{MSU LIBRARIES —_—- RETURNING MATERIALS: PIace in book drop to remove this checkout from your record. FINES wiII be charged if book is returned after the date stamped beIow. ______._—-——-—-.. A MORPHOMETRIC AND FUNCTIONAL STUDY OF THE EARLY EVENTS OF HUMAN NEUTROPHIL ACTIVATION By Barbara Gahan Heerdt A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Anatomy 1986 ABSTRACT A MORPHOMETRIC AND FUNCTIONAL STUDY OF THE EARLY EVENTS OF HUMAN NEUTROPHIL ACTIVATION By Barbara Gahan Heerdt The purpose of these studies was to investigate the early morphological and functional responses of human neutrophils to activation. Cells were exposed to the synthetic chemoattractant N-formylmethionyl leucyl phenylalanine (fMLP), the calcium ionophore- A23187 or a temperature transition from AC to 37C. Neutrophils were fixed and evaluated with light microscopy for shape change. Cell-free supernatants were harvested from activated cells and assayed for evidence of primary, secondary and tertiary extracellular granule release. Quantitation and characterization of intracellular granules and morphometric measurements were made on transmission electron micrographs of unactivated and activated cell profiles. In addition, ' light and electron microscopic evaluations were performed on fMLP and A23187 treated neonatal neutrophils. The results from these investigations suggest that 1) neutrophilic peroxidase negative granules may be heterogeneous in terms of composition, 2) peroxidase negative granules may undergo sequential or selected release, and 3) the release of these granules is coincident with the earliest visible indication of cellular shape change and may be associated with a transient, apparently regulated~ increase in exposed Cell surface membrane. The following sequence of events is proposed by these studies to Barbara Gahan Heerdt occur early in adult neutrophil activation: 1) Brief activation of neutrophils mediates cellular ruffling presumably associated with the selected release of a population of peroxidase negative granules containing large quantities of lactoferrin. 2) These granules merge with the plasma membrane resulting in granule-plasma membrane fusion thereby mediating an increase in exposed surface membrane. 3) An activation induced increase in cellular volume, seen in the ultrastructural morphometric determinations made in these studies as an increase in cross-sectional area, subsequently occurs. This volume increase provides a "compensation" for the increase in exposed surface membrane which, by the methods used in this study, appeared to be regulated and returned to basal levels. To my Mom, Dad and siblings who may not have always agreed Or understood, never gave up. ii ACINOHLEDGEHENTS I would like to thank CIBA-GEIGY Corporation for providing me with the opportunity to complete this research at their Ardsley facility. Additionally, I thank my committee members for agreeing to support my research off-campus. Thanks to Jim Wasvary for cheerfully and consistently providing me with isolated cells.‘ I am also grateful to Michael Kolotila and Bruce Seligmann for their helpful comments, criticisms and support. Special thanks to John Rediske for encouraging me to consider alternative explanations, and his unwavering friendship. I am especially indebted to Dick Patrick for his selfless support, encouragement and patience through the years. Without his gentle persuasion this experience would never have been begun much less have been completed.. And finally, I am most grateful to those who helped me enjoy a life beyond graduate.school. "If the study to which you apply yourself has a tendency to weaken your affections, and to destroy your taste for those simple pleasures in which no alloy can possibly mix, then that study is certainly unlawful, that is to say, not befitting the human mind." Mary Shelley, Frankstein. iii TIBLBIOF CONTENTS LIST OF TABLES LIST OF FIGURES SECTION : INTRODUCTION LITERATURE REVIEW I. Events of Neutrophil Activation A. Shape Change B. Enhanced Adherence C. Migration D. Extracellular Granule Release 1. Primary Granules 2. Secondary Granules a. Lysozyme b. Lactoferrin c. Collagenase 3. Tertiary Granules a. Gelatinase 4. Subpopulations of Neutrophilic Granules E. Plasma Membrane Modifications 1. Qualitative membrane changes: Up-regulation of membrane components . 2. Quantitative membrane changes: Increased plasma membrane expression II. fMLP as an Initiator of Neutrophil Activation III. The Calcium Ionophore A23187 as an _ Initiator of Neutrophil Activation IV. Temperature Transition as an Initiator of Neutrophil Activation A. Actin Polymerization B. Enhanced Adherence C. Extracellular Granule Release D. Plasma Membrane Medifications 1. Qualitative membrane changes: Up-regulation of membrane components V. Neonatal Neutrophils A. Shape Change B. Adherence C. Migration D. Extracellular Granule Release iv vii ix 31 32 34 35 35 35 35 36 38 38 39 39 40 E. Plasma Membrane Modifications 1. Quantitative: Up-regulation of membrane components MATERIALS AND METHODS Reagents Neutrophil Isolation: Adult Neutrophil Isolation: Neonatal Isolated Neutrophils: Purity and Viability Neutrophil Morphology: Light Microscopy Shape Change Determinations Adherence Chemotaxis Extracellular Granule Release Granule Component Assays Primary Granule: Beta-Glucuronidase Secondary Granule: Lactoferrin Secondary Granule: Lysozyme Secondary Granule: Collagenase Tertiary Granule: Gelatinase Electron Microscopy: Neutrophil Processing Ultrastructural Evaluation of Granule Release: Intracellular Granule Characterization and Quantitation Extracellular Granule Release/Intracellular Granule Loss Ultrastructural Evaluation of Morphology: Morphometric Determinations ° Presentation of Data RESULTS ' fMLP Mediated Shape Change: Dose Response: fMLP Mediated Shape Change: Kinetic Response: fMLP Mediated Adherence: fMLP Mediated Chemotaxis: fMLP Mediated Extracellular Granule Release: fMLP Mediated Extracellular Granule Release: Effect of Cytochalasin B: Early fMLR.Mediated Extracellular Granule Release: Early fMLP Mediated Extracellular Granule Release: Effect of Cytochalasin B: Ultrastructural Evaluation of Early fMLP Mediated Activation: Intracellular Granule Quantitation: fMLP Mediated Extracellular Granule Release/ Intracellular Granule Loss: Ultrastructural Evaluation of Early fMLP Mediated Activation: Morphometric Determinations: A23187 Mediated Shape Change: . A23187 Mediated Extracellular Granule Release: Effect of Cytochalasin B on A23187 Mediated Extracellular Granule Release: Ultrastructural Evaluation of A23187 Mediated Granule Release: Intracellular Granule Quantitation: A23187 Mediated Extracellular Granule Release/ Intracellular Granule Loss: Ultrastructural Evaluation of A23187 Mediated Shape Change: MorphOmetric'Determinations:- 4O 42 42 42 44 44 45 46 47 47 49 49 50 51 52 53 54 56 57 57 S8 59 59 59 63 63 63 64 68 7O 73 73 75 80 8O 85 85 89 89 'Neonatal Neutrophils: fMLP Mediated Shape Change: Neonatal Neutrophils: Ultrastructural Evaluation of Early fMLP Mediated Activation:Intracellular Granule Quantitation Neonatal Neutrophils: Ultrastructural Evaluations of Early fMLP Mediated Activation: Morphometric Determinations: Neonatal Neutrophils: A23187 Mediated Shape Change: Neonatal Neutrophils: Ultrastructural Evaluation of Treatment with A23187: Intracellular Granule Quantitation: Neonatal Neutrophils: Ultrastructural Evaluation of Treatment with A23187: Morphometric Determinations: Temperature Transition Mediated Shape Change: Temperature Transition Mediated Extracellular Granule Release: Temperature Transition Mediated Extracellular Granule Release: Effect of Neutrophil Isolation Technique: Temperature Mediated Extracellular Granule Release: Combined Results from Neutrophils Isolated in the Cold: Temperature Transition Mediated Lactoferrin Release: Effect of Various Chemicals: ' Temperature Transition Mediated Extracellular Granule Release: Effect of Cytochalasin B: The Effect of Various Chemicals on Temperature Transition Mediated Shape change and Cell Viability: Ultrastructural Evaluation of the Temperature Transition: Quantitation of Intracellular Granules: Ultrastructural Evaluation of the Temperature Transition: Morphometric Determinations: Summary of Results: DISCUSSION Evidence Suggesting-Heterogeneity of Peroxidase Negative Granules Differing in Composition and Release Dynamics Evidence Suggesting that Granule Release is Coincident with Shape Change and an Apparent Regulation of Membrane Exposure SPECULATIONS Early Lactoferrin Containing-Granule Release and Coincident .Up-Regulation of Surface Membrane Moieties Mechanism for Apparent Regulation of Increased Exposed Surface Membrane CONCLUSIONS APPENDIX A Neutrophil Isolation by Counterflow Centrifugal Elutriation APPENDIX B Merphometric Determinations Made on Hypothetical Neutrophils Utilizing Digitizing Tablet REFERENCES vi 94 : 94 97 101 101 101 103 107 113 117 122 124 124 126 126 128 137 138 148 157 157 161 167 170 172 179 LIST OF TABLES TABLE 1 SUBCELLULAR LOCALIZATION OF GRANULAR COMPONENTS 2 fMLP MEDIATED SHAPE CHANGE: DOSE RESPONSE 3 fMLP MEDIATED SHAPE CHANCE: KINETIC RESPONSE. a fMLP MEDIATED EXTRACELLULAR GRANULE RELEASE 5 THE EFFECT OF CYTOCHALASIN B ON fMLP MEDIATED EXTRACELLULAR GRANULE RELEASE 6 EARLY fMLP MEDIATED EXTRACELLULAR GRANULE RELEASE 7 THE EFFECT OF CYTOCHALASIN B ON EARLY fMLP MEDIATED EXTRACELLULAR GRANULE RELEASE 8 ULTRASTRUCTURAL GRANULE QUANTITATION OF EARLY fMLP MEDIATED ACTIVATION 9 fMLP MEDIATED EXTRACELLULAR GRANULE RELEASE/ INTRACELLULAR PEROXIDASE NEGATIVE GRANULE LOSS lO ULTRASTRUCTURAL ANALYSIS OF EARLY fMLP MEDIATED ACTIVATION: MORPHOMETRIC QUANTITATIONS 11 A23187 MEDIATED SHAPE CHANGE 12 A23187 MEDIATED EXTRACELLULAR GRANULE RELEASE 13 THE EFFECT OF CYTOCHALASIN B ON A23187 MEDIATED EXTRACELLULAR GRANULE RELEASE 14 ULTRASTRUCTURAL GRANULE QUANTITATION: A23187 TREATED NEUTROPHILS 15 A23187 MEDIATED EXTRACELLULAR GRANULE RELEASE/ INTRACELLULAR PEROXIDASE NEGATIVE GRANULE LOSS 16 ULTRASTRUCTURAL ANALYSIS OF A23187 TREATED NEUTROPHILS: MORPHOMETRIC QUANTITATIONS 17 NEONATAL NEUTROPHILS: KINETIC SHAPE CHANGE RESPONSE TO~1nM7fMLP vii Page 17 60 61 65 66 69 71 74 76 78 81 83 86 88 9O 92 95 18 19 20 21 22 23 24 25 26 27 28_ 29 30 31 32 33 B 1 B 2 NEONATAL NEUTROPHILS:‘ULTRASTRUCTURAL GRANULE QUANTITATION OF EARLY fMLP MEDIATED ACTIVATION NEONATAL NEUTROPHILS: ANALYSIS OF EARLY fMLP MEDIATED _ACTIVATION: MORPHOMETRIC QUANTITATIONS NEONATAL vs ADULT NEUTROPHILS: EARLY fMLP MEDIATED -ACTIVATION: MORPHOMETRIC QUANTITATIONS NEONATAL NEUTROPHILS: ULTRASTRUCTURAL GRANULE QUANTITATION OF A23187 TREATED CELLS NEONATAL NEUTROPHILS: ULTRASTRUCTURAL MORPHOMETRIC QUANTITATIONS OF A23187 TREATED CELLS NEONATAL vs ADULT NEUTROPHILS: EXPOSURE TO A23187: MORPHOMETRIC DETERMINATIONS TEMPERATURE TRANSITION MEDIATED SHAPE CHANGE TEMPERATURE GRADIENT ESTABLISHED DURING TEMPERATURE TRANSITION TEMPERATURE TRANSITION MEDIATED EXTRACELLULAR GRANULE RELEASE: NEUTROPHILS ISOLATED IN THE COLD BY ELUTRIATION TECHNIQUE TEMPERATURE TRANSITION MEDIATED EXTRACELLULAR GRANULE RELEASE: NEUTROPHILS ISOLATED IN THE COLD BY FICOLL/HYPAQUE TECHNIQUE TEMPERATURE TRANSITION MEDIATED EXTRACELLULAR GRANULE RELEASE: NEUTROPHILS ISOLATED AT ROOM TEMPERATURE TEMPERATURE MEDIATED EXTRACELLULAR GRANULE RELEASE: COMBINED RESULTS FROM NEUTROPHILS ISOLATED IN THE COLD BY ELUTRIATION OR FICOLL/HYPAQUE TECHNIQUES THE EFFECT OF VARIOUS CHEMICALS ON TEMPERATURE TRANSITION MEDIATED LACTOFERRIN RELEASE THE EFFECT OF CYTOCHALASIN B ON TEMPERATURE TRANSITION MEDIATED EXTRACELLULAR GRANULE RELEASE ULTRASTRUCTURAL GRANULE QUANTITATION: TEMPERATURE TRANSITION ULTRASTRUCTURAL MORPHOMETRIC QUANTITATIONS: TEMPERATURE TRANSITION HYPOTHETICAL CELL MORPHOMETRIC DETERMINATIONS: MEMBRANE REDISTRIBUTION HYPOTHETICAL CELL MORPHOMETRIC DETERMINATIONS: MEMBRANE ADDITION‘ . >-- ‘ viii 96 98 100 102 104 106 110 112 114 116 118 120 123 125 127 135 174 174 LISTOPFIGURQ FIEWHE 10 11 12 13 14 15 16 17 NEUTROPHIL SHAPE CHANGE: LIGHT MICROSCOPY NEUTROPHIL SHAPE CHANGE: TEM fMLP MEDIATED SHAPE CHANGE: KINETIC RESPONSE EFFECT OF CB ON fMLP MEDIATED EXTRACELLULAR GRANULE RELEASE EFFECT ON CB 0N EARLY GRANULE RELEASE ULTRASTRUCTURAL EVALU ACTIVATION A23187 MEDIATED SHAPE A23187 MEDIATED EXTRA KINETIC RESPONSE EFFECT OF CB ON A2318 GRANULE RELEASE ULTRASTRUCTURAL EVALUATION OF A23187 TREATED NEUTROPHILS NEONATAL NEUTROPHILS: KINETIC SHAPE CHANGE RESPONSE TO lnM fMLP ULTRASTRUCTURAL EVALU ACTIVATION ULTRASTRUCTURAL EVALUATION OF A23187 TREATED NEUTROPHILS fMLP MEDIATED EXTRACELLULAR ATION OF EARLY fMLP MEDIATED CHANGE CELLULAR GRANULE RELEASE: 7 MEDIATED EXTRACELLULAR ATION 0F EARLY fMLP MEDIATED NEUTROPHILS FIXED IN THE COLD: LIGHT MICROSCOPY TEMPERATURE TRANSITIO EFFECT OF ISOLATION TEMPERATURE ON TEMPERATURE MEDIATED N MEDIATED SHAPE CHANGE EXTRACELLULAR GRANULE RELEASE TEMPERATURE TRANSITIO N MEDIATED EXTRACELLULAR GRANULE ix Page 8, 10, 12 62 67 72 79 82 84 87 93 95 99 105 108 111 119 121 18 19 20 B 1 B 2 ULTRASTRUCTURAL EVALUATION OF TEMPERATURE TRANSITION NEUTROPHILS FIXED IN THE COLD: TEM PROPOSED EARLY EVENTS OF NEUTROPHIL ACTIVATION HYPOTHETICAL CELL: MEMBRANE REDISTRIBUTION HYPOTHETICAL CELL: MEMBRANE ADDITION 136 130, 132 169 175 176 - INTRODUCTION By migrating to a site of injury and clearing foreign material and tissue debris, activated neutrophils represent one of the major defensive cell types of the human body. Substances generated from bacterial invasion or from damaged tissue can act as activating agents, or chemoattractants, which bind to select neutrophil membrane receptors initiating activation (Murphy, 1970). Ig_yi££g, activation can be dissected and studied by exposing isolated neutrophils to purified or Synthetic chemoattractants. The binding of the synthetic chemotactic tripeptide N-formylmethionyl leucyl phenylalanine (fMLP) to specific neutrophil membrane receptors has been found to induce morphological and functional cellular alterations which are defined as activation. Activation events have been shown to include Cellular shape changes (Smith et.a1., 1979; Zigmond, 1978), enhanced adherence to substrata (Hoover et.al., 1980; Smith et.al., 1979; Tonnesen et.al., 1984), chemotaxis (Zigmond, 1978) extracellular granule release (Brentwood and Henson, 1980; Dewald et.al., 1982; Hibbs et.a1., 1983; Petrequin et.a1., 1985; Wright and Gallin, 1979) and modifications in the quality, in terms of increased expression of membrane components, (Arnaout et.al., 1984; Berger et.al., 1984; Fearon and Collins, 1983; Fletcher-and Gallin, 1980; Lanier et.al}, 1985; Zigmond et.al., 1982) 2 and quantity-of exposed plasma membrane (Hoffstein et.al., 1982). Intracellularly, a consequence of chemoattractant binding is an increase in cytosolic free calcium through mobilization of intracellular calcium stores as well as increased calcium influx from the external environment (Naccache et.a1., 1985). The calcium ionophore A23187 has been reported to increase intracellular calcium Ilevels by both of these mechanisms while by-passing receptor ligation. Exposure of neutrophils to A23187 has been found to mediate cellular shape change (unpublished personal observation), enhanced adherence (Gallin et.al., 1978), extracellular granule release (Hoffstein and Weissmann, 1978) and alterations in the quality of exposed plasma membrane (Arnaout et.al., 1984; Todd et.a1., 1984). Several of the cellular alterations describing activation can be mediated in the absence of conventional activating agents or calcium ionophores. Temperature transitions have been reported to spontaneously induce actin polymerization (Howard and Oresajo, 1985), enhance adhesion (Charo et.al., 1985), mediate apparent extracellular granule release (Corcino et.a1., 1970; Dewald et.al., 1984; Goldstein et.a1., 1974; Wright and Gallin, 1979) and cause an increase in the expression of surface membrane components (Berger et.a1., 1984; Fearon and Collins, 1983; Springer et.al., 1986). The studies presented in this report investigated morphological and functional cellular responses occurring after a brief exposure of neutrophils to fMLP, following treatment with A23187 and in response to a temperature transition. LITERATURE REVIEH The purpose of this section is to provide foundational information concerning the morphological and functional events of neutrophil activation addressed in this study. The events of activation are discussed first. The synthetic chemotactic tripeptide fMLP and the calcium ionophore A23187 as initiators of activation are'examined in parts II and III, respectively. Part IV discusses a temperature transition from 4C to 37C as an initiator of neutrophil responses. Because neutrophils isolated from umbilical cord blood are reported to exhibit select deficiencies in functional responses to activation, they were investigated in this study. Neonatal neutrophils are discussed in part V of this section. 4 I. Ebents of Neutrophil Activation A. Shape Change ‘ In most tissues, the polarity of individual cells is reflective of the overall architecture and function of the tissue. Neutrophils respond to chemoattractants generated in yiyg_(through bacterial invasion, cleavage of complement proteins or tissue degradation) by adhering to endothelial cells adjacent to the origin of the stimuli and migrating out of the microcirculation into specific extravascular sites of tissue destruction and infection (Murphy, 1970). While cells in the process of directional migration exhibit an apparent acquired functional and morphological polarity (Zigmond, 1978), polarity in neutrophils has been reported to be an intrinsic characteristic independent of both adhesion and directed migration (Smith et.a1., «1979). Viewed with phase contrast optics (Figure 1), nonactivated neutrophils in suspension are spherical. Within seconds after exposure to chemotactic factors the cells begin to exhibit shape change responses. The first visual indication of shape change is a ruffling of the cell membrane. Thin, sheet-like membrane protrusions, or pseudopods, are randomly extended over all regions of the cell surface giving the cells a ruffled appearance. With continued exposure to the chemoattractant the cells may lengthen to exhibit bipolar configurations displaying defined front and rear regions. Pseudopods are restricted to the front end of the cell, the other end is smooth, constricted and may have an knob-like structure or uropod. The mid-region is also smooth, without membrane extensions or ruffles ‘ _(Zigmond, 1978). _The fact that a bipolar Shape change response can Figure 1 NEUTROPHIL SHAPE CHANGE: LIGHT MICROSCOPY Upper panel: Unactivated isolated adult neutrophils. Middle panels: Activated neutrophils (fMLP); three ruffled cells and one bipolar cell. Note knob-like uropod on bipolar cell seen on lower portion of the middle panel. Lower panel: Bipolar configurations with uropods. Bar = 10um take place in the absence of a substratum or'a chemotactic gradient suggests that neutrophils have an intrinsic polarity which can be expressed when cells are activated in suspension (Smith et.al., 1979). Shape change responses are maintained while the cells are exposed .to the chemoattractant but are reversed by washing the cells free of the activator. Active glucose utilization is required for shape change but the effects of blocked glucose metabolism are also reversible. Shape change responses are reported to be cation (Ca++ and Mg++) independent but to require intact miCrofilaments (Heerdt et.al., 1984; Smith et.a1., 1979). Activation of neutrophils has been Shown to cause polymerization of actin, the major component of microfilaments (Rao and Varani, 1982). Detergent insoluble cytoskeletal preparations from unactivated cells are composed almost exclusively of cross-linked actin filaments, actin binding proteins and a small amount of myosin. Within seconds after chemoattractant receptor binding the quantity of polymerized actin increases, appearing to be localized to the developing pseudopods (Fechheimer and Zigmond, 1983; Howard and Oresajo, 1985). Activation induced actin polymerization is chemoattractant dose and time dependent, subsiding within approximately 60 seconds (Rao and Varani, 1982; White et.al., 1982). Ultrastructural changes accompany activated neutrophil shape change (Figure 2). Microfilaments in nonactivated spherical neutrophils are typically arranged in a uniform submembranous meshwork. Granules appear to be randomly distributed throughout the cytoplasm. The centriole is generally localized between the nuclear Figure 2 - NEUTROPHIL SHAPE CHANGE: TEM (Figure 2 is comprised of three pages) . 