrane (Buyon et al., 1997). It is unclear if phosphorylation is correlated with binding activity as these studies were performed in the absence of figands. 35 Other studies have elucidated a role for a novel and partially characterized intracellular lipid (Hermanowski et al. 1992; Detmers et al., 1994; Klugewitz et al., 1997). The lipid, integrin modulating factor-1(IMF-1) is produced by human PMNs upon exposure of the PMN to LPS or to the PMN chemoattractant lL-8. Production of IMF-1 significantly augments CD18-ligand binding in both intact membranes and in a cell-free system which employs a soluble form of Mac-1. Another intercellular mediator which can potentiate CDI8 binding is a recently discovered protein called cytohesin -1. Although not tested directly on Mac-1, the protein does interact with the [)2 subunit of LFA-1 and enhances its binding to lCAM-1 (Kolanus et al., 1996). Besides its endothelial ligands, Mac—1 has specific recognition and binding sites for fibrinogen, LPS, factor X coagulation protein and complement protein C3i (Wright, et a., 1988; Ross and Vetvicka, 1993; Flaherty et al., 1997). Thus, during some inflammatory scenarios many ligands for Mac-1 may be present at the same time. For example, during endotoxemia activation of the complement cascade results in generation of C3i proteins and increased expression of endothelial ligands. Fibrinogen and factor X are normally present in blood. Thus, CD18-mediated PMN functions might become dysregulated during endotoxemia and thereby affect normal migration processes. 2. Intracellular Adhe_sjon Molecule - 1 The complementary endothelial ligand for Mac-1 is intercellular adhesion molecule-1 (lCAM-1; CD54), an immunoglobulin (Ig)-like molecule (Fig. 2) that exhibits low constitutive presentation on endothelial cell membranes but is markedly induced by exposure of ECs to 36 inflammatory cytokines (Gerritson and Bloor, 1993; Hashimota et al., 1994; Klein et al., 1995; Scholz, et al., 1996; ligo et al., 1997). lCAM-1 is also found on tissue epithelial cells including Type I pneumocytes (Barton et al., 1995; Burke-Gaffney and Hellewell, 1996). LFA-1 can bind to lCAM-I, but it has higher affinity to a related protein, lCAM-2, a ligand to which Mac-1 binds poorly. LFA-1:ICAM-2 and other interactions are critical to endothelial transmigration of monocytes and lymphocytes. Thus, in concert with the specificity contributed by L-selectin binding, integrin-ICAM interaction provides a second point of control for the type and number of inflammatory cells to be activated during inflammation. As mentioned earlier, mutiple lines of evidence suggest that selectin activation and binding is required before firm adhesion can occur via Mac- 1:ICAM-1 interactions (Springer et al., 1991; von Andrian et al., 1992; Ley, 1995). To assist the smooth shift from rolling to adhesion, selectin- mediated signals are incorporated with other extracellular inflammatory stimuli to regulate the timely expression and engagement of PMN integrins (Crockett-Torabi and Fantone, 1995; Crockett-Torabi, 1998). Studies in vitro have demonstrated that cross-linked activation of L-selectin on PMNs can lead directly to BZ-integnn-mediated adhesion in both static assays (Simon et al., 1995; Steeber et al., 1997) and in shear-flow models (Gopalan et al., 1997). Furthermore, removal of L-selectin from PMNs renders them incapable of firmly adhering to endothelial monolayers in either static (Zouki et al., 1997) or shear-flow systems (Endemann, et al., 1997). It is not surprising that L-selectin is required for the capture and rolling of PMNs in a flow system; L-selectin binds more rapidly and at higher shear rates than CDIB integrins (Taylor et al., 1996). However, the requirement for L—selectin in static systems suggests that it is needed for 37 more than physical capture and perhaps promotes integrin activation via intracellular pathways. Adhesion induced by ligated L-selectin is blocked by inhibitors of protein tyrosine kinase (PTK) and protein kinase C (PKC) activities, suggesting that these signal transduction pathways link L-selectin to Mac-1 upregulation (Steeber et al., 1997). It makes sense that as integrin mechanisms are invoked, the control by selectin mediated adhesions must subside. One example of “bond- trading” between selectins and integrins is the redistribution of PSGL-I and concommitant loss of P-selectin binding when PMNs are treated with integrin-activating chemoattractants (Lorant et al., 1995). In addition, L- selectin binding is down-regulated at the same time as CD18 up-regulation. Thus, the step-wise progression of transendothelial migration is mediated in part by intercellular signaling initiated by adhesion molecule:ligand interactions. C. Transmigm Egress of PMNs through EC monolayers occurs preferentially at tricellular junctions (Burns et al., 1997). Although the mechanism of extravasation is imperfectly understood, it is clear that selectin involvement has been down-regulated at this time and an lgG-type adhesion molecule, platelet endothelial cell adhesion molecule-1 (PECAM-1;CD31) becomes critical for the actual passage of PMNs between ECs (Fig. 1) (Newman, 1997; Dejana et al., 1995; Vaporciyan et al., 1993). Found on PMNs, platelets and endothelial cells, PECAM-1 is unusual among the inflammatory adhesion molecules in that it is its own ligand and forms homodimers with molecules on opposing cells (Fig. 2). PECAM-I is evenly distributed over the surface of circulating PMNs and is 38 concentrated at intercellular junctions of unstimulated endothelial cells (Newman, 1997). By virtue of its junctional location, PECAM-I is hypothesized to be a homing receptor to locate the transendothelial portal for the migrating PMN. Treatment of PMNs or endothelial monlayers with an antibody to PECAM-1 blocks transmigration in vitro (Muller et al., 1993; Muller, 1995), and similar antibodies have been effective in inhibiting PMN migration in rat models of peritonitis and alveolitis (Vaporciyan et al., 1993 ; Bogen et al., 1994; Muller, 1995). In both whole animal and cell systems, PECAM-I antibodies does not block adhesion. Activation of PECAM-1 on PMN by either cross-linking with PECAM- 1 antibody or binding to F(ab) fragments can increase the activity of Mac-1 (Berman and Muller, 1995), suggesting an intracellular link between the two events. Because the trailing end of the migrating PMN must detach from endothelial cells as the leading uropod attaches, up- and down- regulation of Mac-1:lCAM-1 interaction is critical for successful diapedesis. lntegrin activation, in turn, can lead to phosphorylation of tyrosine residues on PECAM-1, and this may be involved in cross-regulation between the two adhesion molecules (Lu et al. 1996). Mac-1-mediated endothelial binding can cause structural changes in endothelial cytoskeletal proteins associated with adherens junctions without causing endothelial cell retraction or injury to monolayers (Allport et al., 1997b; Del Mascio et al., 1996). The endothelial junctional proteins plakoglobin, a and I3 catenin and cadherin disappear within minutes of PMN binding to endothelium (Del Mascio et al., 1996). It is unclear if this reorganization is required for transendothelial migration. After long exposures of endothelial monolayers to large concentrations of TNF and lFN-y, PECAM-1 becomes diffusely distributed throughout the cell 39 membrane and away from intercellular junctions (Romer et al.,1995). At the same time, lCAM-I and ligands to L-selectin redistribute from random expression to localization at cellular junctions (Bradley and Pober 1996). It is unknown what role this plays in PMN emigration; in each system, neither ICAM-1, PECAM-1 nor the L-selectin ligand was bound by PMN counterparts. The presence of PMNs or soluble counterreceptors may result in a different redistribution behavior. Once the PMN has entered the subendothelial matrix, PECAM-1 expression is down-regulated (Christofidou-Solomidou et al., 1997). In summary, the steps from PMN capture to transendothelial migration are a carefully orchestrated continuum of adhesion molecule up- and down-regulation. The intracellular components responsible for cross- talk and modulation of adhesion molecules has been partially explained and is the subject of ongoing investigation (Crockett-Torabi and Fantone, 1995; Rosales and Juliano, 1995; Walzog et al., 1996; Todd and Petty, 1997; Crockett-Torabi, 1998). D. Mediators of Migration Pro-migratory stimuli can generally be classified as either non- chemotactic cytokines or chemoattractants. Canonical inflammatory cytokines such as TNF and lL-1 can engender expression of adhesive proteins on PMNs and ECs, but are not themselves chemotactic to PMNs. For example, TNF and IL-1, along with other inflammatory mediators, promote the firm adhesion of PMNs to endothelium in systems in vitro (Schleimer and Rutledge, 1986; Huber et al., 1991; Burke-Gaffney and Hellewell, 1996; Komatsu et al., 1997). However, egress and migration of PMNs into extravascular spaces requires the presence of chemoattractants 40 which cause the directed migration of PMNs through tissue. Some chemoattractants can promote expression of adhesion molecules on PMNs similar to the responses elicited by lL-1 and TNF. Selected mediators of migration are summarized in Table 1. 1. Cytokines A variety of inflammatory mediators can engender the firm adhesion of PMNs to ECs. Two of the most important pro-adhesive cytokines that are present during most inflammatory responses are TNF and IL-1. Both are pluripotent factors, and their mediation of promigratory function for inflammatory cells is just one of their many actions during pathologic conditions. The macrophage/monocyte is the primary cellular source of TNF, and LPS is perhaps its most important inducer (reviewed in Tracey and Cerami, 1993, 1994; Bemelmans et al., 1996; Di Girolamo et al., 1997). Among its many physiologic effects are shock, cytotoxicity and cachexia. The effects of TNF on PMNs and ECs are induced by binding to two different TNF receptors present on each cell. PMNs can respond to TNF by activating and expressing integrins, producing PAF and other mediators, and releasing granule contents. Likewise, ECs mobilize selectins, up- regulate ICAM and activate procoagulative pathways in response to TNF exposure. During endotoxemia, PMNs release from their membranes a soluble TNF receptor that can bind to and effectively inactivate circulating TNF. Prolonged exposure to TNF can activate apoptotic pathways in both PMNs and ECs. Interleukin-1 has been known under various names for more than 40 years as an important mediator of inflammation and fever (reviewed in 41 WWam their Effects inclines..— TNF IL- 1 IFN-y PAF LTB, FMLP C5a _thQHmL— Interleukin-8 CINC (CINC-1; KC) MIP-Z (CINC-3) _Snmmary 0f Effects Induces P-selectin, ICAM-1, IL-8 in ECs, shedding of L-selectin, expression of CD18 on PMNs, inhibition of PMN chemotaxis Induces P-selectin, ICAM-1 on EC8, shedding of L-selectin, expression of CD18 on PMNs Induces P-selectin, ICAM-1 on EC5, shedding of L-selectin, expression of CD18 on PMNs PMN chemotaxin, induces EC, PMN adhesion molecules PMN chemotaxin, enhances EC, PMN adhesion PMN chemotaxin, induces EC, PMN adhesion molecules PMN chemotaxin, induces PMN adhesion molecules, desensitizes PMNs to other chemoattractants Chemotactic for human, rabbit PMNs, shedding of L-selectin, expression of CD1 8 on PMNs, inhibits PMN migration in vivo Chemotactic for rat PMNs Chemotactic for rat PMNs, shedding of L- selectin, expression of CD18 on PMNs 42 Kampschmidt, 1984; Movat, 1987; Le and Week, 1987; Moldawer, 1994). Its cellular sources and physiologic effects are similar to those of TNF, and the two are often found together in a variety of inflammatory scenarios. Like TNF, lL-1 induces selectin and ICAM expression on ECs and promotes integrin activation on PMNs. During inflammation, PMNs and macrophages express on their membrane a receptor antagonist (lL-1ra) that binds to lL-1 but it is not linked to signal transduction machinery. A similar decoy receptor has not been identified in ECs. Exposure of EC monolayers or PMNs in vitro to TNF (Romer et al., 1995; Bradley and Pober, 1996; Burke-Gaffney and Hellewell, 1996) or lL-1 (Schleimer and Rutledge, 1986; Scholz et al., 1996) causes time- and dose-dependent expression of selectins and integrins. In addition, TNF and lL-1 treatment in vivo induce ICAM-1 in lung and small intestine (Komatsu et al., 1997). As noted above, LPS, TNF and IL-1 are not chemotactic for PMNs, but their exposure to ECs can elicit transendothelial migration in vitro. This phenomenon is dependent on cytokine-stimulated production of endothelial-derived chemoattractants which can be detected in the culture medium during migration assays in vitro (Huber et al., 1991; Kuijpers et al., 1992; Smart and Casale, 1994; Burns et al., 1997b). Studies in vivo confirm that chemoattractant production is often dependent on an earlier appearance and activity of cytokines. 2. Chemoattractants PMNs have at least five different receptors for chemotactic stimuli. Unique receptors for PAF, complement protein C5a, leukotriene B4 (LTB4), and bacterial peptides such as FMLP mediate most PMN migratory responses (Table 1). In addition, chemokines and their specific receptors 43 have been recently characterized as important players in inflammatory responses of the PMNs (Furie and Randolph, 1995). Chemoattractants for PMNs can be produced by a wide variety of cells, including endothelial and epithelial cells, macrophages, monocytes, lymphocytes, platelets, and PMNs. In models in vitro, chemoattractants can activate PMNs or ECs to express adhesive proteins in a similar manner as TNF or IL-1. Thus, redundant pathways for adhesive and migratory processes probably occur in vivo (Detmers et al., 1990, 1991; Huber et al., 1991). PAF is an acetylated phosphoglyceride derived from lipids of cell membranes (Saito, 1996). It can be produced by ECs, platelets, PMNs and macrophages and promotes both pro-inflammatory and pro-adhesive processes. PAF is produced during both systemic sepsis and bacterial pneumonia where its presence augments PMN responses (Makristathis et al., 1993; Mathiak et al., 1997). Infusion of PAF into animals can reproduce effects similar to endotoxemia. In addition to chemotaxis, PAF may also promote adhesion (Zimmerman et al., 1996b). EC-derived PAF can be localized on the endothelial cell surface where it has access to PAF receptors on rolling PMNs. LTB4 is produced by PMNs and macrophage/ monocytes from arachidonic acid (reviewed in McMillan and Foster, 1988; Borgeat and Naccache, 1990; Brooks and Summers, 1996). It is present in most inflammatory foci where it is 10-1000 more effective than PAF at eliciting chemotactic responses of PMNs. In addition, LTB4 can elicit PMN adherence to ECs and to artificial surfaces. Evidence suggests that EC adherence is due in part to direct action of LTB4 on the endothelial cell, however, an endothelial receptor for LTB4 has not been identified (Nohgawa et al., 1997). 44 05a is generated from the cleavage of complement protein 5. Circulating 05a is produced during the activation of the complement cascade in blood and, once formed, it immediately binds to circulating PMNs (Kohl and Bitter-Suermann, 1993). 05a can also be produced in the extravascular compartment where it can promote gradient-dependent PMN migration. Proteins of the alternative complement pathway, which includes 05, can be produced by tissue macrophages and specialized epithelial cells, including Type II pneumocytes (Strunk et al., 1988). Interaction of 05a with its PMN receptor can promote secretory and oxidase pathways as well as chemotaxis. FMLP is the most potent of several formylated bacterial peptides which result from the cleavage the N-terminal portions of common bacterial proteins (Thelen et al., 1993; Bokaoch, 1995). Similar formylated peptides are not found in mammalian cells. FMLP receptors are expressed on unstimulated PMNs, but activation with FMLP or other agents can cause the mobilization of secretory vesicles in which 2-5 fold more receptors are sequestered. FMLP can promote PMN degranulation, oxidative burst, cytoskeletal changes, chemotaxis, and priming for enhanced response by other activators. 3. Chemokines Chemokines are a group of about 30 small proteins (6 to 15 kD) with similar, cysteinyI-containing structures. The most well studied PMN chemokine is perhaps lL-8, which is the primary stimulus for PMN migration in most inflammatory responses in humans and rabbits (Table 1). Chemokines were first identified in vitro and initially thought to be produced only by activated macrophages and monocytes. However under the proper 45 experimental conditions, their production and release can be elicited from neutrophils, endothelium, epithelium, platelets and a variety of paranchymal cells (Huang et al., 1992; Xing et al. 1994; Crippen et al., 1995; Furie and Randolph, 1995). Structurally, all chemokines possess four cysteine residues in their amino terminal end that form disulfide bridges. Chemokines are classified by the sequence of the two most N-proximal cysteines. In aor C-X-C chemokines, the cysteines are separated by an amino acid (X). In [3 or C-C chemokines, the cysteines are adjacent to one another. The structural difference is related to their ability to elicit distinct leukocyte migration. In general, PMNs responses are invoked by a—chemokines, and macrophage/monocytes respond most strongly to p-chemokines. Chemotaxis by basophils and eosinophils can be elicited by both types. 4. Rat Chemokines In rats, PMNs respond to a family of car-chemokines which are similar yet distinct from human and rabbit terms. As mentioned earlier in this chapter, rats do not have lL-8, but instead produce a structurally similar CINC (Watanabe et al., 1993; Nakagawa et al., 1994). A second rat a-chemokine which is found in many rat models of inflammation is MlP-2, which was named for its similarity to the rat B-chemokine MIP-1. Recently 2 more CINC-related proteins have been identified and a new nomenclature based on structural similarities was suggested: CINC-1 (also referred to as CINC -or KC), CINC-2a, CINC-2B, and CINC-3 (MlP-2). All four are released from LPS-stimulated rat macrophages in vitro and possess similar abilities to elicit chemotaxis and degranulation of PMNs (Shibata et al., 1995; Nakagawa et al., 1996). 46 Results from binding studies with ClNCs suggest that rat PMNs have a high affinity receptor for MlP-2 and a shared receptor for the other three CINCs (Murakami et al., 1997). That a CINC-1 receptor antagonist is ineffective against MlP-2-induced chemotaxis further supports that separate receptors are operant on rat PMNs (Zagorski and Wahl, 1997). Cross-desensitization between CINC receptors and other chemottractant receptors has not been reported. Critical roles for ClNCs have been demonstrated in various rat models of inflammation. Both CINC-1 and MlP-2 are involved in pulmonary PMN responses during bacterial pneumonia, ozone and silica dust inhalation, and immune complex deposition (Huang et al., 1992; Driscoll et al., 1993; Frevert et al., 1995a, 1995b; Driscoll et al., 1996; Shanley et al., 1997; Koto et al., 1997). Secretion of MIP-2 has been demonstrated in epithelial of the small intestine (Ohno et al., 1997) and tubulointerstitial cell from kidney (Tang et al., 1997) and has been implicated in models of arthritis (Schimer et al., 1997), allergic inflammation (Xiao et al., 1997), and liver injury ( Jaeshke et al., 1996). Much more work has been performed with human PMNs and lL-8 than with rat PMNs and chemokines. However, observations in rats and humans suggest that lL-8 and ClNCs play similar roles in models of inflammation. Thus, results from lL-8 studies may lend insight into the response of rat PMNs to MlP—2 and CINC-1. 