I"Il'glll‘ll“Till\lllllll This is to certify that the dissertation entitled THE EFFECTS OF CHEMOTACTIC FACTORS OR COLCHICINE ON NEUTROPHIL BINDING OF PROTEIN COATED PARTICLES presented by Thomas Lee York has been accepted towards fulfillment of the requirements for Ph . D . degree in Anatomy fl: 1 oZflL (5761ng; r ajor professor Date 11-3-83 MSU is an Affirmative Action/Equal Opportunity Institution 0.12771 MSU B§IURNING MATERIALS: Place in book drop to LJBRARJES remove this checkout from m your record. FINES will be charged if book is returned after the date stamped below. ' K} It a . THE EFFECTS OF CHEMOTACTIC FACTORS OR COLCHICINE ON NEUTROPHIL BINDING OF PROTEIN COATED PARTICLES £5 VITRO BY Thomas L. York MS A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY 1983 © Copyright by THOMAS LEE YORK 1983 ABSTRACT THE EFFECTS OF CHEMOTACTIC FACTORS OR COLCHICINE ON NEUTROPHIL BINDING OF PROTEIN COATED PARTICLES IE‘VITRO BY Thomas L. York Environmental or cellular changes may alter neutrophil adherence to protein coated substrata. Since neutrophil binding to a variety of protein coated surfaces is essential for margination, motility, and phagocytosis, we investigated the effects of chemotactic factors and colchicine on neutro- phil binding of bacteria, proteins, and protein coated particles. We hypothesized these agents may affect neutro- phil adhesiveness by altering cellular binding site topographies or promoting the release of cellular substances that alter protein coated surfaces. Our results demonstrate the following. Unlike isolated neutrophils, which were spherical, chemotactic factor or colchicine pretreated cells possessed distinct front or tail ends. The polarized cells bound complement, immunoglobulin, and albumin coated particles only at their front or tail ends. Regardless of pretreatment, neutrophils bound lectins and bacteria randomly on their surfaces. Time was a critical factor since neutrophils rapidly cleared surface bound particles from their attachment sites. Some particles were ingested by neutrophils immediately after binding and swept away internally from Thomas L. York their attachment and ingestion sites. Ingestion of particles was often dependent upon whether neutrOphils were stimulated with chemotactic factors. Other protein coated particles not ingested by neutrophils were selectively redistributed in a rearward direction across the neutrOphil's exterior surface. Different particles traveled at different rates on the neutrophil surface and the stimulation(s) required for surface redistributions varied. The localized binding, selective ingestion, and surface redistributions of several particles were observed to occur simultaneously on the cell. The mechanisms for maintaining very distinct binding site distributions and control of particle removal from their attachment sites remain unknown. We also reported that neutrophil adherence to glass was almost totally prevented by coating it with a variety of human proteins. Chemotactic factors enhanced neutrophil adherence to protein coated glass as did pretreating the protein coated surface with proteolytic enzymes. Chemotactic factors may activate proteolytic enzymes on the neutrophil surface and cause neutrophils to release other proteolytic enzymes into their environment, augmenting cell adherence to protein coated glass. NeutrOphil adherence to protein coated glass was inhibitable by the addition of two different specific enzyme inhibitors. Dedicated to my wife, Jane Caswell York, and to my daughters, Katherine and Elizabeth, whose moral support, understanding, and encouragement made this work possible. ii ACKNOWLEDGEMENTS I wish to express my heart-felt appreciation to my academic advisor, Dr. C. Wayne Smith. His intellectual enthusiasm and sensitive appreciation of research have enriched my life greatly. His encouragement over the past several years will always be remembered. I would also like to extend my sincere gratitude to the members of my committee, Dr. Dennis Steidler, Dr. Robert Echt, and Dr. John Wang for their constructive critical review of my research and manuscript. Their interest and encouragement was meaningful. It is with sincere appreciation that I also thank Mr. James Hollers for his advice in statistical analysis, research design, and graphics. His encouragement and friendship will always be remembered. Lastly, but not the least, I wish to express my gratitude to my wife, Jane Caswell York, for her enduring patience, encouragement, editing, and many hours of typing. iii TABLE OF CONTENTS Page INTRODUCTION. 0 O O O O O O O O I O O O O O 0 O O O O O O O O 0 C I O O O O O O 0 O I O O O O O 0 O 1 REVIEW OF LITERATURE O O O O O O O O O O O O I O O O I O O O O O O O O O O O O O O O O O O O 4 I. Physiology and Physiopathology of the Human 4 Neutrophil-0.0.0.........OOOOOO00.0.0...0.0.0... II. NeutrOphil-Surface Interactions: NeutroPhil l4 AdherenceOO0.000.000.0000.0.000.000.0000....... A. Evaluation of NeutrOphil Adherence......... 14 B. NeutrOphil Membrane Attachment Sites....... 19 C. The NeutrOphil Cytoskeleton: Role in AdherenCeOIOOIOOOOOOOO......OOOOOIOOOO0.... 27 D. Factors that Influence or Modulate Neutrophil Adherence....................... 30 MATERIALS AND METHODS.I....0...00............OOOOOOCOOOO 38 I. Isolation of Human NeutroPhils................. 38 II. Neutrophil Pretreatment........................ 39 III. Neutrophil Binding of Lectins, Bacteria, and Protein coated Spheres.........OOOOOOIOOI0.0... 39 IV. Neutrophil Adherence to Glass or Protein coated GlaSSOOOOOOOOOOOOOOOO......OOOOOOOOOOOOO 44 V. Presentation and Analysis of Data.... ....... ... 45 RESULTSOOIOOOOOCOOOOOOO00.00.000.00.........OOOOCOOOOOOO 46 I. The Influence of Albumin or Immunoglobulin on NeutrOphil Adherence of Latex Spheres.......... 46 A. Sphere Pretreatment and NeutrOphil Binding of Albumin or Immunoglobulin Coated SphereSOOOOOOOOOO00............OOIOOOOOOOOO 46 iv TABLE OF CONTENTS--continued Page II. III. IV. E. Chemoattractant or Colchicine Modulation of Neutrophil Adherence of Immunoglobulin Coated Spheres............... ....... ....... 50 C. Evaluation of the Ability of Neutrophils to Simultaneously Adhere Albumin and Immunoglobulin Coated Spheres........ ...... 59 D. Redistribution of Neutrophil Bound Immunoglobulin Coated Spheres....... ....... 67 Effects of Chemoattractants and Colchicine on Neutrophil Attachment and Redistribution of Lectins and Lectin-Like Substances........ ..... 78 A. Neutrophil Binding of Lectins as Influenced by Chemoattractants and COIChiCineOOOOOOOOOOO......OOOOOOOO ........ 78 B. The Effects of Chemoattractants and Colchicine on Neutrophil Binding of Piliated Strains of Gram Negative Bacteria................................... 91 The Effects of Chemoattractants and Colchicine on NeutrOphil Binding of Serum and Complement coated Partiales.........OOOOOOOOOOOOOOOOOOI... 97 A. Particle Preparation.................. ..... 97 B. Verification of the Presence of C b on the Surface of the Bacteria and Latex Spheres and its Effect on Neutrophil Binding of the Particles..................... ......... 98 C. The Effects of Chemoattractants and Colchicine on Neutrophil Binding of C b Coated Particles.....................§ ..... 102 D. The Effects of Colchicine and Time on Neutrophil Binding of C3b Coated Particles............................. ..... 108 E. The Binding Distributions for C b Coated Particles on fMP Stimulated NeugrOphils.... 120 The Effects of Enzymes and Chemoattractants on Neutrophil Adherence to Protein Coated Substrata............................ .......... 136 TABLE OF CONTENTS--continued Page A. The Effects of Chemoattractants on Neutrophil Binding of Albumin, Serum, and C3 Coated Spheres.............. ....... 136 B. The Effects of Chemoattractants on Neutrophil Adherence to Protein Coated Glass.OO.0.0.000.......OOOOOOOOOOOO...OOO.139 DISCUSSIONOOCOOOOCOOI......OOOOOOOOOOOOOOO......0...... 158 I. Neutrophil Binding of Protein Coated Partial-e800.........OOOOOOO......OOIOOOO. ..... 158 A. Neutrophil Binding of Immunoglobulin and/or Albumin Coated Spheres..... ........ 159 B. NeutrOphil Adhesion and Surface Rearrangements of Membrane Bound Lectins and BacteriaOOOOOOOOOOOOO.......00.......0 163 C. Preparation and Neutrophil Binding of C3b Coated Latex Spheres........... ....... 166 II. The Effects of Chemoattractants on Substrate Adhesiveness.................................. 170 A. Neutrophil Adherence to Protein Coated Glass.00.0.00.........OOOOOOIOOOOOOOO..... 171 SUMMARYOOOOOO......OOOOO‘O00............OOOOOOOOOOOOOOOO 174 BIBLIOGRAPHY. ........ . ......................... . ....... 178 vi TABLE 1. 10. 11. LIST OF TABLES Page Neutrophil Adherence of Latex Spheres, Albumin Coated Latex Spheres, and Immunoglobulin Coated Latex Spheres.O......OOOOOOOOCOO......COOOOOIOIO. 49 The Effects of Colchicine or Chemoattractants on Neutrophil Binding of Immunoglobulin Coated Spheres (Ab-ACLS)coonon.ooooooooooooooooooooooooo 53 Effects of Colchicine or Chemoattractants on Neutrophil Binding Distributions for Immunoglobulin Coated Spheres (Ab-ACLS)..... ..... 55 Neutrophil Adherence of Albumin Coated Spheres (ACLS)..........OOOOOOOOOOOOOOOOO ........ 61 Neutrophil Adherence of Immunoglobulin (Ab-ACLS) and Albumin (ACLS) Coated Spheres................ 64 Distributions of Albumin (ACLS) and Immunoglobulin Coated Spheres (Ab-ACLS) on Neutrophils 15 Minutes after Attachment.... ...... 71 The Effects of Time on NeutrOphil Attachment and Ingestion of Immunoglobulin Coated Spheres (Ab-ACLS)ooncoo-cocoa...00000000000000.0000...on. 84 The Effects of Chemoattractants, Colchicine, and Time on Neutrophil Binding of Lectins and Immunoglobulin Cross-Linked Lectins.............. 87 Conditions that Influence Neutrophil Attachment and Redistribution of Cell Bound Salmonella typhimuriumOOOOOOCOO............OOOOIOOOOOOOOOOOO 94 Neutrophil Binding of Complement and Serum Coated Particles................................. 101 Blocking Neutrophil Binding of C3b Coated Spheres with C3c Specific Antisera... ..... ............... 104 vii LIST OF TABLES--continued Page 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. The Effects of fMP or Colchicine on the Numbers of C b Coated Particles Bound by Neutrophils.............. ........ ...... ......... 107 Topographical Binding Distributions for C b Coated Particles on PBS or Colchicine Pre reated Neutrophils as Influenced by Time ........ .. ..... 113 The Effects of Time on the Binding and Ingestion of C b Coated Spheres by Neutrophils Pretreated with3fMP (lo-3M).............. ........ ... ...... . 124 The Effects of Time on the Binding Distributions for C b Coated Particles on fMP Polarized NeutrgphilSOOOOOOOO ...... .... 0000000000 ... ...... 131 The Effects of Chemoattractants on Neutrophil Binding of Latex Spheres Coated with Human C3, Albumin, or Heat Inactivated Serum.............. 138 Neutrophil Adherence to Protein Coated Glass.... 143 The Effects of the Cell Supernatant or fMP Pretreatment on Neutrophil Adherence to Protein coated GlaSSOOOOOOOOOOOOOOO0.0.0.000...0.0.0.... 148 The Effects of Enzymes on NeutrOphil Adherence to Protein Coated Glass ......... ..... ..... . ..... 152 The Inhibition of Chemoattractant Induced Neutrophil Adherence to Protein Coated Glass by aTrypSin Inhibitor ...... ......OOOOOOOOOOOOOOOOO 155 The Inhibition of Chemoattractant Induced Neutrophil Adherence to Protein Coated Glass by Trypsin and Chymotrypsin Inhibitors .......... 157 viii FIGURE 1. 2. 10. 11. LIST OF FIGURES Page Neutrophil Binding of Immunoglobulin Coated Spheres (Ab-ACLS)0.000000000000000...00.00.0000. 58 NeutrOphil Binding of Albumin Coated Spheres (ACLS)...................... ..... ........ ....... 66 Simultaneous Neutrophil Adherence of Albumin (ACLS) and Immunoglobulin Coated Spheres (Ab-ACLS)ooooooooooooooooooooooo oooooo no... ..... 69 Redistribution of Immunoglobulin Coated Spheres on or Inside Spherical Neutrophils.............. 75 Redistribution of Immunoglobulin Coated Spheres (Ab-ACLS) on or Inside Polarized NeutrOphils.... 77 Examination of Neutrophils Fixed 10 Minutes After the Binding of Immunoglobulin Coated Spheres (Ab-ACLS) Using TEM............. ........ 80 Neutrophil Binding of Immunoglobulin Coated Spheres (Ab-ACLS) as Observed Using Light Microscopy with White Light and UV Light Sources........... ............. . ...... .. ........ 82 Neutrophil Binding of Lectins or Piliated BacteriaOOOOOOOOOOOO...... ........... 0.0.0.0.... 89 NeutrOphil Binding of Bacteria and Immunoglobulin Coated Spheres................. ..... ............ 96 The Binding Topographies for C b Coated Particles on PBS and Colchicine Pretreatgd Neutrophils.... 110 Random Binding of C b Coated Spheres on PBS Pretreated Neutrophils................ ...... .... 115 ix LIST OF FIGURES--continued Page 12. 13. 14. 15. l6. 17. 18. 19. 20. The Effects of Colchicine and Time on NeutrOphil Binding Distributions for C b Coated Spheres........................?.. ..... .. 117 The Binding, Ingestion, and Redistribution of C.b and Immunoglobulin Coated Spheres on Calchicine Pretreated Cells................ ..... 122 Neutrophil Binding of C3b Coated Particles After Pretreatment with 10'8M fMP..................... 126 The Effects of Polarizing Doses of fMP and Time on NeutrOphil Binding Distributions for C b coated SphereSOOOOCCOCOCCCOOO......CCCCCC? ...... 129 Simultaneous Adhesion, Ingestion, and Redistribution of Immunoglobulin and C b Coated Spheres on fMP Stimulated Neutrophils........... 135 Diagram of a Tissue Culture Chamber........ ..... 142 The Effects of Chemoattractants on Neutrophil Adherence to Protein Coated Glass............... 145 Summary of Binding Patterns for Albumin and Lectins or Piliated Bacteria and their Subsequent Redistribution on Control, CF, or Colchicine Pretreated Cells................................ 176 Summary of Binding Patterns for C b or Immunoglobulin Coated Particles agd their Subsequent Redistribution on Control, CF, or Colchicine Pretreated Cells..................... 177 INTRODUCTION Human neutrOphils are phagocytic leukocytes absolutely essential for human survival. They seek out, ingest, kill, and degrade particulate matter throughout the body. The first critical step for particle ingestion is the attachment of the particle to the neutrOphil. NeutrOphil binding of many particles can be greatly enhanced by the presence of certain proteins on the particle surface. The proteins of most interest to clinicians and researchers alike have been complement components (i.e., C3b), immunoglobulins, and lectins. Immunoglobulins and C b are produced by specific 3 and non-specific immune mechanisms and, when bound to the particle surface, "tag" that substance for removal by human phagocytic cells. Lectins are often produced by micro- organisms and are incorporated into structural components on their surface. Phagocyte attachment of lectins appears to be a very primitive cell-surface recognition system and the presence of lectins on some strains of bacteria may determine their pathogenicity 1. NeutrOphils can recoqnize particles with surface bound C b, immunoglobulin, or some lectins because neutrophils 3 possess selective domains on their cell membrane that specifically bind these proteins 1' 2’ 3' Very little is known about the chemical nature of these surface receptor or binding sites or the forces that enable such selective binding to C3b, immunoglobulin, or lectin coated particles. Assuming that these receptors and binding sites are chemically and physically distinct units embedded in the neutr0phil's membrane, they should obey the principles of the mosaic model of a lipid membrane. Therefore, they should be free to diffuse throughout the cell membrane and appear randomly distributed on the cell surface. Indeed, several reports have demonstrated that immunoglobulin, C b, 3 and lectin coated particles bind on the neutr0phil surface in an evenly distributed manner 4’ 5' 6. Agents (i.e., colchicine or chemotactic factors) morphologically polarize neutrophils or cause them to assume a shape with a recognizable front and tail end. Recent evidence indicates that chemotactic factors (i.e., CF) may also influence neutr0phil binding site t0pographies for immunoglobulin, lectins or albumin coated particles independent of particle attachment 4' 7' 8. This report investigates the effects of chemotactic factors and colchicine on neutr0phil attachment, surface redistributions, and ingestion of lectins, bacteria, and particle bound C b or immunoglobulins. Emphasis was given 3 to determining whether colchicine and CF could functionally polarize neutrophil receptors or binding site tOpographies for C3b, immunoglobulin, or lectin coated particles. A variety of cells have been shown to perform specific functions only at one region on their surface 9. Previous reports indicate that colchicine pretreated phagocytes ingest a variety of protein coated particles only at one small region on the cell 10. Conditions that influence receptor or binding site mobility on human neutr0phils could cause specific regions on these cells to become "endocytotic windows". A secondary issue of this study was to investigate the conditions necessary for enhancing neutr0phil adherence to protein coated substrata in yitrg. Chemotactic factors are agents that influence the ability of neutr0phils to release lysosomal enzymes 11, possibly activate neutrophil membrane 12, and alter neutr0phi1 adherence to protein coated substrata 7’ 13' 14. Therefore, a technique was bound enzymes devised to evaluate the effects on neutr0phil adherence to protein coated substrata and enzyme modified protein coated substrata in_yi£rg. Particular attention was given to the effects of CF on the release and activation of neutr0phil enzymes that alter a variety of protein substrata and neutro- phil adherence to these surfaces. REVIEW OF LITERATURE There has been an explosive growth of interest and research activity regarding human neutrophil physiology. The purposes of this review are to I) briefly introduce information enabling some understanding of the physiology and physiOpathology of human neutr0phils and 2) review the literature pertaining to neutr0phil adherence interactions important for "normal" neutrophil function. I. Physiology and Physiopathology of the Human NeutrOphil Human blood is basically composed of two components; a cellular fraction and plasma. The cellular fraction contains several types of cells that are highly specialized to perform certain roles within the human body. The primary role of the blood neutr0phi1 is to seek out, ingest, and destroy obligate extracellular pathogens which cause most of the acute infections in humans. Decreased numbers or func— tionally impaired neutrophils are incompatible with life. At the time of birth, neutrophils are made almost exclusively in the bone marrow and the marrow will perform this role throughout the lifespan of the adult. Within the marrow, there exists classes of stem cells which are committed to granulocytic, monocytic, erythroid, and mega- karayocytic differentation. Factors regulating stem cell input into these cell lines remain poorly understood 15. Cells destined to become neutr0phils begin a mitotic phase. Following three mitotic divisions, which last approximately five days, a single stem cell produces eight differentiating cells. Following replication (i.e., mitotic phases) the cells undergo a maturation process, lasting approximately two days 16. There are enough maturing neutr0phils in the marrow, should the marrow stOp functioning, to supply adult daily neutr0phil requirements for.about one week 17. Various tables and figures have been provided by several authors that summarize neutr0phil development in the bone marrow, staining prOperties, and cell morphologies during this develOpment 18’ 19. Release from the marrow into blood sinuses is ordinarily related to the age of the cell. This process may simply be regulated by marrow anatomy. Stem cells lie deep within marrow cords and as these cells replicate and mature, they seem to be pushed onto the sinusoid surface. . . . . 