1. Examples of unactivated isolated adult neutrophils. Note the nuclear lobes in the center of cells, electron dense peroxidase positive granules and more electron lucent peroxidase negative granules dispersed throughout the cytoplasm. Bars a lum 10 Figure 2 (continued) 2. Examples of fMLP ruffled adult neutrophils. Note nuclear lobes in central region of the cells, agranular zone of pseudopod, peroxidase positive and negative granules dispersed around the nuclear lobes. Bars - lum 12 Figure 2 (continued) 3. Examples of A23187 ruffled adult neutrophils. Note nuclear lobes and centrioles in each cell section and peroxidase positive and negative granules. Also notice that the membrane extensions and agranular zones exhibited on A23187 ruffled cells are finer and not as expansive as those mediated by fMLP. Bars 3 lum 14 . lobes and from here microtubules radiate toward, but rarely make contact with the cytoplasmic side of the plasma membrane. As cells begin to change shape the newly formed pseudopods appear as agranular zones containing a fine meshwork of filamentOUs material. In the activated neutrophil, as in the spherical cell, the centriole remains within the central region of cell, between the nuclear lobes. Microtubules radiate toward but do not contact the plasma membrane (Anderson et.a1., 1982; Oliver and Berlin, 1982). As neutrophils become bipolar, the agranular-zone is localized to the front of the cell, the nucleus to the mid-region with the granules dispersed generally around the lobes. Neither the nuclear lobes nor the granules extend into the uropod. Using anti-tubulin antibody and indirect immunofluorescent techniques, Anderson and coworkers (1982) demonstrated that microtubules lengthen along the axis of cellular polarity and shorten perpendicular to polar axis. The mean number of microtubules in chemotactically activated neutrophils has been reported to be significantly greater than the number seen in the nonactivated cells, suggesting microtubule involvement in activated shape change. Microtubule assembly appears to be transient. Several minutes after activation the number of microtubules returns to baseline levels (Goldstein et.a1., 1973). The uropod of bipolar cells is organelle-free, composed of microfilaments and frequently a core of intermediate filaments (Oliver and Berlin, 1982). B. thanced Adherence Neutrophil adherence to artificial substrata or to cultured endothelial cells is glucose, temperature and extracellular Ca++ dependent but is independent of polymerization of microfilaments 1.5 (Charo et.al., 1985; Smith et.al., 1979). Chemotactic factors have been reported to enhance adhesion not only by increasing the number of cells adhering to a surface (Hoover et.al., 1980; Smith et.al., 1979; Tonnesen et.al., 1984) but also through increasing the strength of cellular adhesion (Charo et.a1., 1985). Smith and coworkers (1979) reported that, in contrast to shape change responses, chemoattractant mediated enhanced adhesion is not reversible. The augmented adhesion induced by exposing neutrophils to fMLP was not removed by washing the cells free of the chemoattractant. In addition, although neutrophils exposed to 2—deoxyglucose (2-DOG) demonstrated a reversible inhibition of shape change responses, normal adhesion functions were not restored when the 2-DOG was removed (Heerdt et.al., 1984), A degree of neutrophil adhesion is a prerequisite for cell motility (Charo et.al., 1986). Cells in the process of adhering appear to first flatten against the substratum and then round up and extend pseudopods oriented in the direction Of eventual migration (Zigmond and Sullivan, 1979). Adhesion sites exhibit polarity forming at the front of the cell and being transported to the tail as translocation occurs. As the cell migrates new adhesion sites are formed at the pseUdopod region and again transported to the rear of the cell (Smith and Hollers, 1981). Ultrastructurally, localized cytoskeletal-adherent membrane associations appear to be generated upon adhesion. In an elegant investigation of the cytOplasmic side of neutrOphil plasma membranes during adherence, Boyles and Bainton (1979) demonstrated that during neutrophil adhesion complexes of dense globular centers with radiating networks of thin branched filaments are formed. These regions were 16 located exclusively on the adherent membranes, never being found on the ventral side of cells. Pseudopods extending out from cells, in the process of forming new adhesion sites, were generally free of these regions. The extent of formation of central regions and the associated filaments was determined to be temporally related to cell adhesion. As Cells became more adherent the branched filaments spread to cover the entire cytoplasmic side of the adherent membrane. The nature of these regions is unknown but the evidence strongly suggests that they are intimately involved with the adhesion of cell activation. C. Migration Neutrophils undergoing directed migration, or chemotaxis, demonstrate morphological and functional polarity based on the concentration gradient of the chemoattractant (Smith and Hollers, 1981; Sullivan et.a1., 1981; Zigmond, 1978). In a chemotactic gradient the majority of cells are oriented in the same direction _ suggesting an acquired polarity.. PSeudopods are directed toward the higher concentration leading the way as the cells migrate up the gradient. If the gradient is reversed so that the higher dose of chemotactic factor is toward the uropods, the cells reorient along the gradient, pseudopods again toward the higher concentration (Zigmond, 1977). Conditions simulating in gigg_chemotaxis have been developed in. 1i££g_by a number of methodologies including migration of neutrophils toward a chemoattractant on plastic (Zigmond, 1977), under agarose, through porous filters (Gallin and Quie, 1978), through ‘cultured endothelial cells (Beesley et.al., 1979) and through 17 endothelial cells grown on porous filters (Taylor et.a1., 1981). Manipulations of these ighyitgg chemotaxis conditions have indicated that maximum neutrophil chemotactic responses require extracellular Ca++, glucose, and polymerized microfilaments and microtubules (Caner, 1965; Wilkson, 1974; Zigmond, 1978). D. Extracellular Granule Release Mature human neutrophils contain an assortment of cytoplasmic granules which have been classified by morphology, cytochemical techniques and enzyme content into at least three discrete types designated as primary (or azurophilic), secondary (or specific) and tertiary (sometimes referred to as C-particles). The granules appear at different stages during granulocyte maturation, contain different enzymes (Baggiolini and Dewald, 1985; Bainton and Farquhar, 1968; Scott and Horn, 1979) and respond differently in terms of release dynamics (Brentwood and Henson, 1980; Dewald et.al., 1982; Hibbs et.al., 1983; Lew et.a1., 1986; Petrequin et.al., 1985; Wright and Gallin, 1979).. A summary of granule components is shown is Table 1. l. Primary_Grann1ea: The primary granules are the first type detectable in maturing granulocytes, appearing in the promyelocyte stage. Mature primary granules contain neutral proteinases (elastase and cathepsin G), acid hydrolases (N-acety1-B-g1ucosaminidase, cathepsins B and D, B—glucuronidase, B—glycerophosphatase and a-mannosidase) and microbicidal enzymes (myeloperoxidase and lysozyme) (Baggiolini and Dewald, 1985; Bainton and Farquhar, 1968). Recently, three small structurally related peptides, referred to as defensins, were localized in the primary granules of human neutrophils (Ganz et.al., l7a mmmmh< nasm :ououm mmmcfimoccmaum ommumnqmonqouousawum mmmwficoususawlm a :qumnumu m :fimqmnumo mmmcficfiammoonawlmlamumumnz uoum>auu< cmmocwammam mmmcfiuoaou mmaacmuc mumwuuoe camuoun menses; Nam enamon> :Huumwouomq mmmvfimoccmalm ommumnmmonmouoomawlm ommvflcouausawlm a enmsmnumo m :fimamnumo ommcficHammousawlmlazuoomlz u :flmaonumo mmmcmwmaaoo ommummam mcfimcomma mahuommq mamuommq ommuaxoumaoammz mmazcmnu humvcoomm mafiacmuu mumafium Lasso mommaoucmm vflo< mommcfimuopm Hmuusmz Hudsonunafiua< uneconaoo mo mqu mezmzomzoo Much model, Boxer and coworkers (1982a) visualized increased :utrophil adherence to endothelial cells following the infusion of lctoferrin. Enhanced adherence was found to coincide with systemic autropenia. By promoting adhesion to endothelial cells and at the Ame time enhancing toxic hydroxyl radical generation, lactoferrin has zen implicated in innziggg endothelial cell damage and in 2212 lema formation (Vircellotti et.a1., 1985). Despite the possible deleterious affects on endothelium, actoferrin appears to plays an important role in the body's defense gainst disease. Several patients suffering from recurrent bacterial lfections have been found to lack lactoferrin. Neutrophils isolated rom these patients showed a variety of defects in cell adherence and rdroxyl radical production. Ultrastructurally, these neutrophils are found to be deficient in secondary (peroxidase negative) granules 1d morphometrically they appeared chemotactically activated and >nresponsive to further activation (Boxer et.al., 1982b; - 'enton-GOrius et.al., 1980; Strauss et.a1., 1974). Collagenase: The neutral protease collagenase is also a constituent of the 21' secondary granules of neutrophils (Murphy et.al., 1977). Intracellularly this metallo-enzyme occurs in a latent form, probably as an inhibitor-enzyme complex. Weiss and coworkers (1985) have reported that approximately 50% of the collagenase secreted from activated human neutrophils is in an active, rather than a latent form. These investigators determined that cells auto-activated the enzyme through the production of hypochlorous acid (HOCl). In the presence of hydrogen peroxide (generated from respiratory burst activity) myeloperoxidase (contained in primary granules) catalyzes the peroxidation of chloride to HOCl. HOCl in turn was found to activate latent collagenase. The remaining latent enzyme was found to be activateable with organomercurials. Activation of latent collagenase with mercurials results in a decrease in molecular weight thought to be caused by removal of the inhibitor from the enzyme-inhibitor complex (Murphy et.al., 1980). . Activated collagenase specifically cleaves native type I collagen into characteristic fragments 3/4 and 1/4 the size of the original collagen molecule (Cawston and Barrett, 1979). Collagenase activity is dependent upon divalent cations, being inhibited with EDTA or 1,10—phenanthrolein (Murphy et.a1., 1980). Cathepsin G and elastase, serine proteases found in primary neutrophilic granules (Baggiolini and Dewald, 1984; Tanaka et.al. 1985), have been reported to destroy collagenase activity (MUrphy et.al., 1980). 3. Tertiary Granules: Fractionation of human neutrophil homogenates by zonal differential sedimentation has revealed the presence of a third j—population of small particles referred to as C—particles or tertiary 22 granules. Although morphologically still unidentified in whole cells, the fractionation technique indicated that tertiary granules are considerably smaller than either primary or secondary granules (Dewald et.al., 1982). Recent evidence suggests that a proton pump ATPase, responsible in part for the acidification of phagolysosomes following neutrophil ingestion of foreign materials, is associated with tertiary. granules (Mollinedo and Schneider, 1984; Mollinedo et.al., 1986). These granules also contain a number of acid hydrolases also found in peroxidase positive granules including cathepsin B and D, beta-glucuronidase, beta-glycerophosphatase, alpha-mannosidase and N—acetyl-beta-glucosiminidase (Baggiollini and Dewald, 1985). Additionally, the metallo-proteinase gelatinase has been reported to be localized exclusively in neutrophilic tertiary granules (Sopata and Dancewicz, 1974). Monocytes contain little if any gelatinase (Dewald et.a1., 1982), and cultured macrophages do not exhibit gelatinase activity (Vartio, 1985). a. Gelatinase: Similar to collagenase, gelatinase is a metallo-proteinase, occurring as a latent enzyme-inhibitor complex. Like collagenase, a portion of the latent enzyme is thought to be auto-activated by neutrophils through the production of HOCl (Peppin and Weiss, 1986). Gelatinase activation with organomercurials is associated with a decrease in molecular weight, suggesting that enzyme-inhibitor complex disruption accompanies enzyme activation. The latent form of I gelatinase is thought to be inactivated by elastase in such a way that it cannot subsequently be activated. Unlike collagenase, the serine I proteinase cathepsin G has been found to activate, rather than prevent 23 lthe activation, of latent gelatinase. Gelatinase activity is inhibited by EDTA or 1,10-phenanthrolein but is not affected by serine proteinase inhibitors (Dewald et.al., 1982; Hibbs et.al., 1985; Murphy et.al., 1980; Sopata, 1982). Once activated gelatinase acts synergistically with collagenase to degrade type I collagen. Collagenase, as mentioned, cleaves collagen at a single site producing 3/4 and 1/4 collagen fragments. These fragments spontaneously denature at body temperature making them accessible to further degradation by gelatinase. Although gelatinase does not directly cleave native type I collagen it is capable of direct degradation of type V collagen (Hibbs et.al., 1985; Murphy et.a1., 1980). Gelatinase secretion from human neutrophils appears to take place prior to secondary or primary granule release. Dose response determinations of extracellular granule release mediated by several different activating agents indicated that gelatinase was released following exposure of cell to low doses of the activators. Secondary and primary granule release were not detected until higher concentrations of activators were used (Dewald et.a1., 1982; Hibbs et.a1., 1983; Petrequin et.al., 1985). Hibbs and associates (1985) have recently reported that secreted gelatinase consists of three molecular weight species. All three forms were found to be immunologically identical and proteolytically active_against gelatin. ‘4. Subpopulations of Neutrophilic Granules There is evidence indicating that subpopulation of the three types of cytoplasmic granules just discussed may exist. Peroxidase 24 positive-primary granules in human neutrophils can be subdivided into populations differing in morphology (Pryzwansky and Brenton-Gorius, 1985), enzyme composition and extracelldlar release dynamics (Kinkade et.a1.,1983; Pember and Kinkade,1983). Ultrastructural studies of DAB treated neutrophils revealed three morphologic forms of peroxidase positive granules; large spherical granules with strong homogeneous peroxidase activity, somewhat elongated granules with weaker heterogeneous peroxidase activity and small peroxidase reactive granules appearing in clusters or chains. By high voltage electron microscopy theSe small granules appeared to be interconnected. (Pryzwansky and Brenton-Gorius, 1985). Subpopulations of primary granules differing in enzyme content have been identified using centrifugation techniques. Continuous sucrose density centrifugation of neutrophilic primary granule fractions has been found to yield two populations of granules with high and low densities (Kinkade et.al., 1983; Pember and Kinkade,_ 1983). Three distinct forms of myeloperoxidase (MPO), reported to differ in enzymatic activity, extractability (Pember et.a1., 1982), amino acid composition (Morita et.al., 1986) and localization have been isolated from the granule subpopulations obtained from a single neutrophil donor. MPO forms II and III were confined to the low density granules while the high density granules Contained all three forms of MPO (Xinkade et.al., 1983; Pember and Kinkade, 1983). Studies of extracellular release of the different forms of MPO suggest that selective secretion of primary granule subpopulations occurs. MPO II and III, contained within low density granules, were found to be secreted by human neutrophils activated with fMLP in the 25 presence of cytochalasin B, while extracellular MP0 I, localized in the high density subpopulation, was not detected (Xinkade et.al., 1983; Pember and Kinkade, 1983). Similar to MP0, the neutral proteinase elastase is a primary granule constituent found to exist in different forms. Garcia et.al. (1985) have identified at least three isoenzymes of elastase, differing in carbohydrate content and extractability. Unlike MPO, the distribution of the different forms of elastase appeared to be equivalent in both high and low density primary granule subpopulations, and the isoenzymes were not selectively secreted in response to neutrophil activation with fMLP plus cytochalasin B. Distinct subpopulations of peroxidase negative granules have been identified in neutrophils isolated from ruminant blood (Baggiolini et.al., 1985; Genarro et.al., 1983). During maturation of these cells three types of granules were identified. As in human neutrophils, peroxidase positive granules formed first (Bainton and Farquhar, 1968). These were followed by an intermediate type of peroxidase negative granule and finally classical peroxidase negative secondary granules were formed. The intermediate granules were reported to be larger than the later peroxidase negative granules, had more acutely defined membranes and contained lactoferrin exclusively. Lysosomal hydrolases, peroxidase and serine- or metallo-proteinases were not detected in the intermediate granules. Similar to secondary granules, these intermediate granules were secreted in response to soluble A stimuli (A23187) independently of primary granule release (Gennaro et.al. , 1983) . There are reports_indicating that distinct forms of secondary 26 granule lactoferrin may be divided into select subpopulations of cells. Broxmeyer and coworkers (1980) reported that a specific form of human neutrophil lactoferrin inhibited the Granulocyte-Macrophage Colony Stimulating Activities (GM-GSA) from endogenous bOne marrow derived GM—CSA producing cells. Further, these investigators found that the type of lactoferrin exhibiting these inhibitory properties was contained in a specific population of circulating cells. Isolated neutrophils were separated into subpopulations based on the capacity of the cells to bind antibody coated erythrocytes (EA). NeutrOphils binding EA were referred to as Fc receptor positive (FcR+) while those not binding EA were FcR-. Compared to FcR- cells, FcR+ neutrophils demonstrated superior adhesiveness, aggregation, chemotaxis and phagocytic capacity of opsonized particles. The difference in FcR expression was not due to cell maturity as aging did not mediate a conversion from one type to the other. In addition, conversion could not be achieved with neutrophil activation (Klemnper and Gallin, 1978). Quantitatively, both FcR+ and FcR- populations contained equivalent amounts of lactoferrin, approximately 3.4 ug/million cells. However, qualitatively the lactoferrin from the subpopulations of -neutrophils differed in function. FcR+ cells were found to contain an active form of lactoferrin which bound to human monocytes and inhibited the GM-CSA. FcR- neutrophils, on the other hand, contained an inactive type of lactoferrin which demonstrated negligible binding to monocytes and did not affect the GM—CSA. Additionally, lactoferrin extracted from FcR- cells interfered with the binding of active lactoferrin to monocytes and consequently blocked the inhibitory _effect ofchR+-lactoferrin on GM-CSA.. 27 Although neutrophils generally function as defensive cells, essential for maintaining health, they Can be involved in the exacerbation of disease states. As mentioned, lactoferrin has been implicated in endothelial cell damage and subsequent edema formation (Vircellotti et.al., 1985). Neutrophils migrating into sites of inflammation may also contribute to tissue destruction by releasing their proteolytic enzymes (Muirden and Leyden, 1984). Gelatinase and collagenase have the capacity to alter extracellular matrix materials. Collagenase is thought to be critical in initiating the extracellular collagen degradation seen in rheumatoid arthritis (Spalding et.al., 1986), and has, in fact, been reported to play a key role in the development of inflammatory disease states affecting every organ system in the body (Weiss et.al., 1985). Acting in concert with collagenase to further degrade the extracellular matrix, gelatinase 'may contribute to tissue destruction. Laskin and coworkers (1986) have recently demonstrated that collagen degradation products have the potential of being as chemotactically active for human neutrophils as fMLP. Therefore, infiltrating neutrophils not only have the capacity to degrade collagen matrixes, but the act of degradation has the potential of recruiting more neutrophils to the site. Igngiggg, limited extracellular granule release has been implicated in increased