5. CflokinelChemoattractant Interactions During Migration Although LPS, TNF and IL-1 are not chemotactic for PMNs, their exposure to ECs can induce the production of chemoattractants and cytokines such as lL-8 and PAF (Huber et al., 1991; Kuijpers et al., 1992; 47 Smart and Casale, 1994; Burns et al., 1997b). Hence, early observations of cytokine-induced PMN migration are explained by the presence of EC- derived chemoattractants. For example, lL-8 released into culture medium from TNF-stimulated ECs can cause CD18 up-regulation on PMNs (Huber et al., 1991). In addition, lL-8 produced from stimulated endothelial monolayers can become localized on the endothelial surface, where it can activate rolling or adhered PMNs (Kuijpers et al., 1992; Rot, 1993; lmaizumi et al., 1997). Indeed, treatment of endothelial cell monolayers with antibody to lL-8 inhibits rolling PMNs from firmly adhering to the monolayer (Rainger et al., 1997), suggesting that localized lL-8 is required for adhesion. The mechanism of lL-8 localization to ECs is unclear. Rot and coworkers (1996) described in situ binding of radiolabeled lL-8 to vascular endothelium in several pig organs including lungs, liver and kidney. However, there is no evidence for an lL-8 receptor or binding protein on cultured or primary endothelial cells in vitro (Petzelbauer et al., 1995; Rot et al., 1996). lL-8 and related molecules can bind to heparan and glycosaminoglycans, two molecules that are associated with the matrix of the vessel wall (Hoogewarf et al., 1997). Thus, localization might not be due to an endothelial cell component, but to extracellular sites near the cell. The ability of chemoattractants to bind to the extracellular matrix allows for migration of PMNS through inflamed tissues. Stimulation of ECs with lL-1 causes lL-8 synthesis and secretion within 1-2 hours, whereas longer exposures result storage of lL-8 in Weibel-Palade bodies (Rot et al., 1996). Furthermore, release from Weibel- Palade bodies could be induced with treatment of cells with phorbol ester or histamine. This observation raises the possibility that comobilization of endothelial lL-8 with P-selectin might modulate PMN adhesion during 48 chronic inflammation. These studies suggest a mechanism that is characterized by a spatial and temporal orderliness for PMNzEC interaction in the face of mutiple and conflicting inputs from inflammatory mediators. Cytokines and chemoattractants may promote redundant and perhaps dysregulated signals for firm adhesion when both types of mediators are present (Detmers et al., 1990, 1991; Huber et al., 1991). For example, premature integrin activation on circulating PMNs by cytokines might be detrimental to the stepwise dependence of EC-mediated migration processes. This is suggested by results from systems in vitro in which pretreatment or cotreatment of PMNs with chemoattractants such as lL-8, FMLP, LTB4 or C53 inhibits adhesion and/or migration across endothelial monolayers (Luscinskas et al., 1992; Moser et al., 1993; Takahashi et al., 1995). Although the effect on integrin expression was not examined, there was a positive correlation between L-selectin shedding and inhibition of migration by the various chemokines (Moser et al., 1993). Similarly, intravenous administration of lL-8 inhibits PMN migration to extravascular sites of inflammation in rabbits (Hechtman et al., 1991; Ley et al., 1993). Similar experiments with regards to rat CINCs have not been performed. However, exposure of PMNs to MlP-2 in vitro mobilizes intracellular calcium and causes degranulation (Shibata et al., 1995). In addition, exposure of isolated blood to MlP-2 can cause PMNs to shed L- selectin and increase surface expression of CD1Ib/CDI8 (Frevert et al., 1995b). These are the same responses seen in human PMNs after exposure to lL-8, TNF, lL-1 and C5a, thus MlP-2 may modulate PMN migration in a similar manner as these mediators. Although the mechanism of chemoattractant- and chemokine- 49 induced inhibition of migration is unclear, results from studies in vitro suggest that receptor desensitization may be involved (Campbell et al., 1997). For example, CSa-treated PMNs demonstrate decreased chemotactic responses (Kitayama et al., 1997), and CSa-receptor activation in isolated PMNs can induce cross-desensitization of receptors for lL-8 and FMLP (Tomhave et al., 1994; Blackwood et al., 1996; Sabroe et al., 1997) Likewise, FMLP treatment of neutrophils will down-regulate IL- 8 receptors without affecting FMLP receptors (Campbell et al., 1997). In addition, TNF can inhibit migration in models ex vivo and in vivo (Otsuka et al., 1990), presumably because the PMN receptor for TNF is functionally linked to chemotactic receptors (Schleiffenbaum and Fehr, 1990; Balazovich et al., 1996). Thus, cross-regulation of chemotactic receptors might be an important modulator of inflammatory cell responses during infection. Studies which address cross-regulation of MlP-2 and CINC receptors with other chmeoattractant receptors have not been performed. E. Summagy Transendothelial migration of PMNs is a carefully orchestrated sequence of cell activation, adhesion molecule expression, and molecular cross talk between receptors on both cell types. As such, there are several opportunities during the migratory process to modulate and modify the response by both exogenous and endogenous factors. Multiple lines of evidence suggest that integrin activation needs to occur at the PMNzendothelial cell interface and not in the circulation. Thus, inhibition of migratory processes like those observed in models of endotoxemia- associated nosocomial pneumonia might occur from premature activation of adhesion pathways. Alternatively, many inflammatory mediators can 50 cause desensitization of PMN chemoattractant receptors. Premature activation of PMN integrins and chemotactic receptors by circulating mediators in vivo might be avoided by a temporally and spatially controlled release and concentration of chemokines at the endotheliumzblood interface as suggested by Rot and coworkers (1993,1996). Given the precise, step-wise nature of transendothelial migration and the multiple modulating factors resent during inflammation, there are ample points where interference could occur, resulting in impaired neutrophil trafficking. 51 V. PMN Migration in the Lungs Most of the known molecular and cellular mechanisms of transendothelial migration have been elucidated from studies in vitro and in systemic vessels in the mesentery and dermis. Recent work in lung, however, suggests that PMN trafficking in the pulmonary circulation is fundamentally different from events in the systemic vasculature. In animal models tested so far, PMN behavior in lungs is particularly unique with respect to (1) the marginated pool of PMNs, (2) the site of transendothelial migration, and (3) requirements of adhesion molecules for PMN extravasation. Although the basic mechanisms of transendothelial migration are still incompletely understood, these three differences suggest that different paradigms are needed to explain the kinetics of PMN migration in different vascular beds. A. Margination Margination of PMNs allows for their escape from the main flux of blood flow where they can sense and respond to inflammatory signals present in vessel wall microenvironment. In the pulmonary vasculature, the concentration of PMNs within pulmonary capillary blood is 35-100 times greater than in large vessels of the systemic circulation (Doershuk et al., 1987; Doerschuk et al., 1993; Gee and Albertine, 1993). In the systemic circulation, margination occurs as rolling in postcapillary venules and is mediated by L-selectin on PMNs and P-selectin on endothelial cells (Spertini et al., 1991; Lawrence and Springer, 1991; Butcher, 1991; 52 Tozeren and Ley, 1992). However, 97 % of pulmonary vascular PMNs are found in the capillary network where vessels are too small to allow rolling (2-15 pm) (Doerschuk et al., 1993). Selectin-mediated rolling still occurs in pulmonary venules (80% frequency), but it makes a small contribution to the total marginated pool in the pulmonary circulation. Despite the lack of rolling in pulmonary capillaries, selectins may still be involved in the PMNzEC interactions. For instance, treating rabbits with fucoidin, a polysaccharide made mostly of sulfated fucose which inhibits binding of all selectins, decreases the frequency of PMN stopping in capillaries by 25% and the duration of stops by 50% (Kuebler et al., 1997). Similar results are seen in fucoidin-treated rats, where PMN localization in alveolar capillaries is decreased by 15% (Yamaguchi et al., 1997). In the same study in rats, an antibody to P-selectin was ineffective in slowing PMN transit through the lung, whereas rolling on systemic venules was significantly inhibited. Because E-selectin is not expressed in uninflamed rat lungs, all selectin-mediated binding is therefore due to the action of L- selectin on PMNs. Furthermore, despite the expression of ICAM-1 in normal rat pulmonary capillaries, treatment with antibodies to ICAM-1 does not affect PMN transit times (Yamaguchi et al., 1997). Thus, L-selectin is the only adhesion molecule shown likely to be involved in pulmonary PMN margination in rat. Another factor to account for the dramatic extent of pulmonary PMN margination may be the discrepancy between neutrophil and capillary diameters (Downey et al., 1990). PMNs which are 7-8pm in diameter must 53 deform to an elongated shape in order to pass through capillaries, which average 5-6pm, and can be as little as 2 urn (Hogg, 1987; Doerschuk et al., 1993; Wiggs et al., 1994; Gebb et al., 1995). Shape change in PMNs takes much longer than erythrocytes (red blood cells;RBCs), and this may account for their longer pulmonary transit times (27s vs. 1.3s) (Hogg et al., 1988; Lien et al., 1991). A dramatic increase in pulmonary margination is accompanied by neutropenia when animals are given intravenous LPS or zymosan- activated serum (i.e., complement activation products) (Haslett et al., 1987; Doerschuk et al., 1992). Capillary sequestration in these models has been hypothesized to be due to lack of PMN deformability; these same agents cause actin polymerization and PMN stiffness in vitro (Erzurum et al., 1992). B. Site of Migration Given the slow transit and intimate contact between PMNs and the capillary endothelium, PMNs are well positioned to respond to inflammatory signals generated within airspaces. Thus, in contrast to migration from postcapillary venules in the systemic circulation, PMNs primarily extravasate from the alveolar capillary network in rabbits, rats and mice (Doerschuk et al., 1989; Walker et al., 1991; Doerschuk, 1992; Downey et al., 1993). It is curious, therefore, that P-selectin is constitutively expressed on pulmonary arterioles and venules in rabbits and not on pulmonary capillary endothelial cells where margination and extravasation occurs 54 (Mulligan et al., 1992b). In rat, by constrast, P-selectin is absent in the normal lung, but ICAM-1 is constitutively expressed on capillary and venular endothelium (Yamaguchi et al., 1997). As discussed previously, ICAM-1 associates with CD18 to mediate the firm adhesion of PMNs and usually requires induction for full responses. While ICAM-1 has a low constitutive level of expression in most tissues, it is 30-fold higher in lung tissue (Panes et al., 1995). Thus, pulmonary Ieukostasis after infusion of endotoxin or zymosan-activated serum (ZAS) may be due to the high level of ICAM-1 in lung (Haslett et al., 1987; Doerschuk, 1989,1992). Both endotoxin and ZAS are capable of mobilizing CD18 on isolated PMNs, and similar upregulation of CD18 in vivo would provide ligands for lung ICAM-1 (Erzurum et al., 1992; Klut et al., 1997). Therefore, by virtue of the density in adhesion molecules and slow PMN transit times, PMN adhesion and extravasation is favored in the pulmonary capillaries over downstream venules. C. Adhesion molecule reguirements 1. lntggrins Another important difference between the pulmonary and systemic circulations is in the mechanics of PMN diapedesis. or egress of the PMN through the vessel wall. In all models tested so far, systemic PMN migration from postcapillary venules always requires CD18 integrins. However, pulmonary PMN migration can occur independently of CD18 55 adhesion molecules. Whether or not migration depends on CD18 varies with the intrapulmonary stimulus (Table 2). lL-1, phorbol myristate acetate (PMA) and Gram negative bacterial stimuli including LPS elicit migration via predominately CD18-mediated pathways (Doerschuk et al., 1990; Hellewell et al., 1994; Qin et al., 1996; Ramamoorthy et al., 1997). By contrast, Gram-positive bacteria, HCI, and C5a elicit pulmonary PMN recruitment that is mostly independent of CD18 involvement. The adhesion molecules needed for CD18-independent migration in lung are unknown. Endotoxin derived from Gram-negative bacteria elicits airway PMN migration which requires CD18 and causes the upregulation of ICAM-1 within pulmonary vessels (Tang et al., 1995; Freeman et al., 1996; Beck- Schimmer et al., 1997). At the same time, expression of L—selectin and CD18 is unchanged on PMNs in the vascular compartment, and only after PMN s arrive into airspaces is L-selectin shed and CD11/CD18 expressed (Burns et al., 1994) . By contrast, during CDI8—independent migration to gram-positive pneumonia there is no change in ICAM-1, yet CD11/CD18 is up-regulated on vascular PMNs prior to migration, and is further expressed on ainrvay PMNs. Thus, an inverse relationship exists between CD18 requirements and expression on vascular PMNs prior to migration. PMNs can express integrins other than CD18 ([32). For example, adherence to cardiac myocytes, endothelial cells, fibroblasts, fibronectin and laminin is mediated through (31 -type integrins (Shang and lssekutz, 1997; Reinhardt et al., 1997). However, studies in vitro of CD18-independent migration have failed to implicate [31 integrins or any other adhesion molecule 56 Table 2. Adhesion Molecule Requirements for Stimuli that Cause PMN Migration into Airways CD18 Dependent CD18 Independent Uncha_rgcterized Gram-negative bacteriazLPS Gram-positive bacteria LTB4 Interleukin-1 Hydrochloric Acid Viral products Phorbol esters (PMA) C5a FMLP IgG complexes Quartz particles Ozone Cigarette smoke 57 examined (lssekutz, et al., 1995). 2. Selectins It is unclear if selectins are responsible for CD18-independent migration in vivo. Studies using knockout mice and blocking antibodies which target selectins have not provided definitive answers. Mice deficient in L-, P- or E-selectins have compromised PMN rolling behavior in both normal and inflamed venules in the systemic circulation (Kunkel and Ley 1996; Johnson et al., 1995; Mayadas et al., 1993). In a model of pneumonia using S.pneumonia organisms (a CDIB-independent stimulus), E-IP- selectin double-knockout mice had 4 times as many ainrvay PMNs as wild type mice (Mizegerd et al., 1996), suggesting that selectins suppress CD18-independent migration. However the mutant mice also had basal neutrophilia and margination of pulmonary PMNs 8-fold greater than wild type mice. Furthermore, knockout mice also had decreased L-selectin on their PMNs (15% of normal), increased hematopoietic cytokines, and failed to thrive normally (Frenette et al, 1996; Bullard et al., 1996). Therefore, caution should be used in interpreting results of knockout studies in consideration of compensatory mechanisms that may occur in these animals. These results do however, suggest that P- and E- selectin are not required for migration of PMN to S.pneumonia, a CD18-independent stimulus. Compared to normal animals, mice deficient in L-selectin are protected against injury and death caused by endotoxemia (Tedder et 58 al.,1995b). The knockout mice had significanty less recruitment of PMNs into the pulmonary vasculature after intravenous LPS. By contrast, pulmonary PMN recruitment in response to S. pneumonia were the same in wild-type and L-selectin knockout mice (Doyle et al., 1997). Thus L- selectin-mediated processes appear to be involved in CD18-dependent migration but not for migration to CDIB-independent stimuli. This is consistent with results from P- and E-selectin knockout studies. That is, selectins appear to be involved in CDIB-dependent migration but not in CDIB-independent processes. This is consistent with the sequential selectin-CD18 relationship presented in section IV. An alternative method of blocking selectins is with sialyl- oligosaccharides which approximate the structure of natural selectin ligands. Lung injury caused by immune-complex deposition injury is CD18-dependent and is prevented by treating with sialyl compounds (Mulligan et al., 1993). Immune deposition injury is characterized by the formation of immune complexes at the alveolarzcapillary interface. Sialyl analogs can also inhibit PMN pulmonary recruitment (by 50%) to airway endotoxin in rats (Hashimoto et al., 1997). These results further support the link between selectin- and CD18-mediated pathways. As seen in other knockout models, studies which employ ICAM-1 deficient mice have provided disparate conclusions concerning CD18- dependent and -independent stimuli. In ICAM-II P-selectin double knockouts, peritoneal PMN migration to S. pneumonia was blocked completely, but PMN migration in response to airway S. pneumonia was 59 unaffected (Bullard et al., 1995). These results are similar to the effect of anti-CD18 antibodies on PMN migration when rabbits are given the same stimulus in the peritoneum and lungs (Doerschuk et al., 1990). However, when Doerschuk and coworkers (1996) infected lCAM-1/ P-selectin knockout mice with Gram-negative organisms (CDIB—dependent stimulus), pulmonary PMN emigration was unaffected. Even anti-ICAM or anti-P- selectin antibodies could not block migration in these mice. Clearly, this double-mutant animal had developed compensatory mechanisms for PMN migration which are not activated in wild-type mice to the same stimuli. D. Models of Pulmonagy PMN Migration PMNs emigrate into pulmonary airspaces of rats in response to a number of different pathogenic and toxic stimuli. Although each stimulus induces the production of chemoattractants and other inflammagens, the qualitative and quantitative profile of these mediators can be different across stimuli. In addition, the adhesion molecule requirement for PMNs varies with the stimulus (Table 2) as does the extrapulmonary effects engendered by each intrapulmonary stimulus. Below are a few of the more well studied models which involve pulmonary PMN emigration. 1. Bacteria and bacterial products Rabbits respond to both Gram-postive and Gram-negative pneumonia with ainlvay production of lL-8 and TNF (Shoberg et al., 1994). However, lL-8 concentrations in BALF are two-fold greater and TNF is 10- fold greater in rabbits with Gram-negative pneumonia compared to animals with Gram-positive pneumonia. The production of CINCs and TNF after 6O Gram-negative ainrvay stimuli in rats is well documented and was detailed earlier. It is unknown if Gram-positive stimuli in rat airways elicit a lesser cytokine response than a Gram-positive stimulus similar to what is observed in the rabbit. In general, PMN airway accumulation in response to Gram-negative bacterial stimuli requires 0018. The extent of inhibition of PMN migration by CD18 antibodies is 75-80% (Doerschuk et al., 1990; Ramamoorthy et al., 1997). Conversely, Gram-positive stimuli elicit PMN emigration which is mostly independent of CD18 pathways, with inhibition by C018 antibodies ranging from 0-45% (Doerschuk et al., 1990; Ramamoorthy et al., 1997). Thus, based on the available data, Gram-positive stimuli seems to be largely CD18-independent and Gram-negative stimuli are predominately C018-dependent. Moreover, Gram-negative stimuli appear to elicit greater TNF and lL-8 production than Gram-positive stimuli. Taken together, the results of C018 antibody studies and the disparate cytokine production elicited by various stimuli suggests that the adhesion molecule requirement for migration might depend on the type and quantity of cytokines and chemokines produced in response to a stimulus. Several mediators and chemoattractants are produced during even minor infections and inflammation. Further work on this hypothesis is required to match mediators definitively with adhesion molecule involvement. 2. Acid Aspiration lnstillation of HCI acid into the ainrvays of laboratory animals is used to model gastric acid aspiration in humans. Neutrophil accumulation in acid-instilled rabbits is dependent on the generation of ainNay-derived lL-8 (Folkesson et al., 1995), but it does not require CD18 adhesion 61 molecules (Doerschuk et al., 1990). However, C018 is required for lung injury in this model. Treatment with antibody to CD18 protects from abnormalities in oxygenation and vascular leak without affecting PMN ainrvay accumulation (Goldman et al., 1995; Folkesson and Matthay, 1997). Acid aspiration can also lead to PMN-mediated injury at sites distal from inoculated lung lobes. For example PMN accumulation occurs in contralateral lobes but, unlike inoculated lobes, PMN emigration is blocked by CD18 antibodies (Goldman et al., 1995). Thus, direct effects of acid on pulmonary PMN migration seem not to require CD18, but indirect effects (e.g., in contralateral lungs) are CD18-dependent. Similar responses occur in acid-instilled rats. That is, PMNs accumulation in acid-instilled ainlvays is CDIB—independent (Motosugi et al., 1998), whereas PMN-mediated injury to remote organs, including heart, kidney, and small intestine, requires CD18 (Goldman et al., 1990; Yamada et al., 1997). In addition, extrapulmonary injury is dependent on complement activation and circulating TNF. Blockade of either of these inflammatory mediators prevents remote organ injury and lung vascular leak in the aspirated lobe, however PMN emigration is unchanged. In summary, acid instillation induces compartmentalized inflammatory responses. Localized responses include CD18-independent pulmonary PMN emigration and CD18-dependent lung injury. The remote component of injury is mediated by TNF and complement products which promote CD18-dependent PMN processes. 