20 Therefore, release may occur on a first in—first out bas1s . However, blood flow 21, blood concentrations of neutro- phils 21, bacterial products 22, and "colony stimulating 23, 24 factors" also influence neutr0phil release from the marrow. Cell maturation and acquisition of certain cell properties (e.g., deformability, membrane fluidity, etc.) may be additional factors that also influence neutrophil release from the marrow. Once in the blood stream, neutr0phils constitute approximately 70% of the blood leukocytes. These neutr0phils have a multilobed, or segmented nucleus, neutral staining granules in their cytOplasm, and are formally identified as polymorphonuclear neutr0phils or PMNs. Other common names include "polys" or "segs". IsotOpe dilution studies 25 reveal two pools of neutrophils. Approximately 2000—7000 neutr0phils per cubic micron are found in the central blood flow while almost an equal number are found to be inter- mittently attached to the vascular endothelium (i.e., marginated). Factors influencing interactions between these two pools remain poorly understood. Exercise, or epine- pherine injected into the vaSculature, have been shown to promote release of the marginated pool of neutrophils from 21: 26: 27 and can approximately double the the endothelium numbers of circulating neutr0phils. Optimal defense against infection requires intact integument and mucous membranes. Humans are in constant contact with a variety of micro-organisms and, according to cell numbers, the human body is approximately 99% prokaryotic. Following penetration of the epithelial front lines of defense by micro-organisms, inflammatory agents or chemical signals are apparently generated. It has been postulated that these inflammatory signals may mobilize and recruit neutr0phils to inflammatory sites. What are the inflammatory signals that account for neutrophil mobilization and accumulation at inflammatory sites? Metchnikoff theorized that products released by bacterial growth within the host attracted leukocytes 28. Since this observation in the late 1800's, numerous chemical substances have been isolated and classified as chemotactic factors (CF). Gallin provides an extensive review 29 of diverse agents that, by various criteria, have been shown to be chemotactic. Most of them are biological products generated by bacteria, immune effector cells or by enzymatic action on a variety of protein substrates. Little is known about the mechanisms by which these chemically diverse substances interact with the neutr0phil and illicit a chemo- tactic response(s) 29’ 30' 31. Specific membrane receptors for several CF have been demonstrated 32’ 33. Some CF analogues compete for receptor sites yet do not trigger identifiable chemotactic responses 31. Therefore, some additional event must accompany receptor—ligand formation to trigger a chemotactic response. Chemotactic factors have been shown to alter neutr0phil morphology, cytoskeletal arrangements, cytOplasmic concentrations of the cyclic nuclotides, depolarize the cell membrane, increase membrane fluidity, induce ion fluxes, enhance phagocytosis, adherence, glycolysis, superoxide generation, and lysosomal enzyme release 31. Following surface stimulation, there appears to be a sequential series of events activated that modulate several neutr0phil responses. There may be one fundamental factor that triggers these mechanisms, but, if so, it has not been determined. Conheimn, in the late 1800's, noted that the initial leukocyte response during inflammation was the margination of these cells along the vasculature near sites of infection 28. The marginated leukocytes were then observed to exit from the vasculature and enter the tissue compartment. Over 30 billion neutrophils are leaving the vasculature daily 21, and while in the tissue compartment neutr0phi1s literally eat themselves to death, forming the host's second line of defense against infection. Neutrophils have never been observed re-entering the circulation and are true end cells with extremely Short half lives (e.g., minutes to hours). Surprisingly, histologic sections of most non-pathologic tissues reveal few neutr0phils on the mucosae. However, tissue sections obtained from infected or inflamed areas obtained in as little as one hour following infection, reveal that neutr0phils emigrate to these sites to the extent that they are often the primary cell type in the region. Such recruitment and prolonged utilization can result in an immediate marked neutropenia. This prompts immature and mature neutrophils to be released from the bone marrow into the blood stream which provides a reserve force of neutr0phils if needed 19' 25' 27. Neutr0phils in the vasculature are basically round intermittently adherent cells. Isolating them using various in yit£g_techniques 34 does not appear to alter their morphology. Yet, within seconds, in a balanced salt solution or minutes in whole blood, neutr0phils "ruffle" and become polarized 35’ 36’ 37 when exposed to various concentrations of CF. While polarized, a distinct front (i.e., lamellapodia) and tail (i.e., uropod) end can be observed on the CF activated cells. When neutrophils migrate on a surface, migration will occur in the direction of the lamellapodia. This morphological polarization allows researchers to evaluate not only cell responsiveness to CF but also 1) cell orientation, and 2) redistribution of substances bound to the surface of the cell's membrane. Neutrophils exiting the vasculature crawl in an amoeboid fashion. Stossel and others 38' 39 have reported the presence of actin and myosin in human neutr0phils which may account for as much as 2.5% of the cell's total protein concentrations. These proteins have been implicated in the motile mechanisms of other non-muscle cells, and neutrophil mobility appears to be generated by their activity with other cellular components which allow for contraction and relaxa- tion of the moving parts of the cell. Agents that interfere with actin polymerization interfere with neutr0phil motility. Energy is needed for cell migration and inhibitors of glycolosis or the hexose monophosphate shunt also impair neutrophil migration 38' 40. 10 The distances neutrophils crawl in response to various CF in yitrg is probably the most commonly used technique to clinically evaluate the functional capabilities of the cell. Chemotaxis, or the directional migration of cells towards increasing concentrations of CF, has fascinated biologists for over a hundred years, and is apparently fundamental in the biological activities of numerous motile cells 40. Although, as numerous researchers note 13' 28' 29’ 40, chemotaxis could explain neutr0phi1 emigration to infection sites and retention of cells at that site, no in 3133 or‘in yitrg evidence have supported this hypothesis 13’ 42. In the last 20 years, numerous in 213:9 techniques have demonstrated that the rate at which neutrophils migrate is stimulated by CF and neutrophils orient and crawl up a gradient of CF‘in yitgg 29’ 41’ 42. Evidence for the establishment of a chemo- tacticgradient or neutrophil chemotaxis in 3332 has not yet been reported. The mechanisms by which CF influence neutrophil mobility and how neutrophils orient towards a chemotactic gradient remain unknown. It may be the nature of the adhesion between the cell and the substrata that facilitates both of these responses. Neutrophils have been shown to migrate up an adherence gradient (i.e., haptotaxis) in yitgg 43' 44. The effects of CF on neutr0phil adherence will be discussed separately, but it should be emphasized that mechanisms controlling cell adherence influence margination, neutrophil mobility and phagocytosis. 11 Sites of infection have often been compared to a battle- field. Pathogens employ exotic mechanisms to immobilize host defenses and, like battles, infections are complex, involving much cell death, and destruction. Once at the inflammatory site, neutr0phils ingest micro—organisms, damaged tissue, cell debris, and foreign substances. Attachment and recog- nition of these substances occurs at the neutr0phil surface and cell-substrata interactions are dependent on the cell's surface binding sites, attachment surface, and the environment. Following attachment, the particles may or may not be ingested. Phagocytosis is also an energy requiring process 'involving the flow of a hyaline pseudopod around a particle. Almost certainly, contractile proteins are important in mediating the cytoplasmic movement and pseudopod forma- tion 29' 40' 45. Surface modifications or opsonization by various serum proteins may be required to make particles more palatable. Attachment apparently triggers a signal for ingestion of the attached particles. Localized areas of the plasma membrane in contact with the particle invaginate, forming a pouch. This pouch, containing the particle, seals and separates from the plasma membrane forming a closed vesicle (i.e., phagosome). Two types of lysosome granules are primarily found in the neutr0phil (i.e., primary and secondary granules) and within these granules, various cata- 46 bolic enzymes have been isolated and characterized . These lysosomes fuse with the phagosome forming a 12 phagolysosome where the enzymes assist in the killing and degradation of ingested matter. Neutrophil killing of micro- organisms may also be mediated by plasma membrane bound ectoenzymes, superoxide radicals, lysosomal enzymes, anions, pH, or combinations of any or all these mechanisms. The success of degradation is determined largely by the properties of the ingested particles. Failure to degrade phagocytized material may be to the host's benefit, since undigested material may be needed to maintain immunologic awareness 47. Whereas neutr0phil phagocytosis is clearly essential for normal microbiocidal activity, events occurring during phagocytosis may also promote host tissue damage. Following, or coincident with plasma membrane closure during phago- cytosis, constituents of lysosomal granules have been found extracellularly. External degranulation may also occur when neutr0phils are adherent to non-phagocytizable substrates (i.e., reverse or frustrated phagocytosis), or following exposure to various soluble stimuli (e.g., cytochalasin B, CF, phorbol acetate, calcium ionophores, and lectins) 47. Phagocytosis also promotes marked changes in neutrophil oxidative metabolism. Neutrophils consume large amounts of 02, generating hydrogen peroxide, superoxide radicals, and 46 hydroxy free radicals , which can also be released into the cell's environment. Lysosomal enzymes and products of O metabolism released into tissue compartments can promote 2 tissue injury. Chronic neutrophil accumulation and release 13 of lysosomal enzymes or oxidation products into the host environment may actually lead to secondary clinical problems such as autoimmune vasculidities, arthridities, and pneumoni— dities. Therefore, intricate controls must exist that normally keep this response in check. NeutrOphils remove CF by endocytosis or by enzymatic degradation 12. Several CF inactivations have also been identified in human plasma 29. Several enzyme inhibitors which, due to capillary permea- bility changes during inflammation, are also in high concentrations at inflammatory sites. These enzyme inhibitors (i.e., alpha-l-antitrypsin, alpha-Z-macroglobulin, and C1 inhibitor) inhibit CF generation 48 49 , neutrophil , and lysosomal enzyme function 47. The actions 46 chemotaxis of various anti-inflammatory drugs (e.g., corticosteroids, colchicine, and CF analogues) on neutr0phil function is also providing insight to man's ability to prevent these clinical manifestations. In 1954, Janeway et al described the first group of patients with recurrent infections later identified as having a phagocytic cell disorder 50. Numerous disease processes have since been reported where this situation exists and the importance of neutr0phil function is aptly illustrated by the high rates of infection and dismal survival statistics of patients with these diseases. A description of these diseases can be found elsewhere 29' 51. Studies involving congenital or acquired neutr0phil deficiencies has greatly attributed to increased understanding of the molecular events involved with several neutrophil functions. However, several 14 important limitations become apparent while reviewing this literature. First, the complexity of the neutr0phil's interaction with other host defense mechanisms makes it difficult to evaluate neutr0phil function as an independent process. Our lack of understanding of these interactions makes predictions of factors causing the abnormality(ies) almost impossible. Second, evaluations of neutr0phil func- tion (i.e., chemotaxis, phagocytosis, microbiocidal killing, adherence, etc.) only confirm diagnosis. These assays are not standardized from lab to lab and seldom contribute to understanding the mechanisms causing the abnormality. Last, ignorance of cell mechanisms that control important cell functions is amplified by our inability to treat these patients. Treatment consists of blood transfusions and preventative measures against infection (e.g., antibiotic therapy). Patients with congenital disorders of neutr0phil function (i.e., chronic granulatomes disease) often suffer prolonged infections in spite of antibiotic therapy. Prog- nosis is almost always uncertain, unless the disease is severe and then the patient almost assuredly dies before age twenty. II. NeutrOphil-Surface Interactions: NeutroPhil Adherence A. Evaluation of NeutrOphil Adherence Neutrophil adherence to other cells, various substrata, or particulate matter appears essential for margination, 15 motility, and phagocytosis, all pre-requisites for neutr0phi1 involvement in host defense. Since blood vessels are lined by endothelium, transitions from blood to tissue compartments require neutr0phil-endothe1ial adhesions. Although the forces influencing this interaction are poorly understood, it appears to be the initial neutr0phil response to inflammatory stimuli. Other neutrophil-cell interactions appear important for selective removal of damaged cells and thrombosis; but other than isolated reports very little research information is available concerning these interactions. Early attempts to evaluate neutr0phi1 adhesiveness utilized techniques that evaluated neutrophil margination or phagocytosis. Most of these techniques have relied on direct inyiyg observations. In the early 1800's, Dutrochet observed leukocyte margination to the vascular endothelium and these observations were later confirmed and expanded upon by Addison and Conheim 28. Elaborate models (e.g., tadpole tail 52, rabbit ear 53) have been developed over the years to better observe blood flow and leukocyte attachment to 28 used a laser capillary endothelium. Grant and Epstein beam to selectively destroy tissue in localized areas around or on vascular endothelium and concluded that localized margination was controlled by alterations of endothelial cell adhesiveness. Advances in tissue culture techniques 34 and endothelial cell isolation 54 and enabling neutr0phil culture provided researchers a means to study this cell-cell interaction‘invitro. Direct observations of neutr0phil 16 attachment to endothelial monolayers have provided data for the establishment of two separate hypotheses. Several 53, 55 investigators contend that inflammatory agents 53’ 6 suggest influence endothelial adherence, while others that inflammatory and anti-inflammatory agents influence this interaction only to the extent of affecting neutr0phil adherence. A third consideration, recognized by both groups is that local alterations of blood flow decreases the shear- ing forces between these cells facilitating leukocyte- endothelial adhesion 57. Although conflicting results, these in yitrg techniques appear instrumental in contributing to our understanding of this cell-cell interaction. NeutrOphils bind to and ingest microbes, damaged cells, and a variety of opsonized or non-opsonized particulate matter. Often, the collision rate between the attachment particle and neutr0phi1 will determine the phagocytic rate; however, attachment