chemotatic peptide binding (Fletcher and Gallin, 1980; Gallin et.al., 1978) and enhanced neutrophil adhesion and migration (Gallin et.al. 1978; Wright and Gallin, 1979). While extracellular Ca++ is not essential for limited granule secretion, 28 secretion does require the mobilization Of intracellular calcium _ stores (Smolen et.al., 1981). Release is inhibited by the presence of 2-DOG but these affects are reversible '(Heerdt et.al., 1984; Smolen et.a1., 1980). Granule release is not modulated by protein synthesis inhibitors (Gallin et.al., 1978). Disruption of microfilament formation with the fungal metabolite cytochalasin B has been shown to enhance activated extracellular granule release (Henson and Oades, 1973; Skosey et.al., 1973; Zurier et.al., 1973). E. Plasma Membrane‘Mbdifications- 1. Qualitative membrane changes: Up-regulation of membrane components A correlation between up-regulation of chemotactic peptide receptors and secondary granule release has been reported, suggesting that these granules may act as intracellular pools for the receptors (Fletcher and Gallin, 1980). Recent reports indicate that neutrophil activation induces up-regulation of other surface moieties as well, possibly also through the mobilization of intracellular pools (Fearon and Collins, 1983; Thrall et.al., 1980; Zigmond et.al., 1982). Activated cells were found to exhibit increased membrane expression of the related glycoproteins (GPs), Mo 1, also known as OKM 1 and Mac—1 (Berger et.al., 1984) and p150,95 (Springer et.al., 1986). These GPs represent a major portion of the components found on the surface of human neutrophils. Structurally the GPs are double chain noncovalently associated alpha-1 beta-1 complexes (Sanchez-Madrid et.a1., 1983). A 95 kilodalton beta chain is common to both while the alpha chain varies. A third structurally related GP, the lymphocyte function associated antigen—1 (LFA-l), shares the common beta chain 29 while expressing a unique alpha chain. Similar to M0 1 and p150,95, LFA—l has been identified on leukocyte membranes (Gallin, 1985; Sanchez-Madrid et.al., 1983).' However, while the LFA-l eXpressed on the surface of monocytes has been reported to increase when these cells are activated (Mentzer et.al., 1986), LFA-l is not up—regulated on neutrophils in response to activation (Lanier et.al., 1985). Functionally the Mo 1, p150,95 and LFA-l glycoproteins are thought to be involved in some form cellular adhesiveness (Anderson et.al., 1985; Anderson et.al., 1986). The alpha chain of Mo 1 is approximately 160KD in size and contains the antigenic epitopes. Monoclonal antibodies directed against specific portions of the alpha chain have been found to block the adherence of iC3b coated particles to granulocytes, suggesting that the Mo 1 antigen is closely related to, if not identical with, the iCBb receptor (CR3) found on granulocyte membranes (Anderson et.al., 1986; Arnaout et.al., 1983; Arnaout et.al., 1984; Dana et.a1., 1984; Sanchez-Madrid et.al., 1983). This GP has also been associated with cellular-substratum adhesiOn and subsequent chemotaxis (Anderson et.al., 1986). In addition to being found on granulocytes, Mo 1 has been reported on monocyte and natural killer cell membranes (Eddy et.al., 1984; Gallin, 1985; Sanchez-Madrid et.al., 1983). The p150,95 GP consists of the common 95KD beta chain and a 150KD alpha chain. p150,95 has been identified on neutrophil and monocyte membranes (Gallin, 1985; Sanchez-Madrid et.al., 1983). Functionally this GP is thought to be involved in adhesion activities of these cells (Anderson et.al., 1986; Harlan et.al., 1985; Lanier et.al., .1985; Springer et.al., 1986). _Wright et.al. (1985) presented 30 preliminary evidence suggesting that p150,95 may be the CR2 receptor expressed on the surface of phagocytic cells. CR2 is reported to bind iC3b, C3dg and C3d cleavage fragments of C3 (Arnaout and Colten, 1984) and act as the receptor for the Epstein-Barr virus (Frade et.al., 1985). LFA—l, identified on the surfaces of lymphocytes, thymocytes and activated macrophages as well granulocytes (Gallin, 1985; Sanchez-Madrid et.al., 1983), has been reported to participate in the adhesion between target cells and cytotoxic T lymphocytes or natural killer cells (Gallin, 1985), homotypic adherence of B lymphocytes (Mentzer et.al., 1985) and monocytes (Mentzer et.al., 1986), T-cell dependent antibody production (Fischer et.a1., 1986; Howard et.a1., 1986) and the interactions between activated macrophages and tumor cells (Strassman et.al., 1986). The characteristic alpha chain of LFA-l is approximately 170KD in size (Gallin, 1985). Although intracellular localization of the GP subunits has not been demonstrated ultrastructurally, Arnaout et.al. (1984) and Todd et.al. (1984) have implicated secondary granule release with increased surface expression of the Mo 1 antigen. Examination of whole cells and subcellular fractions using electrophoretic and immunological techniques indicated that Mo 1 was associated predominately with secondary granule membranes. These investigators proposed that during activated secondary granule release Mo 1 was transported to the plasma membrane thrOugh granule-plasma membrane merger. O'Shea and coworkers (1983) also presented evidence suggesting that secondary granules may act as the intracellular pool for CR3 by reporting that a patient with Isecondary granule_deficiency was unable to increase cell surface 31 eXpression of the CR3 receptor in response to neutrophil activation. Additional evidence indicating that secondary granules may represent the intracellular source of the Mo 1 antigen was provided by the myloid cell lines KGl and THP-l. These cells do not contain secondary granules and do not express Mo 1 on their surfaces (Arnaout et.al., 1984). Petrequin and associates (1985) recently presented evidence suggesting that the Mo 1 GP may be more closely associated with tertiary than with secondary granules. Neutrophils activated with 1nM or 100nM fMLP for 5 minutes were reported to exhibit a 3 fold increase in cell surface Mo 1 expression and concomitant extracellular gelatinase release without significant release of secondary granule vitamin 312 binding protein or primary granule myeloperoxidase. 2. Quantitative membrane changes: Increased plasma membrane expression Evidence suggests that activated granule release may not only produce qualitative alterations in neutrophil plasma membrane, in terms of up—regulation of certain moieties, but may also contribute to a quantitative alteration in exposed surface membrane. Hoffstein and coworkers (1982) reported that new membrane was added to neutrophil surfaces following 10 seconds exposure of cells to 100nM fMLP. The rapidity of membrane addition suggested that an intracellular pool of preformed membrane was being mobilized to the cell surface. . Furthermore, quantitation of intracellular granules at an electron microscopic level suggested that the source of this pool was the secondary granules. 3.2 II. fMLP as an Initiator of Neutrophil Activation The tripeptide N-formylmethionyl leucyl phenylalanine (fMLP) was originally synthesized based on the finding that low molecular weight peptides from rapidly growing bacteria activated leukocytes (Keller and Sorkin, 1967; Schiffmann et.al., 1975a; Schiffmann et.a1., 1975b; Schiffmann et.al., 1978; Showell et.al., 1967). Competitive binding experiments using synthetic peptides and bacteria culture filtrates indicated that fMLP was structurally related to the naturally occurring bacterial substances, both activating cells through a common leukocyte receptor (Aswanikumar et.al., 1976; Aswanikumar et.a1., 1977; Schiffmann et.al., 1975a). Membrane receptors for fMLP have been identified on pulmonary macrophages (Spilberg et.al., 1981), monocytes (Weinberg et.a1., 1981) and,neutrophils (Aswanikumar et.al., 1977; Sha'afi et.al., 1978; Vitkauskas et.al., 1980; Williams et.al., 1977; Zigmond and Sullivan, 1979). The receptors on human neutrophils have been isolated and were found to be glycoproteins approximately 40,000 to 70,000 daltons in size (Goetzl et.al., 1981; Niedel et.al, 1980). As a population, isolated neutrophils are reported to exhibit heterogeneity in terms of fMLP binding characteristics and subsequent functional responses (Berkow and Baehner, 1985; Gallin, 1984; Klempner and Gallin, 1978; Seligmann et.al., 1981; Seligmann et.a1., 1984). Exposure of neutrophils to fMLP mediates a number of morphological and functional cellular responses indicative of activation. These responses include shape change (Smith et.al., 1979; Zigmond, 1978), enhanced adhesion to a substratum (Hoover et.al., _ 1980; Smith et.al.,.1979; Tonnesen et.al., 1984), migration (Zigmond, 33 1978) extracellular granule release (Brentwood and Henson, 1980; Dewald et.al., 1982; Wright and Gallin, 1979), modifications in the expression of surface membrane moieties (Arnaout et.al., 1984; Berger et.al., 1984; Fearon and Collins, 1983; Lanier et.al., I985; Zigmond et.al., 1982) and alterations in the quantity of exposed surface membrane (HOffstein et.al., 1982). 34 III. The Calcium Ionophore A23187 as an Initiator of Neutrophil Activation Ionophores are small hydrophobic mOlecules which dissolve into the lipid bilayer of cell membranes and transport specific ions down their concentration gradients into the cell cytosol (Alberts et.al., 1983). The ionophore calcimycin, or A23187, specifically transports divalent cations such as Ca++. Increased cytosolic free Ca++ concentration is one of the first measurable events to occur following neutrophil receptor-ligand binding. After a lag of approximately 3 seconds, cells treated with 100nM fMLP have been reported to rapidly take up extracellular calcium (Korchak et.al., 1984) as well as mobilize intracellular Ca++ stores (Naccache et.al., 1985; von Tschnarner et.al., 1986). Thus, the result of A23187 treatment of neutrophils is a receptor independently inddted increase in intracellular Ca++. In neutrophils, as in other cells, calcium is believed to act as a secondary messenger in activation. Neutrophils treated with A23187 have been found to change shape (unpublished personal observation), exhibit enhanced adhesion (Gallin et.al., 1978), extracellular granule secretion (Hoffstein and Weissmann, 1978) and up-regulation of the CR3 receptor (Arnaout et.al., 1984; Todd et.al., 1984). 35 IV. Thmperatnre Transition as an Initiator of Neutrophil Activation . Neutrophils exposed to an increase in temperature have been reported to exhibit responses somewhat analogous to the morphological and functional events of cellular activation. A. Actin Polymerization. As mentioned previously, neutrophil activation has been shown to induce polymerization of actin (Oliver and Berlin, 1982; Anderson et.al., 1982), and microfilament formation is required for cellular shape change (Smith et.al., 1979). Howard and Oresajo (1985) recently reported that temperature mediated actin polymerization takes place in human neutrophils. The relative F-actin content of cells incubated at various temperatures ranging from 4C to 37C was assessed by fluorescence activated cell sorter analysis of phallicidin stained cells. Compared to cells held at 4C, these investigators detected a 4 fold increase in F-actin content following 37C incubation. B. Enhanced Adherence Charo et.al. (1985) reported that a temperature increase from 4C to 37C induced enhanced neutrophil adhesion to albumin coated plastic or cultured endothelial cells. The response was determined to be temperature dependent, as cells exposed to either substrata and maintained at 4C did not demonstrate augmented adherence. C. Extracellular Granule Release Apparent temperature mediated extracellular granule release has been reported. Goldstein and associates (1974) and Wright and Gallin (1979) detected lysozyme in the cell-free supernatants of neutrophils . incubated at 37C.for 30 minutes, with no additional release seen after 36 60 minutes incubation. Release was determined to be dependent upon extracellular Ca++, plateauing at 1mM calcium chloride. Spontaneous temperature dependent vitamin 812 binding protein release has been reported by Corcino et.al. (1970). These investigators found significant amounts of extracellular vitamin 312 binding protein in supernatants from neutrophils incubated at 37C for 5 minutes. Vitamin BlZ binding protein was not detected in supernatants from cells incubated up to 5 hours at 4C. Dewald and coworkers (1982) also reported that vitamin B12 binding protein was released from cells_ after 5 minutes incubation at 37C, but only from neutrophils which were isolated from buffy coat cells held at 4C for 24 hours. Freshly isolated neutrophils did not spontaneously release vitamin 312 binding protein. Neutrophils isolated from buffy coats aged for 24 hours at 4C were also found to spontaneously release gelatinase when warmed at 37C. Dramatic, highly significant release occurred within 5 minutes incubation at 37C. As was determined for spontaneous vitamin B12 binding protein release, neutrophils isolated from freshly drawn blood did not demonstrate-spontaneous temperature dependent gelatinase release (Dewald et.al., 1982; Baggiloini and Dewald, 1984). I In contrast to activation induced extracellular granule secretion, cytochalasin B has been reported to have no affect on temperature mediated granule release (Skosey et.al., 1973; Zurier etual., 1973). D. Plasmm.MEmbraneIModifications 1. Qualitative membrane changes: Up—regulation of membrane components . ‘ Recent evidence indicates that qualitative alterations in 37 neutrophil membrane can occur without exposing the cells to conventional activating agents. Incubation of human neutrophils at '37C has been reported to produce spontaneous increases in surface expression of CR1, the C3b receptor, (Fearon and Collins, 1983) CR3 (Berger et.al., 1984; Fearon and Collins, 1983) and the p150,95 GP (Springer et.al., 1986). 38 V. Neonatal Neutrophils Human newborns are more susceptible to frequent and serious bacterial infections than adults. The evidence suggests that this susceptibility is, at least in part, due to depressed neutrophil functions. Depressed activity is not the result of an aberration in chemoattractant sensory mechanisms but is thought to be related to decreased responses of neonatal cells to activation (Anderson et.al, 1981; Anderson et.al., 1984; Ambruso et.al., 1984; Becker-Freeman et.al., 1984; Krause et.al., 1982). Recently, Xrause and coworkers (1986) reported that isolated neonatal human neutrophils represent a heterogeneous population of cells. These investigators identified a subpopulation of cells which, like adult cells, expressed quantitatively less of a specific surface membrane component and were less motile than the major population of cells. Compared to isolated adult cells, this subpopulation represented a significantly larger portion of isolated neonatal cells. Xrause et.al. (1986) further suggested that the depressed functional responses of neonatal neutrophils may be partially due to the relatively larger proportion of this subpopulation of cells. Depressed responses are transient, subsiding within several months after birth. Krause and coworkers (1982; 1986) reported that the activities of neutrophils isolated from neonates and from infants 3 days to 2 months of age were significantly diminished when compared to adult cells. However, between 2 and 4 months of age cell activities were markedly increased and continued to increase until approximately.17 years of age. . A-I Shape Change._ 39 Neonatal neutrophils were found to express relatively normal intrinsic polarity, but defective acquired polarity. Neutrophils isolated from umbilical cord blood and activated in suspension exhibited ruffled configurations within seconds. Although bipolar forms were seen with continued exposure to the chemoattractant, compared to adult shape change responses, fewer neonatal cells exhibited knob-like tail structures or uropods (Anderson et.al., 1981). On the other hand, neonatal neutrophils demonstrated a marked depression in the ability to orient, or express acquired functional polarity. Anderson et.al. (1984) reported that while more than 90% of adult cells demonstrated proper orientation in a chemotatic gradient by exhibiting pseudopods directed toward the higher concentration Of chemoattractant, only 70% of neonatal cells oriented properly. Additionally, when the gradient was reversed, those neonatal cells that did orient were unable to reorient. B. Adherence Neonatal neutrophils also exhibit abnormal adherence associated activities. While baseline adhesion of newborn neutrophils was reported to be comparable to adult levels, chemoattractant mediated enhanced adhesion was diminished (Anderson et.al., 1981; Becker-Freeman et.al., 1984). An inability of newborn cells to modulate adherence sites may be associated with depressed activation mediated adhesion. In suspension, neonatal neutrophils eXposed to chemotactic.factors did not demonstrate a normal capacity to transport latex bead-bound adhesion sites from pseudopods to the tail of. polarized cells (Anderson, et.al. 1981). C-.Hisrdt.1¢n.g 40 The abnormalities in newborn neutrophil orientation and adhesion are reflected in impaired motile responses. The motility of neonatal cells has been found to be significantly reduced in response fMLP, C5 cleavage products and low molecular weight chemoattractants isolated from §;“ggli_culture fluid (Anderson et.al. 1981; Krause et. al. 1986; Nunoi, et.al.1983). D. Extracellular Granule Release Neutrophils isolated from umbilical cord blood have been reported to contain myeloperoxidase and beta-glucuronidase and lysozyme in quantities comparable to those found in adult neutrophils. On the other hand, lactoferrin levels have been found to be significantly less (Ambruso et.al., 1984; Becker-Freeman et.al., 1984). The levels of lactoferrin in lysates prepared from neonatal cells are reported to be approximately SOZ'Of those found in adult lysates. Compared to adults cells, neonatal cells exposed to fMLP, A23187 or the tumor promoter PMA secreted equivalent amounts of myeloperoxidase and lysozyme but approximately 50% less lactoferrin (Becker-Freeman et.al., 1984). E. Plan Membrane Modifications l . Quantitative: Up—regulation of membrane components Compared to adult cells, neonatal neutrophils have been reported to exhibit depressed baseline levels of the adhesion associated GP Mo 1. Neonatal neutrophils demonstrated dose dependent up-regulation of the alpha and beta subunits following activation with fMLP or exposure to A23187 or PMA, however, the degree of expression was significantly diminished when compared to activated adult neutrophils (Beckeerreem n et.al., 1984). 41 As discussed above, Mo 1 and lactoferrin have been implicated in neutrophil adhesion and hydroxyl radical production. The low levels of Mo 1 expression and lactoferrin content in neonatal cells may contribute to the increased susceptibility and severity of bacterial infections seen in newborns (Becker-Freeman et.al., 1984). MWTERIALS AND METHODS Reagents The following reagents were routinely used throughout these studies. Unless otherwise indicated, complete PBS, phosphate buffered saline containing 1.0mM calcium chloride, 0.15mM magnesium chloride, pH 7.4 (Gibco) made 5mM glucose was used as reagent dilution buffer and neutrophil suspension buffer. Elutriation buffer was PBS, O.25mM EDTA. Stock solutions of 1mM fMLP, cytochalasin B at Smg/ml (Sigma) and 5mM A23187 (Calbiochem) were made in 100% dimethyl sulfoxide (DMSO) and stored at —35C until use. The highest concentration of DMSO present in reactions with neutrophils, 0.012 (v/v),_had no affect on the cells or the assay systems. Cells were fixed for light and electron microscopy with diluted electron microscopy grade glutaraldehyde (Ladd). Neutrophil Isolation: Adult For these studies, human neutrophils were isolated from whole blood obtained by venipuncture, with informed Consent, from male and female donors ranging from 21 to 68 years of age. Blood was anticoagulated with buffered sodium citrate and neutrophils were purified using one of three methods; cold counterflOw centrifugal elutriation, cold ficoll/hypaque discontinuous density gradient or ficoll/hypaque discontinuous density_gradient performed at room 42 43 temperature (see Appendix A). The counterflow centrifugal elutriation technique utilized a JElO-X elutriation rotor, J6M centrifuge (Beckman) and Masterflex Pump with 7016-20 head (Barnstead). Blood samples subjected to elutriation were mixed with Hespan (6.02 hetastarch in 0.92 sodium chloride, American Hospital Supply) to sediment erythrocytes. Leukocyte rich plasma was diluted with Ca++,Mg++-free PBS (Gibco) and pumped into the elutriation chamber at approximately 45ml/minute, centrifuging at 1420 rpm, 10C. After the sample had been loaded, cold elutriation buffer was pumped into the chamber at 45ml/minute for 10 minutes. The pump speed was then increased to approximately 55ml/minute for 10 minutes followed by 64ml/minute for 30 minutes. Samples from chamber effluent were taken at 2.5 minute intervals for Coulter Counter determinations of erythrocyte content. When erythrocyte counts fell below 40,000/ml, the pump was stopped and centrifuge speed decreased to 1000 rpm. After 4 minutes the centrifuge was stopped, the separation chamber removed and the isolated neutrophils were transferred to tubes pretreated with normal human serum.