3. lnterle_ukin-1 Production of lL-1 in rat airways is required for full PMN migratory responses in rat models of immune complex deposition or 62 inhalation of LPS, quartz dust, or diesel exhaust particles (Warren, 1991; Kusaka et al., 1990; Ulich et al. 1991a; Yang et al, 1997). lnstillation of IL-1 itself is sufficient to induce PMN emigration which is dependent on ainrvay production of the neutrophil chemokines, CINC and MlP-2 (Xu et al., 1995; Hybertson et al., 1996). Treatments with PLAz inhibitors, antiinflammatory prostanoids, or inhaled nitric oxide inhibit PMN accumulation after IT lL-1 without affecting chemokine production (Leff, et al., 1994; Guidot, et al., 1996; Lee et al., 1997). lL-1 instillation also causes PMN-dependent vascular leak and is linked to oxidant injury (Guidot et al., 1994; Leff et al., 1994). In rabbits treated IT with lL-1, pulmonary PMN emigration is significantly reduced by antibodies to CD18 (Hellewell et al., 1994). Similar studies using C018 antibodies have not been performed in lL-I-instilled rats. 4. Complement Protein C5a Complement protein C5 is found in the lavage fluid collected from healthy humans and animals. Proteolytic cleavage of CS during inflammation produces the highly chemotactic fragment, 05a. In rabbits, instillation of C5a into ainrvays causes emigration of PMNs into airspaces which is not significantly affected by antibodies to CD18 (Hellewell et al., 1994). In studies using C5-deficient mice, PMN emigration during Gram- positive pneumonia did not occur, whereas the PMN migratory response to Gram-negative pneumonia is only delayed by an hour (Larsen et al., 1982; Toews and Val, 1984). This is consistent with the C018 requirement for each stimulus. That is, C5a is associated with Gram-positive pneumonia 63 and both stimuli elicit CD18-independent migration. Conversely, C5a is not as critical for PMN responses to Gram-negative bacterial pneumonia, which is C018-dependent. 5. Immune-complex Deposition Immune complex deposition injury in lungs Is induced in rats by administering bovine serum albumin (BSA) intravenously and anti-BSA antibodies (IgG) intratracheally. Immune complexes which form at the vascularzairway interface serve as the focus for inflammatory responses which include airway PMN recruitment and vascular leak (Johnson et al., 1984 ). PMN emigration is due to airway generation of CINC-1, MlP-2 and C5a (Shanley et al., 1997). In addition, production of lL-1, TNF, and PAF contribute to both PMN emigration and vascular leak (Mulligan and Ward, 1992; Warren, 1992). Blocking either PMN influx or oxygen-derived free radicals, which are generated from activated PMNs or alveolar macrophages, protects from edema and vascular leak (Mulligan et al, 1992a). Airway PMN emigration in response to immune complexes relies on multiple integrins. Antibodies to C011a/C018, C011b/0018 or [31 (0029) integrins block PMN emigration to various degrees (Mulligan et al., 1993; Mulligan et al., 1995). Furthermore, the antibody to C011blC018 is more effective when administered intratracheally than intravenously, whereas the other antibodies have the opposite relationship. The mechanism for the differential inhibition is not known. E. Summapy The pulmonary circulation is unique in comparison to the systemic circulation in that there are large numbers of marginated PMNs and migration preferentially occurs from capillaries. Moreover, the involvement of adhesion molecules varies with the stimuli. For example the involvement of CD18 during PMN emigration depends on the stimulus and is likely differentiated by the particular cytokines, chemokines, and other chemoattractants produced in the airways in response to each stimulus. Studies to verify this hypothesis have not been performed. In addition, the mechanism of CD18-independent PMN emigration has yet to be characterized. 65 VI. Animal Models of Endotoxemia IV administration of LPS in animals is used to model the systemic inflammatory response syndrome (SIRS) and related sepsis syndromes in humans. SIRS is described as elevated body temperature, tachycardia, tachypnea, and an elevation in circulating leukocytes (Bone et al., 1989; Bone et al., 1992) and can occur in the absence of overt bacterial infection. Sepsis syndrome includes the presence of infection and at least one hypoperfused organ. Septic shock occurs when subjects experience additional systemic hypotension (Knaus et al., 1992). SIRS and sepsis syndromes are often associated with endotoxemia, and administering LPS to animals can duplicate symptoms of human SIRS and sepsis. Different aspects of SIRS and sepsis have been modelled in baboons, monkeys, dogs, sheep, pigs, rats and mice. Recently, the production and release of inflammatory cytokines (e.g., IL—1, lL-8, and TNF) and PMN activation have been of particular interest in many clinical studies (Glauser et al., 1991; Simms, 1995;Christou, 1996; Slotman et al., 1997; van der Poll et al, 1997). Rats given endotoxin have similar hemodynamic responses and mediator production as those seen in humans, as well as similar organ injuries and failure (Hewett and Roth, 1993; Pearson et al., 1995). As such they provide an attractive and relevant animal model to study endotoxemia and SIRS. A. Rat Responses During Endotoxemia The response of rats and other mammals to endotoxemia generally follows a similar pattern: an initial, hyperdynamic stage (0-6 hours) characterized by the activation of Inflammatory, coagulative, anti- inflammatory and fibrinolytic pathways, which is followed by a hypodynamic 66 phase marked by cellular injury, degeneration and organ injury (Taylor, 1994). Depending on the dosing regimen in rats, the most affected organs include liver, lungs, kidney and pancreas (Ruetten et al., 1996; Ruetten and Thiemermann, 1997). 121-LIE! The primary sequelae of IV LPS-induced endotoxemia in Sprague Dawley (SD) rats is hepatic parenchymal cell injury which is associated with mortality by 16 hours (Hewett and Roth, 1993). Along with the spleen, the liver preferentially accumulates LPS immediately after administration, and it is still detected there for at least 28 days (Ge et al., 1994). Kupffer cells, the resident liver macrophages, remove LPS from the circulation and at the same time can become activated to produce TNF, IL- 1, NO and prostanoids, among other inflammatory products. Hepatic parenchymal cell injury occurs by 6 hours as evidenced by the increase in plasma activity of liver cytosolic enzymes (Hewett et al., 1992). PMNs that accumulate in the liver are critical cellular mediators of tissue injury. Although LPS can directly activate circulating mononuclear cells and extrahepatic tissue macrophages to secrete reactive mediators, the liver, and putatively the Kupffer cell, is the primary source of plasma TNF during endotoxemia (Asari et al., 1996). Plasma TNF, hepatic accumulation of platelets and PMNs, activation of the coagulation cascade, and Kupffer cell activation have all been implicated in the development of liver injury in this model (Hewett and Roth, 1993; 1995; Pearson et al., 1995; Shibayama et al., 1995). 67 2. Lungs Hepatotoxic doses of IV LPS do not cause overt lung injury in SD rats (Brown et al., 1997). This is in spite of a substantial degree of pulmonary vascular sequestration of PMNs within 15 minutes of endotoxemia that persists for at least 3-4 hours. New and immature PMNs are mobilized from bone marrow by 3-4 hours and preferentially sequester in the lung vasculature (van Eeden et al., 1997). Thus, large marginated pool of PMNs over the course of endotoxemia may be due to different PMN populations. In species that are more sensitive to LPS than the rat, pulmonary edema is a common development by 1-2 hours of endotoxemia (Brigham et al., 1979; Brigham and Meyrick, 1986). Edema and accumulation of ainrvay PMNs also occurs in the endotoxemic rat when chronically infused, given repeated or large doses, or when given secondary treatments with PMN activators such as PAF or FMLP (Worthen et al., 1987; Miotla et al., 1998). However there is not a marked edemagenic response with bolus, hepatotoxic doses of LPS in SD rats (Brown et al., 1997). It is interesting, therefore, that treatment of rats with gadolinium chloride to inhibit Kupffer cell function results in PMN and protein accumulation in ainlvays of rats given hepatotoxic doses of LPS (Brown et al., 1997). One explanation is that a downstream effector from activated Kupffer cells inhibits PMN airway migration in endotoxemic rats. Thus, inhibiting Kupffer cells inhibits the effector. Alternatively, gadolinium chloride might reach ainrvays where it alters the activity of AMs to promote PMN migratory pathways (Pendino et al.1995) At least one study suggests that the pulmonary compartment is relatively insulated from leakage of LPS and cytokines from the vascular 68 compartment (Ghofrani et al., 1996), yet other researchers report that alveolar macrophages are activated during endotoxemia (Wizemann and Laskin, 1994; Smith et al., 1994). In this regard, macrophages collected from ainrvays of endotoxemic rats are primed for enhanced production of NO and for secretion of lL-1, TNF and prostanoids. Thus, the lack of pulmonary PMN emigration during endotoxemia in the rat occurs in spite of enhanced inflammatory potential of airway macrophages. Endotoxemia in rats is associated with systemic hypotension by 3-4 hours. At the same time, however, pulmonary artery pressure and pulmonary vascular resistance are increased (Newman, 1994). Nearly every known vasoconstrictor has been found to be elevated during endotoxemia. It is unclear which mediator is responsible for the changes in pulmonary circulation or if the increased pressure affects PMN sequestration. In non-pathologic conditions, increased pulmonary blood flow by epinephrine releases marginated PMNs into the circulation (Martin et al., 1982). This result should be interpreted with consideration for the effects of epinephrine on [32 adrenergic receptors of PMNs, since increases in intracellular cAMP can inhibit PMN adhesion (Bazzoni et al., 1991; Derian et al., 1995). Nevertheless, it is possible that increased pulmonary pressures might affect PMN adhesion and migration during endotoxemia. A spike in pulmonary NO production occurs about the same time (15-30 minutes) as PMN sequestration after rats are infused with LPS (Gryglewski et al., 1998). Although localized NO would decrease pulmonary pressure and potentially facilitate PMN adherence, NO also has the ability to inhibit adhesion between PMNs and ECs (Banick et al., 1997). Thus, the role of NO and vascular pressure in pulmonary PMN localization during endotoxemia is unclear. 69 B. Summapr Endotoxemia in rats mimics human responses during SIRS and sepsis particularly with respect to the production of inflammatory mediators, PMN activation and multiple organ injury (Hewett and Roth, 1993; Simms, 1995; Christou, 1996). PMN behavior during endotoxemia in rat is characterized by the accumulation of these cells in organs with large capillary beds, mostly in lungs and liver but also in kidneys and spleen. The involvement of PMNs in tissue injury during endotoxemia in rats is not completely understood. Of particular interest is why the lungs are spared injury despite the PMN localization, and why, despite the presence of airway inflammagens, PMNs fail to migrate into lung tissues. In the next section, several effectors of PMN migratory and adhesive functions which are present during different stages of endotoxemia will be described. Prior to identifying these indirect effectors of LPS in vivo, however, the direct effects of LPS on PMNs in vitro will be surveyed. 70 VII. Effects of LPS exposure on PMNs LPS exposure to PMNs engenders a broad array of immediate, delayed, direct and indirect effects. Most of these diverse responses are believed to be mediated through interactions with the LPS receptor (i.e., CD14), which is found on tissue macrophages and monocytes, as well as on PMNs (reviewed in Viriyakasol and Kirkland, 1995; Holst et al., 1996; Mayeaux, 1997). Membrane bound CD14 (mC014), is a glycosylphosphatidylinositol-linked glycoprotein and is positioned in the lipid bilayer such that it lacks a cytoplasmic component. Signal transduction pathways after C014 binding are unclear, although internalization of LPS after binding to mCD14 has been demonstrated in human PMNs (Luchi and Munford, 1993; Gegner et al., 1995; Detmers et al., 1996). An accessory membrane-bound protein has been hypothesized to link CD14 to signal transduction pathways, which include MAP-kinases (Nick et al., 1996), phospholipases C and 0 (Yamamoto et al., 1997) and NF-KB among others (Delude et al., 1994; Sweet and Hume, 1996). Binding of LPS to mCDI4 is greatly enhanced in the presence of LPS binding protein (LBP), a liver-derived plasma glycoprotein, concentrations of which increase in the circulation during infection (Su et al., 1995; \firyakosol and Kirkland, 1995). LBP:LPS complexes represent a plasma pool of LPS, and serve as a shuttle for LPS from the circulation into the plasma membrane (Wurfel and Wright, 1997). Binding of LPS to other plasma lipoproteins, including low-density lipoprotein (LDL) and high- density lipoprotein (HDL) in human blood, may represent an important mechanism for LPS clearance (Baumberger et al., 1996; Schlichting et al., 1996; Netea, 1998). C014 is stored in secretory granules of PMNs, and can be mobilized to the surface by common PMN activators such as FMLP, 71 TNF and LPS (Rodeberg et al., 1997; Borregaard and Cowland, 1997). Another plasma protein that binds to LPS is a soluble form of the CD14 receptor (sCDI4). Complexes of LBP, sC014 and LPS can mediate the activation of cells which lack mCD14 such as vascular endothelial cells and ainrvay epithelium (Pugin et al., 1993; Vita et al., 1997). Increasing evidence suggests that LPstC014 can also modulate LPS-mediated responses in cells bearing mCD14, including human PMNs. For example, PMN responses to LPS are modified in the presence of sC014; both inhibition and enhancement of responses occur depending on concentrations of $0014 and LBP (Hailman et al., 1996; Troelstra et al., 1997). Because antibodies to mCD14 inhibit sC014:LPS-induced activation of macrophages and PMNs, it has been hypothesized that sCD14 acts to transfer LPS to mCDI4 in a similar manner as LBP (Hailman et al., 1996). Conversely, others researchers have described a putative receptor for sCD14zLPS complexes which is separate from mCD14 on transformed cell lines, monocytes, and PMNs (Vasselon et al., 1997;\flta et al., 1997). In some systems, activation of cell responses by sCDI4zLPS complexes is even stronger when mCD14 is removed by phospholipase activity. Thus the characterization of $0014 and mCDI4 behavior may depend on the particular experimental model. Clearly, these studies in vitro show that LBP and sC014 are capable of modulating both mCDI4-dependent and -independent inflammatory responses and may have similar effects in endotoxemic humans and animals (Martin et al., 1997). Despite the profound responses of PMN in the endotoxemic rat, mCD14 has not been identified on rat PMNs. Furthermore, mCD14 on rat macrophages has only 64% homology with human CD14 (Takai et al., 72 1997). Compared to humans and rabbits, the mouse and rat are less sensitive to endotoxin, a difference which is reflected in the binding characteristics of LPS:CD14 in each species. Because plasma sC014 can originate from the shedding or secretion from activated monocytes and macrophages, it may be a critical modulator of rat PMN activity during the inflammatory response in vivo (Haziot et al., 1993). That high concentrations of LPS can stimulate cells in the absence of mCD14 or $0014 may suggest a third mode of action of LPS on PMNs. In this regard, LPS has been hypothesized to immerse into the cell membrane where it can mimic the effects of endogenous, membrane- derived ceramide (Wright and Kolesnick, 1995). However, supporting evidence for this premise is indirect. Liberation of ceramide by sphingomyelinase activity elicits LPS-like responses in macrophages, and LPS can cause ceramide-dependent kinase activities in myeloid cells (Joseph, et al., 1994; Barber, 1996). In addition, ceramide pathways are defective in an LPS-resistant mouse strain (Barber et al., 1995; Thieblemont and Wright, 1997). Taken together, these results suggest that ceramide pathways might be important to macrophage responses in endotoxic animals, yet it isunclear if LPS-induced ceramide pathways are important in cells which do not have mCD14 and are not responsive to $0014. A. Direct LPS-induced PMN Activation LPS can directly stimulate PMNs to change shape, express adhesion molecules and other receptors on the membrane, and synthesize cytokines and chemoattractants (Figure 3). Most of these responses are influenced by the presence of serum, which is a putative source for $0014 and LBP. 73 .uagaohso: .5 mm: .«o 38.5— 325 .m PSME 55.5.5 5 5:5 ozumExo voocusco BEE—tn m_xBoEo:o 9 \ cos—Ego... o_o_mo> \ :ozosuoa act—030 >>\\ mnj :ofiocum mmoctzm 74 In general, responses are pro-inflammatory. However cytoskeletal changes caused by LPS can inhibit some responses that depend on cell mobility or that involve cytoskeletal proteins. 1. Cfloskeleton Exposure to LPS in vitro causes assembly of filamentous actin and reorganizes cytoskeleton in PMNs (Erzurum et al.,1992). These effects render the PMN stiff and resistant to deformation. Dysregulation of actin kinetics can potentially alter the responses of actin-dependent pathways of PMNs. lntravital microspcopy shows circulating PMNs as fairly circular both in the shear flow and rolling along vessel walls (Kuebler et al., 1997; Yamaguchi et al., 1997). In the lungs, circulating PMNs of ~ 8 um diameter must traverse pulmonary microcapillaries which have average diameters of 5.5 to 6 um (Doerschuk et al., 1993). Migration of PMNs through 5 pm filters in vitro has been demonstrated to induce shape change and net actin filament assembly (Kitagawa et al., 1997). Lack of deformability induced by LPS has been proposed as one mechanism for the rapid sequestration of PMNs in microcapillary networks in lungs after LPS injection in animals (Haslett et al., 1987; Erzurum et al., 1992). Dysregulation of actin polymerization can lead to other defective responses of PMNs. For example, exposure of PMNs to chemokines causes them to flatten and migrate in vitro, processes which require simultaneous and coordinated actin polymerization and depolymerization (Ehrengruber et al., 1995; Merry et al., 1996). Reorganization of actin filaments is also necessary for phagocytosis, oxidative burst and 75 degranulation (Bengtsson et al., 1991,1993). 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 (Wonderling et al., 1996). As discussed above, LPS can enhance the effectiveness of PMN activators for superoxide and vesicular mobilization. Thus, modulation of PMN actin may mediate inhibition of some functions but enhance others. 2. Adhesion Exposure to LPS causes both shedding of L-selectin and a transient increase in expression of membrane Mac-1 (Lynn et al., 1991). This phasic transition is hypothesized to be a necessary sequence for firm adhesion of PMN to physiologic surfaces. Cleavage of L-selectin is believed to be via the action of a metalloprotease (Kishimoto et al., 1995; Walchek et al., 1996). Although LPS can upregulate peptidase activity on the PMN surface, similar upregulation of metalloprotease activity has not been confirmed (Fagny et al., 1995). A prerequisite for LPS-induced mobilization of Mac-1 is the internalization of either the LPS:mCD14 complex, or of LPS alone into endocytic vesicles (Detmers et al., 1996). In addition, LPS-exposed PMNs produce IMF-1, a lipid which promotes the binding ability of Mac-1 (Detmers et al., 1994). In this manner, LPS may also up-regulate basal, inactive Mac-1 already on the PMN surface. LPS also can induce the retraction of CD18 from the PMN membrane surface and into azurophilic granules in vitro (Simms and D’Amico, 1995). Thus, LPS could potentially downregulate PMN adhesion during some inflammatory processes. However in most studies of human or animal endotoxemia, isolated blood PMNs display up-regulation of CD18 76 on PMNs (Duigan et al., 1986; Frevert et al., 1994;Wakefield et al., 1993). Thus, it is uncertain if CD18 downregulation occurs in vivo. It is notable that LPS has a specific binding site on CD18 through which it activate signal transduction pathways (Flaherty et al., 1998). It is uncertain what effects this may have on adhesion. 