does not guarantee recognition and ingestion. Suggested mechanisms of recognition by phago- cytotic cells have been extensively reviewed elsewhere 9. Although early observations of phagocytosis were primarily concerned with evaluations of the ability of neutr0phils to ingest and kill microbes, these experiments broadened researchers' understanding of the diversity of surfaces neutrophils bind to. In addition, by examining the early events of phagocytosis (i.e., attachment), information was obtained to evaluate various mechanisms of attachment. Binding affinities, scatchard plots, and calculations of l7 association and dissociation constants were calculated. Comparisons were made to enzyme kinetics and adsorbtion isotherms to evaluate these interactions 58. Preliminary data was obtained by direct observations of attachment in ZEEEE and later fluorescent and radio-labeled tags accelerated and improved these calculations. Characteriza— tion of numerous types of neutrophil-surface interactions, mechanisms that control attachment and recognition, and evaluation of agents influencing these mechanisms have resulted from these studies. Research involving several of these interactions will be reviewed later. ‘In_yiyg studies (i.e., "clearance assays") have proven less useful and unreproducible. Neutrophils not only attach to endothelium, but have been observed migrating across and between cytOplasmic processes of these cells 27. In addition, neutr0phils must crawl on various substrata through extravascular spaces to sites of infection. Translocation across a surface implies that neutr0phils must not only possess mechanisms for attachment but also detachment. Adhesion appears to provide the frictional forces required for movement. If hyperadhe- sive, cell locomotion would be minimal. Altering motile cell adherence to a substrata has been shown to influence the rate and direction of cell locomotion 44. Electron micro- graphs of motile cells reveal localized plaques where the cell membrane comes in close contact (e.g., within 30 nm) with the substratum. Within these plaques are electron 18 dense zones of oblique and longitudinal tracts of micro- fibrils 59. Little is known about the chemical nature of the membrane components that are closely associated with the attachment surface. Most of the data used to evaluate neutr0phil motility have been obtained using in 21339 assays. Likewise, the resurgence of interest in neutr0phil surface interactions has also been stimulated by the develOpment of numerous in vitro assays to evaluate this parameter and its effect on motility, phagocytosis, margination, etc. Yet, in_vivo techniques are difficult to quantitate, evaluate factors influencing the cell response, and often impossible to use on humans. In addition, cell manipulation and exposure to isolated factors are often impossible in yiyg. There are also distinct problems involved with evaluation and inter- actions. With the exception of evaluating neutr0phil interactions with microbes or other cells, neutr0phil adherence assays, like'in;yitrg assessments of neutrophil motility and phagocytosis, utilize non-biological surfaces which are foreign to the cell. Glass bead columns 69, capillary tubes 61, coverslips 35, plastics 62, chambers 63, and latex 7 are but a few of the surfaces used to evaluate neutrophil adherence. Comparison of results between adherence assays are difficult not only because of different attachment surfaces, but other reasons as well. Different medias are used to sustain the cells (e.g., serum, buffered l9 salt solutions plus proteins, etc.) which may have different effects on the attachment surfaces of the cells. Different detachment forces are used (e.g., gravity 35, centrifugation 64, rotation 65, etc.) which confuse issues; for example, is one measuring attachment or detachment? Some techniques use whole blood, which perhaps better reflects in yiyg events, but complicates interpretations with a multitude of factors influencing the cell or substrata (e.g., platelets, serum proteins, etc.). Complications also arise using isolated systems such as removing cells from their natural environment and harsh treatments required for cell isolation. B. Neutrophil Membrane Attachment Sites. Two concepts are generally used to explain cell adhesion. First, adhesion is mediated by macromolecules that can be solubilized or secreted at the cell surface which acts as a glue or cement. Second is a recognitive system where complementary molecules on the cell and surface form "lock and key" attachments. The only differences in the theories are the number of substances involved in the interactions; the glue hypothesis involves a single substance whereas the complementary theory involves several recognition substances (i.e., receptors or binding sites). The cell membrane is a fluid lipid bilayer interspaced with protein, glycoprotein, and glycolipid molecules which appear to provide binding or receptor sites for various substances. Selective attachment of appropriate ligands to these sites thereby provides the 20 "radar shield" by which the cell communicates with its 'environment through recognition of selected substances. Electrostatic zeta potential, chemical bonding, hydrophobic and hydrophillic forces contribute to the attachment and repulsion forces between the ligand and cell 66. Selective attachment is determined by the molecular structure of the attachment site and the ligand. The neutrophil membrane appears to contain certain domains for the selective attach— ment of certain foreign or protein coated surfaces. Although the chemical nature, turnover, and modulation of these domains are poorly understood, they certainly are important for host defense. Before elaborating on these attachment sites, a clear understanding of the terms "binding sites" and "receptor sites" must be developed. Following attachment of a ligand to a cell surface, the cell may or may not recognize the attachment process. High affinity and saturable attach- ment sites for selective ligands describes a membrane binding site. If such an attachment invokes a cell response, this membrane domain is referred to as a membrane receptor 9. Ingestion, cell shape change, submembrane structural altera— tions, metabolic changes, membrane fusion, lysosomal enzyme synthesis and release, and stimulation of microbiocidal systems are examples of neutrophil responses used to evaluate cell recognition of a ligand. Initial suggestions of a complementary-molecular theory of adherence promoted the concept of glycoproteins on cell surfaces interacting with complementary glyc0proteins on 21 other cell surfaces as the governing force for cell adhesion. Isolated glycoproteins from different cell types do agglutinate when mixed. Proteins derived from certain plants (e.g., leCtins), are able to bind with high affinity to carbohydrate or carbohydrate containing substances.. They appear to be the recognition mechanisms for attachment between plant and nitrogen fixing bacteria 67. Plant lectins also bind to the surfaces of eukarotic cells, including neutr0phils, probably through interactions with various terminal sugar residues on glyc0proteins, glycolipids, and glycosaminoglycans on the eukarotic cell surface. Pilated strains of gram negative bacteria attach to neutro- phils in the absence of serum which appears to be dependent 1, 68 on the presence of lectins located on the bacteria's pili 69' 70. Although other lectin based recognition mechanisms have not yet been identified for neutr0phils, it may explain their affinity for substances like zymosan. Neutrophil affinities for bacterial lectins appears rela- tively non-specific and can be reversed by the addition of the appropriate monosaccharide. Such interactions, as pointed out by McKeever and Spicer 70, may contribute to understanding of the ability of neutr0phils to interact with so many "non-specific" surfaces. The presence or absence of serum proteins on particle surface is widely recognized as the most important factor governing neutrophil attachment to that surface. Perhaps most notable are complement components (C3b) and antibodies. 22 These proteins are referred to as opsonins (i.e., to prepare food) since coating non-phagocytizable substances with them enhances their palatability. Antibody opsonization, discovered in 1903, reconciled the controversy between Metchnikoff's followers and those that advocated the importance of antibodies in host defense against infection 71. Antibody attachment to an antigen greatly 72 of immunoglobulin for increases the affinity (i.e., 4000K) phagocytic cells. Phagocytic cells appear to possess a selective domain on their unit membranes for binding of the Fc segment of several IgG and IgA immunoglobulins sub— classes 72’ 73. Attachment of the antibody to an antigen appears to alter the Fc segment on the immunoglobulin allowing for phagocyte recognition 74. The membrane receptors on neutr0phils for antibody are probably similar to those described for macrophages, although little has been described to confirm this hypothesis. Antibody attachment to macrophages is very tight (i.e., 10-40 nm), and the bound immunoglobulin may sit in a "receptor pocket" on the membrane surface 75. Phagocytic cells appear to have an abundance of 5-106/cell) 70 Fc receptors (i.e., 10 and depletion of these receptors following phagocytosis does not totally exhaust their number, suggesting a marginal pool of receptors. Regeneration, requiring protein synthesis, of these receptors has been documented 76. The chemical nature of the antibody receptor is unknown. Protease treatment of neutr0phils has been reported to enhance their 23 antigenfantibody binding capabilities 77. Attachment does not appear to be energy dependent and aldehyde fixation appears to destroy the Fc redeptors 4. Several techniques have been used to evaluate the Fc receptor. Various cells (e.g., RBC, bacteria,.etc.) or particles (e.g., latex beads) may be coated with immuno— globulin and neutrophil binding of these cells or particles have been evaluated. Neutrophil binding of antigenfantibody complexes, in particular ferritin-antiferritin or peroxidase labeled antibody complexes, have been extremely useful for observing Fc mediated attachments using electron microscopy. A group of eleVen heat labile serum proteins (i.e., complement) comprising 10% of all serum proteins, functions in effecting immune and non-immune destruction of foreign substances 78. These proteins appear to circulate in whole blood in unactivated forms. When activated, by either of two mechanisms, a cascade of enzyme-substrate reactions occurs, 'generating proteolytic fragments that influence several host defense mechanisms. Illustrations of this enzyme cascade have been presented in several reviews 78’ 79. Activation of this system may occur when either Cl reacts with an antibody-antigen complex (i.e., classical complement pathway) or when an ill-defined protein called "initiating factor" interacts with various particulate surfaces (i.e., alter- native complement pathway). Regardless of the modes of activation, both systems merge at a common point, the ‘generation of C3 convertase. Following 2.4 the formation of this serine esterase trypsin-like enzyme, the pathways are identical. Although highly specific, trypsin-like enzymes generated in the blood clotting, fibrinolysis, and kallikrein systems also act on various complement components 80. Several products generated in the complement cascade have a pronounced effect on inflammation (e.g., C3a, Csa) leukocyte responsiveness (e.g., C3a, C3b, 81 . 789) . Hav1ng C5a, C567 complex) and cytolysis (e.g., C already briefly discussed the role of complement components as CF, the following is a review of the literature pertaining to the complement component (i.e., C3b) that contributes toward immune adherence. Several recurrent infections have been observed in 82 3 ' 3 with C3 convertase, trypsin 83 or trypsin-like enzymes interacts 80, 84 I patients with depressed levels of C When C a small fragment (MW=7000) is cleaved from the amino terminal of the alpha chain and this fragment is called C3a. The large fragment (MW=170,000), identified as C b, has two 3 recognized binding sites. The first enables C3b attachment to RBC membranes, bacteria, and other particles. These binding sites are labile and if attachment does not occur, they spontaneously decay 81. An additional binding site is .generated on C following proteolytic cleavage. This one is 3 stable, although-serum inactivators can functionally destroy it 85. It facilitates C3b or particle bound C3b attachment to specific receptors on "responder cells". Responder cells include human erythrocytes, platelets, B lymphocytes, and 25 mononuclear and granulocytic phagocytes. Formal proof for the specificity of neutr0phil C3b receptors has not been presented; however, it avidly binds C3b coated particles, a fact not demonstrated for other complement components, excluding Csa 33, for which a separate receptor apparently exists. Neutrophil attachment of particle bound C3b appa— rently invokes a cell response 5’ 86 7O unlike macrophage attachment , suggesting a true receptor exists for C3b on the neutrophil. Binding studies on macrophages reveal fewer C3b receptors/cell than Fc receptors; however, better techniques are needed to confirm this observation. These receptors are trypsin sensitive, and require Mg++ for ligand attachment 70. Recently, neutr0phil receptors for C b have 3 been isolated, characterized, and antibodies prepared that 87' 38 which should enable appear specific for this receptor clarification of numerous conflicts in the literature concerning neutrophil C3b receptors. Two methods of evaluating C3b mediated attachment of phagocytic cells have been used in the past. The C3b- opsonization of various particles was first performed using fresh serum as a source of complement 5’ 89’ 90. Complement deficient serum or agents that prevented activation of classical or alternative complement cascades have been used as controls for these experiments. Since other serum proteins may have influenced results, two similar approaches, using purified complement components, have become popular techniques for studying neutr0phil attachment of C3b coated 26 particles. The first employs exposing antibody coated particles sequentially to purified C1, C4, C2, and C3, generating a particle-Ab-Cl423b complex 2. The major dis- advantages to this approach are time and expense whereas the advantages are built in controls (e.g., particle-Ab-Cl42) and comparisons of binding studies with particles only coated with antibody (e.g., IgM or IgG). An alternative approach has been to incubate particles with purified C3 and C3 convertase, trypsin, or trypsin like enzymes 91. These enzymes rapidly convert C3 to C3b which attaches and opsonizes various surfaces. The bulk of most phagocyte C3b receptor studies have employed C3b opsonized erythrocytes as attachment particles. However, the relative size of the RBC to the phagocytic cell prevents interpretations of receptor numbers distribution, affinities and to some extent, phagocytosis. Only a few subclasses of antibodies are recognized by human neutrophils. However, IgM and two subclasses of IgG when bound to an antigen, activate the complement cascade. Since complement is found in the blood and extravascular compartments, it would not be unreasonable to assume most antibody-antigen complexes are also C3b opsonized. Unlike the mononuclear phagocyte, distinct roles for complement and antibody attachment and recognition respectively have not consistently been observed for human neutrophils. Both antibody and complement opsonization have been reported to enhance particle attachment, and cell recognition (e.g., phagocytosis) 3’ 88. 27 Neutrophils bind and ingest a wide variety of foreign substances. For lack of better terminology, the attachment of these substances has been termed "non-specific" and their membrane attachment sites as "non-specific" receptors. This appears to be a primitive recognition system, operating via mechanisms quite distinct from those mediated by the PC and C3b receptors, although, as previously noted, some similarity has been reported to glycoprotein recognition mechanisms. These receptors appear to have low levels of specificity and attachment may be by some non-specific force such as charge 92. Of particular interest to researchers concerned with neutrophil surface interactions should be the complica- tion of attachments via this process interfering with attach— ments wished to be studied. Numerous particles (e.g., latex beads, glutaraldehyde fixed RBCs, bacteria, etc.) may attach to neutrophils via non-specific receptors. Coating these surfaces with serum proteins, lectins, etc., may not totally prevent non-specific cell—particle interactions. In addition, neutr0phil or serum enzymes may cause the erosion of serum or lectin coats from a particle's surface enabling non- specific interactions to again occur. C. The Neutrophil Cytoskeleton: Role in Adherence Networks of spectrum and actin are present within erythrocytes which appear to anchor membrane components. Nucleated cells appear to have a similar cytoskeleton composed of micro-tubules and micro-filaments. Although 28 cytoskeletal components are difficult to preserve and observe, recent electron microscopic and immunofluorescent techniques have allowed researchers to evaluate them in several cell types, including the neutrophil 6’ 93. There are two main components of the neutrophil cyto- skeleton. The first are microfilaments which are approxi- mately 60 angstroms in diameter, usually present just under the cell membrane and are most prominent at regions where the membrane has been perturbed by surface contact 6’ 94. There appears to be at least three categories of microfilaments, which Stossell 95 has shown to be comprised primarily of actin, myosin, and other poorly defined components. Micro- tubules, the second element of the neutr0phil's cytoskeleton, are composed primarily of tubulin. This protein appears quite primitive phlyogenetically. Tubulins polymerize, in_yiyg or + in vitro, in the presence of Ca+ and GTP assembling into a tube with a hollow core which may be several microns in length 96. The bulk of the reports concerning neutrophil cyto- skeletal components have used pharmocologic agents that promote assembly or dissolution of these structures. Numerous pharmacologic studies indicate that membrane dependent processes of neutrophil chemotaxis, adherence, phagocytosis, and lysosomal enzyme release are influenced by the cell's cytoskeleton. 