- Cells were adjusted to 10 million/ml in complete PBS and held on ice until use. For ficoll/hypaque isolation of neutrophils in the cold, citrated blood was diluted with an equal volume of Ca++,Mg++-free PBS at room temperature and carefully layered over one-half the volume of cold ficoll/hypaque solution (Histopaque type 1083, Sigma) in glass tubes. Tubes were centrifuged at 1000 rpm for 20 minutes at 4C resulting in the formation of three discrete regions. The upper plasma region and mid-ficoll/hypaque interface region, containing mononuclear cells, were aspirated and discarded. The erythrocyte-granulocyte pellets 44 were resuspended to twice their volume with cold Ca++,Mg++-free PBS, transferred to normal human serumscoated tubes containing Hespan, at one-tenth of the cell volume, and mixed. The cell isolation tubes were maintained at 4C for approximately 30 minutes to allow erythrocyte sedimentation. The upper granulocyte-containing layer was removed and washed twice by centrifugation and resuspension of cell pellet with cold Ca++,Mg++—free PBS. Any contaminating erythrocytes were removed by hypotonic lysis. One ml of ice cold sterile distilled water was added to the cell pellet while mixing for 20 seconds. An equal volume of cold 2X saline was then added to restore tonicity, followed by Ca++,Mg++-free PBS. Isolated neutrophils were washed once with Ca++,Mg++-free PBS followed by two washes with complete PBS. Cells were adjusted to 10 million/ml and held on ice until use. NeutrOphils isolated at room temperature were obtained from ficoll/hypaque gradients performed as above with the exception that all centrifugations and reagents were at room temperature. Isolated cells were maintained at room temperature until use. Neutrophil Isolation: Neonatal Neonatal neutrophils used in these studies were isolated from anticoagulated whole blood samples obtained from umbilical cords with informed parental consent. Neutrophils were isolated in the cold using the ficoll/hypaque gradient technique and held on ice until use. Isolated Neutrophils: Purity and Viability _ Isolated cells were approximately 95% neutrophils (determined by polychromatic dye stained smears) containing no more than 3% erythrocytes. Viability of the cells, determined by trypan blue exclusion, exceeded 972.. An aliquot of isolated cells was mixed with 45 an equal volume of 0.04% trypan blue (w/v) in PBS and held for approximately 5 minutes at room temperature. At least 200 cells were examined with light microscopy for excldsion of the dye. Neutrophils were used within two hours of isolation. Neutrophil Morphology: Light Microscopy Shape Change Determinations Determinations of neutrophil shape change responses were made with light microsCOpy on cells fixed in suspensions with an equal of volume 22 buffered glutaraldehyde (v/v diluted with Ca++,Mg++-free PBS). At least 200 fixed cells were examined with phase contrast or Normarski optics and classified as "spherical", "ruffled" or "bipolar" as described (Smith et.al., 1979). Round cells with a smooth contour were considered to be spherical. Neutrophils that were basically round in shape but exhibiting membrane protrusions, or pseudopOds, were classified as ruffled. Bipolar cells appeared ovoid or triangular in shape (Figure 1). After warming, approximately 90% of fixed isolated adult and neonatal neutrophils exhibited round configurations. Dose response fMLP mediated cell shape change was determined. Neutrophil suspensions were prewarmed for 5 minutes at 37C. Warmed fMLP was added, with mixing, to obtain final concentrations from 1pM to 100nM. Thirty-seven degree incubation conditions continued for an additional 5 minutes at which time the cells were fixed with glutaraldehyde. Kinetic shape change responses were determined on neutrophils activated with fMLP. Cell suspensions were warmed by incubation for five minutes at 37C. Prewarmed fMLP was then added, with mixing, to 46 achieve final concentrations of 1nM or 100nM. At 10—20 second intervals for up to 5 minutes aliquots of the cell-activator mixture were removed and immediately fixed with glutaraldehyde. Neutrophil shape change in response to exposure to the calcium ionophore A23187 was determined by prewarming the cells far 5 minutes at 37C, followed by the addition of warm A23187 to obtain final concentrations of either 100nM or 250nM. Samples of treated cells were fixed 30 seconds, 1,3,5,7 and 10 minutes after the addition of ionophore. Cells exposed to a temperature transition were isolated in the cold transferred from an ice bath to a 370 water bath. At time points from O to 60 minutes at 37C, sample aliquots were taken and cells were fixed. Results of shape change determinations are expressed as the percent of total cells evaluated exhibiting spherical, ruffled or bipolar configurations. Adherence The effect of fMLP on adherence of adult neutrophils to a serum coated substratum was evaluated as described (Smith et.al., 1979). Briefly, the adherence chambers consisted of two milled brass plates supporting two 25mm round cover glasses separated by a rubber gasket. One of the cover glasses was pretreated with 22 normal human serum (v/v in complete PBS) for 2 minutes and washed with complete PBS. The other cover glass was simply washed with PBS. Suspensions of 1 million neutrophils were exposed to 1nM or 100nM fMLP at room temperature and immediately loaded into the chambers. Cells were allowed to settle onto the serum treated glass for 400 seconds and 47 random fields were counted using inverted phase contrast optics to obtain "total number of cells". The chambers were flipped over. Cells not adhering to the serum coated Substratum fell off and become attached to the untreated glass. \The chambers were flipped again and random fields of cells adhering to the serumetreated glass cover slip were counted again to obtain "number of adhering cells". Percent adherence of fMLP activated and nonactivated cells was calculated as follows: (Total number of cells - Number of cells adhering) Total number of cells X100 Chemoattractant mediated adherence is expressed as percent over nonactivated baseline adherence. Chemotaxis Evaluations of fMLP mediated cell motility were made using blind-well chambers (Neuroprobe). The lower compartments were filled with 10nM fMLP, made 22 (w/v) HSA (essentially globulin-free, Sigma), or 21 HSA alone to determine baseline migration. The compartments were~covered with a 13mm, 3um pore-size nitrocellulose filter (Schleicher and Schuell). Two hundred ul of isolated adult neutrophils, at 2 million/ml in complete PBS made 22 HSA, were loaded into the upper compartment. The chambers were incubated for 30 minutes at 37C. Filters were removed, stained, cleared (Maderazo and WOrnick, 1978) and motility was evaluated with light microscopy by determining the maximum distance migrated by at least 2 cells, the "leading front" technique (Zigmond and Hirsch, 1973). Chemotaxis is expressed as percent migration over baseline level. 48 Cell-free supernatants were assayed to indicate extracellular release of granule components from adult neutrophils in response to fMLP activation or exposure to A23187 or a temperature transition. Granule release mediated by fMLP was determined on supernatants from neutrophils activated with 1nM, 10nM or 100nM fMLP. One million neutrophils/ml were preincubated for 5 minutes at 37C. Warm fMLP was added and the incubations were continued for 20 seconds or 10 minutes. A23187 mediated extracellular granule release determinations were made on supernatants harvested from 1 million neutrophils eXposed to the ionophore. Prewarmed cell suspensions (5 minutes at 37C) were exposed to 100nM or 250nM A23187 for 1 to 10 minutes at 37C. Extracellular granule release conditions were terminated by immersing the reaction tubes into an ice bath for approximately 5 minutes. Baseline release determinations were made on supernatants from nonactivated cell suspensions incubated for 5 minutes at 37C. Granule release in response to a temperature transition was determined on neutrophils isolated in the cold or at room temperature. Cells were adjusted to 1 million/ml in complete PBS and incubated at 37C. At several time points, ranging from 1 to 60 minutes, the reaction tubes were transferred to an ice bath. Baseline line release was determined on supernatants from cell suspensions maintained on ice or at room temperature. Cell-free supernatants were harvested from fMLP, A23187 and temperature mediated granule release reaction tubes by centrifugation at 1000 rpm for 10 minutes at 4C. The total cellular content of granule components were determined from cell lysates. “Neutrophils_were treated with 0.12 triton X-lOO, 49 sonicated approximately 20 seconds on ice (Heat Systems-Ultrasonics) and freeze—thawed at least once. Lysates were centrifuged at 5000 rpm for 60 minutes at 4G to remove cellular debris before assaying. Granule Component Assays Cell—free supernatants were assayed to indicate extracellular release from primary, secondary and tertiary neutrophilic granules. Primary Granule: Beta—Glucuronidase Beta-glucuronidase was assayed as an indicator of primary granule release. The assay used is based on the enzymatic hydrolysis of phenolphthalein glucUronide at acid pH (Fishman, 1965). The phenolphthalein liberated as a result of the reaction generated a pink color at alkaline pH which was quantitated spectrophotometrically at 540nm. Standard curves were generated with solutions of phenolphthalein (Sigma) diluted in complete PBS to provide 10ug to 200ug of phenolphthalein/ml. One hundred ul of standard solutions, cell—free supernatants or lysates were added to 0.8ml 0.1M acetate buffer, pH.4.5. One hundred ul of substrate, 100mM phenolphthalein glucuronide, pH 4.5 (Sigma), was added, the tubes were mixed and incubated at 37C for 17 to 20 hours. The pH of the reaction mixtures was then adjusted to approximately 10:3 by the addition of 2.5m1 glycine solution, (217mM glycine, 217mM NaCl and 300mM NaOH, pH approximately 11.20) 1.0ml 52 (w/v) trichloroacetic acid (TCA) and 1.5ml distilled water. The tubes were mixed and the Optical density (O.D). was read at 540nm sgainst a water blank. Beta-glucuronidase activity is expressed in Fishman units, defined as the amount of enzyme which liberates lug phenolphthalein from 100mM substrate solution in 1 hour at pH 4.5. Extracellular primary granule 50 beta-glucuronidase release is expressed as percent of total Fishman units detected in neutrophil lysates prepared from 1 million cells. Secondary Granule: Lactoferrin The glycoprotein lactoferrin was assayed as an indicator of secondary granule release. An indirect double-sandwich enzyme-linked immunoassay (ELISA) was used (Hetherington et.al., 1983). Sodium phosphate buffer (0.01M, pH 7.4) containing 0.5M NaCl was used throughout as the assay buffer. Assay buffer made 0.05% Tween 20 (polyoxethylenesorbitan monolaurate, Sigma) was used as the plate washing buffer. Except were indicated, all incubations were performed at room temperature for 30 minutes. Ninety-six well, flat bottom polystyrene plates (NUNC Immunoplate I, Gibco) were incubated with 100ul/well of goat anti-human lactoferrin (Nordic) diluted 1:2000 in 0.1M carbonate/bicarbonate buffer, pH 9.0 (coating buffer), at 4C overnight. Coated plates were washed 3 times for 3 minutes each with washing buffer. Washed, coated plates could be held in plastic bags at 4C for up to one week prior to use without affecting the lactoferrin ELISA. One hundred ul/well of diluted sample was added to prepared plates and incubated. Plates were washed (3 times 3 minutes each wash), and 100u1/well of the IgG fraction of rabbit anti-human lactoferrin (Cappel) diluted 1:3000 in assay buffer was added. Plates were incubated, washed and 100ul/well of peroxidase conjugated swine anti-rabbit IgG (Dako) diluted 1:1000 with assay buffer was added. After incubation and washes, a peroxidase substrate was added. Two hundred ul of freshly prepared 0.4mg/ml orthophenylene diamine hydrochloride (Kodak) made 0.0252 hydrogen peroxide in 0.05M citrate buffer, pH 5.0, were added to each well. The peroxidase reaction was 51 allowed to develop for 30 minutes at room temperature, then stopped by the addition of SOul/well 2.5N sdlfuric acid. ELISA plates were spectrophotometrically read on a microplate reader, Dynatech or Titertek, at 490nm or 492nm respectively. Lactoferrin standard curves were generated for each granule release assay using human milk lactoferrin (Sigma) at dilutions ranging from 6.25ng/ml to 200ug/ml and plotting ng of lactoferrin/ml against 0.D. at 490nm or 492nm. The amount of lactoferrin released into the supernatants or contained in cell lysates was determined from the standard curve correcting for initial sample dilutions. Extracellular release results are expressed as percent of total lactoferrin detected in lysates prepared from 1 million neutrophils. Secondary Granule: Lysozyme In addition to lactoferrin release, evidence of extracellular secondary granule release was evaluated by assaying cell-free supernatants for the enzyme lysozyme. The lysozyme assay is based on kinetic bacteriolytic activities of the enzyme (Neurkar, 1981). Suspensions of substrate bacterium, Micrococcus lysodeikticus . (Sigma), were made in 0.066M potassium phosphate buffer, pH 6.24. The amount of bacteria/ml of buffer was adjusted so that the 0.D. at 450nm, read against air, was between 0.600 and 0.700. One hundred ul of lysozyme standard (egg white lysozyme Grade I, Sigma) providing from 12 units/ml to 100 units/ml or 100ul of supernatant or cell lysate was added to 2.5ml of substrate in a polystyrene cuvet and mixed. 0.D. readings of the mixture were made one minute after the . addition of test solutions and at one minute intervals thereafter for five minutes. The average change in 0.D./minute mediated by the 52 lysozyme standards were calculated and plotted against units/ml to generate standard curves. The amount of lysozyme contained in assay ‘ samples were determined by calculating the average change in 0.D./minute and interpolating units/ml from standard curves. Extracellular granule release of lysozyme is expressed as the percent of total enzyme detected in cell lysates prepared from 1 million neutrophils. Secondary Granule: Collagenase Some cell-free supernatants were also assayed for collagenase to indicate extracellular secondary granule release. Collagenase activity was assessed using a soluble [14C]-collagen substrate. Labeled collagen was prepared from calf skin type I collagen as described below for use in determination of gelatinase activity, with the exception that dialyzed labeled collagen was lyophilized and redissolved to half the volume with 0.01M acetic acid. The collagenase assay employed was a combination of several published methods (Cawston et.al., 1979; Murphy et.al., 1980; Sellers and Reynolds, 1977). Supernatants and lysates were first treated with 1mM phenylmethanesulfonyl fluoride (PMSF, Sigma) exactly as described below for the material used in gelatinase determinations. Prior to assaying, latent collagenaSe was activated by incubating PMSF-treated samples with 1mM p-chloromercuricbenzoate (Sigma) and 10mM CaC12 in a total volume of 225ul at pH 8.4, for 60 minutes at 37C. The samples were cooled to room temperature and soluble [14C]-collagen, also at room temperature, was added to provide approximately 45,000 cpm per reaction tube. Samples were incubated at room temperature for 24 hours. Uncleaved substrate was pelleted by centrifuging assay tubes 53 at room temperature for 15 minutes at 5000 rpm. Cpm were determined on 100ul of assay supernatant added to 10 ml scintillation fluid (Hydrofluor, National Diagnostics). One unit of collagenase is defined as the amount of enzyme that degrades lug of substrate/minute. Collagenase release is eXpressed as percent of total activity found in lysates from 1 million neutrophils. Tertiary Granule: Gelatinase The tertiary granule component gelatinase was assessed by the ability of cell-free supernatants and cell lysates to enzymatically cleave labeled gelatin. [14C]-gelatin was prepared by heat denaturation of labeled calf skin type I collagen (Calbiochem). Briefly, type I collagen was solubilized in 0.01M acetic acid at 1mg/ml with stirring at 4C. The pH was adjusted to 9.0 with 0.1M sodium borate buffer and 1mCi of [14C]-forma1dehyde (ICN)/72mg collagen was added. The mixture was stirred on ice for 60 seconds followed by the addition of four 225u1 aliquots of 0.01M sodium borohydride. After an additional 30 minutes of mixing on ice and one 500ul aliquot of 0.01M sodium borohydride, the mixture was loaded into dialysis tubing. The [14C]-collagen was dialyzed exhaustively at 4C against 0.01M acetic acid to remove any unbound [14C]. The final preparation was stored at 4C in the presence of 0.02% sodium azide. The gelatinase assay used combined several published methods for determination of the enzyme (Dewald et.al., 1982; Hibbs et.al., 1985; Murphy et.al., 1980). Harvested supernatants or prepared lysates were first treated with 1mM PMSF in 50mM Tris-HCl, 50mM NaCl, pH 7.6, for 60 minutes at 37C to block Serine proteinases. Treated samples were .frozen at e35C until they were assayed. Latent gelatinase was 54 pre-activated by incubating PMSF-treated samples with 1mM p—aminophenylmercuric acetate (Sigma) and 10mM CaCl 2 in a total volume of 225ul at pH 8.4, for 1 hour at 37C. Immediately before use [14C]-collagen was denatured by heating at 60C for 20 minutes. A portion of labeled gelatin providing at least 10,000 cpm was added to each assay tube at 37C and the incubation was continued for 1 hour. The reaction was terminated by precipitating uncleaved [14C]—gelatin with ice cold TCA. 50u1 of 90% (w/v) TCA was added to each assay tube, the tubes were mixed and incubated on ice for 30 minutes. The tubes were then centrifuged in the cold at 5000 rpm for 15 minutes and 100ul samples of assay supernatants were removed, added to 10ml scintillation fluid and counted for 0.1 minute. One unit of gelatinase activity is defined as the amount of enzyme degrading lug of substrate/minute. Extracellular gelatinase release is expressed as percent of total gelatinase detected in lysates prepared from 1 million neutrophils. Electron Microscopy: Neutrophil Processing Ultrastructural evaluations of neutrophil morphology and intracellular granule characterization and quantitation were made on micrographs of adult and neonatal neutrophils activated with fMLP or exposed to A23187 and on adult cells exposed to a temperature transition. To standardize the population of cells to be used in the morphometric measurements it was essential that the region of each cell beifig sectioned was representative of the whole cell regardless of the orientation of Sectioning. Therefore, it was critical that the cells were not bipolar in configuration but were spherical or ruffled 55 in,shape. Chemotactic peptide mediated ruffling conditions, determined with light microscopy of adult and neonatal neutrophils, were used to °activate cells for transmission electron microscopy. Cells were prewarmed for 5 minutes at 37C in complete PBS. Warm fMLP was added to obtain final concentrations of either 1nM or 100nM and the incubation conditions were continued for 20 seconds. Adult and newborn cells were exposed to the calcium ionophore to induce cell ruffling. Prewarmed neutrophils were incubated for 5 minutes at 37C with either 100nM or 250nM A23187. Spherical nonactivated control cells for fMLP and A23187 morphometric determinations were neutrophils incubated at 37C for 5 minutes. Adult neutrophils treated with a temperature transition were isolated in the cold and incubated at 37C.for 5 or 30 minutes. Baseline control cells were maintained on ice. For transmission electron microscopy, cells were fixed with warm fixative, 1.8% (v/v) glutaraldehyde buffered with 0.1M cacodylate, pH 7.4 (Electron Microscopy Sciences), made 1.0% (w/v) sucrose. Fixation