3. Cytokine Production TNF and IL-1 are two important, proximal mediators produced by macrophages during endotoxemia in humans and animals. Modest amounts of both cytokines can be elicited from PMNs after incubation with LPS (T iku et al., 1986; Dubravek et al., 1990; Haziot et al., 1993). Furthermore, both PMNs and macrophages produce TNF and lL-1 via similar NF-xB-mediated pathways (McDonald et al., 1997). Because macrophages can synthesis 50-100 times more lL-1 or TNF from the same stimulus, the physiologic importance of synthesis by PMNs is not clear. LPS exposure to PMNs can also induce production PMN chemoattractants such as PAF and lL-8 (Makristathis et al. 1993; Wertheim et al., 1993). These factors, including TNF and IL-1, are usually produced by PMNs at the inflammatory focus and thus serve to promote further PMN involvement in the inflammatory response. 4. Vesicle Mobilization PMNs possess a host of cytocidal enzymes and inflammation- mediating proteins in their numerous azurophilic, specific, and gelatinase granules. LPS has not been reported to directly induce degranulation. However LPS can cause the mobilization of secretory vesicles to the plasma membrane (Sengelov et al., 1996). Secretory vesicles do not 77 contain the roster of inflammatory enzymes that granules do, but they express on their membranes important receptors including CD14, Mac-1, the complement receptor (CR1) and the FMLP receptor (Borregaard and Cowland, 1997). Incorporation of the secretory vesicle into the plasma membrane clearly makes the PMN competent to react to bacterial products (through CD14 or via FMLP) and pathogens opsonized with complement (through Mac-1 and CR1). Vesicles also contain alkaline phosphatase, an enzyme capable of dephosphorylating critical phosphate groups on LPS which can diminish its activity (Poelstra et al., 1997). 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 has been shown to hydrolyze FMLP (Fagny et al., 1995) and has been implicated in the cleavage of the IL-8 receptor (Bhattacharya et al., 1997). Thus, these peptidases may be critical to modulate PMN responses during inflammation. Indeed, LPS exposure to PMNs can inhibit the expression of lL-8 receptors on human PMNs (Khandaker et al., 1998). LPS can cause the expression of IL-8 receptors, this process is rapid and peaks 30 minutes after exposure (Bhattacharya et al., 1997). Based on the kinetics, it is possible that these receptors as well as many other uncharacterized modulators of inflammation are harbored in secretory vesicles. 5. Chemotactic Responses Pretreatment of PMNs with LPS can inhibit PMN chemotactic ability in vitro by unknown mechanisms (Bignold et al., 1991). Several possibilites for this effect, including modulation of actin assembly, 78 chemotactic receptors, and CD18 have been described above. 6. Priming Effect of LPS on PMNs PMNs responses to activators and secretogogues may be enhanced by a prior exposure of PMNs to LPS. Probably the most often cited example of the “priming” effect of LPS on the PMN response is the enhanced production of superoxide in response to “trigger” stimuli such as phorbol esters and FMLP (Aida and Pabst, 1990; Shapira et al., 1995; Aida et al., 1995; Hughes et al., 1995). The ability to augment this oxidative burst by common PMN activators in vitro has been proposed as the mechanism for PMN-mediated injury in animal models of sepsis and ARDS (Mulligan et al., 1992a). PMNs isolated from endotoxemic humans and animals display enhanced oxidant capacity (Wakefield et al., 1993; Solomkin et al., 1994; Kajdacsy-Balla et al., 1996). Exposure of human PMNs to LPS causes the membrane localization of flavo-cytochrome b553, a subunit of the superoxide-generating enzyme, NADPH-oxidase (De Leo et al., 1998). Coincidentally, D558 is co-Iocalized with Mac-1 in secretory vesicles (Sengelov, 1996). In this light, the C018- mediated enhancement of oxidative burst induced by TNF, FMLP, or phorbol esters may be due to the mobilization to the membrane of both CD18 and cytochrome D553 (Shappell et al., 1990; Ottonello et al., 1994; Liles et al., 1995). Thus, a single stimulus which mobilizes secretory vesicles would potentially promote both adhesion and oxidase functions. B. Summary The response of PMNs to LPS is mostly to promote inflammatory pathways. During endotoxemia, the PMN is first exposed to LPS and then to several secondary mediators which are produced minutes to hours later. 79 Thus, observations in vitro might accurately portray the initial activity of PMNs exposed to LPS in vivo. In this regard, in vitro studies which test the priming behavior of LPS on the PMN may effectively mimic the PMN responses to secondary mediators present later during endotoxemia. The sum of the effects described above produce a PMN that is hyperadhesive and responsive to oxidative bursts, yet prone to depressed phagocytic and chemotactic ability. This "endotoxemic" profile of the PMN might enhance some aspects of host defense responses while compromising others. This conclusion is supported by observations in humans and animals where endotoxemia is associated with both PMN-dependent tissue injury and with immunosuppression. 80 VIII. Effectors of PMN Fufnction During Endotoxemia In addition to the direct effect of LPS, PMN activity is modulated by several soluble and cellular factors that are present at different times during endotoxemia (Table 3). The following is a brief survey of agents that can modify PMN migratory activity, listed by their relative temporal appearance in the rat after IV LPS administration. A. Early Effectors (0-1 hOIfl) Administration of IV LPS to rats causes immediate inflammatory responses including effects on PMNs. Some of these responses were discussed earlier, such as PMN adhesion molecule expression and pulmonary PMN vascular sequestration. In addition, the mobilization of platelets, activation of complement, and production of TNF are early events that can impact on the PMN’s ability to respond to stimuli. 1. Platelets ‘ Blood platelets are anuclear cells which when aggregated together or adhered to endothelial cells promote thrombus and blood clot formation. After IV injection of LPS into rats, platelets colocalize with PMNs in pulmonary and hepatic capillary networks (Itoh et al., 1996). Platelets can bind to both endothelial cells and PMNs via glycoprotein adhesion molecules including PECAMs, and by doing so may regulate PMN adhesive responses to cytokines and other inflammatory stimuli. For example, PMN adhesion and transendothelial migration are modified in the presence of adhered platelets in vitro (Diacovo et al., 1996). In addition to having physical interactions with PMNs, platelets can secrete a variety of products that directly affect PMNs. Within a-granules of platelets are PMN 81 Table 3. Effectors of PMN Function During Endotoxemia $1326 of Endotoxemia Effector Early (0-1 hours) Platelets Complement activation products Tumor necrosis factor Intermediate (1 -3 hours) Interleukin-l Interleukin-6 Platelet activating factor Interleukin-8 ClNCs, MIP-2 Colony stimulating factors Interleukin-1 0 Late ( 3-6 hours) Prostaglandin E2 Nitric oxide Coagulation factors Fibrinolytic factors 82 activators such as platelet factor 4 (Aziz et al., 1997), the PMN-specific chemokine lL-8 (Su et al., 1996), and ligands for CD18 such as fibrinogen and fibronectin (Kaplan, 1986). Accordingly, granular release from platelets could directly affect PMNs colocalized within the pulmonary vasculature. Also, activated platelets release adenine nucleotides which, in addition to promoting platelet aggregation, inhibit PMN adhesive interactions with endothelial cells (Oryu et al., 1996). Thus, the platelet is 1) capable by a number of mechanisms to inhibit PMN migratory responses and 2) anatomically positioned to exert these activities. 2. Complement Activation Productjs) 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 CSb—C9) on cell membranes of pathogens and susceptible host cells. The appearance of C5a in blood occurs as soon as 5 minutes after IV LPS administration in rats and reaches a plateau by 30 minutes (Smedegard et al., 1989). This early increase in 05a is matched with the rapid upregulation of PMN adhesion molecules shortly after exposure of rats to LPS (Witthaut et al., 1994). Infusion of rabbits with ZAS, a source of 05a, causes pulmonary Ieukostasis in a similar manner to that induced by IV LPS in rats (Doerschuk, 1992). Although this suggests that complement activation during endotoxemia may be responsible for vascular pulmonary PMN localization, the results of Haslett and coworkers (1987) suggest that sequestration is due to direct LPS:PMN interactions. In addition, activation of PMN by C5a can lead to desensitization to other stimuli. CSa-treated PMNs demonstrate decreased chemotactic 83 responses in vitro (Kitayama et al., 1997), and activation of the C5a- receptor on isolated PMNs can induce desensitization of receptors for chemoattractants and other inflammatory mediators (Tomhave et al., 1994; Blackwood et al., 1996). C5a that is produced early in endotoxemia may bind to PMN receptors and render circulating PMNs insensitive to chemotactic signals that appear by one hour on the pulmonary endothelial surface. ,1. Tumor Ncfirosis Factor TNF appears in plasma by 30 minutes and peaks 1.5 hours after LPS is injected into rats (Pearson et al., 1995). Administration of large, nonphysiologic doses of TNF can reproduce the inhibition of PMN migration caused by IV LPS (Mason et al. 1997). Although this supports a role for TNF in modulating pulmonary PMN trafficking, it leaves unconfirmed that endotoxemia-associated TNF concentrations could impart similar effects. For example, studies in which TNF production is blocked have not been reported. Studies in vitro show that the TNF receptor is linked functionally to chemotaxin receptors in PMNs (Balazovich et al., 1996) and that TNF- pretreated PMNs have depressed migratory responses both ex vivo and in vivo (Otsuka et al., 1990). Thus, circulating TNF might modulate chemotaxin receptor activity on PMNs and lead to alteration of PMN responses to intrapulmonary stimuli during endotoxemia. B. Intermediate Effectors (1-3 hours) Plasma levels of TNF during endotoxemia peak at 90 minutes in most mammals. By 1-2 hours, the synthesis of secondary mediators 84 elicited by TNF and LPS begin to appear in plasma and include pro- inflammatory interleukins (IL-1, lL-6) and chemokines such as lL-8 in humans and CINCs and MlP-2 in rat. In addition, anti-inflammatory factors such as IL-10 and prostaglandin E2 (PGE2) begin to appear by 2-3 hours. 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, ECs, and smooth muscle. Thus, factors are present which might act in synergy or in opposition may be modulating the responses of PMNs during this time. 1. IL-1 As described previously, IL-1 has similar effects to TNF on ECs PMNs, and macrophage/monocytes. Unlike TNF however, lL-1 has not been demonstrated to desensitize PMNs to chemotactic stimuli (Otsuka et al., 1990; Balazovich et al., 1996). lL-1 can enhance the response of PMNs to TNF and other stimuli present during endotoxemia. In this regard, lL-1 may act as a cofactor which contributes to the modulation of PMNzEC interactions. 2. IL-6 IL-6 in plasma is associated with the severity and mortality of endotoxemia and has a broad array of pro-inflammatory effects on PMNs and macrophages in vitro. For example, exposure of PMNs to lL-6 promotes phagocytic and oxidant production capabilities (Mullen et al., 1995). In addition, IL-6 can cause PMNs to produce PAF (Biffl et al., 1996). 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 85 other important PMN modulators. LPAE PAF receptor antagonists are protective in a variety of endotoxemia models (Mathiak et al., 1997). Studies suggest that PAF promotes not only the accumulation and activation of pulmonary PMNs during endotoxemia but also causes hemodynamic changes (Rabinovici et al., 1993). Exposure of PMNs to PAF causes loss of L-selectin and C018 upregulation (Filep et al., 1997), but it does not desensitize PMNs to other chemoattractants (Luscinskas et al., 1992). Thus, by virtue of its chemotactic ability and effect on PMN adhesion molecule expression, PAF might contribute to the modulation of PMN responses during endotoxemia. 4. lL-8/ClNCs Concentrations of the primary PMN chemokines, lL-8 (humans, primates, rabbits, and sheep) and CINCs (rat), peak in blood by 2-3 hours after LPS administration (Martich et al., 1991; Ohira et al., 1995). Sources of plasma CINC-1 and MlP-2 in rat are not known, but these chemokines are produced in both the liver and lung during endotoxemia (Rose et al., 1994; Zhang, et al., 1995). Injection of lL-8 into rabbits blocks PMN emigration to extravascular sites of inflammation (Hechtman et al., 1991). Similarly, treatment of PMNs with IL-8 inhibits their migration through endothelial monolayers (Luscinskas et al., 1992). That lL-8 causes both the expression of chemoattractant receptors and CD18 integrins in vitro, suggests that activation of these membrane proteins on circulating PMNs may be detrimental to transendothelial migration (Roberts et al., 1993). That is, 86 activation of adhesive steps while the PMN is in circulation rather than in contact with endothelial cells may result in dysregulated or aborted adhesion. MIP-2 has similar effects on increasing CD18 on rat PMNs as IL- 8 has on human PMNs. Thus, the presence of ClNCs in plasma may negatively affect PMN migration to intrapulmonary stimuli. 5. Colony Stimulating Factors Neutrophilia which occurs by 4-6 hours 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 hours after LPS exposure (Kuhns et al., 1995). Treatment of isolated PMNs with G-CSF mobilizes CD18, enhances transendothelial migration and has no effect on adhesion (Yong, 1996). In addition G-CSF can modulate IL-8 receptors on human PMNs (Lloyd et al., 1995). Cotreatment of humans with endotoxin and G-CSF has different results depending on the time of dosing. When G-CSF is given 2 hours before LPS it enhances production of TNF, IL-6 and IL-8, whereas a 24 hour pretreatment generally inhibits the LPS responses, including pulmonary PMN sequestration (Pajkrt et al., 1997). The latter effect is associated with an increase in anti-inflammatory mediators. Thus although G-CSF can affect PMN functions in vitro, it is difficult to predict how the endotoxemia-elicited production of G-CSF affects PMN migratory behavior in the face of other inflammatory mediators. 87 6. lL-10 IL-10 appears in plasma by one hour after endotoxemia and peaks by 3 hours. It works in concert with PGE2 to turn off the production of inflammatory mediators by macrophages and PMNs (Cassatella et al., 1993; Wang, et al., 1994). That lL-10 can also reverse agonist—induced upregulation of CD18 suggests that it might modulate the adhesive and migratory capabilities of PMNs during endotoxemia (Laichalk et al., 1996). C. Late Effectors (3—6 hours) Production of mediators such as IL-10 and PGE2 actually occurs during the first 3 hours of endotoxemia but peak by 3 hours or later. Thus they may have effects on PMNs that are not clearly demarcated by the time frames used in this outline. In addition nitric oxide concentration spikes rapidly in lung at the start of endotoxemia and appears again later with different kinetics at 3-6 hours. In addition, the importance of these factors might be viewed in the context of what other mediators are present at the same time. For example, the PMN may be more responsive to PGE2 in the absence of TNF or IL-8, and thus exert its effects more strongly by 3-6 hours. 1. PGE2 PGE2 is an anti-inflammatory prostanoid produced by the enzyme cyclo-oxygenase 2 (COX-2), which is upregulated during endotoxemia and preferentially produces PGE2 and other arachidonic acid- derived products (Yu et al., 1993). PGE2 inhibits the production of inflammatory cytokines by negative regulation of NF-xB pathways. It can directly inhibit PMN chemotaxis (Armstrong, 1995) and aggregation (Wise, 88 1996). It appears in plasma about 2-4 hours after IV LPS. 2. 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 and smooth muscle cell among others (Liu et al., 1997; Aono et al., 1997). The hypotensive course during endotoxemia in rats is marked by an intial (10-20 minutes) drop in mean arterial pressure (MAP) from 120 down to 40-50 mmHg (Ruetten et al., 1996; Hock et al., 1997). By 2 hours MAP recovers to 100 mm Hg, only to fall gradually to 60 mmHg by 6 hours. That nitro-arginine -L-methyl ester (L-NAME, effective against constitutive NOS) blocks the initial drop in MAP and the iNOS inhibitor aminoguanidine reverses the latent hypotension confirms that NO mediates both events. Plasma concentrations of nitrite and nitrate, the metabolites of NO, are not evident for at least one hour after endotoxemia in rats, after which they increase 10-fold by 5-6 hours (Nava et al., 1992; Paya et al., 1995; Ruetten et al., 1996). This suggests that early NO release by endothelial cells is localized to the vascular wall or is quantitatively sufficient to promote vasodilation without producing detectable nitrite and nitrate in plasma. This distinction is important in consideration of studies in vitro, in which NO inhibits CD18-dependent PMN adhesion to endothelial cells in a dose-dependent manner (DeCaterina et al. 1995; Banick et al., 1997). In this context, NO might have a phasic inhibitory effect on PMNs in vivo in a response similar to hypotensive changes. That is, effects of NO on PMN adhesion may occur shortly after LPS administration and again 2-3 hours later. 89 Induction of iNOS in rat lungs occurs by 2 hours of endotoxemia, and by 3 hours the levels of nitrite and nitrate in lungs are greater than in other organs (Hock et al., 1997). In iNOS-deficient mice, PMN rolling in systemic venules and sequestration in lungs is significantly increased during endotoxemia. Taken together, NO production during endotoxemia likely has effects on PMN responses, but its role in pulmonary airway recruitment of PMNs is unknown. 3. Coagulation/flbrinolvtjc Fgctors Activation of coagulation processes as indicated by the appearance of prothrombin fragments and thrombin-antithrombin complexes in serum begins by 0.5 hours, but peaks much later at 5-6 hours. Despite this, major decreases in plasma fibrinogen do not occur until 2-3 hours after LPS administration to rats (Pearson et al., 1995). The appearance in plasma of plasminogen activators occurs by one hour and peaks rapidly by 1.5-2 hours during endotoxemia, suggesting the activation of fibrinolytic pathways (van der Poll et al., 1997). Fibrinogen and factor X are two components of the coagulative/fibrinolytic pathways that can bind to CD18. These interactions allow for initiation of coagulation on the surface of macrophage/monocytes which express CD18 during inflammation (Plescia and Altieri, 1996). Hypothetically, CD18 which increases on PMNs during endotoxemia could bind to fibrinogen and factor X and interfere with CD18:ICAM-1 interactions and inhibit PMN migration (Rozdzinski et al., 1995; Mesri et al., 1998). 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 (Altieri 90 et al., 1988; Ross and Vetvicka, 1993). Furthermore, fibrinogen inhibits PMN chemotaxis in vitro (Higazi, et al., 1994), and a peptide derived from factor X can bind to CD18 on PMNs and inhibit transendothelial migration (Rozdzinski et al., 1995). In this scenario, the disappearance of fibrinogen from plasma between 2 and 6 hours of endotoxemia would hypothetically free CD18 for ICAM-1 binding (Hewett and Roth, 1995) and thus promote adhesion and migration after fibrinogen is depleted. However, with the decrease in fibrinogen comes an increase in the circulation of fibrin degradation products (FDPs), and these products promote CD18-dependent chemotaxis by PMNs (Gross et al., 1997). In this context, the presence of FDPs in the circulation could modulate chemotactic responses of PMNs to chemotaxins generated in the pulmonary airspaces. D. Summam Taken together, endotoxemia presents a number possible culprits which could contribute to PMN migratory dysfunction. Some factors such as fibrinogen, factor X and LPS could bind to CD18 and potentially block adhesion, while others could upregulate CD18 in a fashion untimely for competent PMNzEC interactions. Cytokines and chemoattractants present during endotoxemia can downregulate chemoattractant receptors or adhesion molecules and either reverse or inhibit adhesive pathways. It is clear from the above description that a better temporal characterization of inhibition of PMN migration by IV LPS would simplify the task of identifying a mediator(s) and a potential mechanism. 