29 Cytochalisns are fungal metabolites which cause structural disorganization and disruption of microfilaments perhaps by preventing gelation of actin with actin binding proteins 97. In addition, this drug inhibits hexose and nucleoside transport 6' 90. Numerous cell functions including phagocytosis, cell migration, and lateral movement of membrane attachment sites 11' 97' 98' 99' 100' 101 are all impaired or totally prevented following exposure to this agent. Most of these functions require active membrane movement, an apparent function of actin-myosin contractions. Although not a direct stimulus for degranulization or Ca++ influx, cytochalisns potentiate stimuli that invoke these 101) and have been responses (e.g., CF or Calcium ionophores used extensively to study them. Microtubule disruption by antitubulins has been shown to promote a polarization of structure and function of a variety of cells, including the neutrophil. Lectin attachment to antitubulin treated or buffer treated control neutrophils appears to be in a random, evenly distributed manner. Yet, unlike control cells, the receptor-ligand complexes "cap" or flow to the uropods of the antitubulin treated neutro- phils 102’ 103. Similar observations have been reported following the attachment of C3b or antibody opsonized particles on antitubulin treated macrophages 10’ 103' 105. Following particle redistribution, macrophage endocytosis of the particles occurred only at the uropod (i.e., polarized cell function) and at the same rate as control cells. 3O Microtubule depolymerization has been shown to facilitate microfilament recruitment to the uropods of antitubulin treated cells, which as Oliver suggests 106, may facilitate polarized endocytosis. Disruption of microtubules also appears to impair the chemotactic but not random migrational responsiveness of leukocytes which may be due to microtubule 106, 107, 108 or the inability of these cells to release cell derived CF 42' 109. dependent orientation mechanisms D. Factors that Influence or Modulate Neutrophil Adherence Adherence is an energy requiring process and the energy for it appears to be derived from glycolysis and possibly oxidative reduction 110’ 111. Adherence appears intrinsic to the cell since the cellular and plasma fractions of blood can be removed without altering the ability of neutrophils to attach to various substrata 111. However, several proteins have been shown to modulate this adherence. It is temperature dependent 110, maximal between 250 and 42°C, and apparently reversible since neutrophils must be able to detach from endothelium and during migration. Anticoagulants have variable effects on neutrophil adherence, and although heparin has no direct influence, it can modify plasma induced alterations of neutrophil adherence 112. In contrast, calcium chelators dramatically impair neutrophil adherence. Although controversial, metal cations are required for adherence 35' 110' 111. Kaverstein 113 has shown a wide pH range (i.e., 6.3-8.8) in which no effects on leukocyte 31 adherence were demonstrable. Only extreme osmolality changes impair neutrophil adherence (i.e., 600 mOsm) 61. As early as 1955, neutrophil surface charge has been a suggested mechanism of adherence 53. Neutrophils have a negative surface charge and modification of this charge has been shown to influence adherence 92. Several plasma proteins, in addition to antibody and complement, also influence neutrophil adherence. These substances alter either cell or substrata adhesiveness and have been extensively studied because of their obvious possible biological roles during the inflammatory response 14’ 114. Serum albumin appears to have no influence on neutrophil adhesiveness; however, it avidly binds to many surfaces inhibiting neutrophil attachment to those surfaces 14’ 35 , possibly by covering up attachment sites for non-specific neutr0phil receptors. Baseline levels must be established to quantitate increases or decreases in neutro- phil adherence in vitro. Researchers can control baseline levels of cell adherence to various substrata in vitro by varying the amounts of albumin or serum used to coat attach- ment surfaces 14. Epinepherine, endotoxin, or exercise induce the production of an uncharacterized plasma protein(s) that impairs neutrophil adhesiveness in yiyg 115. A tremendous amount of research has focused on isolating and developing substances that reduce neutr0phil adherence. These substances may provide a source for potential anti- inflammatory drugs. 32 Plasma proteins, other than opsonins, also augment neutrophil adherence. Of particular interest, are CF which may be generated at inflammatory sites (e.g., C a, bacterial 5 filtrates, formylated peptides, etc.). Although the effects of CF on neutrophil adhesivenesS'in vitro is controversial 56, most researchers agree CF enhances neutrophil adherence either 35, 54 116, 117, 118 by influencing the cell ,yor substrata adhesiveness. Agents shown to influence concentrations of cyclic nucleotides, calcium fluxes, microtubule organization, or charge also have been shown to modulate neutr0phil adherence. Chemoattractants and other drugs or agents that promote 108 microtubule assembly , decrease the net negative surface charge 92, promote calcium influxes 119, elevate intra- cellular concentrations of cGMP 119, also increase cell adhesiveness. In contrast, agents that decrease neutr0phil adherence have been shown to increase cAMP concentrations (possible yin-yang relationship?) or inhibit calcium influx 119. Chemoattractants enhancement of neutrophil adhesiveness appears reversible until the concentrations of CF used to pretreat the cells are in the "deactivating" ranges; then the enhanced adherence is sustained following removal of the CF 35. Deactivation has been described as a condition where neutrophils fail to migrate in response to high concentrations of CF 120. Several authors have suggested this apparent IOSS‘Of mobility may in fact be due to the sustained enhanced adhesiveness of the cell 35' 120' 121. 33 Curiously enough, "priming" the cells with low concentrations of CF also dramatically impairs or alters a "normal“ neutro- phil response to a second higher dose of CF. Unlike deactivation, which perhaps by saturating the cell with CF overloads the cell's responding mechanisms, priming the cell with low doses of CF appears to desensitize the cell. Examples of neutrophil desensitization have been provided by several recent reports. Neutrophils primed with sub-optimal concentrations of CF fail to degranulate or aggregate 122’ 123 following a second exposure to optimal doses of the CF. Neutrophil adherence also fails to be enhanced in response to CF when first primed with low concentrations of CF 35. Indeed, their adhesiveness was reported as being reduced below levels observed for cells having never seen the CF. Therefore, this reduced adherence doesn't appear to be due to a desensitization in a classical sense. Priming appears to facilitate a response to the second CF (e.g., actually decreasing adherence and, although not yet discussed, pro- moting a redistribution of surface binding sites) not observed if the cells were only challenged with a single CF 7. Desensitization must concern investigators of neutr0phil adherence for two reasons. First, isolation techniques provide a harsh environment for neutr0phils and this process may liberate CF which could prime control cells. This may explain why some researchers have reported that CF reduces neutrophil adherence. Second, if two CF exposures (i.e., a primary and a challenge dose) reduce neutr0phil 34 adherence, is this reduced adherence due to the cell's failure to release enzymes or a direct effect upon the cell's adherence mechanisms (e.g., a redistribution of binding sites). What are some of the mechanisms by which a CF could alter neutrophil adherence to a surface? Chemoattractants, by decreasing the net negative surface charge, may decrease the repulsion forces between the cell and its attachment surface 92. This would appear to allow for more collisions, possible recognition and attachment. Chemoattractants also alter the numbers or affinity of membrane receptors for various ligands. Kay et al have reported that CF enhances 124 the numbers of C b and not Fc receptors on neutrophils . 3 Likewise, CF apparently enhances the number of neutr0phil binding sites for protein coated surfaces 7' 35. The enhanced attachment of C3b or protein coated particles did not appear to require protein synthesis. If indeed CF has increased the numbers of receptors, two conditions may eXplain these events. Either new membrane, with additional receptors, has been inserted into the cell or a marginal pool of "hidden" receptors were uncovered following CF stimulation. Chemoattractants have been shown to increase neutrophil volume and membrane fluidity 125. These agents also promote degranulation and granule membranes appear to not only become incorporated into the cell membrane, but also , . 109 possess surface receptors common Wlth the cell membrane . 35 Chemoattractants also appear to polarize certain neutrophil membrane receptors and restrictions of this receptor topography would appear to influence cell function, including adherence. Walter et al 4 have demonstrated that Fc receptors, normally evenly distributed on neutr0phils, redistribute to the lamellapodia of neutrophils responding to a gradient of CF. Lectin binding sites, again normally evenly distributed, also appear to occur at the lamellapodia of neutr0phil orienting in a gradient of CF. The bound lectins are then observed to flow along the cell surface and "cap" on the uropods of these neutrophils 8. Smith and Hollers have reported that a single chemotactic stimulus enhances the numbers of albumin coated particles bound by neutrophils. These particles were bound on the cell in an evenly distributed manner. However, unlike the reports on ligand capping, these binding sites, redistributed to the uropods on neutrophils exposed to sequentially increasing concentrations of CF and this redistribution was independent of ligand attachment 7. Although the mechanism(s) by which CF promotes neutro- phil membrane binding site and binding site—ligand redistributions remains speculative, it is probable that cytoskeletal alterations will eventually be linked to these processes. Chemoattractants may also alter the attachment substrata. CF may bind to various substrata, which provides a recognizable ligand on the substrata for neutrophil attachment via their chemotactic receptors. The activation 36 or release of neutrophil enzymes also occurs following cell stimulation by CF, which may influence cell or substrata adhesiveness. Potent inhibitors of serine esterases, some of which exist in yiyg and in high concentrations at inflammatory sites, inhibit phagocytic chemotaxis, phagocytosis, enzymes released during degranulization, and super-oxidegenera- tion 8' 49’ 126’ 127. Wilkinson suggested that neutrophil enzymes decrease cell attachment and mobility on protein coated surfaces possibly by cleaving attachment proteins from the substrata 118. Enzymatic activity may also generate sub- stances recognized by neutr0phi1 membrane attachment sites. Neutrophil enzymes cleave fibrinogen in 31339, and apparently in yiyg, in a plasmin like manner 80. Neutrophils adhere and migrate on strands of fibrin, a property formerly used to isolate these cells. Neutrophil proteases may also influence the formation and degradation of Chemoattractants. These enzymes may also loosen interstitial spaces facilitating penetration. Enzymatic modulation of neutr0phil adherence may also be a result of protease activity on the cell membrane. MacroPhage affinity for antibody coated particles was shown to be enhanced following pretreatment of the cells with proteases 126. NeutrOphil adhesiveness to endothelial cells was also reported to be decreased following protease pretreatment 129. Several surface enzymes appear to be 120 activated by CF and their role in influencing cell adherence remains unknown. Chemoattractants activated 37 Chymotrypsin like ecto—enzymes may be involved in the chemo- tactic activation of the cell 12. Therefore, neutrophil ecto-enzymes or endo-enzymes, released or activated by CF or other stimuli, may influence neutr0phil adherence by altering attachment surfaces, affinities of surface attachment sites, modulating concentra- tions of and/or responsiveness to CF, and perhaps by serving as receptors for appropriate substrates on the cell membrane. MATERIALS AND METHODS I. Isolation of Human NeutrOphils Human blood (5 ml) was obtained from healthy adult donors by venipuncture and immediately placed in buffered citrated vacutainer tubes (Bectin-Dickinson, Rutherford, New Jersey). The citrated blood was diluted with an equal volume of Delbecco's phosphate buffered saline (PBS) and overlaid on 3-4 ml of Ficoll-Hypaque (2.16 grams ficoll, 3.396 grams hypaque; q.s. 34 ml distilled water) (Sigma Chemical Company, St. Louis, Missouri). This solution was centrifuged (1500 rpm for 20 minutes) and the supernatant removed by suction. The cell button, consisting of granulocytes and erythrocytes, was resuspended in 9 ml of CA";+ free PBS and erythrocytes sedimented from the suspension by dextran sedimentation as previously described 7' 14' 35. Briefly, 1.0 ml of a 6% (W/V) solution of dextran (70,000 MW; Sigma) in PBS was added to the suspension, incubated 45 minutes at 22°C, the granulocyte rich supernatant removed and washed with an equal volume of PBS. The cells were then resuspended in PBS and counted. Rare platelets or mononuclear cells were observed in the suspension and the granulocyte- erythrocyte ratio was usually greater than 5:1. Greater than 95% of all observed granulocytes in these preparations 38 39 were neutr0phils and greater than 98% of these cells remained viable hours after separation (determined by eosin exclusion). All isolated cells (107 cells/ml of PBS) were refrigerated (4°C) when not used. II. NeutrOphil Pretreatment NeutrOphils (106 cells/0.1 ml PBS) were incubated with 0.9 ml of a specified concentration of Formyl-L—methionly-L- phenylalanine (fMP) (Sigma), colchicine (Sigma), or PBS for a selected time interval and temperature. The treated cells were then either used while in the pretreatment solutions or centrifuged (400 rpm for 5 minutes), the supernatant decanted, and tests performed on the pelleted cells. When cells were to be exposed to multiple doses of pretreatment reagents the conditions for treatment are carefully described within the results section for each experiment. All cell pretreatments were carried out in glass test tubes previously coated with 50% human serum and washed in PBS. Coating the glass with serum proteins was found to greatly reduce cell adherence to the glass during the time intervals required for treatment. III. Neutrophil Binding of Lectins, Bacteria, and Protein Coated Spheres Concanavalin A (ConA) and FITC labeled ConA were obtained from Sigma Chemical Company. Succynylated conCanavalin A (SConA) was a generous gift from Dr. John 40 Wang's laboratory, Michigan State University. Lectins (long/ml) were incubated with pretreated neutr0phils (106 Cells/ml) for varying time intervals and the cells fixed with a mixture of 4% paraformaldehyde and 0.1% glutaraldehyde (Sigma) for 30 minutes. The fixed cells were then washed X3 with PBS. If the lectins had not been previously labeled with fluoroscein isothrocyanate (FITC), the cell bound lectins were labeled using an immuno- histochemical "sandwich" technique. Briefly, the fixed cells with ConA attached to their surface were incubated in solutions containing rabbit anti-ConA for 1 hour at 37°C, washed in PBS, and reincubated with solutions containing FITC labeled goat anti-rabbit immunoglobulin overnight. The cells were then washed in PBS. All antisera were purchased from Behring Diagnostics, North Somerville, New Jersey. To examine for the presence of FITC labeled lectins on the cell surface, the cells were mounted on glass slides, coverslipped, and examined using a Leitz microscope (100X objective) with UV illumination. Gram negative bacteria (a generous gift of Dr. Robert Moon's laboratory, Michigan State University) were grown (18-24 hours at 37°C in a 5% CO atmosphere) in Tryptic Soy 2 Broth (Difco Laboratories, Detroit, Michigan), heat killed (56°C for 30 minutes) and labeled with FITC as previously described 5’ 89. Briefly, 1010 bacteria/ml of saline were incubated 30 minutes at 37°C with FITC (lmg/ml saline adjusted to a pH of 11.0 with 1N naOH) and washed x3 in PBS. 41 The organisms used were Eschericia coli and piliated or non-piliated strains of Salmonella typhimurium. NeutrOphils (106 cells) were incubated for various times at 22°C with varying concentrations of the bacteria and then fixed in 2% glutaraldehyde (10 minutes at 22°C). To remove bacteria not bound by neutrophils, the suspension was centrifuged (1500 rpm for 20 minutes) through a Ficoll—Hypaque solution (same concentration used for cell isolation). Neutrophils with attached bacteria were pelleted through the Ficoll-Hypaque suspension when centrifuged at 1500 rpm for 20 minutes, leaving the unattached bacteria in suspension. Several experiments utilized bacteria treated with human C (Cordis) 3 and fresh or heat inactivated (56°C for 30 minutes) human serum prior to incubation with neutr0phils. Human serum was obtained by centrifugation of human coagulated blood. Bacterial specific immunoglobulins were absorbed from serum 9 heat killed as previously described 5. Briefly, 10 bacteria were incubated with human serum 15 minutes at 0°C, centrifuged for 10 minutes at 2000 rpm (0°C) and the serum decanted off the bacteria. The conditions for treating bacteria with human proteins and concentrations of human proteins used are clearly specified for each experiment within the Results section. Following treatment, the bacteria were washed and resuspended in the various neutro- phil pretreatment solutions before incubation with neutrophils. Microscopic examination of bacteria attached to the neutrophil surface was performed using several techniques. 