continued for 1 hour at 37C followed by up to 96 hours at 40. After washing with 0.01M sodium cacodylate, pH 7.4, made 7.0% (w/v) sucrose, cells were resuspended in Karnovsky's diaminobenzidine (Graham and Karnovsky, 1966), 3-3'diaminobenzidine tetrahydrochloride (DAB, Sigma) at 0.5mg/ml in 0.05M tris-HCl, pH 7.6, made 0.01% (v/v) hydrogen peroxide and 1% (w/v) sucrose. Following 30 minutes room temperature incubatiOn with DAB, neutrophils were washed 3 times with 0.05M acetate-veronal buffer (A-V buffer), pH 7.4 and postfixed with 1% . (v/v) osmium tetroxide (Electron Microscopy Sciences) in A-V buffer 56 made 5% sucrose, for 1 hour. Cells were washed twice with A-V buffer and stained enbloc for 1 hour with 0.5% (w/v) uranyl acetate dissolved in A-V buffer made 4% sucrose. After dehydration in graded ethanol and transition with propylene oxide, samples were embedded in either Epon/Araldite or LX-112 resin (Ladd). Gold-silver sections were cut using glass or diamond knives, picked up on acid-alcohol cleaned copper grids and stained with saturated aqueous uranyl acetate counterstained with Reynold's lead citrate. Sections were examined using a JEOL 1008 or 10OCX transmission electron microscope at 60KV or a Siemens Elmiskop 101 electron microscope at 80KV. All microscopes were initially and periodically calibrated for magnification using a shadow cast replica of diffraction line grading mounted on a copper grid (Pellco). In an attempt to standardize the ultrastructural granule quantitations and morphometric measurements, negatives were taken only if a sectioned cell represented the central region of a neutrophil. Sections were considered to be through the central region if the cell profile contained at least three nuclear lobes or the centriole. Cells fulfilling these criteria were photographed at approximately 7,000X magnification. Ultrastructural Eraluation of Granule Release: Intracellular Granule Characterization and Quantitation Intracellular granule characterizations were made based on cytochemical staining. As indicated above, neutrophils prOcessed for use in ultrastructural analyses were treated with DAB and hydrogen peroxide prior to osmium tetroxide postfixation. The DAB reaction stained primary granules electron dense making them distinguishable 57 from the relatively more electron lucent secondary granuls (Figure 2). Peroxidase positive (primary) and negative (secondary) granules were counted on electron micrographs frOm neutrophils activated with fMLP, exposed to A23187 or a temperature transition and nonactivated control cells. Intracellular granule quantitation results are expressed as the average number of peroxidase positive and negative granules per neutrophil profile. Extracellular Granule Release/Intracellular Granule Loss To associate extracellular and intracellular evidence of granule secretion, release was expressed as a ratio of the amount of a. particular granule component detected extracellularly per intracellular granule lost. To calculate the ratios, extracellular release was first re-expressed on a per neutrophil basis. The mean quantity of each granule component detected in cell—free supernatants was converted from percent of total to ng or units and divided by 1 million to generate an average extracellular release per cell. The release per cell values were then divided by the average number of granules lost (peroxidase positive or negative depending on the granular origin of the component) in responSe to the particular treatment of the cells as determined by intracellular granule counts made on micrographs. Since tertiary granules have not been identified ultrastructurally, the assumption was made that they may be peroxidase negative and gelatinase released/peroxidase negative granule was calculated. The resulting extracellular granule release/intracellular granule loss ratios were then corrected for nonactivated baseline release.- 58 lknqlnmlnndc:Determinations For ultrastructural evaluations of neutrophil morphology, measurements of micrographs were made on a Houston graphics tablet interfaced to an Apple IIe computer equipped with software by Optomax. The graphics tablet was first calibrated so that the final magnification of each electron micrOgraph was equivalent to 18,000X. Perimeter measurements were made directly from micrographs by tracing the cell profile with a cursor. The Optomax program calculated the area from the perimeter measurements. Surface-to-volume ratios (S/V) were determined to describe morphometric measurements of two dimensional sectioned cells in three dimensional terms. S/V were calculated from perimeter measurements and calculated area based on the following geometric constants for circles and spheres: Circle: I Sphere: Perimeter . ZTTT Surface Area = 4TTr2 Area - TTrz Volume s 4TTr3/3' S/V-3/2(P/A) Exposed surface membrane was then calculated from S/V using 397nm 3 as the mean neutrophil volume (Korchak and Weissmann, 1978; Hoffstein, et.al. 1982). Perimeter and area measurements are expressed in arbitrary units. Presentation of Data Results are expressed as mean i.1 standard deviation. I Statistical significance was determined utilizing the Student's t-test using pooled estimator of common variance 2 and (n 1 + n 2) - 2 degrees Of freedom. Deviations from baseline determinations were . considered significant if the calculated p values were (0.01. I RESULTS Two series of experiments were performed using the chemotactic tripeptide fMLP as a specific, receptor mediated, initiator of neutrophil activation. The first experiments examined the events of cellular activation in terms of visible shape change from spherical to bipolar forms, enhanced adhesion, chemotaxis and extracellular granule secretion. The second series of experiments specifically investigated those events occurring early in fMLP mediated activation. fMLP Mediated Shape Change: Dose Response: Neutrophils were found to be very sensitive to fMLP in terms of a visible shape change. Results of 5 minutes dose response shape change experiments are shown in Table 2. The cells responded to concentrations of fMLP as low as 1pM by exhibiting ruffled configurations. Approximately 70% of cells activated with 1nM fMLP for 5 minutes at 37C had become bipolar. The percentage of bipolar cells remained at about 60% to 70% with increasing doses of fMLP. fMLP Mediated Shape Change : Kinetic Respénse : Not only were neutrophils morphologically sensitive to pM amounts of fMLP, they were also found to respond rapidly t6 the chemoattractant. Results of kinetic shape change determinations made .on cells treated.withan P are shown in Table 3 and Figure 3. 59 60 Table 2 p ‘ fMLP MEDIATED SHAPE CHANGE: DOSE RESPONSE nM fMLP % Ruffled forms % Bipolar forms .3 None 12:5 0+1 20 0.001 76:8 2:2 5 0.005 75:3 2:1 5 0.01 77:] 2:2 5 0.05 82:8 3+2 3 0.1 71:33 S:7 4 0.5 56:}3 42:16 4 1.0 25:8 70:8 8 5.0 24:8 72:4 3 100 42:3 60:3 '2 Neutrophils were prewarmed to 37C, exposed to fMLP at various concentrations for 5 minutes at 37C. Results are expressed as mean:standard deviation. Table 3 61 fMLP MEDIATED SHAPE CHANGE: KINETIC RESPONSE Sedonds Exposure: 100nM fMLP 10 20 30 40 60 300 % Ruffled forms 68+30 93:3 90:10 57:14 36:38 14:3 11+7 25:8 89+8 95:2 91 82:13 66:34 42:3 Baseline : Cells warmed to 37C 12:5 % Bipolar forms 4:4 4:3 6:9 38:16 63:19 84:2 86:8 70:8 8:10 2:2 8 15:14 19:10 60:3 0:1 I: 0‘ WNWU'ISND—‘V NUDUOD-‘VUJ Neutrophils were prewarmed 5 minutes at 37C prior to exposure to fMLP. Results expressed as mean:standard deviation 62 1 NM FMLP xtorlinis 8 8 '1 Lg ; ‘ o no 20 an 40 scans AT me 100 NM FMLP XUCELS O 10 20 3O 4O SEEMS AT 37C 0 MNWIED o BUWIAR Figure 3 fMLP MEDIATED SHAPE CHANGE: KINETIC-RESPONSE Data from Table 3‘ = ' ' ' 63 Approximately 90% of the cells exposed to 1nM fMLP exhibited ruffled configurations within 20 to 30 seconds. By 40 seconds approximately 40% of the cells had progressed to bipolar forms. After 2 minutes, 90% of the cells had become bipolar. Neutrophils activated with 100nM fMLP for 10 to 40 seconds also exhibited ruffled configurations. However, with longer incubation periods, cells activated with the higher dose of fMLP tended to remain ruffled rather than progressing to bipolar forms. fMLP Mediated Adherence: Compared to nonactivated baseline adhesion, neutrophils exposed to fMLP at room temperature demOnstrated enhanced adherence to an artificial substratum. 21.34 : 4.20% or 29.24 : 6.77% more cells adhered to serum coated glass surfaces following approximately 6.5 minutes treatment with 1nM or 100nM fMLP, respectively (mean : standard deviation, n24). fMLP Mediated Chemotaxis: Chemotaxis was performed and analyzed by the "leading front" technique as described in the Methods section. Neutrophils in chemotaxis chambers containing 10nM fMLP (in 2% HSA) in the lower compartment were found to have migrated 24.36 :_8.46% farther into the filter then control cells in chambers with 2% HSA in the lower compartment (mean : standard deviation, naS). fMLP Mediated Extracellular Granule Release: The Chemotactic tripeptide was also found to mediate extracellular granule release. Neutrophils were prewarmed for 5 minutes at 37C and exposed to various concentrations of fMLP for 10 .minutes.at.37Ca-aCellrfree supernatants.were harvested and assayed for 64 primary, secondary and tertiary granule components. The results of dose response fMLP mediated granule release are shown in Table 4 and Figure 4. fMLP induced lactoferrin and gelatinase secretion in an apparently dose responsive fashion. Lactoferrin release was significant following 10 minutes exposure to 1nM, 10nM and 100nM fMLP. Significant amounts of gelatinase were detected extracellularly following 10 minutes activation of cells with 10nM or 100nM fMLP. Neither lysozyme nor beta-glucuronidase were detected in significant quantities. fMLP Mediated Extracellular Granule Release: Effect of Cytochalasin B: The effect of cytochalasin B (CB) on fMLP mediated extracellular release from primary, secondary and tertiary granules was then investigated. Neutrophils were pretreated with 5ug/ml of GB for 5 minutes at 37C before fMLP was added. The incubations continued for 10 minutes at 37C, and cell-free supernatants and lysates prepared from CB treated cells were assayed for granule components. CB did not interfere with any of the component assays (data not shown). As seen in the lower portion of Table 5, the total content of granule components detected in lysates prepared from CB treated cells did not differ significantly from the amounts detected in lysates from cells without CB pretreatment (shown in the lower portion of Table 4). Table 5 and Figure 4 show that, compared to release without pretreatment, CB had no influence 1nM fMLP mediated lactoferrin, gelatinase or beta-glucuronidase release. 0n the other hand, CB appeared to enhance 1nM fMLP mediated lysozyme secretiOn. .Pretreatment of cells augmented 10nM and 100nM fMLP induced lysozyme, 65 rehearse he Ho.ova * Aoumowansv cw mucmafiumexm mo umcaszv ohm um mousefia m ensue: mHHoo “oceammmm cofiumfi>oe upmacmunHemo: on: mean: 05.nflwm.ma "mmmvficoususamlm mane aaaapuaeo.ahemm.o ataaaeaaeau one rhea: .03 x ae.o+ -Ofiux me.m ”aaaeamaseau u: muss: N .¢<+9wam~ “mamnommq care we me.o+es.~ "aaaaaeaaeaa ”maficqouuso: :OHHHHE a scam moummma as emefimucoo massed mo canoes ecu no woman Hmuou mo aeounoe mm commouexo ma ommoaou oasenno .mmuscfls OH new eosefiueoo meofiumcsoea .eoeem mm: m42m .mmusewa m now Ohm um ensumsona who: magnoonusoz Amvme.~Hwe.e *Aevom.nmwe.me Amvem.nmwm.m rflmvma.mmwm.a OOH Ans o *AmeH.OeHea.ke Amvee.ewoo.e *Amvme.ewme.e on Adv o Aavom.e+em.ee Amvmk.o+aa.a *Amvoe.e+mm.e a u. u. I. maze 2a ANV o AmeA.m+em.m Aevem.e+em.e Amevee.o+os.m aeaeaaam ommeficoususawlm ommcaumHmu mmammmwmw cfisnmmouomq “eaeaaaeao "neoconseo oaseouu ommoaom mmme unneemunfidmoz .mo Ha\m=m cue: ohm um mousefia m mom emumnsocfl mHHmU umcaammmm Nue mane: mm.mmwo.m~ ”aaaaaaaaeeaswum Nu: muse: mfiJDWNmm.o “omoewumaou on: mass: mm.m~+omwmo~ "osmnomaq nae ma he.o+es.~ “eaaaaeoeaae .mo Ha\wsm mo mocmmmua ecu cw mecqouusm: :ofiaawa H Boom eosmamuq mmummha cw eOuOOqu neoconeou we uesoam one :o comma Hmuou mo unmonmq mm eommmuaxm ma ommOHmm . .mouscfia o~ now emsefiueou measumcsoefi .emeem mm: mAZM .omm um nuances m now mo «0 Ha\w=m cue: cmumcsoewouq mum: maficnouusmz x~vae.mema.woe Amvem.ammmo.ae Amvmm.w+oo.on Amvem.euam.os mmmcfiumaou Amvse.mmo~.es Amvmm.mmem.- vaoo.oape.ee Amvsm.m4mo.ae Amvam.o+me.a x~vsm.e+~e.m Assam.afl~m.m Aevmm.auak.e maunomna :finuomouomg "neocoaaoo oasemso coy OH H qum z: oaaaommm "meoauweeou paradox mmU mo huwhmm flab m beers 67 1 NM FMLP l-GLUC. 10 NM FMLP PEAK. 100 NM FMLP ““64! n Data from Tables 4 and.5... ._ Figure 4 H7 E 7' EFFECT OF CB 0N fMLP MEDIATED EXTRACELLULAR GRANULE RELEASE 68 lactoferrin and gelatinase release. The primary granule enzyme beta-glucuronidase was deteCted extracellularly following activation of CB pretreated cells with 100nM fMLP for 10 minutes. The data presented above, from the first set of experiments investigating fMLP as an initiator of activation events, concurs with the well documented finding that the synthetic chemotactic tripeptide mediates visible changes in neutrophil morphology from spherical to bipolar configurations (Smith et.al., 1979), enhanced adherence (Smith et.al.,l979), chemotaxis (Zigmond, 1978) and extracellular granule release which is augmented by pretreating the cells with CB (Dewald et.al., 1982). Those events occurring early in fMLP mediated activation were examined in the next series of experiments. The earliest visible indication of cell activation, ruffling, was used as the criteria for selecting the early fMLP activation conditions. The fMLP mediated shape change determinations shown above indicated that neutrophils began to ruffle within seconds following exposure to low doses of fMLP (Tables 2 and 3, Figures 3). To determine whether functional events of activation were detectable early in fMLP mediated activation, cell-free supernatants from fMLP ruffled cells were analyzed for extracellular granule release. Early fMLP Mediated Extracellular Granule Release: Extracellular release of secondary granule lactoferrin was detected early following exposure of neutrophils to fMLP. Cells were prewarmed for 5 minutes at 37C and treated with 1nM,10nM or 100nM fMLP for 20 seconds to produce Cell ruffling. Cell-free supernatants were harvested and.assayed for-evidence of extracellular granule 69 ocwHommc ou cosmosoo Ho.ove *. AmueoeHnoexo mo nocaszv eoeHauouon uoz u.n.z eofiuoa>oe enmeeouaudmoz .onm um mousefie m eosnms mHHoo “oeHHommm .Ao oHnt oomv mHHcoouuso: cOHHHHe H scum eouooona moumth.:H coewmucoo oammeo «0 scream ocu co comma Hmuou mo ueounoq mm vommounxo onm muHsmom .mveooom om How concaueou maceumcsoea .eoeem mm: HASH AmVeL.HHwa.o ALL I. o Amvee.s+ma.o Ame o ommeweousosHmHm. Amva~.mmwm.m Aevue.ewae.m Amvs~.~+ma.m Aroma.nwem.m ommeHuoHou Hmvam.mfloe.m nua.z Anv~H.o+em.e “mesa.HHam.~ ommeommHHou .onm no mousefia m now coaumzonq ouo: mHHcoouusoz Aevmm.mnme.m . *Aavam.owmm.e cos Amvem.emee.e *Amva.HHee.m o3 Aevoa.s+mm.e rxavmm.o+ea.m H I. I. mean 2: Aevem.~+em.e Amevae.o+o3.~ aeaeaaam oaumomuq ewuuomouomq . "meoauweeou “aeoeoaaoo oHsemno omooHom mm4Moc eumeemunflemo: .mo mo Ha\w=m nus: Own an mouscea m new woumcsocw mHHoU ”oefiHomom .Am oHcoe oomv no mo Ha\wsm mo oocomouo ocu :H mHchonusoe =OHHHHE H Boom woumeono moummzH cw coeHmucoo neocooeoo mo unseen Hmuou mo ueouuoa mm uommouexo mH ommoHou oHsemnc .meeooom om you eoseHueoo m:OHuoc=o:H non woven mm: HHZH .Unm um mouscHe m now no mo Ha\wsm cud: eoumcsoeHona ouo: mHfineouusoz Amoco. HHON. om Amvmm. NHem. an a~vkm. HHmo. m Aevm~.Qst.H :Huuomouuma uucoeooaoo oHsemnw zsooH zeoH zeH @42m 2: oefiHommm Am=OHuHesoo omooHom mmHmU mo Bowman nah h oHnt 1 N" FMLP 72 _ W%%////////////za Tables 6 and 7 5 EFFECT OF CB 0N EARLY fMLP MEDIATED EXTRACELLULAR GRANULE RELEASE from Figure Data 73 of ruffled neutrophils to confirm the biochemical results indicating extracellular granule secretion by demonstrating a depletion in intracellular granules. - Ultrastructural Eraluation of Early fMLP Mediated Activation: Intracellular Granule Quantitation: Neutrophils were warmed to 37C and exposed to 1nM or 100nM fMLP . for 20 seconds. Activation was terminated by the addition of TEM fixative. Fixed cells were processed and photographed as described in the Methods section. Intracellular granules were characterized and counted based on peroxidase staining. Granule quantitations made on electron micrographs of control and fMLP activated cells are shown in Table 8 and Figure 6 A. The early - events of fMLP mediated activation induced a significant decrease in the average number of peroxidase negative granules in cells treated with either 1nM or 100nM fMLP (p<0.005 vs baseline for both concentrations of fMLP) while not significantly affecting the number of intracellular peroxidase positive granules (p>0.25 and p<0.10 for 1nM and 100nM fMLP, respectively). The average number of peroxidase negative granules/cell profile lost following activation with either 1nM or 100nM fMLP for 20 seconds was almost identical, 11.4 and 12.0, respectively (not significantly different, p>0.25). ' fMLP Mediated Extracellular Granule Release/Intracellular Granule Loss: To further investigate the granule secretion occurring early in activation, calculations associating extracellular granule release with intracellular peroxidase negative granule depletion were made as described in the Methods_section. The results.of early fMLP mediated 74 Table 8 - ULTRASTRUCTURAL GRANULE QUANTITATION 0F EARLY fMLP MEDIATED ACTIVATION ' Granules/Cell Profile: Peroxidase Positive Peroxidase Negative Baseline (40) 74.2:13.7 ,60.2:11.1 nM fMLP ‘ 1 (22) 72.6:14.5 48.9:9.8* 100 (25) 79.5:11.3 48.2:10.0* Neutrophils were prewarmed for 5 minutes at 37C. fMLP was added, incubation continued for 20 seconds. Pooled data from 2 neutrophil donors. Baseline: Nonactivated cells which were incubated for 5 minutes at 37C before fixing. Mean:standard deviation (Number of micrographs analyzed) * p<0.01 vs baseline determination 75 neutrophil granule release/granule loss are shown in Table 9. Correcting for spontaneous release, neutrophils activated with fMLP for 20 seconds released an average 4.84.X 10’6 ng of lactoferrin/granule. The results from assays of cell-free supernatants for lysozyme (Table 6) indicated that 20 seconds activation with either dose of fMLP did not cause significant lysozyme release. Expressed as corrected release/granule (Table 9), 1nM fMLP did not induce lysozyme release above baseline levels while 100nM caused the release of 0.72 X 10‘6’units/granule. fMLP mediated extracellular collagenase release also did not differ significantly from baseline release (Table 6). Correcting for background release, activation of neutrophils with fMLP for 20 seconds caused an average release of 0.71 X 10"11 units of collagenase/granule. If tertiary granules are peroxidase negative, none of the granules released early in fMLP activation contained gelatinase. These results suggested first, that the lactoferrin detected in supernatants from ruffled cells was granular in origin and second, that these granules contained lactoferrin exclusively or relatively high concentrations of lactoferrin. To investigate a correlation between the alteration in cell shape and the secretion of granules seen early in fMLP activation, quantitative morphometric determinations were made on electron micrographs of fMLP ruffled cells. Ultrastructural Evaluation of Early fMLP Mbdiated Activation: ‘Morphometric Determinations: Neutrophils were activated as described above for ultrastructural intracellular_granule.quantitation.l Morphometric measurements were 76 .owm um mouscHe m voumcsocfl mHHoU ”ocHHommm .:0Huoom meonuoz :H eocHsomoe mm woumH=UHmo mo: oHscmuw o>Humwo= omoeonuoa\ommoHou uoHsHHoomuuxm ashes. cuoH x em.m auaaeaaaeao mane: ouch x me.m "aaaeawcaeao ease: enoH x mm.N “earners; . we _IoH x on.~ "cannomouomq .eoHuoom meocuoz :H eocHuumoe mo eoumH=OHoo mm: Hacmwduso:\ucocoosoo Hmuou owmuo>< .meeooom om now eosewucoo eOHuocsoeH .eoeeo mo: easy .Umm um mouscws m eoEumzoua odor mHHoU a: OH x NH.OI a: OH x n<.H m: OH x mm.H 2cooH m: OH x oH.OI m: OH x mm.H m: OH x om.H st _ onH x No.H ocHHomom AmuHca owmno>mv “ ommeHumHou .n HHI oH x ~m.0 HHI OH x em.H oHIoH x mw.H zoooH HHI oH x Ha.o HHIoH N om.H cHIoH x cm.m zeH I OH x NN.H osfiHommm Hmumma owmno>ov A ommsommHHoo .0 cIoH x Nn.o cl oH x mn.H mIoH x oH.~ zeooH oo.o onoH x ao.H mIoH x «N.H zeH mtoH x «N.H ocHHommm Amuwca owmuo>ov " oaNNomNH .m cIoH x 0H.m oIoH x @¢.o nnoH x oo.NH zcooH @10H x mm.q oIoH N oo.a mIoH x mo.oH st mloH x oh.m ocfiHommm Aw: ommuo>ov u cHuuowouumH .