91 IX. Research Goals During endotoxemia, PMNs become hyperadhesive and sequester in capillary beds of lung and liver, while at the same time they are primed for exaggerated inflammatory responses. Despite their “activated“ state during endotoxemia, PMNs are unable migrate from the pulmonary vasculature in response to an airway stimulus. This inability of PMNs to respond to respiratory infections or to toxicant inhalation that requires PMNs for resolution can be life threatening. There are clear associations between endotoxemia, PMN function and the predisposition for nosocomial pneumonia. Moreover, the occurrence of subclinical, environmental endotoxemia might predispose individuals to compromised respiratory defense responses which require PMNs (Roth et al., 1997). Current understanding of the mechanism of endotoxemia-associated PMN migratory dysfunction is limited. Therefore, it is critical to extend the findings in existing animal models of endotoxemia, especially with regard to pulmonary inflammatory responses. Understanding PMN behavior during clinical and environmental endotoxemia may suggest preventive strategies and therapies for individuals at risk for developing nosocomial and community-acquired respiratory infections. The first goal of the research in this dissertation is to test the hypothesis that inhibition of pulmonary PMN migration by IV LPS administration in rats was dependent on the timing of endotoxemia. Previous studies have established that PMN migration is inhibited by 5 hours after endotoxemia. However, production of several inflammatory mediators and activation of vascular and circulating inflammatory cells have occurred by this time. Thus, several mediators and inflammatory events could account for the inhibitory effect on PMN emigration. That 92 these mediators are present at different times during endotoxemia makes it possible to focus on different phases of endotoxemia and potentially limit the investigation to the mediators present during certain temporal windows. Accordingly, the temporal relationship between eliciting (airway) and inhibiting (intravenous) administrations of LPS in rats was altered and the effects on PMN migration evaluated. In this manner, the time of onset and duration of inhibition could be determined. For these investigations, PMN accumulation in BALF and histologic location of PMNs in lung tissues was compared between groups that received IV and IT LPS at different times. The second goal of this dissertation research was to test the hypothesis that inhibition of PMN migration is mediated by specific factor(s) present during endotoxemia. Based on the results from the first research objective, factors present very soon after N LPS administration were proposed as mediators of inhibition. Specifically, platelets or platelet products, TNF and activated complement proteins were selected because of their ability to modulate PMN adhesive and migratory functions. Consistent with Koch's postulates, a step in testing this hypothesis was to eliminate or inhibit the potential mediator or its activities. In this regard, rats were depleted of platelets, TNF or complement protein prior to being given LPS IT and/or IV. The effect of these treatments was evaluated on the ability of IV LPS to inhibit the accumulation of BALF PMNs. The last objective of the research was to examine the role of altered adhesion molecules in the mechanism of inhibition. PMN migration in response to intrapulmonary LPS requires CD18. PMNs from endotoxemic animals and humans have altered CD18 expression and chemotactic responses, and similar effects are seen with PMNs exposed to LPS in vitro. Thus, a hypothesis was proposed that IV LPS-induced inhibition of PMN 93 migration was due to alterations in CD18 pathways. Accordingly, the effect of IV LPS on pulmonary PMN migration in response to CD18-dependent and -independent stimuli was evaluated. Taken together, results from studies described in this dissertation provide important new information regarding the mechanisms responsible for altered PMN migratory behavior during endotoxemia. Future research on endotoxemia-associated nosocomial pneumonia can concentrate on early events and mediators during endotoxemia, or perhaps on LPS itself. That systemic endotoxin exposure is commonplace suggests that altered PMN function might also be important in community-acquired infections. In addition, this work provides critical insights into the modulation of adhesion molecules during disease, and adds to the growing body of research on trauma, sepsis and inflammatory syndromes for which adhesion molecules and cytokines are potential targets for therapy. Chapter 2 PULMONARY LEUKOSTASIS AND THE INHIBITION OF AIRWAY NEUTROPHIL RECRUITMENT ARE EARLY EVENTS IN THE ENDOTOXEMIC RAT 94 95 Chapter 2 SummaLy Neutrophil (polymorphonuclear leukocyte; PMN) migration into pulmonary airspaces is a prerequisite for clearance of bacteria in nosocomial pneumonia. Patients at risk for nosocomial pneumonia often experience endotoxemia, and altered neutrophil dysfunction is associated with endotoxemia in both humans and animals. Using a rodent model of endotoxemia-associated pneumonia, I characterized the altered kinetics of pulmonary PMN trafficking. In male, Sprague-Dawley rats made endotoxemic with intravenously (IV) administered endotoxin (lipopolysaccharide; LPS), recruitment of PMNs into the lung airspaces in response to intratracheally (IT) instilled LPS was inhibited. Numbers of airway PMNs were significantly elevated by 2 hours after receiving IT LPS (0.5 mg/rat), and immunohistochemical evaluation revealed PMNs in alveolar airspaces, alveolar walls, and in perivascular and peribronchiolar interstitial spaces. lV LPS (2 mglkg) caused neutropenia and pulmonary PMN sequestration within 15 minutes of administration. Inhibition of airway PMN accumulation to IT LPS occurred by 30 minutes and lasted for 6 hours after IV LPS. The location of pulmonary PMNs in rats receiving IV and IT LPS were similar to that observed in rats given IV LPS and IT saline. Thus, pulmonary Ieukostasis and inhibition of pulmonary PMN migration are early events in the endotoxemic rat and may be caused by the same mechanism. Furthermore, the kinetics of both events suggests that an early mediator present during endotoxemia or LPS itself is responsible for PMN migratory dysfunction and pulmonary sequestration. 96 INTRODUCTION Existing models of endotoxemia-induced inhibition of pulmonary PMN recruitment allow endotoxemia to develop for 2 hours before challenging airways with bacteria or endotoxin (Nelson et al., 1990, Frevert et al., 1994; Mason et al., 1997). In addition, PMNs do not appear in pulmonary airspaces for up to 1.5 hours after instillation of endotoxin stimuli into the lungs. Thus, at least 3.5 hours have elapsed between the onset of endotoxemia and the first signs that inhibition of pulmonary PMN recruitment has occurred. Systemic exposure of rats to endotoxin results in a cascade of soluble mediators which appear in blood at various times after endotoxin exposure, and many of these factors can alter PMN migratory function (Hewett and Roth, 1993; Burrel, 1994; Kuhns et al., 1995). As a first step toward elucidating the mechanism for inhibition imparted by IV LPS administration, I characterized the time course of inhibition of PMN migration. By altering the temporal relationship between administrations of the inhibiting (IV) and eliciting (IT) doses of LPS, I examined the kinetics of endotoxemia-induced inhibition of pulmonary PMN migration. MATERIALS AND METHODS Animals Male, Sprague-Dawley rats (CD-Crl:CD(SD)Br;Charles River, Portage MI), weighing 225-275 grams were used for all studies. Animals were used in accordance with guidelines set forth by the All-University 97 Committee on Animal Use and Care at Michigan State University. Rats were maintained on a 12 hour light/dark cycle under controlled temperature (70-72 °F) and humidity (40-60 mm Hg) and breathed HEPA-filtered air until experimental protocols commenced. Food (Harlan Teklad 22/5 Rodent Diet 8640; Harlan, Madison, WI) and tap water were supplied ad libitum. Treatment Protocols lntratracheal lnstillation of LPS: Rats were anesthetized with ketamine (100 mg/kg i.p.), an incision was made to expose the trachea, and 0.5 ml of 0.09% sterile saline or LPS (Escherichia coli, serotype 0128:312, Lot # 90H4012, activity: 24 x 106 EU/mg, Sigma Chemical Co., St. Louis, MO) dissolved in saline (1 mg/ml) was instilled into the trachea via a polyethylene catheter. Catheters were removed from the trachea and rats were allowed to recover before being returned to their cages. At various times after LPS administration as dictated by the experimental design, rats were anesthetized with sodium pentobarbital (50 mglkg, i.p.), the trachea was exposed and cannulated, a midline Iaparotomy was performed, and animals were killed by exsanguination. After opening the thoracic cavity, the heart/lung was carefully removed en bloc. Selective lavage of the right lung lobes was accomplished by clamping the left bronchus. Five ml of sterile saline were instilled through the tracheal cannula and withdrawn to recover bronchoalveolar contents of the right lung lobes. A second lavage with 5 ml of saline was performed and combined with the first. After lavage, the left lung clamp was released, and lung airways were infused through the tracheal cannula with 10% buffered formalin (at 30 cm of water pressure). After 2 hours, the trachea was 98 ligated and the lungs were immersed in the same fixative for at least 24 hours. Total leukocytes in the bronchoalveolar lavage fluid (BALF) were determined using a hemocytometer, and BALF PMN numbers were determined from the fraction of PMNs in a cytospin sample of BALF. Protein content in lavage fluid was determined using the BCA assay (Pierce, Rockford, IL). Intravenous Administration of LPS: Saline or LPS dissolved in saline at concentrations dictated by the protocols was injected (1 ml/kg) into tail veins of rats. At various times thereafter, animals were killed as described above, except that in some animals blood was drawn from the descending vena cava into sodium citrate (0.38%, final concentration) before exsanguination. Enumeration of total white blood cells, and platelets was performed on samples using an automated cell counter. PMNs were determined by a differential count of blood smears. Coadministrations of Intravenous and lntratracheal LPS: Time and sequence for administration of IV and IT LPS were altered depending on the experimental design (Figure 4). Specifically, saline or LPS was injected IV at 6, 3, or 1.5 hours before or 1, 1.5, or 2 hours after IT administration of saline or LPS. For all experiments using lV/IT dosing protocols, animals were killed for BALF and tissue analysis 3 hours after IT. Histologic lmalvsjs; Beginning at the lobar bronchus, the main axial ainlvay was microdissected so as to expose the openings to the branching airways. Airway generations were identified by the method of Harkema and Hotchkiss (1992). Briefly, lateral ainNay branches were numbered 99 .wnfi .E Eta p.50: m 035.com 903 $95.5 =< .wn: >— EOEEEUm 9.03 99. ma.— .: 5cm p.502 N 3 m... ._. ._o .989. mé .5 m .0 55m .230... o u 6E: H mm.— .: 82002 £9 =< 6.829 E953: mm... .25. no 9:an .v 0.59“. was: 32. Lo m.zo .5 :35 on... >_ nqlnlzlllz IITIIILIITIIIITIIIIILIIIIIIT 23o; n N m... _. c m._... m- o- a a $3.2» .8 an... E 8.5. 5% 82.8 100 consecutively beginning with the left bronchus (generation1) and successive numbers were assigned to each airway branch down the main axial ainlvay. Three consecutive transverse tissue blocks (approximately 4- 5 mm thick cross-sections through the main axial airway) of the fixed left lung lobe were excised, embedded in paraffin, and cut at 5 mm in thickness. The first lung block was taken at the fifth airway generation along the main axial airway. The second and third transverse tissue blocks were taken at approximately 5 and 10 mm distal to the first along the main axial airway, respectively. Sections 5 mm were stained with hematoxylin and eosin (H&E) for morphologic evaluation. PMNs within the lung sections were immunohistochemically identified using a rabbit-derived antibody to rat PMNs (Dahm et al., 1991). Briefly, formalin-fixed tissue sections were digested for 10 minutes in a pepsin:HCl solution before incubation overnight with rat PMN anitiserum. These immunohistochemical sections were then incubated with rabbit IgG conjugated to alkaline phosphatase which reacted with a substrate (Vector Red Substrate Kit; Vector Labs, Burlingame, CA) to form a red stain as imaged under a light microscope. The samples were counterstained with hematoxylin. Positively stained PMNs were identified not only by the red color, but also by other distinct morphologic features, including cell size, and a multilobular cell nucleus. In addition, reaction of antibody with rat PMN was confirmed by positive staining of rat peritoneal PMNs in cytospin preparations and by ability of the antibody preparation to deplete circulating PMNs when injected into rats (Dahm et al., 1991). 101 Morphometm: Pulmonary sequestration of PMNs was determined in immunohistochemical sections under light microscopy at 400x magnification. One entire section from the tissue block cut at airway generation 5 was scanned and every other field (12-16 fields per section) which contained only parenchymal cells were displayed on a color monitor using an image analysis system previously described in detail (Harkema and Hotchkiss, 1992). Briefly, slides were imaged a light microscope with an attached charge-coupled device camera and displayed on acolor monitor. Images were optically overlaid with a lattice-point grid (total area . approximately 75 mmz) and the number positively stained PMNs within the borders of the grid were counted (Stereology Tool Box, Morphometrix, Davis, CA). To ensure that the changes in PMN numbers between groups were independent of the area of tissue in the section, the surface density of tissue in each image was determined and comparisons were made among groups (Weibel and Cruz-Orive, 1997). Specifically, 125 test points within the lattice grid intersected with either tissue or airspace, and the percentage of points intersecting with tissue relative to the total points in the grid was calculated (points on tissue divided by 125, x 100). This value differed less than 2% between groups. Data for pulmonary PMN sequestration are expressed as the numerical density of PMNs in 100 mm2 of parenchymal tissue. Statistical Analysis: Results are expressed as mean 1 SEM. Data were analyzed using a completely randomized ANOVA. Multiple comparisons were made by the Least Significant Difference (LSD) post hoc test. When data were not normally distributed, analysis was performed on transformed data. Criterion for significance was taken as p < 0.05. 102 RESULTS Time course of the BALF PMN accumulation after IT LPS: Introduction of LPS into airways caused a time-dependent increase in the number of PMNs recovered in BALF. This increase was statistically significant by 2 hours compared to animals receiving saline (Figure 5). lnstillation of sterile saline caused a transient increase in airway PMN infiltration that peaked between 1 and 3 hours, but did not reach statistical significance. IV LPS- induced neutropenia and pulmonau seguestration of PMNs: After IV-LPS treatment, blood PMN numbers were significantly depressed and remained so for at least 90 minutes (Figure 6). This confirms results from previous work from this laboratory, in which neutropenia persisted for at least 2 hours, and numbers of mature, segmented PMNs did not return to normal until 6 hours after administration (Pearson et al., 1995). Morphometric analysis revealed that pulmonary sequestration accompanied neutropenia (Figure 7), since the number of PMNs within alveolar walls was significantly elevated by 15 minutes and remained elevated for at least 90 minutes after treatment with IV LPS. At no times were PMNs found within alveolar airspaces after IV LPS. IV LPS administered before IT LPS: Pretreatment of rats with IV LPS resulted in inhibition of PMN recruitment into pulmonary airspaces in rats in response to subsequent IT LPS (Figure 8). LPS (2 mglkg) given IV 1.5, 3 or 6 hours before the IT eliciting dose of LPS (Figure 4) prevented the increase in PMNs in BALF that occurred in rats given saline IV. Animals IT 103 A a 0 '° IT TREATMENT Z 4 ' [:3 saline I a 500 pg LPS _1 < m 3 E D I.” a: 2 |.l.| > O U |.l.l 0: 1 on 2 E O. 0 0.5 1 2 3 HOURS AFTER IT TREATMENT (0. 5 ml) or 500 pg LPS dissolved 1n 0. 5 m1 saline were instilled intratracheally into rats. At times indicated, animals were killed and lungs were lavaged with saline. PMNs were enumerated in BALF as described in Materials and Methods. Results are expressed as mean : SEM; n = 4; a= significantly different from saline controls. 'A 104 1750 'D o 1500 2 f 1250 :3. m 1000 :3 E 750 O E 500 D I.l.l z 250 - 5 15 30 45 60 90 MINUTES AFTER IV LPS Figure 6: evel m t f tr enia after VLP dminis ' Rats were treated intravenously with 2 mg/kg LPS and killed at the times indicated. Blood was collected and neutrophils were quantified as described in Materials and Methods. Results are expressed as mean : SEM; n = 4-5; a= significantly different from values at 0 hours. 105 20 (D 13 a 2 a 3 1e . . N a E14 o 12 ' c : 10 a in E 3 - a. :11 ° = (D 4 o 1’ I'- 2. 0 05 15 30 60 90 MINUTES AFTER IV LPS Figure7: 11'-1‘01‘u‘r 0.410111 '.-.0 - ' 1, -. ai‘ l _'- 11-111-12.01 Rats were treated intravenously with 2 mg/kg LPS and killed at the times indicated. Lungs were fixed and processed for PMN immunohistochemical staining and alveolar wall PMNs were enumerated as detailed in Materials and Methods. Results are expressed as mean : SEM; n = 3; a= significantly different from values at 0 hours. 106 .1: mm: 2:382 95% 25832 89¢ 822156 2:585:me .1. n .>_ 2&8 2:382 macaw 258%2 80¢ 62253 388$in n a £4 n : ”me H :88 mm 32298 Em $33M .mwofio—z was $2882 5 concave mm BEHSBU 203 235 28 .253 53, comma: 22> mwS: fiooctomm 20>» $2 E2525 mm; .2 Sta 2:0: 82:. .mmq 1: BBEEEE mass 282. 2:0: 0 co .m 64 was 8 258m 5? >685ng @285 225 9mm dorm—E a” m . L. - . U...“ . m .. HEB . «.... . ...m .3 - . .J. .. a .m . .. ... -, "w 0.5»?— 107 PMNs RECOVERED IN BALF .34 3m .34. .3. 4.623038" 2 \ :. U 3.5» \ mm=so E rpm \ 2:50 a «~53 \ rum I Cum \ Cum a a a ...m w m ICC—am wmgmmz _< >20 3. Cum ...—~m>._.z_mz._.m 108 instilled with LPS and given IV saline had significantly more PMNs in BALF than all others groups, and the recovery of PMNs in BALF was the same regardless of the time between IV and IT LPS treatments. Histologic Analysis: No abnormal histologic alterations were identified in lung sections from rats given saline IT and IV (Figure 9A). Alveolar parenchyma and conducting airways were devoid of pulmonary inflammation or treatment-related alterations. PMNs were observed in capillaries of alveolar septa, with occasional identification outside alveolar walls in perivascular or peribronchiolar interstitial spaces, in alveolar airspaces, or in lumens of conducting airways. Of all lungs that were histologically examined, those from this group of rats had the fewest numbers of PMNs in the lung sections. By comparison, the degree of cellularity in the alveolar septa and pulmonary parenchyma were markedly increased in rats given IV LPS and IT saline (Figure 9B). This increased cellularity in the alveolar parenchyma was due to an intracapillary accumulation of PMNs. Otherwise, sections from these animals were indistinguishable from control rats (saline lV; saline IT). lnstillation of LPS into rat airways resulted in acute bronchopneumonia that was histolologically characterized by a marked influx of PMNs in the centriacinar regions of lungs (terminal bronchioles and surrounding alveolar parenchyma; Figure 10A), and slightly lesser numbers of PMNs in preterminal bronchioles and in the main axial ainNay. Marked accumulations of PMNs were present in airway and alveolar walls and extended into their associated airspaces. The influx of PMNs was often accompanied by protein material (i.e., exudate). Neutrophilic influx was 109 600% 35225-2: 503:: 5205285 -2: ”222; 50003 ->: .Amv 250m 5 in: >— 28 As 25% .5 \258 >5 :53 @225 22 .50 2:52 0:002: 05 5 50:00 E 2:022:50: £525 358% 3:03:85 200502 :0: mfitofiE E 50:20: me 2553 ZSE 20.5 50020002: can :25 203 mwcfi :0: .5005: 203 22 225020 252 5 :05: 2:0: 025. .258 .5 cozw 250: 2050: 2:0: m: mm: .5 250m :23 3305222: @202: 20>» 205 .m. . .. 5: > :8 25. a. 95.... . . .53.: .3 .. 41.2., _ .. "a 0.5m:— 111 000% 35222-2: 203:: 2:020:05 L: ”2020‘, :02: ->: 0:03 2200:: 2 >20 mZSE 30:m Amv ma: .5\mm: >5 ::3 @200: 2am .95 mm: 53:22 >5 ::3 c2002 22 E .2020.» 5000—: 28 233:: 3:030:02: :00320: 300% 32:20:: E 2.. :03 mm £00322 :5: 2:00 H2003: _: 2025:: €200 2 5022:0w02v mZSE “02:03 430350: 200502 28 222202 .: “00:20: 20 2:206 235 20.: 32002: 28 :26 203 mw::_ ca: 60:2 203 22 E0222: mm: .5 :25: 2:0: 02:... .mm: .5 :0>:m 9:0: 2050: 2:0: 0. mm: :0 0:22 ::3 3305322.: @200: 203 235 ... . .5 . 4 . . m - :3 E; .. . 2.5, . 3: .0., .3 .. 