42 The aldehyde fixed cells were washed in PBS X3, placed on a glass slide, a coverslip attached and the cells examined using the Leitz microscope (50-100X objectives) with a white light or UV illumination. The aldehyde fixed cells with attached bacteria were also prepared for scanning electron microscopic (SEM) examination as previously described 7. Briefly, the aldehyde fixed cells were washed in distilled water and applied to poly-L-lysine coated glass coverslides (10mm diameter). The coverslip was rinsed in tap water, dehydrated in a graded series of ethanol, and critical point dried in a C02 liquid-gas transition (1300 psi at 42°C). The coverslips were then attached to aluminum stubs and "sputter coated" with gold (approximately 200nm in thickness). The stub was placed in a JEOL 135 C SEM and the cells examined at 3000-10,000X magnifications. Latex spheres (Sigma) of varying diameters (0.6-1.1u) were washed X3 in PBS and treated with a variety of human serum proteins. The conditions for these treatments are carefully described for each experiment within the Results section. The reagents used include human serum, human albumin (Sigma), rabbit or goat anti-human albumin (Behring Diagnostics), and human C3, a generous gift of Dr. Richard Patrick's laboratory (Sterling Winthroupe Research Institute, New York); and Cordis Chemical Corporation (Miami, Florida). Following all treatments, the spheres were washed in PBS and stored at 4°C. All treated spheres were resuspended in the same concentrations of solutions used to pretreat the 43 neutrophils. Neutrophils (106 cells/0.1ml of pretreatment solution) were mixed with 0.1ml of the treated spheres and incubated for various times before fixation (2% glutaralde- hyde; 10 minutes at 22°C). Spheres not bound by the neutro- phils were removed after aldehyde fixation using a Ficoll- Hypaque density gradient as previously described. Several microscopic techniques were also used to evaluate neutrophil-sphere attachments after each experiment. First, aldehyde fixed cells were washed in PBS, mounted on glass slides, coverslipped and examined using a Leitz microscope (SO—lOOX objectives) with white light or UV illumination. Neutrophils were also prepared for SEM examination as previously described. When cells were prepared for transmission electron microscopic (TEM) examination, the aldehyde fixed cells were washed in PBS and post fixed (10 minutes at 22°C) in a 0.1M phosphate buffer solution containing osmium tetroxide (1.0%). The fixed cells were then washed in PBS, incubated for 3 minutes in a graded series of ethanol (50%, 75%, and 100%), and immediately covered (255mm thickness) with a 50% (W/V) solution of LX 112 Resin with hardener (Polysciences, Inc., Warrington, Pa.) in absolute acetone. After a 3-hour incubation at 22°C, the resin was trimmed into 2mm squared and sectioned using an ultra microtome with a glass knife. The sections were mounted on copper grids, stained with lead citrate and uranyl acetate, and examined in a Phillip's 201 TEM at 3000-10,000X magnifications. 44 IV. Neutrophil Adherence to Glass or Protein Coated Glass Multiple well tissue culture chambers (Tissue Tech) fixed onto glass microscope slides were used to evaluate neutrophil adherence to glass. Each chamber was composed of 10 individual wells. Each well held 0.4ml of fluid and provided approxi- mately 5mm2 of a uniform substrata (glass) on which neutrophils could settle and attach. The glass surface could easily be treated with a variety of solutions. Solutions containing human fibrinogen (Kabi, Grade L, Stockholm, Sweden, 98% clottable; a generous gift of Dr. Douglas W. Estry, American Red Cross, Lansing, Michigan), Knox gelatin, human collagen (Sigma), human C3 (Cordis), human serum and PBS were incubated in these chambers for various time intervals and washed with PBS. Neutr0phils (105 cells/0.3ml) pretreated as previously described were then injected into each well, incubated (37°C) for various times, and then the chamber inverted. The wells of the chamber were immediately detached from the glass slides. The glass slide was then immersed in methanol, and attached cells stained with Wright's stain. The slides were then air dried, and neutrophils fixed onto the glass slide were counted using an image analyzer (Optimax; Hollis, New Hampshire). The protein coated glass was, on occasion, exposed to a variety of enzymes (e.g., porcine pancreatic trypsin or Chymotrypsin, Sigma) prior to use. The conditions for these treatments are specified for each experiment within the Results section. 45 On several occasions, specific enzyme inhibitors were also used to terminate enzyme action on these surfaces. These included L-1-tosylamide-2-pheny1-ethylchloio-methyl ketone (TPCK) and n-alpha-tosyl-L-lysine chloromethyl ketone (TLCK) (Sigma). The conditions for their use are also specified for each experiment within the Results section. V. Presentation and Analysis of Data The data are expressed in terms of the mean 1 standard error of the mean; n represents the number of experiments. Students' test was used to assess significance. Those items compared with p values <1 0.005 were considered significantly different; those with p values :> 0.50 considered strongly related. Most often comparisons were made to a "control pOpulation" response identified within each experiment. RESULTS I. The Influence of Albumin or Immunoglobulin on Neutrophil Adherence of Latex Spheres A. Sphere Pretreatment and NeutrOphil Binding of Albumin or Immunoglobulin Coated Spheres Latex spheres are uniform, available in different sizes ranging from 0.6-1.1u in diameter, and easily observed using light microscopy, SEM, or TEM. Their ability to bind proteins enabled evaluation of neutrophil adherence of particles coated with purified human proteins. Latex spheres were washed (X3) and reconstituted in PBS (0.1% packed sphere volume), aliquoted and used or stored at 4°C. Aliquots of the spheres were then incubated with 10% human serum albumin (HSA) in PBS for 30 minutes at 37°C. The spheres coated with HSA (albumin coated latex spheres or ACLS) were washed in PBS. Aliquots of ACLS were stored at 4°C or reincubated with rabbit anti-human HSA (antibody coated spheres or Ab-ACLS) for 30 minutes at 37°C, washed in PBS and stored at 4°C. Isolated human neutrophils (lo6 cells/ml PBS) were centrifuged (400 rpm for 10 minutes) into a cell pellet within serum coated 12X75 mm glass test tubes. The supernatant was discarded and the cells gently 46 47 resuspended. Washed latex spheres, ACLS or Ab-ACLS were added (0.1ml) to the neutrophil suspensions, gently mixed, and incubated for one minute at 22°C. Neutrophil adherence of the spheres was terminated by diluting the suspensions with 2ml of PBS and immediately fixing the cells with an equal volume of 2% glutaraldehyde for 10-30 minutes at 22°C. Spheres not bound by neutrophils after aldehyde fixation were removed using a Ficoll-Hypaque densitygradient and the number of spheres bound by neutrophils determined using phase-contrast microscopy. The data in Table 1 demonstrates that neutrophils avidly adhere latex spheres, and HSA coating of the spheres almost totally abolished this adherence. The HSA coated spheres were refrigerated and used over a span of several days with little variations to the results. The reduced adherence did not appear to be a direct effect of HSA on cell adherence mechanisms, since pretreating neutrophils with 10% HSA (10 minutes at 22°C) did not impair their ability to adhere (11 i 1.0 latex spheres/neutrophil, n=2) latex spheres (0.1% packed volume). Incubation of ACLS in solutions containing rabbit anti-human albumin agglutinated ACLS, yet following the removal of the antiseras, the spheres were easily dispersed by sonification. Immunoglobulin was demonstrated as being antigenically present on the surface of the spheres days after their preparation using FITC labeled anti-rabbit immunoglobulins (n=3). The attachment of immunoglobulin to ACLS significantly (p<: 0.001) enhanced the ability of Table l. * *‘k 48 Neutrophils (106 cells/0.1ml PBS) were incubated with the pretreated latex spheres (0.1% packed volume, 0.8u in diameter) for 1 minute at 22 C, diluted with 2 ml PBS and fixed with equal volumes of glutaraldehyde. A 1.0 volume of PBS, HSA (10% in PBS) or a variety of antiseras (1:10 titer in PBS) was used for each pretreatment. Spheres (0.05 ml of a 10% packed sphere volume) were washed in PBS after each pretreatment. After the last pretreatment the spheres were reconstituted to a 0.1% packed sphere volume with PBS. 50-100 neutrophils/test were examined using phase-contrast microsc0py (lOOX objective), attached spheres counted and divided by the number of cells observed. n = the number of individual experiments performed. 49 Cessnad cmeom Ame om.o H m.e + «mm naucm pennmm amm CHHDQOHmOGSEEH GHESQH4 spasm Rev Ho.o H m.o + pannmmnaucm umow lauds panama 4mm mu cmfism Ame mo.c H H.o u: nu ufluca pannmm 4mm cwfisnad cmESm Ame om.o H m.» + u: nfluca pannmm smm Ame no.0 H «.0 -- -- -- ems . I . II I: II Amwumnmm xmumqv Amy mm o + N m mmm HHH HH H mAcv Haflcmonusm2\mmuonmm coflummmummm «emucmEumwnpmum muwnmm xmumq umnfisz mmmuo>m mumnmm «.mmnwnmm xmumq pmumoo cfiaononoanEH paw .mmumnmm xoumn pmumou cwesnad .mmnmnmm xmumq mo mocmumnpd Handouusmz .H wanna 50 neutrophils to bind the treated spheres (Table 1). This enhanced adherence persisted when the antisera was heated (56°C for 30 minutes) before use (n=3). Rabbit anti-human C3 failed to agglutinate ACLS and spheres treated with this antisera were poorly bound by neutrophils. Although goat anti-human albumin agglutinated ACLS, it failed to enhance neutr0phil adherence of the spheres. Removal of albumin from ACLS by components within the albumin specific antisera could not account for the enhanced neutrophil adherence of Ab—ACLS since refincubation of Ab-ACLS in HSA failed to markedly diminish sphere adherence to neutrophils (Table 1). The Ab-ACLS were also used days after their preparation with little variation to the results. Sonification of ACLS did + not alter neutr0phil adherence of ACLS (0.2 - 0.03 ACLS/ neutrophil, n=3). B. Chemoattractant or Colchicine Modulation of Neutrophil Adherence of Immunoglobulin Coated Spheres Since Chemoattractants and antitubulins influence neutr0phil adhesiveness to various substrata, their effects on neutrophil adherence of Ab-ACLS were studied. Neutrophils were preincubated in PBS, colchicine (2X10-6M), or fMP for 15 minutes at 37°C. Single or multiple concentrations of fMP were used to pretreat the neutrophils. The cells were either 6M fMP for 10-15 minutes or exposed to 6 exposed to lO-BM or 10- 8 M for 5 minutes and then re-exposed to 10- M fMP for 10 6 lo' minutes'(10-8-lo- MfMP). All treated cells were then 51 centrifuged into a cell pellet, and the supernatant discarded. The cells were then gently mixed with 0.1ml of Ab-ACLS and incubated at 22°C for 1 minute. The cell mixtures were then diluted 20 fold in their respective cell pretreat- ment solutions, immediately fixed with an equal volume of 2% glutaraldehyde, and incubated 10-30 minutes at 37°C. Spheres not adhering to neutrophils were removed using a Ficoll- Hypaque density gradient and sphere binding evaluated using light microscopy or SEM. The data in Table 2 demonstrates that the percentages of cells binding Ab-ACLS did not appear to be influenced by col- chicine or fMP pretreatments. Chemoattractants or colchicine also did not appear to enhance neutr0phil adherence of Ab-ACLS even when the concentrations of Ab-ACLS were increased 10 fold (Table 2). Decreasing Ab-ACLS concentrations to 0.01% packed volume almost totally abolished sphere adherence to neutro- phils (0.55 i 0.07 Ab-ACLS/neutrOphil, n=2). The neutrophil pretreatments also did not appear to alter the fraction of cells adhering Ab-ACLS. The t0pographica1 surface distributions of adhering Ab—ACLS were also evaluated on fMP, colchicine, or PBS treated neutrophils. The data in Table 3 demonstrates that two adherence distributions were routinely observed using light microscopy or SEM to examine the pretreated cells. Consistently greater than 90% of the cells pretreated in PBS 8 or 10- M fMP remained spherical after pretreatment (n=11) and the majority of them bound Ab-ACLS at a single region on Table 2. 52 * 106 n utrophils were incubated (10 minutes at 37 C) in various pretreatment solutions, centrifuged into a pellet, and the super- natant discarded. ** Ab-ACLS were reconstituted in the pretreat- ment solutions to a concentration of 0.1% or 1.0% packed sphere volumes. To each cell button, 0.1 ml of the spheres were added, incubated 1 minute, diluted with 2.0 ml of the pretreatment solution and immediately fixed with 2.0% glutaraldehyde. 50-100 neutr0phils were examined and the number of spheres adhering to these cells counted and this number divided by the number of cells counted. 100 cells were examined for each test and the number of cells adhering spheres divided by the number of cells examined. n = the number of individual experiments performed. 3 .DANV n0.0 H n0.0 10v 00.0 H 00.0 Ame 00.0 H 00.0 10V 00.0 H 00.0 Ame 00.0 H 00.0 Ame 00.0 H 00.0 Ame 00.0 H 00.0 10v no.0 H 00.0 Ame 00.0 H 00.0 10V 00.0 H H0.0 00.H Ace 0H.0 mxcv meehmrea mews mAU¢Inm nonpomz Ho coHHomnm «4 HHBQOhusmz\mqoann< 100 0.0 H 0.0 10V H.0 H 0.0 Ame 0.0 H 0.0 100 0.0 H H.0 E 0.0 H 0.0 E 0.0 H 0.0 100 0.0 H 0.0 rev 0.0 H 0.0 100 0.0 H 0.0 10V «.0 H 0.0 .Asv wo.H Ace wH.o 120:0Hn0u0HV 02H Az0u0HV mam as nose mzH AEmloaxmv mcHOHaoaoo mmm « ucwEumeHonm HHoU Ho mchch HHnmonpnmz so mucmuomuuHMOEmso Ho mcHoHnoaoo mo muommmm one Amqoaunae mwuwnmw emumoo :HssnonocsssH .m magma Table 3. 'k ** 54 106 cells were pretreated with PBS, colchicine, single or multiple doses of fMP for 10 minutes at 37 C and centrifuged into a pellet. Ab-ACLS (0.1%) suspended in each pretreat- ment solution were added (0.1 ml) to the pretreated cells, incubated 1 minute, diluted with 2 mls of the pretreatment solution and immediately fixed with an equal volume of 2.0% glutaraldehyde. Following glutaraldehyde fixation and removal of unattached spheres, the cells were examined using phase-contrast micro- scopy. The spheres attached on spherical cells were categorized as being random or localized. Sphere attachment on cells with distinct front and tail ends was categorized as being random, lamellapolar (front binding), or uropolar (tail end binding). For cells to be placed in a lamellapolar or uropolar category, that cell must have bound greater than 90% of all spheres exclusively at the front or tail end respectively. 100 cells were examined for each test and the percentages of cells placed within each category determined. n = the number of individual experiments performed. 55 Amy m.m H om n.m H m I: m.H H em Amy H.H H no II II N.H H mm Amy m.m H mm - m.m H HH In m.H H mm Amy I: I: m.m H mn o.H H mm Ame II II m.H H Hn N.H H mm Ace Hmaommaawsmq w HmHomonb w pwuHHmooq Eocemm w 4* mqumlnd Ho mcoHponHuumHo mcHUch HMUHnmmHmomoa 120-0H- -0H0 e20 0 As nose mzm is nosxmv mcHoHnoaoo 0 120:0HV 02H mmm an #CTEHMTHHQHQ HHGU Ameoaueae mwemgmm ememoo eflaeeoflmoesesH hoe mcoHuanuumHo maHchm HHsmouuooz co muCMHUMHHHMOEmno no mcHoHnoHou Ho muommmm .m magma 56 their surface. Since the cells were spherical and possessed no discernable front or tail ends, this type of binding was classified as localized (Table 3). Colchicine 6M or 10-8-10-6M) pretreatments caused the and fMP (10' majority of the neutr0phils (greater than 65%, n=15) to change shape, forming a polar configuration. These cells possessed a distinct front and tail end that was easily discernable using light or scanning electron microsc0py. When Ab-ACLS were incubated with the polarized cells, they were observed to bind only at one pole on the cells regard- less of cell pretreatment. That region was the front end or lamellapolar region of the polarized neutr0phil (Table 3). When neutrophils were polarized, ruffling occurred primarily at the front of the cells and this was the principle region for attachment of Ab-ACLS (Figure la). Spherical neutrophils binding of Ab-ACLS at a single region was often observed to cause the cells to ruffle near the Ab—ACLS attachment sites (Figure lb). An attempt was made to enhance the ruffling on spherical cells and attachment of Ab-ACLS re-evaluated. Neutrophils 6M fMP (0.1ml) for 10 (106 cells) were incubated in 10- seconds and Ab-ACLS immediately added. Neutrophil adherence of the spheres was terminated 30 seconds after the addition of the spheres by aldehyde fixation. Although this short incubation period with fMP did not induce shape change, the majority of the cells (greater than 90%, n=3) were observed Figure l. 57 106 neutrophils were pretreated with PBS, colchicine, or various concentrations of fMP for 10 minutes, then incubated with 0.1 ml Ab-ACLS (0.1%) for 1 minute before being fixed in 2. 