< oHssmuu\ommoHom eouuouuou oHscmuo.woz.sonom\ommoHom HHoU\ommoHom mmoa nonzero m>He0.25), it did produce a significant increase in calculated cross-sectional area. Area determinations made on profiles of cells activated with 100nM fMLP for 20 seconds averaged 14.6% greater than baseline (p<0.005). In contrast, the average area determination of cell profiles from 1nM fMLP treated cells was not significantly different than the baseline value (p<0.025). Within 20 seconds following exposure to 1nM or 100nM fMLP, neutrophils were found to have changed shape, released significant quantities of lactoferrin extracellularly and lost a significant number of intracellular peroxidase negative granules. Additionally, in responSe to the lower dOse of fMLP, cells were found to have significantly more surface membrane, while cells exposed to 100nM fMLP 78 Barometer *HH.eaH+HH.mee Ho.m~HHeo.Hmm -.a:vo:munaoS auaaHauHao aeHHaaaa he Ho.ova * AeoNHHmcm mcHoLwOHOHE mo nocaszv :OHumH>oe euoeemuafidmoS .wcHxHH ouomon ohm um mouseHa m woumnsocH mHHoU .oeHHommm .muocov HHcHouuso: m scum mums eoHOOH .Unm um mesooom on new eosefiucoo coHumnsueH .voecm mm: HASH He.emwe.H rae.o+mc.H HH.QH¢H.H .HAw Amuflmmfimk cm.mH+am.aH ma.HHHoH.me mou< .Unm um mouseHa m now coaumzouH ono: mHHcHouusoz ram.mnmwm.ms . HNav ooH *HN.mH+eH.mH Hume H daze :e mc.mHHem.Hc AHHV aeHHaaam uouoaHuoH monHHBU< QMHASA Ho.OVH N AmocoeHHono mo Hocaszv woefiauouon nozwwn.z sowumfi>oe enmeemum+emoS .on um mouseHa m woumcsocH mHHoo “ocHHomom .Ae oHcmB oomv Hmuou mo ueoouoH mm eommoHon mH ommoHoH *HHVma. mMmm. HN HNVHN. NMaH. ON *Havca. mMaa. HN HNvoc. mHmm. aH HNvaa. HMaN. m HNvNN.o+Ho.c *Hmvma. cMNN. NN HNvam. mMco. 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NHoo. m HHHVae.QHcH.N sHHHomouooA Humo:OHeoo oHscmHu .eoceo HmeN< .on um mouscHs m eosnmonH mHHSHonusoz o v-4 MOOQOO SdomN o v-fl Hmunxo OHMWINO 000.00 .cws m. o.SdooH HmHmmd ocHHommm HmcoHuHceoo ommoHom mmmu unoccmuoflwmo: .on um mouscfle m no mo Ha\w:m nag: wouonsocfi mHHoU Hocflammmm .Am magma oomv Hmuou mo acouuom mo ummmmuaxo ma omooaom .maamu may :a vmumowvcfi mm wmscwucoo who: mcofiumnsocfi .uouum mm: HmHm~< .on um mouscfia m now no mo Ha\w:m nu“: vmumnsocfimua who: mafinaouuamz HNVHH.H+om.mm . n..n.z HNVmH.¢N+HN.HH .n.z .a.z .n.z HNva.mH+mo.Ho a..n.z HNvNN.Hm+HN.mo .a.z .o.z .n.z HNvom.eHmm.oH QQQGHUwHUU HNvom.mHHm.Hm HNvoo.oHHm.oo HNVHN.mHmN.No HNvmo.HHHH.om HNvHH.Hme.He HNvoH.N+H¢.o¢ HNvoq.mHmH.oo HNvo.qfiHmqum HNvmm.»HHo.Nm HNva.mHoo.mq HNVHH.mHHN.H< HNva.o+Ho.Nm HNvmm.mHNm.m maunomnu HNvHH.oH+ON.NN n..a.z HNVHN.m+oo.qN .a.z .o.z .a.z HNVHH.N+HH.mN u..a.z HNvNH.H+no.oH .a.z .a.z .a.z HHVHN.QHHN.H :Huuomouumq Hucocoqaoo mascouu I O O H Hmmho o—ammHo .cfia . zeomm O O O H CDCDCDCJCD USCDCDCDCDCD .:Ha m.onz=ooH HmHm~< mafiaommm Hmcofiuavcoo ommoamm mm0.25). Exposure of neutrophils to 100nM or 250nM ionophore caused an average loss of 6 or 14 peroxidase negative granules/cell profile, respectively. The granule depletion mediated by 250nM A23187 was significantly greater than that induced by 100nM A23187 (p<0.005). A23187 Mediated Extracellular Granule Release/Intracellular Granule Loss: To associate the results from ionophore mediated extracellular granule secretion and intracellular granule depletion, ratios of release/granule loss were calculated as described in Methods. Table 15 indicates that 100nM A23187 induced the secretion of approximately two fold more of each secondary granule component/peroxidase negative granule then cells treated with 250nM A23187. Neutrophils exposed to‘ 100nM A23187 for 5 minutes released an average of 1.8 times more lactoferrin, 2.1 times more lysozyme and 2.0 times more collagenase than cells treated with 250nM A23187 for 5 minutes. Making the assumption that tertiary granules may be peroxidase negative, 100nM treated cells lost 1.5 times more gelatinase/granule that 250nM A23187 treated cells. These results appear to suggest that the granules secreted from A23187 ruffled neutrophils contain lactoferrin, lysozyme, collagenase and gelatinase. Further, the granules secreted from cells ruffled with the lower dose of A23187 appear to contain quantitatively more of each component. . , Ultrastructural Ebaluation of A23187 Mediated Shape Change: QHorphaletric DetenndnatiOns: 90 .Uhm US mwufidfia 0 ROM fiQUGfiflUn—fi 0H0} QHHGU «QGfimeflm .:owuo0m mvonu02 :H v0nfiuum0v 00 e0u0H00H00 0H0: mmoa 0H::0Hm\0000a0m .Aa 0Hnoh 00mv muonu02 :H v0nwuum0v 00 c0u0H00H00 003 Hficaouu=0:\u:0:oaaou Houou 0wmu0>< .UHm 00 m0u::Ha m 00::Hu:ou m:ow00a:0:a .0000: 003 whHm~< .UHm um 00u=:Ha m :0au030uq 0H0: mnaou TH: H 8H 9.2 H 3.2 0.. 0H N «0.0m 0102 x MNJH 0H::0H0\0000H0m v0u00uuo0 mug N min mu0H x 3.: 2:03 0:2 N 00.2 mug x 5.0 2:02 mI0H x No; 0:300me Amu«:= 0m0u0>0v H 000:Hu0H00 .0 3.. 0H N H0.N 07.3 02 >05 2:0mm 3:02 02 mad 0H: 0H 02 0m.m 2:02 01.2 x «N; 0:30000 AmuH:= 000H0>0v H 000:mwwHHou .0 cl 0H x HEN mu 0a a aim 2:03 0.. 0H 02 $6 mI0H x 0H6 2:03 ml 0H N «N; 0:.30000 Amua:0 0wou0>0v «.mmfimmmum. .0 0:02 N 2:3 n.0H x $.00 2:03 0.02 x :50 wk: x 3.3 2:02 9.2 M 05m 0:3003 Am: 0mou0>0v H :Huu0mou004 .c 0H::0u0.m0z.xou0m\0000a0¢ HH00 0000H0m mms 21523.0 m>HH :o.ova * Av0umam:0 mammuwouofla mo u0naszV :ofiumfi>0c 0:00:000H:002 .w:wxww 0:000: ohm 00 000::Ha m v0umn:0:fi 0HH00 "0:fiH0mam .muo:ov Hangouua0: m Sony 000: v0Hoom mu .m0u::Ha w you 00::Hu:oo :ofluma:0:fl .00000 003 hwfimm< .Unm um 000::Ha n how u0aumz0u: 0:03 mawzaouua0z c~.mfimm:fi.moo m~.gmwm.: oq.oflmw:.mh *Ha.mflmwo.ma Acme omm eo.-+wm.~om m:.o+w~.: *oo.m+qm.fim *w~.¢ +e~.wo Afimv co: I l. I . 382 z: Ho.m~_+co.mmm Nm.o+om.: me.m:+o~.wo mo.m:+om.~o AHBV mcaflwmam Nmsdv0:0una02 .NNW. m0u< .mmmmmwmmm. umumH=UHmo monH0.25 for 1nM and 100nM fMLP, respectively). Neonatal cells_treated with 1nM fMLP did not exhibit a highly significant 95 Table 17 NEONATAL NEUTROPHILS: KINETIC SHAPE CHANGE RESPONSE TO 1nM fMLP Seconds Exposure 1 Ruffled forms '2 Bipolar forms I: 20 84:] l3} 5 30 . 8513 6:9 5 40 65:] 25:}0 5 Baseline : Cells warmed to 37C 131l0 l1} ' S Neutrophils were prewarmed 5 minutes at 37C prior to exposure to fMLP Results are expressed as meangstandard deviation 1 NM FMLP: NEONATAL IUCELLS O 20 N ‘0 ‘ SEEMS AT 37C 0 men . BIPGJR Figure 11 NEONATAL NEUTROPHILS: KINETIC SHAPE CHANGE RESPONSE TO 1nM fMLP Data from Table 17 96 Table 18 NEONATAL NEUTROPHILS: ULTRASTRUCTURAL GRANULE QUANTITATION 0F EARLY fMLP MEDIATED ACTIVATION Granules/Cell Profile: Peroxidase Poéitive Peroxidase Negative Baseline (38) 84.5115.4 59.Qil0.1 nM fMLP . . 1 (40) 80.6:16.7 53.2il3.2 100 (32) 84.21fl2.2 50.2:8.4* Neutrophils were warmed for 5 minutes at 37C, fMLP was added, incubation continued for 20 seconds. Pooled data from 5 neonatal neutrophil donors. Baseline: Cells incubated 5 minutes at 37C before fixing. Meanistandard deviation (Number of micrographs analyzed) * p<0.01 vs baseline determination. 97 decrease in peroxidase negative granules (p<0.025). However, cells exposed to 100nM fMLP for 20 seconds did exhibit a significant reduction in peroxidase negative granules/cell profile (p<0.005). Neonatal Neutrophils: Ultrastructural Evaluation of Early fMLPNHediated Activation: Morphometric Determinations: Next, to determine whether the intracellular granule loss detected following 20 seconds activation of newborn cells with 100nM fMLP correlated with morphometric alterations, electron micrographs of neonatal neutrophils were measured. As shown in Table 19 and Figure 12 B, none of the morphometric quantitations made on cell profiles of fMLP ruffled newborn cells differed from the baseline determinations. Comparisons of the results from the ultrastructural morphometric measurements of neonatal and adult cells exposed to fMLP are shown in Table 20. Nonactivated neonatal neutrophils were found to be comparable in perimeter, significantly larger in area and to expose significantly less plasma membrane than nonactivated adult cells. Following activation with fMLP, the mean area determinations of neonatal and adult cells were comparable, but the exposed plasma membrane of newborn cells was still significantly less. The average perimeter measurements of 1nM fMLP activated neonatal neutrophils were significantly less than those of identically treated adult cells (p<0.005), while the mean perimeter determination of newborn neutrOphils activated with 100nM fMLP for 20 seconds were comparable to those of adult cells. As described above, adult cells treated with the calcium ionophore exhibited responses similar those seen early in fMLP activation. _Morphologica1 studies were made of neonatal cells treated 98 sowumswauoumc meadows; m> Ho.ovq * Acoumamcm mzqmquHUHa mo monaszv coaumfi>ov unmeamuouwomz .wcfixww opommn ohm um moussfia m voumnsuca mHHoU “ocfiaomom .muocov Hangouusm: Hmumsoo: m scum meme cmHoom .mucoomm ON pow voscwucou :owumnsucw .uoccm mm: mAZM .Umm um moussfia m cmsumzouq mum: mawnaouusoz Snowing.» snow: ohmmsam wa.:mmo.$ 8.8 o2 mm.nm+mo.omq «L.o+mi.~ oe.o+em.qs mq.m+mo.mm Aomv L n. .u .. .. mazm z: mm.mos+m~.qsq -.o+mfl._ ms.sfi+w~.ms ~o.-+ms.fio Ammv mafiammmm flea oceansoz a so: 3 smuosausao monBHBU< nme0.25 and p<0.25 for 100nM and 250nM A23187, respectively). Cells treated with 100nM A23187 lost an average of 6 peroxidase negative granules/cell profile, while cells treated with 250nM ionophore lost, on average, 9 granules/profile. Neonatal Neutrophils: Ultrastructural Evaluation of Treatment with A23187:3H0rphametric Determinations: Morphometric measurements were then_made on electron micrographs 102 Table 21 NEONATAL NEUTROPHILS: ULTRASTRUCTURAL GRANULE QUANTITATION OF A23187 TREATED CELLS Granules/Cell Profile Peroxidase Positive Peroxidase Negative Baseline (38) 84.5+15.4 S9.Q310.1 nM A23187 100 (32) 82.8:17.0 52.719.7* 250 (40) 80.2:15.0 SO.1¢9.3* Neutrophils were warmed for 5 minutes at 37C, A23187 was added, incubation continued for 5 minutes. Pooled data from 5 neonatal neutrophil donors. BaSeline: Cell incubated for 5 minutes at 37C before fixing. Meanistandard deviation (Number of micrographs analyzed) * p<0.01 vs baseline 103 of ionophore treated newborn neutrophils to investigate a correlation between A23187 mediated visible ruffling, intracellular granule depletion and morphometric alterations.- The results of these measurements are shown in Table 22 and Figure 13 B. Compared to baseline measurements, 5 minutes exposure to 100nM or 250nM A23187 caused significant increases in cell perimeter (p<0.01 and p<0.005, respectively) and exposed surface membrane (p<0.005 for both 100nM and 250nM A23187). Comparisons of the results from morphometric determinations made on A23187 treated adult and neonatal cell profiles are shown in Table 23. As indicated above, nonactivated neonatal cells were found to be significantly larger in area and expose significantly less surface membrane than adult neutrophils. Compared to ionophore treated adult cells, identically treated neonatal neutrophils were comparable in perimeter, calculated area and exposed plasma membrane determinations. As indicated previously, temperature has been reported to cause functional responses in neutrophils (Berger et.al., 1984; Charo et.al.,l985; Corcino et.a1., 1970: Dewald et.al.,.1984; Fearon and Collins, 1983; Goldstein et.al., 1974; Howard and Oresajo, 1985; Springer et.al., 1986; Wright and Gallin, 1979). In this investigation, neutrophils were exposed to a temperature transition to determine whether temperature alone could induce functional and morphological responses similar to those seen early in fMLP mediated activation and following exposure of cells to A23187. Tamperature»Tranaition Mediated Shape Change: A The effect.of a temperature transition on neutrophil shape was (investigated.with.light microscopy. Neutrophils were isolated in the- 104 coaumcwauouoc mafiaomma m> Ho.ovm * Awmumamcm magnumonofia mo nonaszv coaumfi>ou unaccounfldmoz .wcwxau onowon ohm um mouscaa m cmumnsocfi mHHoo .mnHHommm .mnoaou Hangouusmc m scum mumv uoHoom .mouscfia m now uosefiucoo :ofiumnsocfi .covum mm: mwfimm< .Unm an mounnfia m coaum3ona one: mafinnonunoz *mq.wnfiwmm.hom om.owmq.H nm.nfimw<.sh woo.~flmwm.~5 Aamv omm *mm.~o+nh.nqm m~.o+om.~ mo.nn+om.ms *eH.oH+Hc.~o Anne con .. I I. .. swam~< z: mm.mon+mh.q~a -.o+on.fi ms.-+m~.m~ No.nn+~n.no Ammv mqnflommm m adv ocmunaoz .MVW mou¢. mmmmmwmmm N vmnmflauamu maqmo mm9 N 100 m A23187 250 m A23187 - PERIDETER V/A AREA [:1 m. mm Figure 13 ; ULTRASTRUCTURAL EVALUATION OF A23187 TREATED NEUTROPHILS Data from Tables 21 and 22 expressed as Z deviation from baseline 106 Table 23 NEONATAL vs ADULT NEUTROPHILS: EXPOSURE TO A23187: MORPHOMETRIC DETERMINATIONS Calculated Perimeter Area. §[!_ surface membrane Baseline a f + + nM A23187 1m I = = = 250 ' a a a a Results are eXpressed as significant differences between mean determinations made from neonatal and adult cell profiles (p<0.01) a: No difference ' 1: Significant increase, neonatal vs adult : Significant decrease, neonatal vs adult Baseline: Cells were incubated for 5 minutes at 37C and fixed 107 3 cold and maintained at approximately 4C until use. A sample of the cell suspension was fixed on ice to serve as the baseline for shape change determinations. Additional samples were transferred to a 37C water bath and fixed at time points ranging from 1 to 60 minutes. Although the cells fixed on ice clearly were not ruffled, approximately 80% of them were found to exhibit membrane extensions. In contrast to ruffles these extensions were short and blunt rather than the thin, spiked, sheet-like membrane protrusions typical of ruffles (Figure 14). Table 24 and Figure 15 show the results of shape change determinations made on cells incubated at 37C. The membrane extensions detected on cold cells disappeared after the cells were warmed for approximately three minutes at 37C (Figure 14). Warmed cells took on a typical spherical appearance, and remained spherical for up to 60 minutes. The temperatures established during the transition were determined to approximate the temperature at which the membrane extension were resolved. Tubes containing neutrophil suspensions were transferred from ice to a 370 water bath. At various time points the temperature was recorded. As seen in Table 25, a dramatic increase was seen in the first 30 seconds with the maximum temperature being reached within about five minutes. At 3 minutes, the incubation time at which 85% of the cells exhibited spherical shapes (Table 24 and Figure 15), the temperature of the cell suspension was approximately 34C. Temperature Transition Hediated Extracellular Granule Release: In contrast to fMLP mediated activation, warming neutrophils _ isolated in the cold induced the resolution, rather than the 108 Figure 14 ' . NEUTROPHILS FIXED IN THE COLD: LIGHT MICROSCOPY Upper panels: Adult neutrophils isolated and fixed in the cold: Note short, blunt membrane extensions occasionally' appearing as blebs. Lower panel: Cells isolated in the cold were warmed for 5 minutes at 37C prior to fixation. Note the loss or resolution of the membrane extensions seen on cells fixed in the cold. Bar = lOum 110 NH OH OH OH ON «a Aasomsm N mHm mwm mwm owes mHmH mwws aims o+o coamwsm N mums ammo muss Hausa ognmm “sums Names Lisa HQUHHQfiqm N :ofiumw>oc cumuamuufiswoz woos co coxflm magnaouusoa “onwaommm fifiifigfigfiifigfifl 4. HH Hm mcoamcouxo oemunaoa cu“: N as me on ma as m m Aucaammamv o own as nausea: muz0.25 for lactoferrin and lysozyme). TauperatnrelTranaitionInadiated Extracellular Granule Release: Affect.o£.leutr0phil Isolation Tachniqne:. 114 cmumquQ moummma :H couumumw NNV o vaas. «Has. a NNvom. NMom. a vaoo. sMom. 0 vamm. NMaN. o vaNN. sHNN. m vaom. aMmo. N Navmq. oMNw. a vawN. oMmN. N vaNo. NMNN. m Novas. NHNo. s ommafiumaoo .a. z Amvmm. H+ow. m $N. z NNVaN. oMNN. m NNva. oHNN. s ommcommaaoo ommmHoN mafiaom Amucmawuo wocfiau :owumfi>mu oNua muNaa IoNo.QHosN.o one muoN N as. oH -oNI N NN. m on: muaca N .m¢+mH. New NNua N: mm. onc. N "coaumfiuusam an vaou on» :H umumaomfl maflnaouusoc vamN.NHmm.m *vaoo. NMNN. a *NsVNN. «MNN. N NNVNN. NMNN. a vamm. NMNN. m vamm. 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Si N N1N 338N330 Nu: NNNNN N1 «NHN9 NNN "maNuomNN NN-: N: N9 oHN1 N NcNatoNouuaN «Hum new om moHnmh mHmuou voaHasoov mHquoNusoe soHHHHa H souw moummmH :« wouoouov unoeoaaoo Houou mo aeouuoa ms commouaxo NH ommoHom .N.z ANN NN.N .N.z NNNNN.HHNN.N NNNNN.QHNN.N NNNNNN NNvNN.HHNN.N NNVN9 NHN9 N NNNNN.H:_9 N *NNNN9 NMo1 N *NNNNNN.HMWN.N oN NNV o NNVN1 NHN9 oN 9 z *NNNN9 NMN1 N *NoNVNN.oHNN.N ms NNV o NNNN9 NMN1 N NNvN9N 9 N NNNN1N NHNN. s *NNNNN1 NMN1 N ON NNN o NNNN9 MHNN. N NNVo9 NM N1 N NNNNN.N NHN9 N *NeNvN9 NMNN. N NN NNV o NNNN9 NHN9 oN NNNNN. N# 9 N NNNN1N .HN1 N *NNNVs9 NMNN. N oN ANN o Neve9 NMN1 N 9 z NNvN9 NHN9 N *NNNNNN. oMo1 N N NNN o NNvN1 NMNN. N NNNN9 NHN NN. N NNVNN.N NHNN. s *NNNNN9 NMNN. N N .N.z Nsvs1 NMN1 N 9 z NNNNN.N N.HN1 N *NNNVN1 NMNN. N N ANN o NNVa9 NMN1 N NNNNN. NMNN. N NNNNN.N N1 N *NNNVNN. NMNN. N N ANN o NNNNN. NHN9 s NNNNN. oHNN. s NNNNN.NHNN.N NNNNNN. oHoN. o NmaNNomoNN o unavHGOHNNU—HHNIQ manning—”flu OmflcmmeHOU QENNOflNA :WMHOMOHWQIH. Nueoeonaoo oHseouu “ohm an moussfiz mm=oH2=UMH HzmHn=\HHoUHm mo zOHH No.0vn * Amucmafiuqum mo Nonsszv wonfiaumuon uoz N.n.z coauma>mu cumucmudemmz .mmuacfia om .ucoaapoaxo map mo coaumNsc onu Now Hmowsoso onu saw: ooH :0 can: mHHoo .osaaommm Na: Na NN. o H N1 N .NNNaNNmNoNoNu NENNNNN Nu: w: No. 0 + m¢. N .omoosflmmxoovlu 23m NnN N: N9 o.H.NN.N "NNNN zaN Nu: N: NN. o + NN.N NNN NNNNu++Nz.++No "ohm um mouscfla 00 Na cmsoaaow oUN :o mouscfia ON Now usmmm umou saw: vmumnaocfi mHHmo :oNHHNa N souw umumqouq moummma :N :wuuowouoma Hmuou mo ucoouma mm commmuaxo mmmoaom . .oow :o mouscas ON Hmowaogo umou :uNB woumnsocfioua mHHmU 123 ANVNN.N+NN N AN vN N1 N ANNNN.NMNN.N ANNNN.NMNN.N ON 9 z ANNN1 N *ANVN1 NMNN. N ANNNN.NHNN.N NN ANVN9 NHN9 N ANNN1 N *ANVNN. NMN1 N ANVNN. NMN9 N NN ANNN1 o+N1 N ANNNN.N ANVNN. NMN1 N ANNNN. NMN1 N NN 9 z ANvoo.N ANNNN. NMo1 N ANNN1 oMo9 N ON 9 z ANNNN.N ANvN1 NMN1 N ANNN9 NMN9 N N ANVNN. NHN 9 N ANVNN N ANvN1 NMN9 N ANvN1 NMN1 N N 9 z ANVNN N ANVN1 NMN1 N ANNN1 NMN9 N N 9 z ANvoo.N ANVN1 NMN9 N ANVNN.NHNN.N N ANNNN.NHN9 o ANVNN.N ANNNN. NHNN. o ANNNN.o+NN.N ANNNNNNNNV N NNNaNmeoNuNN NNNNNNN NNN-N zaN NNNN :aN NNNN1++N2.