4:. . _ . "3 0.53% 113 also conspicuous in the perivascular and peribronchiolar interstitium of the affected pulmonary regions. In contrast, when LPS-instilled rats were pretreated with IV LPS, infiltration of PMNs was restricted to the alveolar septa in the lung parenchyma similar to that in rats treated with IV LPS and IT saline (Figure 103). There was no extension of neutrophilic influx into the adjacent interstitial tissues (peribronchiolar or perivascular) or into the luminal air spaces of the conducting always or alveolar parenchyma. The lack of airway PMNs was consistent with low numbers of PMNs in BALF from these animals (Figure 8). IV LPS a_dministered after IT LPS: The ability of IV LPS to inhibit ainlvay PMN accumulation was tested in rats which had already received the eliciting dose of IT LPS, that is, in rats in which presumably ainNay inflammatory signals had been initiated and in some cases where airway PMN accumulation was already evident (Figure 11). When IV LPS was given 2 hours after IT LPS, at a time when significant numbers of PMNs were recovered in BALF, there was no inhibitory effect of the IV LPS treatment on BALF PMNs collected one hour later (at 3 hours post- instillation) (Figure 11, compare two bottom bars). In contrast, PMN numbers in BALF 3 hours after IT LPS were significantly decreased in rats that received IV LPS at 1 or 1.5 hours after IT treatment (Figure 11, top two bars). BALF PMN accumulation was not completely inhibited immediately after administration of IV LPS. Rather, significant increases in BALF PMN numbers occurred after treatment with IV LPS. 1 114 2:02:02: 2 00 02: 0200 05 :0 325300 25:0:w 03:00:02 20: 2205::“0 3200:5220 u : ”2:02:00: 20:00 .«0 00:8:0 0:: 2 mm: .5 00:0 2:0: m 3:032:30 22m “5.2m 20.: 22050:: b200w::w:m H0 ”v u : ”Sam M :002 00 32298 20 23005 .A.0:0:. 3_0:0_ :0: 0.0053 2:02:00: mm: 2 .«0 00:0m:0 05 2 ma: .5 :03 2:0: m :0:0_:2:000 ZSE ”53m 2:00.022 :20 was .5 5:0 32002 22 .«0 30% 0:0 .A20: :33 2:0: m 20 3:3 :20 mm: 53 >5 3:002 0: 0: :0 A20: 0.00::V :0:202::0 ZSE ”Him 20.: 3:2 0: 0: 205:0 3::02m 203 22 .mm: .5 2020 2:0: 0. N :0 m. : o. : 3. .mm: 50 33:50:: 30:00:22: :0 33002 22 :0 2:0: o 02: a. .. .... -.....>. . ‘. 3.::00 .... .. :1 .4). ..J... . ... z .3952“: 115 r. 2 x V "_.__ .o 2:: a mst 5%. I 2o.» 0.... t 3:0 2:2. n 022.. 5 4 minutes), antibodies to adhesion molecules can reverse the retardation of PMN transit (Cooper et al., 1989; Erzurum et al., 1992). In further support of a direct effect of LPS, infusion of PMNs treated ex vivo with LPS results in pulmonary Ieukosequestration with similar kinetics as seen in animals given IV LPS (Haslett et al., 1987). If inhibition of PMN migration coincides with the rapid (5-15 minutes) vascular sequestration of PMN in the pulmonary capillaries after IV LPS 118 (Figure 7), I would expect this to be reflected in decreased numbers of BALF PMNs almost immediately. However, migration appears to continue for at least 30 minutes after treating rats with IV LPS (Figure 11), or at least 15 minutes after significant PMN sequestration is evident with IV LPS alone (Figure 7). One explanation is that inhibition of PMN migratory function in fact does occur within minutes, but PMNs which are in transit through pulmonary interstitial spaces at the time of IV LPS administration or are already intimately associated with the vessel wall might escape the inhibitory effect. Thus, PMNs would increase in BALF due to continued movement into airspaces of PMNs in mid-migration from the vasculature. This is supported by studies of PMN migration kinetics in rabbit lungs in which the appearance of ainlvay PMNs lagged behind the appearance of PMNs in the interstitium and interalveolar septum by about 60 minutes (Downey et al., 1993). Thus, effects on intravascular PMNs might occur very rapidly in rats treated with IV LPS, but such effects do not become evident as a reduction in BALF PMN numbers for at least 30 minutes due to PMNs already in the interstitium (Figure 10A) which are able to continue migrating toward airways, unaffected by circulating LPS. In rats and mice, pulmonary vascular PMN numbers peak 1-2 hours after N LPS and then decline to 50% of maximum at 5-6 hours (Chang, 1994; Hirano, 1994). My results showed that by 6 hours after IV LPS the inhibitory effect on aiwvay PMN accumulation begins to subside (Figure 8). Presumably, PMNs are released from sequestration sites in the lung at this time, and these may be capable of migrating into airspaces if stimuli are still present. In addition, numbers of circulating PMNs at 4 and 6 hours after IV LPS are supranormal and consist of 50% band or immature PMNs (Pearson et al., 1995) which have been released from bone marrow in 119 response to an inflammatory stimulus. Recent studies show that younger PMNs have altered sequestration kinetics in the pulmonary vasculature and are slower to respond to inflammatory signals (Lawrence et al., 1996; van Eeden et al., 1997). Both human PMNs isolated from endotoxemic subjects (Solomkin et al., 1994) and PMNs flushed from the pulmonary vasculature of endotoxemic rats (Williams et al., 1993) display altered responses to stimuli, suggesting that PMNs which were once sequestered in pulmonary capillaries of rats might respond differently to a second, adhesion-promoting stimulus. Thus, the circulating PMNs 4 to 6 hours after IV LPS might comprise two distinct populations, those recently released from bone marrow and those recently released from sequestration in pulmonary capillaries, and both groups may be hyporesponsive to pulmonary inflammatory stimuli. In summary, these results provide evidence that IV LPS can prevent pulmonary PMN migration, and the mechanism of inhibition may be related to the pulmonary vascular sequestration during endotoxemia. Furthermore, the mechanism of inhibition may be related to pulmonary Ieukostasis of PMNs after IV LPS. Because inhibition occurs rapidly after endotoxemia is initiated, either LPS itself or an early mediator may be responsible for inhibition in this model. In addition, because inhibition is long lasting, more distal events and mediators mediators of endotoxemia may prolong inhibition of pulmonary PMN migration. That multiple mechanisms may be involved at different times after LPS requires further testing. Expanded efforts in studying this model may provide insights into basic mechanisms of inflammatory signaling and cellular responses and may also suggest preventive therapies in patients who are at risk for developing nosocomial pneumonia. Chapter 3 AN EVALUATION OF EARLY MEDIATORS OF ENDOTOXEMIA ON PULMONARY NEUTROPHIL MIGRATORY DYSFUNCTION 120 121 Chapter 3 Summam Patients at risk for nosocomial pneumonia often experience endotoxemia, and neutrophil dysfunction is associated with endotoxemia in both humans and animals. Pulmonary Ieukostasis and inhibition of airway recruitment of neutrophils (polymorphonuclear leukocyte; PMN) occurs early in endotoxemic rats. The mechanisms of either Ieukostasis or inhibition of PMN migration are unknown. Using a rodent model of endotoxemia-associated pneumonia, l addressed the roles of platelets, tumor necrosis factor (TNF) and products of complement activation as potential mediators in the modulation of PMN migratory function. In male, Sprague-Dawley rats made endotoxemic with IV endotoxin (lipopolysaccharide; LPS), recruitment of PMNs into the lung airspaces in response to intratracheally (IT) instilled LPS was inhibited. Inhibition of ainlvay PMN accumulation occurred by 30 minutes after N LPS. Factors present or activated after 30 minutes of endotoxemia were hypothesized to mediate the inhibitory effect of IV LPS. I found that pretreatment of rats with cobra venom factor to deplete complement (and 053 production) or immunodepletion of platelets or TNF did not affect the ability of IV LPS to inhibit pulmonary PMN recruitment or to cause pulmonary Ieukostasis. In summary, the results show that neither TNF, C5a nor platelets are sufficient to mediate the inhibitory effects of IV LPS on PMN trafficking and pulmonary Ieukostasis. 122 INTRODUCTION In rat models of pneumonia in which bacteria or endotoxin is introduced into airways, PMN migration into airspaces is inhibited during endotoxemia by unknown mechanisms (Nelson et al., 1990; Frevert et al., 1994). Several mediators are present during endotoxemia that could potentially modulate PMN functions, and only one, TNF, has been the subject of critical investigation (Mason et al., 1997). l have established that LPS exerts inhibitory effects on PMN migration within 1 hour of administration (Chapter 2). Thus, LPS itself or a mediator present during early endotoxemia may be responsible for inhibition in this model. In the present study, factors were identified which have the ability to modulate PMN migratory function and are present at the time of onset of inhibition. TNF, complement fragment 05a, and platelets or platelet-derived products were selected because they are present in the pulmonary circulation at the appropriate time and have been demonstrated to alter PMN adhesive and migratory functions. By blocking or depleting rats of platelets, TNF or complement, l characterized their role in endotoxemia-induced inhibition of pulmonary PMN migration. MATERIALS AND METHODS Animafi Male, Sprague-Dawley rats (CD-Crl:CD(SD)Br;Charles River, Portage MI), weighing 225-275 grams were used for all studies. Animals were used in accordance with guidelines set forth by the All-University Committee on Animal Use and Care at Michigan State University. Rats were maintained as described in Chapter 2. 123 Treatment Protocols lntratracheal and Intravenous Administrations of LPS: Rats were anesthetized with ketamine (100 mg/kg i.p.), an incision was made to expose the trachea, and 0.5 ml of 0.09% sterile saline or LPS (Escherichia coli, serotype 01282312, Lot # 90H4012, activity: 24 x 106 EU/mg, Sigma Chemical Co., St. Louis, MO) dissolved in saline (1 mg/ml) was instilled into the trachea via a polyethylene catheter. After catheters were removed, saline or LPS dissolved in saline was injected into the tail vein (1 ml/kg, 2 mglkg). Three hours later, animals were sacrificed and lungs, lavage fluid and blood samples were collected and processed as described in Chapter 2. Inhibition of Mediators: In some studies, blood samples were taken from the tail prior to LPS administrations to determine platelet numbers or complement activity as dictated by the protocol. The summary of protocols for mediator studies is depicted in Figure 12. In studies which addressed the role of complement products, rats were depleted of serum complement activity by intraperitoneal treatment with 15 units cobra venom factor (CVF) 48 and 24 hours prior to LPS administration (purified CVF was the kind gift of Dr. Gerd Till). Depletion of complement activity in rat serum was verified using the complement hemolytic (CH50) assay, which tests the ability of serum complement to lyse opsonized sheep red blood cells (Sigma, St. Louis, MO). Hemolysis requires the presence of serum 05b complement fragments that are generated by activation of complement pathways. Because C5b production is stoichiometrically related to 053 production (1 :1), we used the assay to estimate the potential 124 002006:— 00:5 8 :0>& 203 00:02:00.: 00:20 20:: 2:0: m .0050: :5 2:0: 0 H 02: 20 mm: .20 09:00 5.3 .5\>_ :000: 203 23:: :< 0:000:05 5005 .20 05.00 2o 00... 095. 0.20 :3. 28:8 :2 “.20 050: m o 092:. .u_._0 2 2 2. .2. 0.59. 0 a 2.0. 0... 092:. .u... O O u.r m 5 0 10 z E n. 104 __ __ saline LPS IV TREATMENT Figure 18: -. I . . -. Wm Rats were treated with CS or anti- TNF serum (1 ml) and immediately given IT LPS and then either saline or LPS IV, as indicated. Three hours after LPS treatments, animals were sacrificed, and BALF PMNs were quantified as described in Materials and Methods. Results are expressed as mean : SEM; n = 4; a= significantly different from respective groups treated with control serum (CS); b = significantly different from respective groups treated with IV saline. . ._ 134 DISCUSSION Pulmonary PMN migratory dysfunction during endotoxemia has been proposed as a mechanism for endotoxemia-associated nososcomial pneumonia in humans (Nelson et al, 1990; Frevert et al., 1994). Using a rodent model that better defines the temporal nature of endotoxemia- induced PMN dysfunction I tested the hypothesis that factors present early during endotoxemia mediate PMN migratory dysfunction in an animal model of pneumonia. My results suggest that three factors present during early endotoxemia, i.e., TNF, complement products and platelets or platelet-derived products, are not necessary for the inhibition of pulmonary PMN migration (Figures 14, 16, and 18). Although each of these factors has the ability to modify PMN migratory responses, individually each is not able to mediate inhibition of pulmonary migratory responses of PMNs in this model. Because platelets colocalize with PMNs in the pulmonary vasculature during the early endotoxemia, l hypothesized that either release of platelet products such as chemokines (Su et al., 1996) and adenine nucleotides (Oryu et al., 1996), or direct cell-cell interactions between platelets and PMNs (Diacovo et al., 1996) might modulate adhesive or migratory responses. However, depleting rats of platelets did not affect PMN migration into airspaces in response to IT LPS or the ability of IV LPS to inhibit migration (Figures 13, 14). Production of C5a from complement activation begins as soon as 5 minutes after N LPS administration in rats (Smedegard et al., 1989), and complement products are implicated in the rapid upregulation of PMN adhesion molecules shortly after exposure of rats to LPS (Witthaut et al., “..— . V_A_‘v. - -‘_s_..._ A #- H..- ...—'4“. 135 1994). Furthermore, C5a-treated PMNs demonstrate decreased chemotactic responses in vitro (Kitayama et al., 1997), and activation of the C5a-receptor on isolated PMNs can induce cross-desensitization of receptors for chemoattractants and inflammatory mediators (Tomhave et al., 1994; Blackwood et al., 1996). However, depleting animals of complement prior to LPS treatment affected neither the PMN recruitment in response to IT LPS nor the ability of IV LPS to inhibit migration (Figure 16). Accordingly, if C5a has PMN-modulating effects on circulating PMNs in rats, they are not sufficient to cause the inhibition of PMN migration into airways during endotoxemia. TNF has been proposed as the common mediator of endotoxin’s effects (van der Poll and Lowry, 1995), including its ability to modulate PMN activity (Hewett et al., 1993; Witthaut et al, 1994; Chang, 1994; Mason et al., 1997). TNF can inhibit PMN migration ex vivo and in vivo (Otsuka et al., 1990), and the PMN receptor for TNF is linked functionally to chemotactic receptors (Balazovich et al., 1996). Plasma TNF activity peaks 1.5 hours after IV LPS and reaches about 5-10% of this peak by 30 minutes (Pearson et al., 1995). Due to its potent effects on PMN function and on other factors that determine such migration, I examined the role of TNF in inhibition of PMN ainrvay accumulation by IV LPS. My results showed that despite complete inhibition of TNF activity in rat plasma, the inhibitory effect of IV LPS on PMN migration was not compromised (Figure 17, 18). This finding differs somewhat from the results of Mason and coworkers (1997), in which PMN infiltration in response to airway bacteria was partially blocked by prior exposure to IV TNF. In that study however, TNF was administered at concentrations 40-50 times that elicited by IV LPS treatment, and these concentrations may exert more profound effects 136 PMNs than are normally seen during endotoxemia in rats (Mason et al., 1997). In addition, TNF was administered 2 hours prior to ainrvay challenge with bacteria, thus allowing for the production of secondary mediators which might influence PMN migration. I observed inhibition of migration by IV LPS at times when plasma TNF is absent or at extremely small concentrations relative to peak plasma levels, and well before the production of mediators secondary to TNF release. Thus, plasma TNF is likely not a critical factor in the early inhibition of PMN recruitment seen in this model. Although neither activated complement, platelets nor plasma TNF alone was sufficient to cause the inhibition of PMN migration, I did not test the possibility that their combined influences may be responsible for altering PMN trafficking. My results demonstrate that LPS or an early mediator can (1) render PMNs less responsive to migration for up to 6 hours, and (2) arrest ongoing inflammatory processes of PMN migration shortly after IV administration. Because many mediators are present at different times during inhibition in this model, it is possible that various factors exert transient and sequential effects on PMNs to inhibit their migration. That is, factors responsible for early inhibition may be different from those that are present and exerting effects hours after endotoxemia is initiated. The onset and duration of inhibition of PMN migration is coincident with pulmonary vascular sequestration of PMNs in lV-LPS treated animals (Chapter 2). This suggests that the nature of sequestration in the capillary bed may be involved in the mechanism by which PMN emigration is inhibited. It is notable that infusion of complement products (05a) or TNF into rabbits can cause a transient pulmonary Ieukosequestration similar to 137 that induced by LPS infusion. This suggests that production of TNF and 05a after IV LPS administration might augment PMN sequestration and migratory inhibition in rat lungs. My results however suggest that TNF and 05a are required for neither the inhibition of PMN emigration nor pulmonary Ieukostasis caused by IV LPS (histologic figures not shown). In summary, removing the effects of certain potential mediators of inhibition, i.e., TNF, activated complement fragments or platelets failed to prevent the effects of IV LPS on both PMN migration and pulmonary Ieukostasis. Taken together, these results suggest that inhibition of migration into ainrvays may be due to a direct effect of LPS on the PMN, at least during the early phase of endotoxemia. The possibilities that LPS directly inhibits PMN trafficking in this model and that multiple mechanisms may be involved at different times after IV LPS require further testing. Expanded efforts in studying this model may provide insights into basic mechanisms of inflammatory signaling and cellular responses and may also suggest preventive therapies for patients at risk for nosocomial pneumonia. Chapter 4 INHIBITION OF PULMONARY NEUTROPHIL TRAFFICKING DURING ENDOTOXEMIA IS DEPENDENT ON THE STIMULUS FOR MIGRATION 138 139 MT 4 §Qmméfl In rat models of Gram-negative pneumonia, pulmonary emigration of PMNs is blocked when rats are made endotoxemic by an IV administration of LPS. To test whether the dysfunctional PMN migratory response in the endotoxemic rat is specific for airway endotoxin, I gave rats intrapulmonary stimuli known to elicit different adhesion molecule pathways for pulmonary PMN migration. Sprague—Dawley rats were treated IV with either saline or LPS and then instilled IT with either sterile saline, LPS from E. coli, interleukin-1 (IL-1), hydrochloric acid (HCI), zymosan-activated serum (ZAS), or lipoteichoic acid (LTA). Three hours later, accumulation of PMNs and protein in bronchoalveolar lavage fluid (BALF) were assessed. BALF PMN accumulation in response to IT treatment with LPS, IL-1, ZAS and LTA was inhibited by 100%, 100%, 40% and 58%, respectively, by endotoxemia. In rats given IT HCI, BALF PMN numbers were unaffected by IV LPS. The pattern of inhibition of migration suggests that N LPS only inhibits migration in response to stimuli for which migration is CD18-dependent. By contrast to PMN migration, BALF protein accumulation was inhibited by IV LPS only when IL-1 or LPS was used as the IT stimulus. To characterize further the differential responses of IV LPS to the various ainrvay stimuli, the appearance in BALF of PMN chemokine, macrophage inflammatory protein -2 (MlP-2), and tumor necrosis factor-a (TNF) was measured. Accumulation of PMNs in BALF correlated with the BALF concentrations of MIP-2 (r=0.846, p < 0.05) and TNF (r=0.911; p <0.05). The ability of IV LPS to inhibit pulmonary PMN migration correlated weakly with MlP-2 (r=0.659; p<0.05) and with TNF(r=0.413; p > 0.05) concentrations in BALF. However, this correlation was strengthened for TNF (r=0.752; p <0.05) when data from lL-1 treated animals were excluded. Thus, the presence in BALF of inflammatory mediators that are known to promote 140 CD18-mediated migration correlates with endotoxemia-related inhibition of PMN migration. Furthermore, the pattern of inhibition of pulmonary PMN migration during endotoxemia is consistent with the CD18 requirement of each migratory stimulus. INTRODUCTION Neutrophils (polymorphonuclear leukocytes; PMNs) are summoned into pulmonary airspaces by a variety of stimuli, including infection by pathogens, inhalation of air pollutants, and aspiration of gastric contents. Cytocidal and phagocytic activities of ainNay PMNs are essential for successful resolution of many bacterial and viral infections (Pierce et al., 1977; Toews et al. 1979; Farone et al., 1995). In addition, PMNs play a critical role in the inflammatory responses to inhaled particles (Li et al., 1997) and for the normal resolution of inflammation and tissue injury in response to irritants such as ozone (Hyde et al., 1997). Dysfunctional PMN migratory responses to airway endotoxin were characterized in Chapters 2 and 3. It is possible that endotoxemia might also affect PMN emigration to other airway stimuli and thereby compromise normal inflammatory processes necessary for the resolution of infection and of inhalation toxicoses. The ability of IV LPS to influence PMN emigration elicited by other ainrvay stimuli has not been reported. PMN migration in rats given IT LPS causes expression of pulmonary ICAM-1, production of the cytokines TNF and lL-1 and chemokines CINC-1 and MlP-2, and requires CD18 on PMNs. Studies using other ainNay stimuli revealed that PMN emigration into ainNays may either require CD18 or work 141 by as yet undefined, CD18-independent mechanisms. For example, ainrvay instillation of Gram-positive bacteria, the chemotactic peptide 05a or hydrochloric acid elicits pulmonary PMN migration which is largely independent of CD18 (Doerschuk et al., 1990; Winn et al., 1993; Hellewell et al., 1994; Folkesson and Matthay, 1997). Conversely, emigration in response to ainrvay administration of Gram-negative bacteria, LPS, IL-1 or phorbol myristate acetate (PMA) is due primarily to pathways involving CD18 (Doerschuk et al., 1990; Winn et al., 1993; Hellewell et al., 1994). In a rabbit model of bacterial pneumonia, lL-8 and TNF production was greater in ainrvays after treatment with a CD18-dependent stimulus when compared with a stimulus that elicits CD18-independent pathways (Shoberg et al., 1994). This result suggested that the profile of specific cytokines and chemokines that is elicited by an intrapulmonary stimulus might influence the adhesion molecule requirements for PMN migration. In this study, I tested the hypothesis that the ability of circulating LPS to inhibit PMN migration into airways depends on the stimulus for migration. The effect of IV LPS on pulmonary PMN migration to stimuli which differed in their requirement for CDIB was evaluated. Finally, I determined whether the appearance in ainrvays of MlP-2 and TNF, two mediators involved in PMN migration, was associated with the stimulus-specific inhibition of pulmonary PMN trafficking by systemic LPS exposure. 