0% glutaraldehyde. The majority of neutrgphils pretreated with colchicine (2 x 10 M), 10 6M fMP or 10‘8M fMP assumed a polarized morphologic configuration such that these cells possessed distinct front and tail ends. These cells were observed to bind Ab-ACLS almost exclusively on their front ends (la). Cells pretreated with PBS or 1078M fMP remained spherical but also bound the Ab-ACLS at one restricted region on these cells. Photomicrographs of cells examined using SEM at 6000-7000 magnifica- tions. RBCs in the background were never observed binding Ab-ACLS. 58 Figure l. Neutrophil Binding of Immunoglobulin Coated Spheres (Ab-ACLS) 59 to ruffle extensively around their entire periphery. These cells also bound most Ab-ACLS at a single localized region (59 i 2% of the cells examined). C. Evaluation of the Ability of Neutrophils to Simultaneously Adhere Albumin and Immunoglobulin Coated Spheres Neutrophils (106 cells/0.1ml) have been shown to adhere ACLS7 when latex spheres were coated with 2% HSA and the sphere concentration was 5-20X greater than used in the previous experiments. Therefore, latex spheres were incubated in 2% HSA, washed in PBS, reconstituted to a 5% packed volume and'neutrophil attachment re-evaluated. Neutrophils (106 cells) were pretreated with PBS, fMP or colchicine as previously described, the supernatant discarded, and 0.1 ml of ACLS (5% packed volume suspended in the appro- priate cell pretreatment solution) added to the cell suspension. The cell-sphere mixture was gently vortexed, incubated 1 minute at 22°C, diluted 20 fold with the cell pretreatment solution, and fixed immediately with 2% glutaraldehyde. As the data demonstrates in Table 4, fMP significantly enhanced the ability of neutrophils to bind ACLS and this enhanced adherence did not appear to be due to fMP increasing the adhesiveness of a separate pOpulation of cells. The patterns of attachment of ACLS on the pretreated neutrophils (Table 4) were also similar to those described previously and very different from neutrophil attachment of Ab-ACLS Table 4. * *‘k 60 106 neutrophils/0.1ml were pretreated in PBS, colchicige, or fMP (0.9ml) for 10 minutes at 37 C, centrifuged into a cell pellet and the supernatant discarded. ACLS (0.1ml) were added to the cell pellet, gently mixed, incubated for 3 minutes at 22°C, and then diluted with 2 ml of 2% glutaraldehyde. Spheres not adhering to the cells were removed using a Ficoll- Hypaque density gradient. The numbers of ACLS adhering to 100 neutro- phils/experiment were counted using light microsc0py and averaged. The numbers of cells adhering more than 3 spheres were also counted and divided by total number of cells examined (100). The attachment of ACLS on the spherical neutrophils was observed to be either in a random fashion or at localized regions on the cells. Polarized neutrophils (i.e., those pretreated with colchicine, 10'6M fMP or 10‘ -10‘5M fMP) were observed to bind ACLS (greater than 90% of all attached spheres) on their front ends (i.e., lamellapolar adherence distributions), on their tails (i.e., uropolar), or randomly across their surface. For each experiment 100 cells were examined, the binding of ACLS on the observed cells examined, and each cell placed in one of the above cate- gories. The ratio of the numbers of cells in each category was compared to total cells counted for each experiment and this ratio expressed as a percentage. n = the number of individual experiments performed. 61 Amy Ame Ame Amy A00 Ace HMHOQWHHmEmQ w =xcv 0 mqoa 0o eccencccd smearmcnmoeon No.0 H an.o «0.0 H mn.o mo.o H mn.o mo.o H an.o «0.0 H on.o Ho.o H mm.o NCHuocom H Awucv mqod mHHmU MO COHHUMHh m.H H cm I: H.N H vH II H.H H we II II om.o H II vo.o H VA h HmHQmons w pmNHHmooq w +| +l +| +I +| + «« Amuce Harmonuamz\mqom m.H H mm o.N H mm «.0 H mm ~.¢ H mm H.m H mm mmmmMMIm 220:0H: uoav dam 0 as noav e20 120-0H x we mcaoaaoaoo iamuoav arm mmm chEHmmHHmum HHOO 120:0Hu 0HV dam 0- 120-0HV mam ran-0ae can Aswuoae dam 120-0H x we mcaoaccaoo mmd c HcmEumwuumum HHoU Amqomv,mmum£mm pmumoo cHfisnad Ho mocmnmnpfi HHnmouusmz .v manna 62 (Table 3). The ACLS were observed to bind in a random fashion around the entire periphery of spherical neutrophils. To the majority of polarized cells, they bound either in a 6M fMP) or random rashion (i.e., those pretreated with 10— almost exclusively (greater than 90% of attached spheres) on their uropolar or tail ends (i.e., those pretreated with col- chicine or multiple doses of fMP, Figure 2). Since very different binding distributions for ACLS and AbeACLS were observed on PBS, colchicine, and fMP pretreated neutrophils, equal aliquots of Ab-ACLS (0.2% packed sphere volume, 1.1u in diameter) and latex spheres coated with 2% HSA (5.0% packed sphere volume, 0.8g in diameter) were mixed in PBS, colchicine, or fMP as previously described. Consistently greater than 50% of all observed cells bound AbfACLS (i.e., the larger of the two populations of spheres) regardless of cell pretreatment and the adherence of ACLS (smaller spheres) appeared unrestricted by the binding of Ab-ACLS (Table 5). Neutrophil attachment distributions of Ab—ACLS and ACLS also remained unchanged on all pretreated cells. Greater than 84% of the neutrophils adhering both Ab-ACLS and ACLS bound Ab-ACLS at their lamellapodia on polarized neutro- phils or at a single ruffled region on the spherical neutro- 8 6 phils. The adherence of ACLS on PBS or fMP (10- M or 10- M) pretreated cells also adhering AbeACLS, occurred in a random fashion on the majority (78 i 3.4%, 76 i 5.3%, and 69 i 4.5% respectively, n=3) of the cells observed. ACLS were observed attached only on the uropods on the majority of colchicine Table 5. 'k ** 63 106 neutrophils were pretreated (10 minutes at 37 C) with various solutions, centrifuged into a pellet, and resuspended in 0.1 ml of mixtures of 0.2% Ab-ACLS (1.1u in diameter) or 5.0% ACLS (0.8n in diameter). Following a l-minute incuba- tion the cells were fixed in 1% glutaralde- hyde and examined using phase-contrast microscopy. 100 neutr0phils were examined and the number of cells adhering 1.1u.spheres counted and compared to the number of total cells examined. When examining cells adhering 1.1p spheres, the numbers of these cells also adhering 0.8p spheres (ACLS) were also counted and compared to the total number of cells adhering Ab-ACLS. All number ratios were expressed as a percentage. n = the number of individual experiments performed. 64 Ame Ame Ame Amv Ame H 15 v.H H mm o.m H Hm m.m H mm m.m H nn 0.0 H en mqofi msHHmnpm mHHsmonpsmz m m.H *« mdudlnd +| +l +l +l + meanmccd maaceonucmz 0 mm Hm mm vm mm 120-0H10u0Hv dam A: node mzH 120-0H x we mcacaccaoo iamuoav mam 0mm 4 ucwEummuumum HHmU Amquav mmuwnmm pmumou CHEDQH< paw Amqumlndv GHHDnonocsEEH Ho mocmnmspd HHsmoHusmz .m manna Figure 2. 65 Neutrophils (106 cells) were pretreated with PBS, colchicine, and various concen- trations of fMP, then incubated with 0.1 ml of ACLS (5.0%) for 1 minute and immediately fixed in glutaral ehyde. Cells pretreated in PBS (a) or 10' M fMP (b) remained spherical and most of these cells bound ACLS in a random fashion around their periphery (a,b). Neutrophils pretreated with 10' M fMP changed shape and ACLS were also observed to bind in a random fashion on the majority of these cell? (c). Colchicine (d) and fMP (10‘8-10’ M) (c) pretreated neutr0phils also formed a polar configuration yet con- sistently appeared to bind ACLS only at their urOpods (c). All photomicrographs obtained using SEM at 6000-7000 magnifications. 66 Figure 2. Neutrophil Binding of Albumin Coated Spheres (ACLS) 67 8 and fMP (10- -10-6M) pretreated cells (71 i 4.2% and 76 i 3.4%, respectively, n=4) also adhering Ab-ACLS. Figure 3 is a collection of SEM photomicrographs which illustrate the simultaneous adherence patterns of Ab-ACLS and ACLS on various pretreated neutrophils. D. Redistribution of Neutrophil Bound Immunoglobulin Coated Spheres Thus far, experiments had been designed to assess neutro— phil adherence and adherence topographies for protein coated spheres. Time intervals following sphere attachment to neutrophils were altered next and the effects of time on neutr0phil redistribution of cell bound spheres examined. Neutrophils were incubated in PBS, colchicine or fMP, centri- fuged into a cell pellet, mixed with Ab-ACLS (0.1%) or ACLS (5.0%) and incubated for 1 minute at 22°C. The neutrophil/ sphere mixtures were then diluted 20 fold in their respective pretreatment solutions to restrict further attachments and incubated for 15-20 minutes prior to the addition of an equal volume of 2% glutaraldehyde. The distributions of adhering ACLS or Ab—ACLS were then evaluated as previously described using light or scanning electron microscoPy. As compared to the data in Table 4, the adherence distributions of ACLS on neutrophils appeared to remain unaltered 15 minutes after their attachment (Table 6). Phase contrast and SEM examina- tion of all pretreated cells failed to provide any evidence of endocytosis of surface bound ACLS during these time Figure 3. 68 Neutrophils were pretreated with PBS, colchicine, or various concentrations of fMP, then incubated with an aliquot (0.1 ml) con- taining equal fractions of ACLS and Ab-ACLS for 1 minute and immediately fixed with glutaraldehyde. Cells incubated with PBS or 10‘ fMP remained spherical and most bound ACLS (0.6u) randomly. Ab-ACLS were bound and ingested at localized regions on the same cells (a). Cells pretreated with 10’6M fMP assumed a polar configuration and most of these cells also bound ACLS randomly with Ab-ACLS binding at theig front ends (b). Colchicine and fMP (10' -10'6M) pretreatment apparently caused most cells to adhere ACLS at their tail ends while simultaneously binding Ab-ACLS at their front ends. All photomicrographs were taken using SEM and 6000-7000 magnifications. 69 Figure 3. Simultaneous Neutrophil Adherence of Albumin (ACLS) and Immunoglobulin Coated Spheres (Ab-ACLS) Table 6. 70 * Neutrophils (106 cells) were pretreated ** with PBS, colchicine or fMP at 22°C for 10 minutes, centrifuged into a pellet and the supernatant discarded. ACLS or Ab-ACLS were added (0.1 ml) to the cell pellet, gently mixed, incubated for 1 minute, diluted X20 with their respective cell pretreatment solution, and fixed with an equal volume of 2.0% glutaraldehyde after a lS-minute incubation (22°C. The fixed cells (100 cells) were examined using light microscopy and the numbers of cells with 80% of the spheres attached only at their front ends (L), tail ends (U), or randomly bound (R) counted. The numbers of cells placed within each category (e.g., L, U, or R) were compared to total number of cells counted and this fraction expressed as a percentage. The category with the highest percentage is given for each experiment. n = the number of individual experiments performed. . 71 Amv Amy Amy Amy Amy Ace «« coHHanHumHo HMUHsmmumomoa Momma m wm H mm we H mm wm H mm we H mm we H mm mAUdIQ< E 5 E E E H a: D wm m wv D wm m wm m wm +l +l +l +l + mqvfl on vb Mb m5 mm 120:0H10u0HV dam A: node dam 120-0H x we mcaoazoaoo 120:0HV e20 0mm « ucofiummuumnm Hawo ucosnomuua Houmm mwuscHz ma mHHnmouusoz co AmAOH80pcmm EsflHsEflnmxp .m H ccsom umsu mawnmoupswz w Amv N H H m h AZmuoaamloHv ASM mfimv m.H H o.m ASmloav mZM Amy m.o H m.m AZmoa x NV mGHUHSOHOU Aoav H.H H m.v mmm =Acv EDHHoEflnmNu .m owumHHHm « ucmEummnumum Hamo ;« mo ucmfinomuufi Hwnmonusmz EDHHsznmmu waawcoaamm pcsom Hamu mo coflasnflnumflcmm can ucmE£UMpam Hangouusmz monosawcH umnu mcofluwocou .m manna 95 15 minutes post attachment. Examination of surface bound S. typhimurium on PBS and lO-GM fMP pretreated cells using light microscopy indicated that the bacteria remained randomly bound by the cells 15 minutes post attachment. Examination of the same cells.using SEM revealed an almost complete absence of bacteria on the exterior surface of these cells, indicating the bacteria were apparently ingested 8-1o'6M fMP during this time frame, yet colchicine and 10- pretreated cells retained bacteria on their exterior surface for up to 30 minutes (n=6) (Figure 8). The apparent redistribution of S. typhimurium across the neutr0phil surface to its tail proceSs may have influenced binding site topography for Ab-ACLS. Therefore, neutrophils were pretreated with colchicine or multiple increasing con- centrations of fMP and incubated with S. typhimurium using conditions previously shOwn to redistribute the attached bacteria. The cells were then centrifuged into a cell pellet, the supernatant removed, and 0.1 ml of Ab-ACLS (0.1% packed volume) suspended in the final cell pretreatment solution added to the cell pellet. Following a l-minute incubation (22°C) the cells were fixed, washed and examined using light microscopy or SEM. A large percentage of the colchicine or fMP stimulated neutrophils (79 i 5% and 84 i 7% respectively, n=5) that redistributed surface bound S. typhimurium rearward also bound Ab-ACLS almost exclusively at their lamellapodia (Figure 9). Figure 9. 96 Neutrophil Binding of Bacteria and Immunoglobulin Coated Spheres (Ab-ACLS) This is a SEM photomicrograph of a colchicine pretreated neutrophil magnified 12,000X. This cell was incubated with S. typhimurium for 15 minutes, Ab-ACLS added for 1 minute, and imme— diately fixed with 2% glutaraldehyde. The attached bacteria were observed to be adhering to the uropods on most of similarly pretreated cells while the spheres were bound almost exclusively to the fronts of the same cells. 97 III. The Effects of Chemoattractants and Colchicine on 'Neutrophil Binding of Serum and Complement Coated Particles A. Particle PreparatiOn Several techniques were used to fix C3b to the surfaces of a variety of particulate matter. First, FITC labeled gram negative bacteria (approximately 1010 S. typhimurium or 1010 S. ggli/ml of PBS) were incubated (30 minutes at 37°C) in an equal volume of fresh or heat inactivated (56°C for 45 minuteS) human serum. All human sera used for these experi- ments were preabsorbed of bacterial specific immunoglobulins 89 before use. The serum treated bacteria were then washed x3 in PBS, resuspended in PBS (109 bacteria/ml), and refrigerated (4°C) until used. Second, latex spheres (1.0% packed sphere volume) were washed in PBS, also incubated (30 minutes at 37°C) in fresh or heat inactivated human serum, washed X3 in PBS, and resuspended in PBS (0.1% packed sphere volume). The serum treated latex spheres were also refrigerated (4°C) until used. The final technique utilized purified human complement as a source of C3b. Latex spheres were again washed X3 in PBS, and then resuspended in 1000 U/ml of human C3 or trypsinized human C3. The C3 trypsinization procedure utilized soluble and insoluble trypsin (100 U) which had been previously shown to almost 5, 91 3 to C3b . Briefly, the trypsinization procedure involved adding the trypsin totally convert 1000 U of human C (100 U) to the C solution and, after a l-minute incubation 3 98 at 370C, neutralizing the trypsin activity by adding soy bean trypsin inhibitor (100 U) or removing the insoluble trypsin by centrifugation. Following latex sphere incubation with the human C3 or trypsinized human C the spheres were washed 3! X3 in PBS, resuspended in PBS (0.5% packed sphere volume) and also refrigerated until used. B. Verification of the Presence of‘CJb on the Surface of the Bacteria and Latex Spheres and its Effects On Neutrophil Binding of the Particles Aliquots containing latex spheres or bacteria previously pretreated with fresh or heat inactivated serum were incubated (30 minutes.at 37°C) with solutions containing rabbit or goat anti-human C3c. Latex spheres pretreated with trypsinized human C3 were treated in a similar fashion. Within 30 minutes, the bacteria or spheres pretreated with fresh human serum or trypsinized C3 were agglutinated from suspension (n=4). No visible agglutination of the spheres or bacteria pretreated with heat inactivated serum occurred using the same conditions (n=4). All test particles were then washed X3 in PBS and resuspended in PBS. Solutions contain— ing FITC labeled anti-rabbit or goat immunoglobulin were then mixed with these particles and incubated overnight at 22°C. The particles were again washed X3 and then observed micro- scopically using UV irradiation. The bacteria and spheres pretreated with heat inactivated serum did not fluoresce when exposed to UV irradiation conditions (n=3). Yet the bacteria or spheres pretreated with fresh serum or trypsinized human 99 C3 fluoresced brilliantly when exposed to the same UV irradia- tion (n=3). Since C3b was shown to be antigenically present on the particles pretreated with fresh serum or trypsinized human C3, they will henceforth be called C b coated particles. 3 The next experiments were designed to evaluate whether the particle bound C3b molecules would influence neutrophil binding of the particles. Neutrophils (106 cells) suspended in PBS were centrifuged into a pellet and the supernatant discarded. Bacteria (109 bacteria/0.1 ml) pretreated with serum or latex spheres (0.1% packed sphere volume) pretreated with serum, human C3, or trypsinized human C3 were then added (0.1m1) to the cell pellet. The neutrophil-particle suspen- sions were then gently mixed and incubated for 3 minutes at 22°C before 2 ml of_g1utaraldehyde (1.0%) were added. This suspension was then incubated (22°C) for 30 minutes, the glutaraldehyde fixed cells washed in PBS, and particles not bound by the neutrophils removed from the suspension using a Ficoll-Hypaque density gradient. The cells were then examined microsc0pica11y and the numbers of particles bound by 100 neutrophils counted and averaged. The data in Table 10 demonstrates that neutrophil bind- ing of the C3b coated particles was 10-50 fold greater than neutrophil binding of particles pretreated with heat inactivated serum or purified human C3. As previously observed when pretreating latex spheres with albumin, the treatment of latex spheres or bacteria with heat inactivated serum or purified human C3 significantly (p‘< 0.001) Table 10 . * ** 100 106 neutrophils/0.1 ml of PBS were incubated (3 minutes at 22°C) with a variety of test particles and then fixed with 2 ml of glutaraldehyde. All particles not attached to the neutrophils were removed by centrifugation through a Ficoll-Hypaque density gradient. Heat killed bacteria (S. typhimurium) were washed X5 in PBS, labeled with FITC, washed in PBS, pretreated with serum and resuspended to a concentration of 109 bacteria/m1 of PBS. Latex spheres were washed in PBS, pretreated with human C or serum, washed in PBS (X3) and resuspended to a concentration of 0.1% packed sphere volume. All particles (1 ml) were incubated with the pretreatment solutions (10 ml) for 30 minutes at 37°C and washed in PBS before use. Following the removal of the majority of unbound particles, the glutaraldehyde fixed cells were mounted on glass slides and examined microscopically. The fluorescing bacteria or latex spheres bound to neutr0phi1s were then counted and averaged (100x objective). n = the number of individual experiments performed. 