++NN “Hmofiamao ummh. "Ohm um awn—2.3”: mm mo Bummmm mmh oN 0NNNN. 124 Under the conditions used, 5mM EDTA demonstrated significant inhibition of lactoferrin release following 30 and 45 minutes incubation of cells at 37C (p<0.005 and p<0.01, respectively). Tauperatnre Transitionlflediated Extracellular Granule Release: Effect of Cytochalasin B: Next, experiments were performed to determine whether CB influenced temperature mediated extracellular granule release. Neutrophils were preincubated with Sug/ml of CB for 20 minutes on ice. The cell suspensions were then incubated at 37C for variouS'time periods. Supernatants and prepared lysates were evaluated for lactoferrin, lysozyme and gelatinase content. As seen in Table 31, CB had no affect on the total cellular content or spontaneous temperature dependent release of lactoferrin, lysozyme or gelatinase. The Effect of Various Chemicals on Temperature Transition Mediated Shape Change and Cell Viability: In terms of visual shape change, none of the chemicals tested altered the morphological responses mediated by the temperature transition. Neutrophils were preincubated with the chemicals for 20 minutes on ice followed by incubation at 37C. Membrane extensions were found on cells fixed in the cold but were resolved if the cells warmed and then fixed (data not shown). To be certain that the lactoferrin detected extracellularly in response to the temperature transition was not due to cell death, viability determinations (by trypan blue exclusion) were made on neutrophils treated with the chemicals or held in complete PBS for 20 minutes on ice followed by incubation 37C for 60 minutes. Cell viability.exceeded.96% for PBS control cells as well as for cells 125 ANvNN.NHNN.N ANvN9 NMNN. NN ANVN9 NMN9 NN ANNN9 NMN1 NN ANVNN. NMNN. NN ANVNN. NMN1 NN ANNN1 NMN1 NN ANvNN. NMNN. oN ANNNN. NHNN. NN ANNNN. NHN1 N ANN c OmflGHUQHQO .n.z ANvNN.NMNo.N ANNNN.N+NN.N ANNN1 NHN9 NN ANNNN. NMN9 N ANNNN. NMN9 N ANNNN. NHN N. N ANNN9N N1 N ANNNN. NW N. N ANNN1 NMN1 N ANVN9 NHN9 N mauuomnq .mo usonufiz mafiaaouusm: scum omnmamu m> No.0va * Amucmsfiumaxo mo Nonaszv cocHaNouon uoz.wwn.z :oNumN>mu uuwcemum+cmmz .mouscfis ow .uamaNumaxm mo cowumuav Now mo «0 Hs\w:m nufiz ooH so was: mHHoo soum ommoaom Nocfiammmm .Am mHanv mu Ha\w=m mo ouammmua mcu :« mHHmo :oNHHNa H scum vmumaoaq moummma Hamo :H cocfimueoo Hmuou co comma omnoaou ueoopoa mm commounxm mum muasmom .moN so mouscfia ON Now mo mo Hs\w:m :ua: woumnsucfimua who: mcofimeoqmsm Hangouuaoz .n.z ANNNN.oHNo.N ANvNN. NMNN. N ANNN9 NMN1 N ANVN1 oMN9 N ANVN9 NMN1 N ANNNN. NMNN. N ANVNN. NMN1 N ANvN9 NMN9 N ANNN1 NMNN. N ANNNN.N+NN.N :Huuomouomq Nucocoasoo oascmuu oo\unm ow mq on ma OH H m m H o Aocaaommmv "ohm um nausea: mm0.25, . respectively). Additionally, although loss of intracellular peroxidase negative granules was detected in response to the temperature transition (approximately 6 granules/cell profile after 5 minutes and 7 granules/profile following 30 minutes incubation), the loss was not highly significant (p<0.025 and p<0.05 for 5 and 30 minutes incubation at 37C, respectively). Ultrastructural Ehaluation of the Temperature Transition: Morphometric Determinations: Ultrastructurally, the membrane extensions seen with light microscopy on neutrophils isolated in the cold appeared to be thin protrusions of plasma membrane containing a meshwork of fine filamentous material. In most cases these structures appeared to bend back toward the cell body forming loops. In some cell sections, 127 Table 32 ' . ULTRASTRUCTURAL GRANULE QUANTITATION: TEMPERATURE TRANSITION Granules/Cell Profile: Minutes at 37C: Peroxidase Positive Peroxidase Negative 0 (Baseline) (17) 70.618.S 59.1i§.3 S (16) 65.4¢§.9 53.41§.7 30 (7) 73.4+10.8 52.Qi§.2 Neutrophils were isolated from a single donor Baseline: Cells fixed on ice. Meanistandard deviation (Number of micrographs analyzed) * p<0.01 vs baseline determination 128 contact between the tip of an extension and the cell body could be seen. Generally the region between the cell body and the membrane loop was devoid of cellular material. however, in some instances there were smaller membrane protrusions contained within the primary loop. These structures were not numerous on an individual cell and seemed to be regionally localized. The remaining plasma membrane was very smooth (Figure 19). To determine whether cells exposed to the temperature transition exhibited quantitative morphometric changes correlating with the detection of extracellular lactoferrin and the modest depletion of intracellular granules, electron micrographs of neutrophils fixed on ice or warmed for 5 or 30 minutes at 37C were measured. The data shown in Table 33 and Figure 18 B indicate that the conditions which mediated temperature dependent shape change and lactoferrin release had no influence on ultrastructural morphometric .determinations. Mean cell perimeter, calculated area, surface-to—volume ratios and exposed plasma membrane determinations made of profiles from neutrophils incubated at 37C for either 5 or 30 minutes were identical to those made from cells held on ice. Summary of Results: The results of these investigations indicate 1) that the morphological event of neutrophil ruffling and the functional event extracellular granule secretion are associated, 2) that these events of activation produce quantitative morphometric alterations and 3) that these morphometric alterations may be specifically related to the . activation-induced release_of a particular type of granule containing 129 relatively high concentrations of lactoferrin. 130 Figure 19 NEUTROPHILS FIXED IN THE COLD: TEM (Figure 19 is comprised of 3 pages) 1. First panel: Grazed cell showing delicate membrane loops. Second panel: Note fine membrane loops on upper and left portion of the cell and blunt protrusion on right side of the cells. Also notice that the remaining cell surface is relatively smooth. Bars 2 lum 132 Figure 19 (continued) 2 and 3. The panels shown on the following two pages are sections of a cell isolated and fixed in the cold. Note the intricate membrane loOping and what appears to be possible membrane shedding. Bars a lum m-—--------—-------—-------- 135 NN.NNMNN.NNN NN.NNHNN.NNN NN.NoN+NN.NNN m asvosmunaoz NNNNNNUNNN nauseouzmmoa oswammmn m> No.0va * AvoNNHmsm mznmpmouuwa mo nonaszv :oNumN>on cumcsmum+smoz .ooN so voxwm mHHoo ”mafiaommm Nocou oawcfim m scum woumaomw who: mflfinqouusoz NN.NMNN.NN NN.NMNN.NN . ANN NN NN.NHNN.NN NN.NHNN.NN NNNV N NN.N+NN.NN NN.N+NN.oN NNNV NNNNNNNNNV o wmh< hmuwafihmm uoNN No mouscwz onHHmz0.25; Table 10 and Figure 6 B). The results from these investigations and the study reported by ,.Hoffstein-et.al..(1982)«suggest that-increased plasma membrane, 150 evidently initiated by the merging of peroxidase negative granule and plasma membranes during activated granule secretion, is a dose dependent, kinetically transient and apparently regulated response to activation. Neutrophils activated for 10 seconds with 100nM fMLP or 20 seconds with 1nM fMLP exhibited significant peroxidase negative granule depletion and increased exposed surface membrane.‘ However, while exhibiting comparable granule depletion, neutrophils activated with 100nM fMLP for 20 seconds did not demonstrate an increase in exposed plasma membrane. The proposed transient and regulated increase in exposed plasma membrane in response to conditions mediating cellular ruffling and granule secretion may also be suggested by the results from ultrastructural morphometric and granule quantitations made on calcium ionophore treated cells. While neutrophils exposed to 100nM or 250nM A23187 for 5 minutes exhibited a significant depletion in intracellular peroxidase negative granules (Table 14 and Figure 10 A), they did not exhibit significant alterations in exposed surface membrane (Table 16 and Figure 10 B). Possibly, during the 5 minutes treatment with ionophore the cells underwent a "regulation"-like response. This response could have returned a transient increase in exposed plasma membrane, proposed to have been initiated by granule-plasma membrane fusion, back to basal levels before the cells were fixed for electron microscopy. The next series of experiments, made on ruffled neonatal neutrophils, may implicate lactoferrin-containing granules in cellular morphology. The results from the ultrastructural evaluations performed on neonatal cell profiles suggest that although visibly 151 identical, neonatal and adult neutrophils differ morphometrically. When viewed with light microscopy fixed neonatal and adult neutrophils appeared to be identical, with approximately 90% of unactivated cells displaying a spherical shape. However, as shown in the baseline comparisons on Tables 20 and 23, unactivated neonatal and adult cells were found to be morphometrically different. While the mean perimeter determinations of unactivated newborn cells did not differ from perimeter measurements made of adult cell profiles, neonatal cells exhibited significantly larger average area and less exposed surface membrane. Since 2 dimensional area measurements may be considered representative of 3 dimensional volume, this finding suggests that unactivated neonatal cells are larger in volume but smoother, possibly due to fewer or less extensive membrane protrusions, than adult cells. In terms of visual assessment of activation mediated shape change, adult and neonatal cells exhibited similar responses. Neutrophils isolated from adults or newborns ruffled within 20 seconds following exposure to 1nM fMLP (compare Table 3 and 17). In addition, the cells were found to ruffle within 1 minute and maintain ruffled configurations for up to 10 minutes following exposure to A23187 (Table 11, neonatal data not shown). However, examination of the ultrastructural data from activated neonatal cells suggests that neonatal and adult neutrophils differ in their early responses to activation and further, that lactoferrin-containing peroxidase negative granules may be intimately involved in alterations in cell morphology. In contrast to adult cells, neonatal neutrophils exposed to 1nM fMLP for 20 seconds did not exhibit a highly significant loss of-intracsllular granules (Table 18 and Figure 12 A) nor an increase 152 in perimeter or exposed plasma membrane (Table 19, Figure 12 B). However, after 20 seconds activation with 100nM fMLP, neonatal cells, like adult cells, demonstrated a significant loss of intracellular peroxidase negative granules (Table 18, Figure 12 A) while not exhibiting significant alterations in exposed surface membrane. Additionally, unlike adult cells, 100nM fMLP treated newborn cells did not exhibit increased average perimeter measurements (Table 19 and Figure 12 B). If increased perimeter is representative of an activated ruffling response (as suggested by the morphometric perimeter measurements made on fMLP and A23187 activated adult cells, Tables 10 and 16), then the lack of significant perimeter alterations in neonatal cells may suggest that, even though visibly comparable, the fMLP mediated ruffling response of neonatal neutrophils differs mechanistically from the adult response. Further, since newborn neutrophils have depressed lactoferrin levels (Ambruso et.a1., 1984; Becker-Freeman et.al., 1894), it could implicate the proposed lactoferrin-containing granules in early activation mediated neutrophil shape change. In summary, comparing the results from granule depletion and morphometric measurements of adult and neonatal cells briefly activated with fMLP suggest that 1) a population of lactoferrin-containing granules exists in normal adult cells, 2) this population of granules is selectively secreted prior to other peroxidase negative granules and 3) these granules are intimately linvolved in fMLP mediated shape change responses. The hypothesis that lactoferrin-containing granules may be involved in the morphometric ,alterations seen in activated normal adult neutrophils appears to 153 corroborated by Boxer and coworkers_(l982b). These investigators continued examinations of 25 year old male initially identified by Strauss and associates (1974). This patient was described to have suffered from severe, recurrent bacterial infections since birth. Functionally, his isolated neutrophils exhibited depressed adherence associated activities (adhesion to cultured edothelium, aggregation and chemotaxis, and an impaired ability to decrease cell-surface charge in response to activation) and abnormal hydroxyl radical production. Ultrastructurally, his neutrophils were found to contain a large number of intracellular granules, the majority of which were peroxidase positive. Secondary granules were reported to develop later in neutrophil maturation that normal and appeared small and generally deficient in content in mature cells. Determinations of total granule components in lysates prepared from the patient's cells revealed normal levels of lysozyme and beta-glucuronidase but only about 3.5% of the normal level of lactoferrin. Morphometric measurements were made on electron micrographs of unactivated neutrophils and cells briefly exposed to 100nM fMLP. Surface—to-volume ratios of normal control cells were reported to increase approximately 36% in response to activation. In contrast, the S/V determined for unactivated and fMLP activated lactoferrin deficient cells were identical. The results presented in Table 19 of this report show that S/V calculated for 20 seconds 1nM and 100nM fMLP activated neonatal cells were also identical to unactivated baseline levels. Therefore, published data and the data presented in this report appear to indicate that lactoferrin-containing peroxidase negative granules may be-involved«in-normal activation mediated 154 morphometric changes in neutrophils. Evidence indicating that a papulation of lactoferrin-containing peroxidase negative granules may be involved in an apparent regulation of exposed plasma membrane is suggested from the results of the ultrastructural quantitations made of calcium ionophore treated newborn cells. As indicated above, neonatal cells exposed to A23187 demonstrated a visual ruffling response and intracellular peroxidase-negative granule loss which was comparable to that found in identically treated adult cells (compare Tables 14 and 21). While ionophore treated adult neutrophils did not exhibit significant changes in exposed surface membrane compared to nonactivated determinations (Table 16, Figure 10 B), identically treated neonatal cells did. Newborn cells exposed to 100nM or 250nM A23187 for 5 minutes exhibited significant increased surface membrane (Table 22 and Figure 13 B). Although unactivated neonatal cells were found to exhibit_less surface membrane than unactivated adult cells, the morphometric determinations of neonatal cells after 5 minutes treatment with 100nM or 250nM A23187 were equivalent to adult determinations (Table 23). Therefore, neonatal cells responded to A23187 exposure by depletion of intracellular granules and, in contrast to adult cells, a coincident increase in exposed surface membrane. Secretion of a population of peroxidase negative granules containing primarily lactoferrin, which in some fashion is associated with regulation of exposed surface membrane, may help to eXplain the finding thatA23187 treated neonatal cells exhibited increased exposed plasma membranes while adult cells did not. -Thus, the~ultrastructural evaluations of morphology and granule 155 quantitation of fMLP and A23187 treated neonatal neutrophils provide circumstantial evidence suggesting an association between the secretion of a population of lactoferrin-containing granules and the generation and apparent regulation of increased exposed plasma membrane. In contrast to fMLP and A23187 mediated cellular shape change, neutrophils exposed to a temperature transition from 4C to 37C were found to loose, rather than generate, membrane extensions. Shape change determinations of cells isolated and fixed in the cold revealed that approximately 80% of the cells exhibited short, blunt membrane extensions. As cells isolated in the cold were warmed, these structures disappeared (Table 24, Figure 15). The resolution of the membrane protrusions was found to coincide with the detection of significant quantities of lactoferrin in cell-free supernatants with both events occurring at approximately 22C. It is tempting to speculate that temperature dependent lactoferrin release was associated with the change in neutrophil morphology seen when cold cells were warmed, analogous to fMLP or A23187 mediated ruffling and granule secretion. However, although neutrophils treated with a temperature transition exhibited visual morphological alterations, unlike fMLP or A23187 treated cells, none of the morphometric determinations made on electron micrographs of cells incubated for 5 or 30 minutes at 37C differed significantly from cells maintained at 4C (Table 33 and Figure 18 B). The results from the ultrastructural morphometric and granule quantitations made on neutrophils exposed to the temperature .transition may indicate that a critical level of 156 lactoferrin-containing granule release is required to produce the proposed transient increase in eXposed surface membrane. As indicated previously, adult neutrophils briefly activated with 1nM fMIP exhibited a loss of approximately 19% of their intracellular peroxidase negative granules, and an apparent concomitant increase in exposed plasma membrane (Figure 6 A and B). The temperature transition had no affect what so ever on the morphometric ‘ determinations (Table 33) and yet caused the loss of approximately 10% of the total intracellular peroxidase negative granules (Figure 18 A). The lactoferrin content of these granules was determined to be comparable to the lactoferrin/granule calculated for adult cells activated with fMLP. If these granules are responsible for initiating an increase in exposed surface membrane in adult cells, perhaps the extent of release in response to the temperature transition was not sufficient to generate such an increase. Alternatively, increased surface membrane may require a true activation initiated response, suggesting that granule secretion alone may not be sufficient to cause alterations in the quantity of surface membrane. SPECULKTIOIS Early Lactoferrin Containing-Granule Release and Coincident Dp-Regulation of Surface Membrane Moieties As indicated previously, recent reports have shown that activation of neutrophils causes increased expression of certain surface membrane components possibly though the mobilization of intracellular pools of these components (Fearon and Collins, 1983; Lanier et.al., 1985; Thrall et.al., 1980; Zigmond et.al., 1982). Secondary granule membranes have been implicated as the intracellular pool for at least some of these membrane moieties (Arnaout et.al., 1984; Fletcher and Gallin,1980; Todd et.al.,1984). Therefore, low level secondary granule secretion, resulting in granule-plasma membrane merger, could be the mechanism for the up—regulation of these components (Arnaout et.al., 1984). A temperature transition from 40 to 37C has been reported to mediate spontaneous temperature dependent increases in CR1, the C3b receptor, and CR3, the iC3b receptor, analogous to the Mo 1 antigen (Berger et.al., 1984; Fearon and Collins, 1983). These investigators found two-fold increases in surface expression of each receptor after neutrophils, which had been isolated in the cold, were incubated for approximately 6 minutes at 37C. Treatment.of cells with protein synthesis inhibitors had no affect on the spontaneous up—regulation of 157 158 the receptors suggesting that the receptors were preformed and 'localized in an intracellular pool (Berger et.al. 1984). Temperature mediated increase did not represent maximal expression of the receptors as additional up-regulation occurred when the warmed cells were exposed to fMLP.(Berger et.al., 1984) or C53 des arg, a chemotactically active cleavage product of the fifth component of complement (Fearon and Collins, 1983). Spontaneous increased expression of CR1 and CR3 was specific for these membrane glycoproteins since the temperature transition had no affect on the concentration of other cell surface components (Berger et.al. 1984; Fearon and Collins 1983). Charo and coworkers (1985) have reported that spontaneous temperature dependent enhanced adherence occurs in human neutrophils. Cells maintained at 4C were allowed to adhere to albumin coated plastic or cultured human umbilical vein endothelial cells at 37C. Enhanced neutrophil adherence was seen after 2 minutes and plateaued after 16 to 20 minutes. Increased adherence was not detected when the experiments were performed at 4C. Since CR3 (Anderson et.al. 1986; Arnaout et.al. 1983; Dana et.al. 1984; Gallin 1985) and lactoferrin (Boxer et.al., 1982a; Boxer et.al. 1982c; Oseas et.al., 1981) have been implicated in neutrophil-substratum adherence activities the spontaneous temperature dependent adherence reported by these investigators may have been mediated by a coincident spontaneous up-regulation of CR3 and extracellular lactoferrin release. Although secondary granule membranes have been implicated as the intracellular pool of CR1 and CR3 (Arnaout et.a1., 1984; Todd et.a1., . 1984)~0‘Shea:and‘coworkers (1985) have'reported evidence suggesting 159 that the CR1 and CR3 antigens are localized in distinct intracellular pools. Neutrophils isolated from a patient with secondary neutrophilic granule deficiency were activated with fMLP and evaluated for expression of the complement receptors.. Increased binding of labeled anti-CR1 was found while the binding of anti-CR3 did not increase. Neutrophils isolated from a normal donor and identically activated with fMLP exhibited up-regulation of both CR1 and CR3. Normal cells were also disrupted and fractionated on sucrose density gradients. The fractions were then evaluated for the ability to bind labeled anti-CR1 and anti-CR3. The plasma membrane fraction reacted with antibodies against both receptors while the secondary granule fraction only bound anti-CR3. Neither antiNCRl nor anti-CR3 bound to the primary granule fraction. These findings suggested that the intracellular pool of CR1 was not only distinct from the pool for CR3, but also suggested that intracellular CR1 may not be associated with classical secondary granule membranes. Berger and associates (1985) have reported that the mechanism for spontaneous CR1 and CR3 up-regulation differs. Temperature dependent increased expression of CR1 was found to be independent of extracellular Ca++ occurring in the presence of 5mM EDTA. However, the mobilization of intracellular Ca++ was determined to be necessary for up-regulation. In contrast, up-regulation of CR3 required both extracellular Ca++ and mobilization of intracellular Ca++ stores. Additional evidence Suggesting that the intracellular pools of CR1 and CR3 may not be identical and further suggesting that lactoferrin may not be directly associated with the pool for CR3 is - provided by the human promyelocytic leukemia cell line, HL—60. When 160 induced with retinoic acid (RA) or dimethylsulfoxide (DMSO) HL-60 cells differentiate into neutrophil-like cells expressing granulocyte surface receptors (Atkinson and Jones, 1984; Hoyle and Freer, 1984; Pommier et.a1., 1984). While both RA and DMSO have been found to induce lysozyme production, neither agent has been reported to mediate the production of lactoferrin (Harris and Ralph, 1985; Olsson et.al., 1981). DMSO has also been reported to induce surface eXpression of Mo 1 (i.e., CR3) (Pommier et.al., 1984). In consideration of the published reports described above, the data presented in this study may suggest that lactoferrin is closely associated the intracellular pool of the CR1 receptor. Kinetically, the reported temperature dependent increased surface expression of CR1 and the lactoferrin release presented here appear to be identical, both occurring and plateauing within 6 minutes at 37C. The reported CR1 up-regulation and the early temperature dependent lactoferrin release presented in this investigation both occurred in the presence of 5mM EDTA suggesting that extracellular calcium was not essential for either event. While some reports propose that both CR1 and CR3 are associated with secondary granules there is evidence indicating that they are localized in distinct intracellular pools. Since the data presented here appear to indicate that a population of selectively or sequentially secreted lactoferrin-containing peroxidase negative granules exists in normal adult neutrophils, such granules. may represent the distinct, yet secondary granule pool of CR1. This hypothesis requires further investigation. 