142 MATERIALS AND METHODS Animals: Male, Sprague-Dawley rats (CD-Crl:CD(SD)Br;Charles River, Portage MI), weighing 225-275 grams were used for all studies. Rats were housed as described in Chapter 2. lntratracheal (IT) lnstillations: Rats were anesthetized with ketamine (100 mg/kg i.p.), an incision was made to expose the trachea, and inflammatory agents dissolved in saline were instilled into the trachea via a polyethylene catheter. Preparation of ainrvay stimuli were as follows; LPS (E.coli; serotype 0128:815, activity: 25 x 106 EU/mg, Sigma Chemical, St. Louis, MO), 500 pg dissolved in 0.5 ml sterile saline (0.9%); IL-1 (human, recombinant, Genzyme, Cambridge, MA), 5 ng, dissolved in 0.5 ml saline; HCI, 0.1 N final concentration in 0.25 ml saline; lipoteichoic acid (LTA) isolated from Staphylococcus aureus (Sigma, St. Louis, MO), 250 pg dissolved in 0.5 ml saline. Blood was drawn from rats and used as a source of serum for zymosan activation and for control serum. To prepare zymosan-activated serum (ZAS), fresh serum was incubated with zymosan (5 mg/ml, Type A, Sigma Chemical Co., St. Louis, M0) for 30 minutes at 37°C and spun in a centrifuge for 10 minutes at 250x 9. The supernatant fluid was collected and spun again to remove any residual zymosan. Control serum (CS) was prepared by incubating fresh rat serum at 70°C for 30 minutes to inactivate complement. Rats were given 0.5 ml of 15% solutions of ZAS or CS in saline. Intravenous (IV) Administration of LPS: Immediately following IT administrations, saline or LPS dissolved in saline (2 mg/ml) was injected into tail veins of rats (1 ml/kg body weight). 143 Jllegtion of Bronchoalveolar Lavage Fluid and Lungs: Three hours after instillation of airway stimuli, rats were anesthetized with sodium pentobarbital (50 mglkg, i.p.), the trachea was exposed and cannulated, a midline laparotomy was performed, and animals were killed by exsanguination. Lungs and BALF were collected and processed as described in Materials and Methods in Chapter 2. BALF TNF: TNF activity in BALF samples was determined with a cytotoxicity assay using WEHI 1640 fibrosarcoma cells (Espevik and Nissen- Meyer, 1986) and compared to a standard curve using human recombinant TNF (R&D Systems, Minneapolis, MN) as described previously (Hewett et al., 1993). BALF MIP-2: BALF MIP-2 (CINC-3) concentration was determined using an ELISA kit which used tetramethylbenzidine (TMB) as a detection method (BioSource International, Camarillo, CA). Dilutions of BALF were compared with a standard curve made of rat MlP-2. Assays were performed in the laboratory of Dr. Kevin Driscoll at Procter and Gamble. §t_a_t_i_stjcal An_alvsis: Results are expressed as means :1: SEM. Data for BALF PMNs and protein were analyzed using a completely randomized ANOVA. Multiple comparisons were made by the Least Significant Difference post hoc test. Pearson Product Moment Correlation was used to evaluate the relationship between BALF concentrations of PMNs, MIP-2, TNF and the ability of IV LPS to inhibit PMN accumulation in BALF. Criterion for significance was taken to be p < 0.05. 144 RESULTS Lipopolysaccharide: lntratracheal LPS caused pronounced PMN accumulation in BALF compared to IT treatment with saline (Figure 19). Increases in BALF PMN numbers were completely blocked when animals received concurrent administration of IV LPS. Interleukin-1: The ability of IV LPS treatment to inhibit pulmonary PMN recruitment was tested in animals in which a PMN-eliciting inflammagen other than LPS was instilled into airways. PMNs are recruited into airspaces of rabbit lungs in response to intrapulmonary administration of lL-1 by mechanisms which require CD18 adhesion molecules (Hellewell et al., 1994). Interleukin-1 (5 ng) caused accumulation of PMNs in BALF after IT administration in rats (Figure 20). In rats treated IT with IL-1, administration of LPS IV completely inhibited the increase in BALF PMN at 3 hours when compared to rats treated IV with saline. onteichoic Acid; Intralobar inoculation of rabbits with Staphylococcus aureus or other Gram-positive organisms having membranous LTA induces ainNay PMN emigration which is partially inhibited by antibodies to CD18 adhesion molecules (Winn et al., 1993; Ramamoorthy et al., 1997). Treatment of rats IT with 250 pg LTA purified from S. aureus induced a significant increase in IALF PMN numbers (Figure 21). When rats were cotreated with IV LPS, PMN emigration was significantly reduced (58%), but not blocked completely. 145 103 - lT Treatment E saline b E LPS 107 10‘3 105 PMNs RECOVERED IN BALF 104 saline LPS IV TREATMENT Figure 19: ' itio IV of ai MN acc ti n elicited airw LP administratipn. Rats were given either sterile saline or 0.5 mg LPS IT and immediately given saline or LPS (2 mg/kg) IV. Three hours later rats were killed, lungs were lavaged with saline, and PMNs were enumerated as described in Materials and Methods. Results are expressed as mean i SEM; n=4; a= significantly different from respective group receiving saline IV. b=significantly different from respective group receiving saline IT. 146 107 - IT Treatment 1:! saline - lL-1 106 105 PMNs RECOVERED IN BALF 104 saline LPS IV TREATMENT Figure20: .' " ' 'Iu .. ' ' ' '- Wm Rats were given either sterile saline or IL- 1 (5 ng) IT and immediately given saline or LPS (2 mg/kg) IV. Three hours later rats were killed, lungs were lavaged with saline, and PMNs were enumerated as described in Materials and Methods. Results are expressed as mean i SEM; n=4; a=significantly different from respective group receiving saline IV; b=significantly different from respective group receiving saline IT. 147 107 - IT Treatment b E saline - LTA a, b 10‘5 105 PMNs RECOVERED IN BALF 104 saline LPS IV TREATMENT Figure 21: -, ‘ - t . . . administratiom Rats were given either saline or 250 pg LTA IT and immediately given saline or LPS (2 mglkg) IV. Three hours later rats were killed, lungs were lavaged with saline and PMNs were enumerated as described in Materials and Methods. Results are expressed as mean 1 SEM; n=4; a=significantly different from respective group receiving saline IV; b=significantly different from respective group receiving saline IT. 148 flydrochloric Acid; PMNs emigrate into airspaces of rabbits in response to intralobar instillation of HCI by pathways which are independent of 0018 adhesion molecules (Doerschuk et al., 1990; Folkesson and Matthay, 1997). Introduction into rat airways of 0.1 N HCI caused significant PMN accumulation in BALF, but to a lesser degree than that caused by IT LPS or IL-1 (Figure 22). When animals were cotreated with IV LPS and HCI, the PMN accumulation in BALF was unaffected. Cytospin preparations of BALF from rats treated with IT HCI appeared to have more erythrocytes when compared with BALF from other groups. In addition, preparations from HCI- treated animals had numerous ciliated cells. Examination of lung sections from these animals showed evidence of mild hemorrhage, specifically, erythrocytes and exudate were located in some alveolar airways. _Zivmosan Activated Serum: lntrabronchial administration to rabbits of complement fragment C5a (human) induces airway recruitment of PMNs that is mostly independent of CD18 (Hellewell et al., 1994). When zymosan activated rat serum was used as a source of 05a, IT administration caused significant elevation in BALF PMN numbers compared to that induced by complement-inactivated control serum (Figure 23). Cotreatment of rats with IV LPS reduced the BALF PMN response to airway ZAS by 40%. Airway Protein Accumulation: lnstillation into airways of LPS, lL-1, HCI, and LTA caused significant accumulation of protein in BALF compared to saline controls (Table 4). Protein concentrations in BALF after CS or ZAS were also significantly elevated compared to saline treated rats. However, inasmuch as CS and ZAS preparations contain 15% serum, the increase in BALF protein can be explained by the serum protein that was introduced into 149 A 0.8 ‘ IT Treatment C: saline = HCI P G l O' 0.4 - 0.2 - PMNs RECOVERED IN BALF (x10'6 P c saline LPS IV TREATMENT Figure22: .c,\ ', one .1 fu «_ 41.1.. 1 1-1... ,. , ' ' ts were given either saline or 0.1N HC1 and immediately given saline or LPS (2 mg/kg) IV. Three hours later rats were killed, lungs were lavaged with saline, and PMNs were enumerated as described in Materials and Methods. Results are expressed as mean i SEM; n=4; a=significantly different from respective group receiving saline IV; b=significantly difl‘erent from respective group receiving saline IT. 150 15i- IT Treatment b 1:1cs -ZAS ...t O 0.5 PMNs RECOVERED IN BALF (x10'5 ) P o saline LPS lV TREATMENT Figure 23: - u . _ _ . admmistration, Rats were given either CSu or ZAS and immediately given saline or LPS (2 mg/kg) IV. Three hours later rats were killed, lungs were lavaged with saline, and PMNs were enumerated as described in Materials and Methods. Results are expressed as mean i SEM; n=4; a=significantly different from respective group receiving saline IV; b=signif1cantly different from respective group receiving saline IV. 151 Table 4. Lavage Fluid Protein 3 hours after IT and IV administrations 8. BALF PROTEIN (nglml) IV Treatment: IT STIMULUS _saline LPS saline 124 i 24 94 i 11 LPS 212:17c 89:9b IL-l 196 :28° 97i16b HCI 485 3: 90 ° 577 i 88 cs 390: 19° 369:35 ZAS 408 i 23 ° 433 i 26 LTA 2471-18c 231:24 “ Rats were given each IT stimulus as described in Materials and Methods and then immediately given saline or LPS (2 mg/kg ) IV. Three hours later they were sacrificed, lungs were lavaged with saline, and BALF protein was determined. b significantly different from respective group receiving IV saline. C significantly different from group receiving saline IT. _‘ ‘ -.u-n‘ 152 the aiwvays. In addition, protein accumulation in HCI-treated rats was greater than that observed in LPS, lL-1 and LTA groups. Evaluation of BALF cytology and of lung sections suggests that mild hemorrhage may have occurred in response to airway HCI administration. Protein accumulation after IT LPS or lL-1 was reduced to control levels by cotreating rats with IV LPS. Elevations in airway protein caused by LTA, HCI, CS or ZAS were unaffected by IV LPS treatment. Inflammatory mediators recovered in BALF: Certain inflammatory mediators may be associated with specific requirements for adhesion molecule expression during PMN emigration into airways. Accordingly, correlation analyses were performed to evaluate the relationship between PMN migratory behavior and MlP-2 or TNF appearance in BALF. lntrapulmonary administration of all stimuli induced production of MlP-2 in airways (Table 5), and concentrations of MlP-2 correlated with the numbers of PMNs recovered in BALF (r=0.846; p < 0.05). TNF activity was evident in BALF from rats treated IT with LPS or LTA, but it was negligible in rats receiving lL-1, HCI, or ZAS. LPS induced more than twice the TNF production of LTA, which corresponded to the greater PMN migratory response. TNF activity in BALF was significantly correlated with BALF PMN numbers (r= 0.911; p < 0.05). Analyses were also performed to determine If inhibition of migration by IV LPS was selective for intrapulmonary stimuli which preferentially result in appearance of TNF or MIP-2. Correlation analysis revealed a significant association between MlP-2 appearance and degree of inhibition by IV LPS (r=0.659; p < 0.05). No association was evident for TNF (r=0.413; p > 0.05); however, omission of data from lL—1 treated rats resulted in a significant 153 TABLE 5. Cell and Cytokine Content of Bronchoalveolar Lavage Fluid 3 hours after IT instillations. % Inhibition of IT BALF PMN BALF MIP — 2 BALF TNF BALF PMN STIMULUS RECOVERED (ng / ml) (ng / ml) accumulation (x 10‘) BY 1v LPS saline 0.04 3: 0.02 0.20 i 0.01 < 0.01 - LPS 4.87 i 0.52 9.36 i 0.78 3.33 i 1.05 100 IL - 1 1.77 i 0.23 6.87 i 1.57 < 0.01 100 HCI 0.45 i 0.10 0.33 i 0.01 < 0.01 0 ZAS 0.93 i 0.19 4.28 i 0.78 < 0.01 40 LTA 2.88 i 0.60 9.68 + 1.60 1.25 _+_ 0.48 58 ‘— r= 0.846“——’ L———— r = 0.911a ———J L—— r=0.659°___l [— r= 0.413 —J Rats were given each IT stimulus as described in Materials and Methods. Three hours later animals were killed, lungs were lavaged with saline, and MIP-2 and TNF-a were determined in BALF as described in Materials and Methods. 8 significant relationship between indicated groups (p< 0.05) 154 association between BALF TNF and inhibition of PMN migration by IV LPS (r=0.752; p < 0.05). The concentration of BALF MIP-2 was also measured in some rats which received IV LPS and an IT stimulus (Figure 24). In rats given IT HCI or ZAS, BALF MIP—2 concentration was significantly increased when animals were also treated with IV LPS. In rats given IT LTA, treatment with IV LPS did not effect MlP-2 concentrations in BALF. 155 A h l IV TREATMENT I==I saline _ LPS ..L N I _\ O l LAVAGE MlP-2(nglml) O, HCI ZAS LTA IT TREATMENT , , ‘ . ul’ 1,! - W Either 0.1N HC1,ZAS or LTA was instilled IT into rats, then saline or LPS (2 mg/kg) was immediately injected IV. Three hours later, animals were sacrificed, lungs lavaged with saline and MIP-2 concentration in BALF determined as described in Materials and Methods. Results are expressed as mean : SEM; n=4; a=signif1cantly different from respective group receiving saline IV. 156 DISCUSSION The results of this study demonstrate that the degree to which endotoxemia inhibits pulmonary PMN recruitment is dependent on the intrapulmonary stimulus. Migration of PMNs in response to airway LPS or lL-1 was blocked completely by IV LPS, emigration after LTA or ZAS was partially inhibited, and the response to HCI was unaffected. Furthermore, the selectivity of inhibition appears to be related to the putative involvement of CD18 adhesion molecules in the migratory response; specifically, IV LPS treatment was more effective at blunting PMN migration which has been shown in other animal models to require CD18. The most extensive characterizations of CD18 involvement in the migration of pulmonary PMNs have been in rabbit models (Doerschuk et al., 1990; Winn et al., 1993; Hellewell et al., 1994; Ramamoorthy et al., 1997). PMN migration in response to airway E. coli (Ramamoorthy et al., 1997), endotoxin isolated from E. coli (Doerschuk et al., 1990; Winn et al., 1993), or lL-1 (Hellewell et al., 1994) was inhibited in rabbits by antibodies directed against CD18. In rats, evidence suggests that PMN migration to intrapulmonary LPS relies on CD18 adhesion pathways (Tang et al., 1995; Beck-Schimmer et al., 1997). By contrast, after HCI aspiration, edema and vascular sequestration of PMNs occurs independently of CD18 (Motosugi et al., 1998). In addition, intrapulmonary lL-1 mediates PMN recruitment during immune complex injury in rats (Warren, 1991), a model in which PMN migration requires CD11a/CD18 and CD11b/CD18 pathways (Mulligan et al., 1995). Furthermore, studies in mice using the same intrapulmonary stimuli as used in rabbits suggests that pulmonary PMN migratory responses might be the same across species (Qin et al. 1996). My results in rats confirm that IV 157 LPS inhibits accumulation of PMNs in airways in response to LPS or lL-1, two stimuli that initiate CD18-dependent migration (Figures 19, 20). Tuomanen and coworkers (1987) instilled various cell wall components of Gram-positive bacteria, including teichoic-acid peptidoglycans, into rabbit airways to create Ieukocytic infiltration. I induced pulmonary PMN emigration in rats by administration of S. aureus-derived LTA and found migration to be inhibited by 60% by IV LPS treatment (Figure 21 ). By comparison, antibodies to CD18 reduced PMN emigration in response to S. pneumonia or S. aureus- induced pneumonia in rabbits by 45% (Winn et al., 1993; Ramamoorthy et al., 1997). Thus, both CD18 antibody treatment and IV LPS inhibit by similar degrees the PMN migration to Gram-positive stimuli in rabbit and rat airways, respectively. In rats instilled with ZAS, two components of airway PMN emigration were observed, one which was reduced by IV LPS treatment (40% inhibition) and one which was unaffected (Figure 23). The chemotactic potential of ZAS is primarily due to complement activation products, especially C5a. In rabbits, an antibody to 0018 reduced only 20-30% of the PMN emigration into airways in response to human recombinant C5a, but this effect was not statistically significant (Hellewell et al., 1994). My observation that N LPS failed to inhibit most of the migratory response to ZAS is consistent with the possibility that IV LPS inhibits CD18-dependent PMN trafficking. lnstillation of HCI into rabbit lungs causes PMN emigration which is unaffected by treatment with antibodies to CD18 (Doerschuk et al., 1990; Folkesson and Matthay, 1997). Likewise, IV LPS had no effect on PMN emigration in response to HCI in rats (Figure 21). Generally, the degree to which IV LPS treatment inhibited PMN trafficking into airways correlated with the degree to which the various IT stimuli caused CD18-dependent migration. 