101 lml oa.a H m.oa is oooac mo amass mmuacamdsua Ame mH.o H m.o in oooac mo amass Amv mm.a H m.m Esnmm ammum Amy mo.o H ~.o Enumm pmpm>fluomcH ummm mmumnmm xmumq Avav Nn.o H v.m Esumm omnHOmn< ammnm Amy mo.o H «.0 Eonwm pmum>fluomcH ummm mwumpomm Imw. H Handcuusw2\©csom mwaowunmm «% usmfiummnumum mauauumm .mwmmmmmm * mwaoflunmm pmumoo Edubm pom ucmEmHmEou mo mcwpcflm Hammonusmz .OH magma 102 impaired neutrophil binding of the spheres or bacteria respectively (p value obtained by comparing data in Table 10 to that in Tables 1 and 9). The C b coated particles were next reincubated (30 3 minutes at 22°C) with PBS, heat inactivated serum, goat anti- C3c or goat anti-human immunoglobulin. Following this incubation, the particles were washed X3 in PBS and resuspended in PBS to their original concentrations. The particles (0.1 ml) were then incubated (22°C) with neutrophils (106 cells/0.1 ml of PBS) for 3 minutes, fixed in glutar- aldehyde and the numbers of particles bound to neutrophils determined as previously described. The data in Table 11 demonstrates that reincubating particles pretreated with fresh serum or solutions containing the trypsinized C ingoat anti-immunoglobulin or heat 3 inactivated serum only minimally reduced the ability of neutr0phils to bind to the particles. However, the reincuba- tion of similarly pretreated spheres with goat anti-C3c almost totally prevented their binding to neutrophils. C. The Effects of Chemoattractants and Colchicine on Neutrophil Binding of Clb Coated Particles It has previously been reported 124, that CF may enhance the numbers of binding sites for C3b on the surface of leukocytes. Therefore, neutrophils (106 cells) were incubated in PBS, various concentrations of fMP, or colchi- cine for 10 minutes at 37°C. The pretreated cells were Table 11. * *‘k 103 106 neutrophils were incubated with spheres (0.1 m1 of a 0.1% packed sphere volume) pretreated as described for 3 minutes at 22°C, fixed in 2%_glutaraldehyde, and unattached spheres removed by centrifuging the cell-sphere mixture through a Ficoll- Hypaque gradient. The spheres were incubated (30 minutes at 37°C) with fresh human serum or trypsinized human C (1000 U) for 30 minutes at 37°C, washed in PBS X3, and then re-exposed to PBS, heat inactivated serum, goat anti- human immunoglobulin (1:10 titer), or goat anti-human C c (1:10 titer) for 30 minutes at 22°C. Thg spheres were then washed X3 in PBS and reconstituted in PBS to a concentration of 0.1% packed sphere volume. The fixed cells were mounted on glass slides and the numbers of spheres bound by 100 neutrophils using a Leitz microscope (100x objective) were counted and averaged. n = the number of individual experiments performed. 104 m H.H +| +l +l +l +l . +l +l + m.m Amncv H HanmonuswC\mmum£mm Um Um U :mEomlwucm o amasmnauca cflasnonocsEEH awasmlflucm QHHSQonocsEEH :mEsmlflpcfl Esumm omum>fluomcH umwm Esnmm ooum>fluomcH umwm mmm mmm ucmfiumwuuwum ocoowm m U Enhmm cmadm Esuwm cmfism Enumm cmfidm Esumm cmfism cmesm nmmnm pmNHCHmmmna cmEsm ammnm Ummwcflmmhue cmfism smmnm pmNHCHmmhne cmfism nmmum pmNflcflmmmHe “Cmgflwwhfiwhm #mhflh «« ZOHBdemmmm mmmmmm « muwmwpcé Damaommm o m u spas mmumamm wmumou n m U mo mcwpcwm Hanmonuooz mcflxoon .HH magma 105 then centrifuged into a pellet and the supernatants discarded. The C3b coated spheres or bacteria (i.e., those pretreated with fresh serum or trypsinized C3) (0.1 ml) were then added to the neutrophil button, the suspension gently vortexed, and incubated for 3 minutes at 22°C. Neutrophil binding of particles was terminated by the addition of 2 m1 of 1% glutaraldehyde and unbound spheres or bacteria removed from the suspension by centrifuging the cell-particle mixture through a Ficoll-Hypaque gradient. The numbers of particles bound by the neutr0phils were then counted and averaged (Table 12). The data in Table 12 demonstrates that colchicine pretreated neutr0phils bound roughly the same numbers of the C3b coated particles as did the control cells (i.e., those preincubated in PBS). The data in this table also demon- 8 strates that low concentrations of fMP (10- or 10-7M) only slightly enhanced the binding of the C 6 b coated particles. 6 3 Yet, pretreating neutrophils with 10- M or 10-8-10- M fMP significantly enhanced (p < 0.001) the numbers.of particles bound by the neutr0phils as compared to the binding of particles by the control cells. Similar percentages of cells were observed binding particles in all experiments (Table 12) . Table 12. 'k *‘k 106 106 neutrophils were incubated (10 minutes at 37°C) with PBS, colchicine or fMP, centrifuged into a cell pellet and the supernatant discarded. 0.1 ml of the various C b coated particles were added to the cell pellet and incubated 3 minutes at 22°C. Neutrophil binding of the particles was terminated by the addition of 1% glutaraldehyde. Latex spheres were incubated with fresh serum (n=8) or trypsinized human C (n=8) for 30 minutes at 37°C, washed in BS and resuspended (0.1% packed sphere volume) in the appropriate neutrophil pretreatment solution. 109 FITC labeled S.'typhimurium (n=5) were likewise incubated in fresh serum, washed, and also resuspended in PBS, colchicine, or various concentrations of fMP. Following neutr0phi1 binding of these particles, and the addition of glutaraldehyde, unattached particles were removed and the numbers of spheres or bacteria attached to the neutr0phils (100 cells examined/experiment) counted. All data for neutrophil binding of C b coated spheres or bacteria were combined and averaged for each experiment. 100 neutrophils were examined/experiment and the numbers of neutrophils binding 1 or more C b coated particles were counted, and expressed as a percentage. n = the number of individual experiments performed. 107 wv H mm m.~ H m.mH ASmIoHImloav mzm wv H mm m.m H 0.5H ASmloav mzw wm H mm N.N H ~.0H Ashloav m2m wm H 55 H.N H m.m A2 Ioav aim wm H mm h.a H w.h AZQIOHxNV mcflownoaou we H vs m.H H m.w mmm Amucv mmaofluumm pmumou nmu mamaucv Hflnmonusm2\mmaofiuumm « ucmEpmwuumum Haou H mafiumnpm maflnmouuowz w *« pmumou Q U mawnmouuswz an ccoom mmaofluumm powwow nmu mo mumnfisz may no mcfiowzoaoo no mzw mo muomwmm one .NH manna 108 D. The Effects of Colchicine and Time on NeutrOphil Binding of‘Cgb Coated Particles Since the binding affinities of colchicine and PBS pretreated neutrophils for C3b coated particles were similar, the adhesion patterns for C3b coated particles on these neutrophils were next eXamined. NeutrOphils (106 cells) were incubated in PBS or colchicine as previously described, centrifuged (400 rpm for 5 minutes) into a cell pellet and the supernatant discarded. To this pellet, 0.1 ml of the 9 C b coated bacteria (10 FITC labeled bacteria/0.1 ml) or 3 C3b coated spheres (0.1% packed sphere volume) were added, gently vortexed, incubated for.3 minutes at 22°C, and the suspension diluted 20 fold with PBS or colchicine. The neutrophil-particle suspensions were then immediately diluted with an equal volume of 2.0% glutaraldehyde. Unbound particles were removed using a Ficoll-Hypaque gradient and the neutrophils examined using a Leitz microscope with white light or UV irradiation, or by SEM as previously described. Virtually all (98 i 2%, n=9) of the PBS pretreated neutrophils remained spherical after treatment. Of these cells, most (92 i 3%, n=9) bound the C3b coated bacteria or spheres in a random fashion on their exterior surface (Figure 10). The majority of the neutrophils formed a motile configuration when exposed to colchicine (71 t 6%, n=15). Of these neutrophils, most (89 i 4%, n=15) were observed with C b coated particles bound only at one localized region, 3 their uropods (Figure 10). It was hypotheSized that either Figure 10. 109 106 neutr0phils were pretreated with PBS or colchicine (2X10'6M) for 15 minutes at 37°C and incubated with C b coated bacteria or latex spheres for 3 minutes before being fixed in glutaraldehyde. Figure 10 illustrates a typical spherical (PBS pretreated) neutrophil binding C b coated spheres in a random fashion. Co chicine pretreated neutrophils are illustrated in Figures b and c with C b coated bacteria (FITC labeled) and sphgres attached only at their uropods, respectively. All micrographs were obtained using a Leitz microscope (lOOX objective) with light (10a) and UV illumination (10b) or by SEM (10c). 110 Figure 10. The Binding Topographies for C b Coated Particles on PBS and Colchicine Pretreated Neutrophils 111 colchicine pretreatment may have modified the adhesion site topography for the C b coated particles or that the colchicine 3 pretreatment may have prompted a rapid redistribution of surface bound C3b coated particles across the neutrophil surface. Therefore, three parameters of this experiment were altered and the experiment repeated. First, the concentra- tion of C3b coated spheres added to the pretreated neutrophils was increased (1.0% packed sphere volume). This alteration was expected to enhance the numbers of sphere-neutr0phil collisions and adhesions that could occur in a short period of time. Second, the incubation time the spheres were in contact with the cells was shortened to 3—5 seconds. Last, after the 3-5 second incubation with the spheres, the neutrophil suspension was diluted 50 fold with PBS or colchi- cine to minimize further attachments and the suspension was either immediately or after a specified time interval fixed with an equal volume of 2.0% glutaraldehyde. The data in Table 13 demonstrates that the PBS pretreated neutrophils were consistently observed binding the C3b coated spheres in a random fashion (Figure 11) regardless of the time interval allowed for attachment and redistribution. Yet, the C b coated spheres were observed to be initially 3 bound almost exclusively at the front ends of colchicine pretreated cells. However, 15 seconds after attachment, most of the neutrophil bound spheres were observed to be attached on the colchicine pretreated neutrophils in a random fashion. Within 60-90 seconds after attachment, the majority of these Table 13. * ** 112 106 neutr0phils were incubated with PBS or colchicine for 15 minutes at 37°C, centri- fuged into a cell pellet, and the super- natant discarded. C b coated spheres (0.1ml) were added t3 the cell pellet, incubated for 3-5 seconds and diluted X50 with PBS or colchicine. The neutrophils were either fixed with 2% glutaraldehyde immediately after their 3-5 second incubation with the spheres, or at various time intervals after being diluted with PBS or colchicine. The fixed cells were then examined using a Leitz microscope and the surface distri- bution of adhering spheres evaluated. Cells binding distributions were classi- fied as random, or when greater than 80% of the cell bound spheres were attached only at the front or tail end of the cells, as lamellapolar or uropolar distributions, respectively. n = the number of individual experiments performed. 113 Away H339.5 wm H «a 0mm coma Ame umaomouo mm H mm 0mm ems Ame umaomoua wm H mm owm om Ame “Macao“: we H mm 0mm om Ame Mademou: as H mm 0mm om lamuoaxml mcaoanoaoo Amy , Eoccmm wm H om 0mm ma Amy umHommaamqu we H om 0mm mam Amy Eonsmm wv H om 00m coma Amy Eoocmm wa H vm owm oma Amy Eoccmm mm H om com om Amy Eoccmm mm H vm 0mm om mmm Amy EOpQMm mm H mm 0mm ma Amv Eopcmm mm H mm owm mum mficv Hcofipsnfluumwa mcflocwm Hmownmmumomoe «a mafia « ucmEummHumum Hangouunmz mafia up pmocmsHmcH mm maflzmonusmz pmummuumum mcfloanoaou no mmm so mmaofluumm pmumou nmu Mow mcoflasnwuumwn mcflpcfim Havanmmnqomoa .MH wanna Figure 11. 114 106 neutrophils in PBS were incubated with C b coated spheres for 5 seconds, diluted X 0 with PBS and immediately or after a 30- minute incubation, fixed with a 2% glutar- aldehyde solution. The photomicrographs (mag. X6600) illustrate the binding of C b coated spheres on the PBS pretreated c 115 5 seconds after the addition of these spheres occurred predominantly in a random fashion (11a). The spheres appeared to be retained on the surface of the PBS pre- treated cells in the same random manner 30 minutes post attachment (11b). 115 Figure 11. Random Binding of C3b Coated Spheres on PBS Pretreated Neutrophils. Figure 12. 116 106 neutrop ils were incubated with colchi- cine (2X10' M) for 15 minutes at 37°C, mixed with C b coated spheres and incubated for 5 secondg. The cell/sphere suspension was then diluted and immediately or after a specified time interval, fixed in 2% glutaraldehyde. These cells were examined using light and scanning electron micro- scopy. The neutrophils were most commonly observed binding the C b coated spheres at their lamellapodia 5 sgconds after the addition of the spheres (12a, b). Within 15 seconds after binding, the most prevalent binding pattern observed was a random pattern (12c). Within 90 seconds, most cells had spheres attached only to their uropods (12d). These spheres remained attached thusly for up to 30 minutes (12e). All photomicrographs were obtained using a Leitz microscope (lOOX objective) with white light illumination or by SEM (12b and e). 117 Figure 12. The Effects of Colchicine and Time on Neutrophil Binding Distributions for C3b Coated Spheres. 118 same cells were observed with only C3b coated spheres bound at their uropods (Table 13, Figure 12). The numbers of spheres bound by the PBS and colchicine pretreated cells were also counted (n=3) and found to be relatively consistent (13 i 5 spheres/neutrophil) from eXperiment to experiment which suggested that binding of spheres after dilution was unlikely. Examination of neutrophils from all experiments using light microscopy and SEM failed to indicate any evidence of sphere internalization by either PBS or colchi- cine pretreated cells. Examination of the PBS or colchicine pretreated neutrophils using SEM also indicated that the C3b coated spheres remained on the cells' exterior surface 30 minutes post attachment (Figures 11 and 12). The next series of experiments were designed to evaluate if colchicine pretreated neutrophils would 1) simultaneously bind antibody and C3b coated spheres at their lamellapodia, and 2) ingest antibody coated spheres at or near their attach- ment sites while selectively redistributing the C3b coated spheres rearward across their exterior surface. NeutrOphils (106 neutrophils) were pretreated with colchicine as previously described, centrifuged into a pellet, and the supernatant discarded. A mixture containing an equal volume of C b and antibody coated spheres (2.0% packed sphere 3 volumes, respectively) was added (0.1ml) to the cell pellet and gently mixed. Two different sized spheres were used in these studies to aid in identification (e.g., 0.6u in diameter spheres were C3b coated and l.lp diameter spheres 119 were antibody coated). In addition, as further evidence for the selective binding of C3b, the antibody coated spheres were prepared differently than previously described in Results Section I. The two populations of latex spheres (0.6u and 1.1u diameter) were first incubated separately in fresh serum. The 0.6u C3b coated spheres were washed X3 in PBS and set aside. The 1.1u C3b coated spheres were also washed X3 in PBS, then reincubated with rabbit anti-C c (30 3 minutes at 37°C), and washed again X3 in PBS. Equal aliquots of both pOpulations of the pretreated spheres (0.6u C3b coated and 1.1u antibody coated) were then mixed. Following the addition of the C3b and antibody coated spheres to the cell pellet, the cell/sphere suspension was incubated for 5 seconds at 22°C and diluted 50 fold with PBS. The neutr0phils were then either immediately or after a specified time interval, fixed with 2% glutaraldehyde and the fixed cells prepared and examined as previously described. Consistently, almost two thirds (65 i 5%) of the cells examined (n=7, 100 cells examined per experiment) bound both the 0.6u and 1.1u diameter spheres 5 seconds after the addition of the spheres. Greater than 90% of these cells (94 i 7%, n=4) were observed with spheres bound only on their lamellapodia during this time period. When the incubation period prior to fixation was extended to 15 seconds, greater than 80% of these cells (82 $ 5%) were observed with 1.1u spheres still bound at their lamellapodia (n=5). However, most of these cells (76 t 8%, n=5) were observed with 0.6u 120 spheres now bound randomly on their exterior surface. Within 90 seconds, 74 i 5% of the same cells were observed with 0.6p spheres bound only on their uropods (n=5). The.uropolar bound C3b coated spheres appeared to be retained on most (63 i 5%, n=3) cells long after (20 minutes) the antibody coated spheres were ingested. Figure 13 illustrates the binding and inges- tion or redistribution of antibOdy and C3b coated spheres respectively on colchicine pretreated neutr0phils. E. 'The Binding Distributions for Cqb‘Coated'Particles on fMP Stimulated Neutrophils '“ We had previously shown that fMP enhanced neutrophil binding of C3b coated particles. Neutrophils were again pre- 8 6M and :Lo"8--1o"6 treated with 10- M, 10' M fMP as preViously described and 0.lml of the C3b coated latex spheres suspen- sion (1.0% packed sphere volume) added to the cell pellet. This mixture was gently vortexed, incubated for 3-5 seconds at 22°C, and diluted 50 fold with the same concentration of fMP used previously to pretreat the cells. An aliquot of the cell suspension was then immediately withdrawn and fixed in 1% glutaraldehyde. Additional aliquots were then removed at specified time intervals and also fixed in 1.0% glutaralde- hyde. Unbound spheres were removed as previously described, the cells examined using light and scanning electron micro- scopy, and the distributions of adhering spheres evaluated. The majority of neutr0phils (90 i 5%, n=16) pretreated 8 with 10_ M fMP remained spherical through all experiments. Figure 13. 