161 Mechania for Apparent Regulation of Increased Exposed SurfacefiMEmhrane The.data presented in these investigations suggest that the release of lactoferrin-containing peroxidase negative granules may be associated with an alteration in the quantity of exposed cell surface membrane. In these studies surface-to—volume ratios were determined to describe the 2 dimensional perimeter and area measurements made from electron micrographs in 3 dimensional terms. The calculated S/V was then multiplied by a mean neutrophil volume determination (Korchak and Weissmann, 1978) to estimate the amount of surface membrane exposed on a whole cell. The results from the ultrastructural determinations made in these studies suggested that an increase in exposed surface membrane may require a critical level of peroxidase negative lactoferrin-containing granule loss, was transient and that neutrophils have a mechanism for apparently regulating the amount of exposed membrane. This apparent "regulation" of surface membrane may actually be a "compensation" response which is initiated by cell activation. This possibility appears to be suggested by examining the perimeter and area measurements made from cell profiles (also see Appendix B). The calculated exposed surface membrane depends upon a relationship between surface area and volume, or in two dimensional terms, perimeter and cross-sectional area. Therefore, a change in perimeter or area relative to one another produces an alteration in the calculated S/V ratio and consequently a change in the calculated exposed surface membrane. For example, the mean perimeter determination made from micrographs of adult neutrophils activated for 162 20 seconds with 1nM fMLP was significantly larger than the determination made from nonactivated cell profiles, while the mean area measurement was not significantly different. Thus, the perimeter increased relative to the area and the calculated S/V was larger than the control value. Consequently, the calculated exposed surface membrane for adult cells activated with 1nM fMLP for 20 seconds was _greater than that for nonactivated control cells (Table 10). In contrast, while the average perimeter measurement of neonatal neutrophils exposed to 1nM fMLP was not significantly different from control, it was less. The mean area measurement of these cells was equivalent to control values. Since the perimeter decreased relative to the area, the calculated S/V was smaller than the baseline value. Thus, the calculated exposed surface membrane of neonatal cells treated with 1nM fMLP was less than that of unactivated control newborn cells (Table 19). Adult cells treated with 100nM fMLP for 20 seconds or 100nM A23187 for 5 minutes might provide an example of exposed membrane "regulation". In these cells, both perimeter and area measurements were significantly larger than control values. Since both perimeter and area increased relative to each other, the calculated S/V ratio was comparable to control and no significant difference was detected in eXposed surface membrane determination (Tables 10 and 16). In each fMLP and A23187 experimental condition examined, adult cells responded by increasing in perimeter (probably reflective of ruffling) and, to a degree, their cross-sectional area (Figures 6 B and 10 B). Therefore, the results from the morphometric determinations could indicate that an initial result of activation 163 mediated neutrophil ruffling and coincident granule release may be granule membrane and plasma membrane fusion resulting in increased exposed surface membrane.' By subsequently increasing in crOss-sectional area neutrophils may compensate for, or apparently regulate, this increase in exposed plasma membrane. The morphometric measurements made on electron micrographs of A23187 treated neonatal neutrophils demonstrates a lack of this proposed compensation response. As shown in Table 22, the mean perimeter determinations of newborn cells treated with 100nM or 250nM A23187 for 5 minutes were significantly increased over baseline measurements, while the average area determinations were identical to baseline levels. Therefore, the S/V increased significantly, as did the calculated exposed membrane determination. In 2 dimensional measurements, as those made on the electron micrographs, area determinations are representative of cell volume. Therefore, an increase in the cross—sectional area of a cell profile over baseline levels may be indicative of an increase in volume. Hsu and Becker (1975) and O'Flaherty and associates (1977) have reported that neutrophil activation causes an increase in cell volume. However, these determinations were made with electronic particle analyzers which do not take into consideration activation mediated cell ruffling or bipolar configurations. Conceivably, cellular shape changes could have been misrepresented by these analyzers as changes in cell volume. A recent report (Grinstein et.a1., 1986) assessed the volume of activated human neutrOphils by three independent methods, forward angle light scattering, isotopic water space determinations and electronic sizing, and concluded that cell swelling did indeed \ 164 occur in response to neutrophil activation with fMLP. Maximum increases of approximately 21% over the basal cell volume were found to occur within three minutes exposure of cells to 10nM fMLP at 37C, with half-maximum increases in about 30 seconds. Activation mediated volume increases were inhibited by amiloride (a K+ sparing diuretic which inhibits extracellular Na+/intracellular H+ exchange) or by replacing extracellular Na+ with K+ or a non-membrane permeating organic cation, N-methyl-D—glucamine+. These results suggested that sodium ions play a critical role in activation mediated neutrophil volume alterations. Exposure of human neutrophils to fMLP has been reported to cause increased Na+ fluxes. Simchowitz (1985a) determined that labeled extracellular Na+ passively diffused in and out of resting human neutrophils through a oubain— and amiloride—insensitive ion channel. (Ouabian blocks the Na+/K+ pump by inhibiting Na+/K+ ATPase.) When the cells were exposed to 100nM fMLP Na+ influx was enhanced approximately 30 fold. Influx was found to be extracellular Na+ concentration dependent, saturable, sustained for at least 60 minutes after exposure of cells to fMLP, membrane voltage independent and oubain insensitive. fMLP was also found to stimulate the efflux of Na+ from cells preincubated with labeled Na+, with 50% maximum occurring at approximately 2.5 minutes following cell exposure to 100nM fMLP. Despite Na+ efflux, the net intracellular Na+ concentration has been reported to increase in response to neutrophil activation. Using flame photometry, Simchowitz (1985a) and Grinstein et.al. (1986) .reported that intracellular Na+ levels increased approximately 2 fold 165 ' and plateaued within 5 minutes after exposure of cells to 100nM fMLP, with 50% maximum concentration reached in about 60 seconds (Simchowitz 1985a). While amiloride had no affect on Na+ fluxes of resting cells, it inhibited activation induced Na+ in- and effluxes. Additionally, monensin, an ionophore which facilitates the exchange of extracellular Na+ for intracellular H+, was found to mimic fMLP induced Na+ fluxes (Simchowitz 1985a). The results from these studies suggested that fMLP activated an amiloride-sensitive Na+/H+ exchange or counter transport mechanism which was dormant in nonactivated, resting human neutrophils. Such a Na+/H+ counter transport system has been reported by Grinstein and Furuya (1984; 1986) and Simchowitz (1985b) to mediate a biphasic cytoplasmic acidification/alkalinization response in activated neutrophils. To measure intracellular pH changes these investigators pre-loaded cells with fluorescein derivatives which are quenched as the pH is lowered. Within 30 seconds following cell activation with 10nM or 100nM fMLP the cytoplasmic pH was found to drop 0.10—0.15 units from a resting level of approximately 7.20. Such cytoplasmic acidification is thought to be associated with respiratory burst activities (Grinstein and Furuya, 1986). Within 60 seconds after cell activation the pH increased by approximately 0.20 units over the resting level, resulting in cytoplasmic alkalinization. Amiloride blocked the alkalinization suggesting that the pH alterations were caused by the same Na+/H+ counter transport mechanism proposed to be mediating the increased intracellular Na+ concentration and volume increases found in activated neutrophils. .', In summary, the studies_described above propose that within 30 166 seconds following exposure of human neutrOphils to nM quantities of fMLP the cell cytoplasm becomes acidic and an amiloride sensitive Na+/H+ counter transport mechanism is activated. Intracellular H+ is exchanged for extracellular Na+ which results in cytoplasmic alkalinization and a concomitant increase in intracellular Na+ concentration. Osmotically obliged water is thought to enter the cells as a result of the increased intracellular Na+, mediating cell swelling. Increased cellular volume was detected as early as 30 seconds after the addition of 10nM fMLP, cytoplasmic alkalinization within 60 seconds (Grinstein and Furuya, 1984; Grinstein and Furuya, 1986; Grinstein et.al., 1986). The activation mediated cross-sectional area increases found in the studies presented in this report may be explained by the Na+/H+ counter transport mediated events proposed above. The area determinations made from profiles of adult neutrophils activated for 20 seconds with 1mM or 100nM fMLP showed average increases of 8.6% or 14.6%, respectively (Table 10 and Figure 6 B). These values are within the 10% half—maximal volume increases reported by Grinstein et.al. (1986) to be reached approximately 30 seconds after exposure of human neutrophils to 10nM fMLP. CONCLUSIONS The purpose of these studies was to investigate the early morphological and functional responses of human neutrophils to activation. The results from evaluations of cellular shape change made with light and electron microscopy, and granule secretion investigated both biochemically and ultrastructurally suggest that 1) a population of peroxidase negative granules containing relatively high concentrations of lactoferrin exists in adult neutrophils, 2) this population of granules undergoes sequential or selected release, and 3) the release of these granules is coincident with the earliest visible indication of cellular shape change (i.e. ruffling) and may be associated with a transient, apparently regulated increase in exposed cell surface membrane. As summarized in Figure 20, the following sequence of events is proposed by these studies to occur early in adult neutrophil activation: 1) Brief activation of neutrophils mediates cellular ruffling and the coincident mobilization of a select population of peroxidase negative granules containing large quantities of lactoferrin. 2) These granules merge with the plasma membrane releasing lactoferrin into the extracellular environment. The resulting granule-plasma membrane fusion provides a mechanism whereby specific surface membrane components (for which the intracellular pool 167 168 is the membrane of secondary granules) are ueregulat d and then quantity of exposed surface membrane is increased. 3) An activation induced increase in cellular volume, seen in the ultrastructural morphometric determinations made in these studies as an increase in cross-sectional area, subsequently occurs. This volume increase provides a "compensation" for the increase in exposed surface membrane which, by the methods used in this study, appears to be regulated and returned to basal levels. 169 PEROX o + . *..‘. LACTOFERRIN IAESCQHHEE (:OLJJUSENHHSE SURFACE RECEPTOR I! 0 4t 4 Figure 20 PROPOSED EARLY EVENTS 0F NEUTROPHIL ACTIVATION rnrn A23187 MICE mud Neutrophil Isolation by Counterflow Centrifugal Elutriation The counterflow centrifugal elutriation isolation technique is based on two opposing forces, centrifugal and centripetal, occurring simultaneously within a separation chamber. The centrifugal field is generated by a spinning rotor, the viscous drag of fluid flowing in the opposite direction creates the opposing centripetal force. 'As the rotor is spinning leukocyte-enriched blood samples are loaded into the separation chamber at a given flow rate. Maintaining this flow rate, elutriation buffer is pumped into the chamber. Due to the opposing forces each cell tends to migrate to a zone where its sedimentation, due to centrifugal forces, is balanced by centripetal force, the flow rate of elutriation buffer through the chamber. By watching the elutriation process through a view port situated above the spinning‘ rotor the formation of an erythrocyte-rich region in the upper portion of the separation chamber can be seen. The flow rate of buffer through the chamber is then increased. As each cell again tends to migrate to the zone where its sedimentation is balanced, lower density erythrocytes are washed from the chamber. By continuing to increase the flow rate of buffer successive populations of larger cells or cells of greater density can be elutriated from the separation chamber. Samples of the elutriant are monitored as the flow rate 170 171 increases and the process is stopped before neutrophils are washed from the chamber. The chamber is removed from the centrifuge and the isolated neutrophils are the collected. APPENDIX B Morphometric Determinations blade of Hypothetical Neutrophils Utilizing Digitizing Tablet Measurements of hypothetical cells were made for two reasons; 1) to assure that the Houston graphics tablet and Optomax software used in the ultrastructural morphometric measurements of this study were sensitive enough to detect subtle changes in neutrophil morphology and 2) to attempt to better understand the process of neutrophil ruffling. Hypothetical cells were paper cut-outs approximating the size of cells ' at 18,000 X magnification. The "cells" were made to structurally represent electron micrograph profiles of unactivated spherical cells and neutrophils in the process of ruffling. Two possible mechanisms of cellular ruffling were mimicked with hypothetical cells; 1) membrane redistribution, where theoretically any "new" membrane expressed on the cell surface as a pseudopod would be at the expense of a loss or redistribution of membrane at some other site and 2) pure membrane addition where "new" membrane was simply added to the cells membrane (for example, through granule and plasma membrane fusion). For each mechanism a series of 12 measurements representing a process of neutrophil shape change from spherical to ruffled were made. The membrane redistribution mechanism was assumed to cause a form of ruffling in which cellular perimeter changed but cellular area did 172 173 not. To this end, pieces were cut out of the initisl hypothetical "spherical cell" and pasted onto the "cell surface membrane". As the cells progressed in the ruffling process, more pieces were cut out and pasted on. The net result was a "cell" with both invaginations and evaginations. Morphometrically this process produced a series of determinations which exhibited an increase in perimeter but remained fairly constant in area (see Table B 1). Figure B 1 shows the computer print out of every third digitized "cell" in the series of measurements. The process of membrane addition as a mechanism of cellular ruffling was thought to roughly simulate the event of extracellular granule release resulting in granule—plasma membrane fusion. To achieve a theoretical representation of this process, pieces of the hypothetical spherical cell were again cut out and pasted on the "cell" surface to mimic pseudopod formation but this time the cut out regions were returned to a smooth contour. By this mechanism, evaginations were created but invaginations were not. Therefore, both cellular area and perimeter should have exhibited an increased. Table B 2 shows the determinations made from the series of hypothetical cells used to mimic neutrophil ruffling caused by membrane addition. Figure B 2 is the corresponding computerized printout of every third ' digitized "cell" in this series. The results from these hypothetical cell determinations indicated several points. In terms of evaluating the methodology used in these studies, the results from the hypothetical cells indicated that subtle morphological alterations in cell shape could be detected with the graphics tablet and software. Additionally, the~results from these Table B 1 HYPOTHETICAL CELL MORPHOMETRIC DETERMINATIONS: MEMBRANE REDISTRIBUTION Figure \OGDVOMJ-‘ri-a r—ar—n—a Nt—‘O Table B 2 Ari 84.18 86.99 86.99 87.68 87.49 88.61 89.05 89.00 88.69 89.11 76.55 76.62 174 Perimeter 34.57 51.99 55.13 69.75 71.02 82.79 95.66 105.20 113.24 120.72 121.80 123.61 gfl 0.615 0.89 0.95 1.19 1.21 1.40 1.61 1.77 1.91 2.03 2.37 2.42 HYPOTHETICAL CELL MORPHOMETRIC DETERMINATIONS: Figure \OCDVO‘Lflwat—I Area 89.29 90.57 91.58 91.75 92.54 93.14' 94.44 95.86 96.08 96.70 96:76 96.93 Perimeter 33.07 37.76 41.18 44.92 47.84 50.95 54.94 60.56 62.06 63.41 65.22 65.73 gfl 0.55 0.62 0.67 0.73 0.77 0.82 0.87 0.95 0.97 0.98 1.011 1.017 MEMBRANE ADDITION 175 onebmHMHmHnmm mz= N m shaman 9 V 176 {'5 onHHna< mz: N m on: Hm 177 determinations, when compared with those made from electron micrographs of actual nonactivated and ruffled cells, seem to provide some insights into the process of neutrophil ruffling. First, the hypothetical cell results appear to indicate that ruffling of adult neutrophils is probably more complex than a membrane redistribution. As seen in Tables 10 and 16, and Figures 6 B and 10 B, brief exposure of cells to fMLP or A23187, respectively, mediated an increase in cross—sectional area as well as a significant increase in cell perimeter. The hypothetical cell determinations representing membrane redistribution on the other hand, show an increase in perimeter without a concomitant increase in area (Table B 1). Furthermore, the results from hypothetical determinations representing membrane addition could to support the hypothesis that activation mediated neutrophil ruffling also mediates an increase in'cell volume'and further, that increased cell volume may be a "compensation" response to a transient increase in exposed surface membrane. As seen in Table B 2, the hypothetical "cells" exhibited increase in perimeter and, albeit slight, an increase in area (8.2% in figure # 12). These changes, relative to one another, resulted in an increase in calculated S/V (85% in figure # 12). In contrast, while both the perimeter and area determinations made on electron micrographs of neutrophils exposed to fMLP or A23187 (Tables 10 and 16 and Figures 6 B and 10 B, respectively) also increased in both perimeter and area, the area increase relative to the perimeter was greater (reflected in S/V compared with baseline spherical determinations) and, with the exception of 1nM fMLP, did not result in an alteration in exposed surface membrane. The increase area detected in activated adult 178 cells, therefore, could have involved more than the addition of surface membrane from membrane fusions. Ionophore treated neonatal cells may represent cellular ruffling due to the membrane addition mechanism. 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