158 Burns and coworkers (1994) have shown that CD18 expression on PMNs in the pulmonary circulation are unchanged prior to migration to a CD18-dependent stimulus in airways. Curiously, CD18 is upregulated on PMNs prior to adhering and migrating toward an airway stimulus which does not require CD18. LPS exposure to isolated PMNs causes shedding of L- selectin and increased expression of CD18 in vitro (Lynn et al., 1991), and similar changes are observed on PMNs isolated from endotoxemic animals (Frevert et al., 1994). Thus, one could speculate that LPS-induced CD18 upregulation on circulating PMNs is a premature step for competent CD18- dependent migration, but is a normal PMN response in the process of CD18- independent pathways. Inappropriate expression of CD18 on circulating PMNs may violate the step-wise sequence of rolling, firm adhesion and diapedesis, and result in aborted or dysregulated interactions between PMNs and endothelial cells. This possibility is consistent with the ability of LPS to inhibit transendothelial migration which requires CD18 in vitro ( Bignold et al., 1991). PMN migration into rat airways is often accompanied by an increase in vascular permeability (Terashima et al., 1996; Hybertson et al., 1997; Motosugi et al., 1998). I measured protein accumulation in BALF as a marker of vascular leak. In rats cotreated with IV LPS and intrapulmonary LPS, IL-1 or HCI, decreases in BALF PMN accumulation were associated with reduced BALF protein concentration (Figures 19, 20, 22, Table 4). That is, protein accumulation was blocked completely after intrapulmonary lL-1 and LPS but was unaffected after HCI. When PMN emigration was partially inhibited by IV LPS treatment in rats given LTA or ZAS, elevations in BALF protein accumulation were unaffected (Figures 21, 23, Table 4). Pulmonary PMNs in the microvasculature and alveoli have been implicated as edemagenic 159 mediators in rat models of ARDS (Windsor et al., 1993; Tsuji et al., 1998). However, the inhibition by IV LPS of BALF protein accumulation after IT LPS or IL-1, was not due to an inhibition of pulmonary PMN localization. Light microscopic evaluation of lung sections revealed that treatment with IV LPS caused sequestration of PMNs within alveolar walls irrespective of IT administration (data not shown). Thus, PMN extravasation may be linked to protein leakage in airspaces, but the mere presence of PMNs in the pulmonary microvasculature is not sufficient to cause leak. At least one study suggests that whether or not CD18 is involved in pulmonary PMN emigration depends on the type of cytokine mediators produced by the stimulus for migration (Shoberg et al., 1994). In this paradigm, inflammatory mediators which activate CD18 on PMNs and/or ICAM-1 on endothelial cells may preferentially induce CD18-dependent migration. Expression of CD18 on PMNs can be induced by either TNF or MlP-2, and ICAM-1 is upregulated on endothelial cell monolayers after exposure to TNF (Sayler et al., 1990; Frevert et al., 1995; Burke-Gaffney and Hellewell, 1996). Accordingly, I measured TNF and MlP-2 concentrations in lavage fluid of rats that were dosed with the various intrapulmonary stimuli used in my model (Table 5). All IT treatments resulted in the appearance of airway MlP-2, and this correlated with the accumulation of PMNs in BALF. Despite the lack of TNF in BALF after lL-1, HCI or ZAS treatments, the appearance of TNF in airways was also associated with BALF PMN accumulation. TNF is a strong promoter of CD18 and ICAM-1 expression and may preferentially mediate CD18-dependent migration into airspaces. When all groups were considered, the appearance of TNF in BALF correlated poorly (r=0.413; p < 0.05) with the ability of IV LPS to inhibit pulmonary PMN emigration, suggesting at first glance that inhibition is not related to the 160 requirement for CD18. However, lL-1 by itself is capable of promoting expression of both ICAM-1 on cultured E05 and CD18 on isolated PMNs. In rats treated with intrapulmonary IL-1 therefore, the presence of TNF may not be necessary to induce CD18 migratory pathways. Indeed, elimination of the results with IL-1 from the analysis resulted in a much stronger correlation between inhibition of PMN migration and BALF TNF (r=0.752; p < 0.05). Concentrations of MIP-2 in BALF also correlated with the ability of IV LPS to inhibit PMN emigration. MlP-2 is a potent PMN chemoattractant that can promote the surface expression of CD18 on PMNs and may preferentially promote CD18-dependent migration. That IV LPS can inhibit MIP-2- dependent migration is supported by the results in HCl- and ZAS-treated rats. Coadministration of IV LPS caused an increase in the appearance of MIP-2 in the airways of rats given HCI or ZAS (Figure 24), yet this increase did not result in further PMN accumulation in BALF. Indeed, PMN migration was reduced in ZAS treated rats (Figure 23) and failed to increase in rats treated with HCI (Figure 22). Thus, the increase in PMNs expected with an increase in MlP-2 in airways was blocked by IV LPS treatment. A recent report suggests that LPS can directly downregulate expression of chemokine receptors on human PMNs (Khanderaker et al., 1998). If MlP-2 receptors on rat PMN are similarly affected, circulating endotoxin could potentially inhibit PMN migratory responses to intrapulmonary stimuli that rely on MlP-2 pathways. Different forms of pulmonary PMN emigration are depicted in Figure 25. During CD18-dependent migration expression of CD18 occurs only after PMNs are associated with the vessel wall, whereas PMNs in the pulmonary circulation have increased CD18 prior to CD18-independent migration. MlP—2 which is produced in the airways can become localized in the vessel wall 161 5355.55 0.8 5058mm: .«o 558 mg 3.558 533 830208 53853 Ea £83525 30550 05,—. .m _ no 2552 505 op 595 88885 .088me $5.55 585585 0.8 Ngaz can .222. $2 $035232 .233 05 no 985532 8 555 :8 5 2053 :53 Emma.» 05 E 53:32 2583 8 50585053 503 was we: .85me? 562363 So was a 50 as 72.42 E 85838.. 28 83.52% 65 2 8%: 68 3.56 $52885 .0955ng owes—58058 55 32.5 858.5 $8.82. 8555 go ahead? .5 5050558; .w 25 0.5552 505 at 555 83385 bowaau macaw mZSE mgag 555 522:3 50 555 235303> haze—£55 05 E mZSE 955530.50 co 53 588.55 a w 25 5859600 is? .3588, 05 553 585683.. 08 M235 .555 5058m55 unwvcwnové _QU W556 mZSE no 588 85 805 w 25 mo aoammoaxm . .. .. . . . 162 .2 «Eu:— was. N-n_=>_ mZ._. LZF 0A.! whoa—6:50.)— wgowEUoS— C90 99 7520. o 0 $3020.: A\ 223255 ...... 3555.55?x :— 9 V 4 f‘- D ..1 u 0. ‘ L .. . vi 5 V “I"! away.“ , I“. ‘3 tag" . -l .x.’ r I «A :4 i ’m an... .. on. . . . . 4 . u 1" u 1 s n I u . . .... u I . u I C ‘ . 1 e . u I n . I o n‘ . . .. .. .. 163 where it can bind to receptors on adhered and tethered PMNs. Each form of migration is likely to be driven by a different complement of cytokines and chemokines, and TNF and MlP-2 are two mediators that may be more important for CD18-dependent processes. Production of specific chemokines has not been systematically correlated with CD18-dependent or -independent ainNay stimuli. My results indicate that distinct profiles of cytokine production occur in response to different intrapulmonary stimuli, and these mediators might dictate specific adhesion molecule requirements for PMN migration. lV LPS in rats strongly inhibited the intraavleolar accumulation of PMNs in response to agents which require CD18 for PMN migration and had no or more limited effects on PMN accumulation with materials that are not CD18-dependent. Migration that is CD18-dependent requires the expression of CD18 in the appropriate amounts and at the appropriate times for successful passage of PMNs through the EC barrier. Selective inhibition by IV LPS may be due to inappropriate expression of CD18 on circulating PMNs, an event that could be detrimental to CD18- dependent migration. Conversely, CD18 expression on circulating PMNs occurs during both endotoxemia and during CD18-independent pulmonary PMN migration. Thus, upregulation of CD18 is a “normal” phenomenon during CD18-independent migration and expression of CD18 caused by endotoxemia does not effect negatively the process of CD18-independent migration. Based on my results and those of others, endotoxemia might be effecting pulmonary PMN migration as depicted in Figure 26. Endotoxemia causes CD18 expression on circulating PMNs where binding may occur between 1) CD18 and circulating LPS (Flaherty et al., 1997), 2) CD18 and fibrinogen which exists in normal serum (Ross and Vetvicka, 1993), and 3) 164 5855585 555585655 5555: 3205 555 m225 55 59588 NA=2 853555355 58 mm: 555655 5 2850556 555 528555 5585 55855555 555 5523 353555 55—3—8555 w555>555 Am no 555555 55%: 55.5 $550958 2 23 5o5€m55 5555505555 25 $5.55 5555885— _ 2_ . .8532 N- 5.5. 535.555.5265 .... 4‘! . . 7“,,” 1 (a: ' . f s. a ‘L \ x.- 4: 5 I‘ V. a; k «r. ‘ h in”. 11" .' £’: 3.. 1 "Ci 11 166 CD18 and Factor X which is generated in blood by the activation of the coagulation system (Rozdzinski et al., 1995). Binding of CD18 by any of these factors may activate the PMN in a manner that is inconsistent with adhesion and migration. As such, appropriate binding of ICAM-1 by CD18 might be inhibited by directly blocking access to CD18 or by modulating the expression or activation of CD18 on PMNs. Taken together, there are multiple points for interference of CD18- dependent processes that take place during endotoxemia. It should be noted that endotoxemia-associated events which effect CD18-dependent pathways (i.e., inappropriate expression of CD18, receptor downregulation, etc.) are not critical features for migration which do not rely on CD18. Thus, immune suppression and PMN dysfunction which occur during endotoxemia in humans might be due to the inability of PMNs to respond to inflammatory stimuli which require competent CD18 function. Elucidating the mechanism of endotoxemia-related PMN dysfunction may suggest therapeutic options for complications such as nosocomial infections and PMN-mediated organ injuries. SUMMARY AND CONCLUSIONS 167 168 Activation and recruitment of circulating PMNs into tissues is a critical component of the inflammatory defense response to acute infections, tissue injury, and toxicant exposure. In lung, many viral and bacterial pneumonias require the presence of airway PMNs to kill and clear invading pathogens. Inhalation of fine particulates, noxious fumes, irritants and air pollutants can elicit a rapid mobilization of PMNs to lung airspaces where their oxidant and proteolytic activity may contribute to tissue injury that is secondary to the initial insult. In these instances however, the PMN also assists in the clearance of particulates and injured pneumocytes as well provides timely signals for the recruitment of other mononuclear inflammatory cells which are necessary for tissue repair. Failure of PMN recruitment into lung airspaces occurs in individuals with Leukocyte Adhesion Deficiency (LAD) disease, a condition in which PMNs lack CD18 adhesion molecules (Kuijpers et al., 1997). These patients are prone to pulmonary infections and have higher mortality than normal patients with the same respiratory diseases. Endotoxemia is characterized by a global activation of inflammatory cells and pathways. PMNs can exhibit different behaviors during experimental and clinical exposure to circulating endotoxin. In some instances, activated PMNs contribute to tissue and organ injury. Alternatively some PMN responses, including chemotaxis, are diminished during endotoxemia and may contribute to immunosuppression which is common during clinical cases of endotoxemia. In experimental models of pneumonia, endotoxemia inhibits pulmonary PMN emigration by unknown mechanisms. Virtually every known inflammatory cytokine is produced and every inflammatory cells activated during endotoxemia. As such, it is difficult to identify the factor or factors responsible for inhibition in these models. Indeed, attempts at identifying a mechanism has been cursory, and results 169 have been inconclusive. Experimental results from Chapter 2 of this dissertation have dramatically simplified the search for a mediator of inhibition. By defining the temporal window of inhibition as the first 30-60 minutes of endotoxemia, a host of candidate mediators can be eliminated from consideration in future investigations. Furthermore, the results from Chapter 3 suggest that three factors which could likely affect inhibition of PMNs during early endotoxemia - platelets, complement products, and TNF- are not necessary for inhibition by IV LPS. Taken together, the data supports the hypothesis that LPS itself is responsible for dysfunctional PMN migratory responses. This hypothesis has yet to be tested in an appropriate animal model. Morphometric analysis of rat lung sections suggests that inhibition of pulmonary PMN migration might be related to the ability of IV LPS to cause vascular sequestration of PMNs in lung tissue. Cytoskeletal changes and adhesion molecule activation have been proposed by others as the mechanism of sequestration, and might also play a role In the inhibition of emigration. Experiments to identify the mediator of sequestration have been unsuccessful, however most of the evidence implicates a direct effect of LPS on PMNs. Thus, LPS may be directly responsible for both pulmonary Ieukostasis and inhibition of emigration, and these two events might be precipitated by the same mechanism. Experiments to separate PMN sequestration from inhibition of migration have not been performed, thus the link between these two phenomenon is circumstantial. Endotoxin can cause the expression of and bind to CD18 adhesion molecules on PMNs. Because CD18 is required for pulmonary PMN emigration to airway LPS, experiments were designed to test the hypothesis that N LPS inhibited migration by altering CD18 pathways. Confirmation of 170 this premise is demonstrated in Chapter 4, where experimental results show that IV LPS inhibition is selective for pulmonary PMN migration which requires CD18, and does not affect migration which is CD18-independent. To further describe the mechanism of inhibition at the cellular or molecular level requires further investigation. For example, it is unclear if premature expression of 0018 or if LPS:CD18 binding interactions is responsible for migratory dysfunction. While some signal transduction pathways after CD18:Iigand binding and LPS:receptor interaction are known, it is unclear how these events might selectively alter PMN migratory processes in vivo. Furthermore, the ability of LPS pretreatment to inhibit chemotaxis of PMNs in vitro may work by the same mechanism which is responsible for effect of endotoxemia in rats. Defining the nature of altered CD18 pathways in this model may require both cellular and molecular approaches. Experimental results in Chapter 4 also demonstrate an association between the presence of TNF and MlP-2 in rat airways with inhibition of migration by IV LPS. Both of these mediators are linked to CD18-dependent pathways of PMN migration in other models. Thus, these data extend further the characterization the CD18 involvement in the mechanism of inhibition. Adhesion molecules responsible for CD18-independent PMN migration are unknown. It is likely that the nature of PMN migration (adhesion molecule requirements) depends on the nature of the inflammatory response. In this paradigm, CD18-dependent stimuli would elicit production of cytokines and chemokines different from those elicited by CD18-independent stimuli. The data in Chapter 4 represent the first attempt in rats to evaluate and compare the qualitative and quantitative nature of soluble inflammatory mediators produced in response to several airway stimuli of known CD18 dependencies. A more thorough approach which analyzes several more 171 cytokines, chemokines, and factors of inflammation present in rat airways is necessary to test fully this hypothesis. For example, the contributions of LTB,,, PAF, C5a, and other interleukins and CINCs to PMN migration after exposure to different airway inflammagens needs to be evaluated. Results from these studies will prove helpful for both basic research and clinical applications. Specific therapies which target selectively the known chemokines or mediators produced by a particular ainlvay stimulus could be used to either augment or inhibit pulmonary PMN migration. E! D' I' I! II Specific knowledge gaps which require further work were briefly described above, including the relationship between vascular sequestration and migration, the identification of non-CD18 processes of migration, and the characterization of the mediator responsible for inhibition of migration. Besides the potential contribution to understanding the basic mechanisms of PMN transendothelial migration, the results described in this dissertation provide an animal model for endotoxemia-associated pneumonia. In addition to nosocomial infection, another association between endotoxemia and respiratory ailments is that which occurs in participants of athletic endurance events. Marathon running, long-distance cycling, and high-intensity, aerobic training can cause endotoxemia, upper respiratory infection and altered PMN Immune and adhesive functions in humans (Bosenberg et al., 1988; Camus et al., 1997, Peters, 1997; Smith, 1997; Miles et al., 1998). Although these reports are isolated and less frequently published than clinical studies of surgical and ICU patients, the observations in athletes are similar to those described in patients with nosocomial infections. Identification of the 172 mediator or mechanism of inhibition would suggest appropriate therapies to prevent nosocomial and endotoxema-related respiratory infections. Understanding the mechanism of LPS-induced modulation of PMN migratory responses may provide the framework for the development of anti- inflammatory agents. Compounds which mimic the anti-migratory effect of LPS on PMN migration, but are without its inflammagenic properties, would provide an effective and specific form of anti-inflammatory therapy. In rats, endotoxemia-associated inhibition of PMN migration is not specific for the pulmonary stimuli. PMN accumulation into the peritoneum after i.p. administration of glycogen is also inhibited in endotoxemic rats (unpublished observation). Thus, an anti-inflammatory compound based on LPS's anti- migratory function might be effective in all extravascular tissue where PMN accumulation is undesirable. The altered behavior of PMNs during mild endotoxemia might also have implications in diverse disease states such as cancer, atherosclerosis, and AIDS. Metastatic cells must undergo similar adhesive and migratory processes as PMNs, but the adhesion molecules and chemokines involved in metastasis are not fully characterized. For example, some tumors produce and secrete the PMN chemokine IL-8 (Aihara et al., 1997). As mentioned earlier, intravascular lL-8 inhibits PMN migration to extravascular sites (Hechtman et al., 1991). Furthermore, injection of LPS into tumor- bearing mice enhances metastasis to lung tissue and adherence to cultured endothelium by unknown mechanisms (Vidal-Vanaclocha et al., 1997; Anasagasti et a., 1997). The killing ability of PMNs is important for removing metastatic cells from humans and experimental animals. Taken together, the inhibition of PMN migration might be a mechanism by which lL—8-secreting tumors and experimental endotoxemia enhance metastasis. It is curious that 173 endotoxemia inhibits PMN migration to the lung but enhances metastasis of tumor cells. These results suggest that tumor cells do not use a CD18- dependent pathway for migration. In addition to potentially inhibiting the ability of PMNs to clear metastatic cells, LPS might be acting to promote adhesive pathways favorable for metastasis. Reports of the co-existence of bacterial infection and tumors are becoming more common (Aihara et al., 1997; Yokota et al., 1997). That LPS is promoting metastasis by both the downregulation of PMN function and upregulation of metastatic cell adhesion pathways requires further research. Last year investigators identified the receptor on macrophages and lymphocytes which recognizes the AIDS virus as a B-type chemokine receptor which also binds to MlP-1 and other monocyte chemokines (reviewed in Dimitrov, 1997). The receptor is required for recognition and infection of these cells by the virus, and some chemokines can compete with the virus for receptor binding (Howard et al., 1998). It is possible that chemokine receptors on PMNs have similar interactions with the HIV virus. Some features of modified PMN function in HIV+ patients are reduced expression of lL-8 receptors and alterations of PMN responses to LPS (Cassone et al., 1997; Gasperini et al., 1998; Meadows-Taylor et al., 1998). The relationship between AIDS and bacterial infections is in humans and animal models complex, but the ability of LPS to alter the responses of PMN and mononuclear cells might play a role in the development of immune- deficiency diseases such as AIDS. Further work in vitro would better describe the relationship between LPS and HIV effects on PMNs. Endothelial cell injury is a popular hypothesis to describe the initiation of atherosclerotic lesions (reviewed in Ross, 1986). Superoxide production from adhered PMNs has been documented in many animal models and 174 systems in vitro to cause cytotoxicity and increased permeability to endothelium (Westlin and Gimbrone, 1993; Bratt and Palmblad, 1997). The enhanced ability of PMNs from individuals with hypercholesterolemia or hyperlipidemia to upregulate CD18 and produce superoxide might contribute to endothelial cell injury and the development of atherosclerotic plaques (Arai et al., 1998). As described earlier, the PMN during endotoxemia become hyperadhesive and have enhanced ability for oxidant production. These two characteristics are consistent with the hypothesis that the endotoxemic PMN may initiate the development of atherosclerotic plaques by injuring endothelial cells (Briner and Luscher, 1994). Indeed, atherogenic lipids which exist in early atherosclerotic lesions render endothelial cells more susceptible to PMN-mediated injury (Sugiyama et al., 1994). That endotoxemia is a contributing factor for the occurrence of atherosclerosis requires further investigation. Taken together, the consequences of altered responses of PMNs during endotoxemia are likely to be more than dysregulated PMN migration into pulmonary airspaces. Endotoxemia-associated blockade of pulmonary PMN migration is an attractive model for nosocomial-, athletic-, and environmental-related respiratory infections. However the studies presented in this dissertation also provide interesting insights into the basic mechanisms of PMN behavior during disease and inflammation. Moreover, the prominent role for PMNs during serious, life threatening diseases - cancer, AIDS and atherosclerosis - and the relationship to endotoxemia can be appreciated. Future research may clarify the importance of altered PMN behavior to the development of these and other diseases. 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