121 Neutrophils were pretreated with colchicine and incubated (5 seconds) with C b coated latex spheres (0.6u in diameter) and immunoglobulin coated spheres (1.1p in diameter) then fixed with 2% glutaraldehyde at various time intervals. Within 5 seconds of binding, the immunoglobulin and C b coated spheres were consistently 0 served to be bound in a localized fashion on these cells' lamellapodia (13a). Within 10 seconds the C b coated spheres were observed binding rgndomly on the same cells. Within 90 seconds, many of the immunoglobulin coated spheres were apparently ingested by the colchicine pre- treated cells. Yet, the C b coated spheres were exteriorly bound on these cells' uropods (130). All photomicrographs were obtained using SEM. 122 Figure 13. The Binding, Ingestion, and Redistribution of C b and Immunoglobulin Coated Spheres on C lchicine Pretreated Neutrophils 123 The data in Table 14 demonstrates that these cells also bound the C3b coated particles randomly across their surfaces, much like PBS pretreated neutrophils. This attach- ment distribution was apparently unaltered 30 minutes after particle attachment. However, unlike PBS pretreated neutro- phils, the fMP pretreated cells did appear to ingest the C3b coated spheres. To quantitate this event, fMP pretreated cells were incubated with the C3b coated spheres for 5 seconds, diluted 50 fold with fMP and immediately or after a 3 or 30-minute incubation, fixed with glutaraldehyde. The cells were then examined using light microsc0py and the numbers of bound spheres counted. This number was allowed to represent the total number of eXternally and internally bound spheres. Two aliquots of the same cell suspension were also either incubated overnight with FITC labeled anti-CBC, washed, and re-examined microscopically using UV illumination or prepared for and examined using SEM. The numbers of spheres fluorescing or visible on the external surface of the cells examined using SEM was then counted and allowed to represent the number of externally bound spheres. The number of spheres initially counted using light microscopy was compared to the number of externally bound spheres and this ratio indirectly proportional to the numbers of spheres ingested by the cells. The data in Table 14 demonstrates that within 30 minutes greater than 90% of all spheres were ingested by the fMP pretreated cells. Figure 14 is a collection of photo- micrographs that best illustrate these events. 124 Table 14. The Effects of Time on the Binding and Ingestion of C3b Coated Spheres by Neutrophils Pretreated with fMP (10'3m)*' ' ‘ ’ ' " % Random Binding % Externally Bound Time ** Distributions’ i Spheres § (n)" 5 sec 97 i 7 103 i 5% (4) 3 min 95 i 6 84 i 11% (4) 30 min 94 i i 3% (4) 6 10 * ** 106 cells were incubated with lO-BM fMP for 10 minutes, centrifuged into a pellet, and the supernatant discarded. Spheres (0.1% packed volume) coated with C b were added to the cell button, gently vortexed and ingubated for 5 seconds. Following a 5 second incubation with the spheres, the cells were diluted X50 with 1058M fMP and immediately, 3 minutes, or 30 minutes later mixed with an equal volume of 2% ‘glutaraldehyde. 100 cells were examined using light microscopy and the binding of the C b coated spheres on these cells categorized. as random or "ot er". Random distribution refers to cells with spheres bound in an evenly distributed manner. The numbers of spheres attached to 100 neutrophils were counted using light microscopy and averaged. This number was allowed to represent the total number of spheres bound/ cell. Aliquots of these cells were also examined by SEM or incubated with FITC labeled anti-C b and examined using UV light. The numbers of spheres visible on 50 cells using SEM were counted and averaged. The number of spheres on 50 neutrophils that fluoresced after exposure to UV light were also counted and averaged. The sum of these numbers was allowed to represent the number of externally bound spheres. % externally bound spheres was obtained by comparing the ratio of the externally bound spheres to total spheres bound X 100. n = number of experiments performed. Figure 14. 125 Neutrophils (106 cells) pretreated with fMP (10'8M) were incubated with C b coated sphgres for 5 seconds, diluted X50 with 10' M fMP and immediately or after a specified time interval, fixed with glutaraldehyde. Neutrophil binding of C b coated spheres was observed to occur in a random fashion 6 seconds after the addition of the spheres using light or SEM (14a and c). These spheres were apparent on the external surface of thecells 3 minutes post attach- ment when observed using SEM or when the ' external spheres were labeled (14b) using a fluorescent label (FITC labeled anti-C3 However, within 10 minutes, most of the cell bound spheres failed to fluoresce when incubated with FITC labeled anti-C c and externally bound spheres were rarely c). -visible on the same cell population when observed using SEM (14d). 126 8C3b Coated Particles after M fMP Figure 14. Neutrophil Binding of Pretreatment with 10. 127 NeutrOphils were next pretreated with shape change concentrations of fMP (i.e., 10'6M or 10-8-10-6M fMP) and incubated with C3b coated spheres using the same experimental conditions. The majority of these cells (76 i 4%, n=9) formed a bipolar configuration after fMP pretreatment and like col- chicine pretreated cells, possessed distinct front and tail ends. When these cells were examined using light or scanning electron microscopy, sphere binding to these cells was observed to be predominantly at their front ends 5 seconds after the addition of the spheres (Table 15). Also like col- chicine pretreatment, the attached spheres were observed to be bound randomly on the fMP pretreated cells within 10 seconds (Table 15). However, when the cells were fixed 3 minutes after the addition of the spheres, a difference was observed between the fMP and colchicine pretreated cell binding of the C3b coated spheres. Observing this difference was dependent on the technique used to examine the cells. When the cells were examined using SEM, the majority of all visible (i.e., surface bound) spheres were attached on these cells' uropods. This binding distribution was again classi— fied as uropolar. Yet, when aliquots of the same cell p0pulation were examined using light microscopy, almost half (48 i 4%, n=4) of the spheres were observed to be bound on or within these cells' bodies. This binding distribution, with roughly half of the observed spheres bound on a cell's tail process and the remaining half on or inside its cell body, was categorized as "mixed" (Table 15). Figure 15 is a Figure 15. 128 Neugrophg ls were pretreated with 10—6M or %M fMP and incubated 5 seconds with b coated spheres. The cell/sphere saspension was diluted X50 with 10‘6M fMP immediately or after a specified time interval fixed with glutaraldehyde and examined using light microscopy and SEM. Initially, the spheres appeared to be bound by these polarized cells at their front ends (15a, b). Yet, 10 seconds after attachment, all spheres were bound in a random fashion (15c, d). Within minutes of attachment, externally visible spheres (visualized using SEM) were predominantly located at the cells' uropods (15e). However, when the cells were observed using light microscopy, many spheres were also observed on or inside these cells' bodies. 129 Figure 15. The Effects of Polarizing Doses of fMP and Time on Neutrophil Binding Distributions for C3b Coated Spheres Table 15. * ** 130 106 neutrophils were pretreated with 10'6M or 10' M fMP and incubated with C b coated Spheres for 5 seconds. The cells were then immediately diluted X50 with 10‘6M fMP and immediately or after a specified time interval fixed with 2% glutaraldehyde. Unattached spheres were removed from the cell suspension and the cells were examined using light microscopy or SEM. Sphere attachment patterns on the cells (lOO/experiment) were classified as random, mixed, lamellapolar, or uropolar when the spheres were bound with no apparent pattern, in equal distributions more than one site, or with greater than 80% of all attached spheres bound at the front or tail ends, respectively. n = the number of individual experiments performed. at 131 Amy Hmaoaous em H as lml emxas em H mm 0mm omH Amy Eoecmu we H en Amy Eopcmn we H an 0mm ma lml anommaHmEma we H mm Ame “waommaamsmfl we H mm 0mm m age seIOHumuoa Ame “Macao“: em H ea Ame emxas we H ma omm oma Amy Eoccmn we H ow Amy Eoecmn we H up now ma Ame umHommHHmEQH we H em Amy HmHommHHmsmH em H mm omm m mam seuoa Acv 2mm Ace mmoomOHOHz uanq we mcoHudeHumHa mcHech mEHB « acmEumwnuwum HHwO m mHHnmouusmz pmNHnmaom mam co mmHoHuHmm omumou Q U MOM wcoHpanuumHo mchch may :0 mEHe mo muowmmm one .mH manna 132 collection of light and SEM photomicrographs that best illustrates the binding patterns for the C3b coated spheres observed on the fMP pretreated cells as a function of time. Similar binding patterns for C b coated spheres were 3 observed on neutr0phils when pretreated with a single (10-6M) 8-1o'6m) of fMP. or multiple increasing concentrations (10- Comparisons of the numbers of spheres, attached to neutro- phils when observed using both SEM and light microscopy indicated that only approximately one half (46 i 3%, n=4) of the spheres remained bound to these cells' exterior surface 1 minute post attachment. Similar data were obtained when the externally bound spheres were labeled with FITC labeled anti+C3c (44 i 7%, n=4). The externally bound spheres (i.e., those labeled with FITC labeled anti-C c or visible using 3 SEM) were almost always bound on these cells' tail process. Large numbers of the externally bound spheres remained exteriorly bound 30 minutes post attachment. The simultaneous attachment and relocation of surface bound antibody and C3b coated spheres on fMP pretreated cells were next evaluated. The spheres were prepared as previously described in Results Section IIIE. Neutrophils were pre— 6 6 treated with 10- M or 10-8-10- M fMP and incubated with the spheres for 5 seconds. The cell mixture was then diluted 50 fold with 10'6 M fMP and immediately or'after a 10-60 second incubation fixed with an equal volume of 2.0% glutaraldehyde. 133 The cells initially bound both populations of spheres as previously described for colchicine pretreated cells. Con- sistently more than half of all fMP pretreated cells (60 i 6%, n=7) bound both types of spheres. The larger (1.1p diameter) antibody coated spheres were observed bound to the lamellapodia on 81 i 3% (n=4) of these polarized cells, regardless of the fMP pretreatment conditions or the time interval before fixation. Most of the cells (56 i 5%, n=8) were also observed with the Smaller (0.6u diameter) C3b coated spheres also bound to the lamellapodia when fixed 5 seconds after the addition of the spheres. However, when the fMP pretreated cells were fixed 10 seconds after sphere attach- ment, 86 : 4%,(n=8)of them were observed with the C3b coated spheres bound in a random fashion on the cell. Within 1 minute 82 i 5%, n=6 of the fMP pretreated cells were observed with exteriorly bound C3b coated spheres only on their uropods. Examination of the fMP pretreated cells 20 minutes post attachment using SEM indicated that the ur0pod bound C3b coated spheres were the only spheres retained on the exterior surface of these cells. Figure 16 illustrates the characteristic binding of C3b and antibody coated spheres on neutr0phils when fixed 5 seconds, 10 seconds and 60 seconds after sphere attachment to the cells. .1! LI! Figure 16. 134 Neugrophils, pretreated with 10'6M or --10"6 M fMP, were incubated with antibody (1.1p diameter) and C b (0.6m diameter) coated spheres for 5 seconds. The cells were then diluted x50 with 10'6M fMP and immediately or after a specified time interval, fixed with glutaraldehyde. Both populations of spheres were observed to bind at the lamellapodia (16a) on most cells. Ten seconds later, the smaller C b coated spheres appeared to be redistribuged rearward (16b) and by 1 minute, were almost exclusively on the cells' uropods (16c). Some C3b coated spheres were apparently being 1ngested (16b) en route to the uropod. Within one minute, most antibody (l.lu diameter) coated spheres were ingested (16c). 135 Figure 16. Simultaneous Adhesion, Ingestion, and Redistribu- tion of Immunoglobulin and C3b Coated Spheres on fMP Stimulated Neutrophils 136 IV. The Effects of Enzymes and Chemoattractants on Neutrophil Adherende to Protein Coated Substrata A. The Effects of Chemoattractants on Neutrophil Binding of Albumiquerum, and C17Coated Spheres Others have shown that neutrophils adhere to a variety 0f albumin (1-2%) and serum (10%) coated substrata 7' 14' 35' 121. These authors have also shown that CF increase neutro— phil adhesiveness to these protein coated surfaces. Yet, we have shown that neutr0phils rarely bound latex spheres coated with human albumin (5%), heated serum (50%), and C3 (1000 U/ml). To determine if CF would enhance neutr0phi1 binding of these protein coated spheres, spheres were coated Iwith 5% HSA, 50% human heat inactivated (56°C for 30 minutes) serum and human C (1000 U/ml) as previously described. The 3 treated spheres were then washed with PBS X3 and recon- stituted to a sphere concentration of 1.0% packed sphere volume. Neutrophils (106 cells) were incubated in PBS, or various concentrations of fMP as previously described and centrifuged into a pellet. The pretreated spheres (0.1m1)- were added to the cell pellet, gently vortexed, and incubated for 3 minutes at 22°C. The cell/sphere mixture was then mixed with an equal volume of 2% glutaraldehyde and the numbers of spheres bound to the cells counted and averaged using light microscopy. The data in Table 16 demonstrates that even relatively high concentrations of fMP (lo-6M) fail to enhance neutrophil binding of these protein coated spheres. Table 16. 'k ** 137 Latex spheres were incubated with 2 m1 of 5% HSA, 50% heat inactivated (56°C for 30 minutes) serum or human C (1000 U/ml). The spheres were then wasfied X3 in PBS and reconstitutgd to a 1% packed sphere volume in PBS, 10' M fMP or 10'6M fMP. Neutrophils (106 cells) were incubated in PBS, or fMP for 10 minutes at 22°C, centrifuged into a pellet and the super— natant discarded. The protein coated spheres (0.1ml) were added to the cell pellet, gently mixed and incubated for 3 minutes. The cell/sphere mixture was then diluted with PBS or fMP and immediately fixed with an equal volume of 2% glutaraldehyde. Spheres not bound by the cells were removed using a Ficoll- Hypaque density gradient and the numbers of Spheres attached on 100 neutrophils counted and averaged using light microscopy (lOOX objective). n = the number of individual experiments performed. 138 m Ame H.o H e.c 12ercav mam Ame H.o H m.o Azmncac ash mo cases Ame H.o H m.o mmm Amy m.o H m.o “Seuoac ESL Ame ~.o H o.H AZmIOHc mam ssnmm cmum>HuomcH pmmm “my H.c H m.c mmm lml «.0 H e.a Aze-eac ash Amy e.c H m.H Azmucav eze «mm wm Amy m.o H v.H mmm Ace + HHmo\mem£mm e «w ucmsummnumum HHmU « ucwfipmmuuwum mnmnmm + (Enumm pmum>HuomcH pmmm Ho .CHEDQH¢ .mu cmanm £HH3 emumou mmumsmm xmumn Ho mchch HHnmoupsmz co mucmuomuuumoamnu Ho muowmmm was .eH OHQMB 139 B. The Effects of Chemoattractants on Neutrophil Adherence to Protein Coated Glass Neutrophils are secretory cells and CF can cause them to release lysosomal enzymes 80. It was hypothesized that these enzymes could act upon protein coated substrata and enzyme induced alterations of the substrata might also allow neutro- phils to adhere to the substrata. To evaluate this hypothesis, a flat surface was required upon which cells could settle and be allowed to act upon the substrata. Multiple well tissue chambers were seleCted to evaluate this process. These chambers are composed of 10 individual wells that are fixed onto a single glass slide (Figure 17). Solutions were prepared that contained heat inactivated serum (50% in PBS, human C3 (1000 U/ml), gelatin (1 mg/ml of PBS), purified human collagen (1 mg/ml of PBS) and purified human fibrinogen (5 mg/ml of PBS). Human serum albumin was not used in these experiments due to our inability to get this protein to consistently adhere to glass. A small volume (0.3 ml) of PBS, gelatin, collagen, human C3, or human fibrinogen was then injected into individual isolated wells within the tissue chamber and the chamber incubated for 10 minutes at 37°C. The protein containing solutions were then removed by suction and PBS injected into all wells. This solution was also removed by suction and the rinsing process repeated X2. NeutrOphils (2 X 105 cells/0.3 ml PBS) were then injected into each of the pretreated wells. The chamber was incubated (10 minutes at 37°C) and then inverted. 140 The individual wells were then gently removed from the glass slide and the slide rinsed in a Petri dish containing PBS. The slide was air dried, immersed in absolute methanol and attached cells stained with Wright's stain. The number of cells adhering to the_glass slide circumscribed by each well was then counted using an image analyzer (10 fields, 6X objective). ' Consistently between 2000 and 4000 cells were counted adhering to the area of glass pretreated only with PBS (Table 16). This count was allowed to represent 100% neutro- phil adherence to the glass. Under the same conditions, neutrophil adherence to glass pretreated with serum, C3, gelatin, collagen or fibrinogen was dramatically reduced (Table 17). Neutrophils were then incubated with various concentrations of fMP for 10 minutes prior to injection into the protein pretreated wells, and the experiment repeated as previously described. Two very different dose response curves were obtained (Figure 18), depending upon the sub- strata used. The effects of fMP on neutrophil adherence to C3, gelatin, and collagen coated glass were very similar. Significant changes in adherence (p.< 0.005) were not observed until the cells were exposed to relatively high concentrations of fMP (lo-GM). Yet, significant (p.( 0.050) changes in neutrophil adherence to fibrinogen and serum coated glass resulted after neutr0phil exposure to 10-7M fMP (Figure 18). Maximal adhesiveness to all protein coated sub— strata occurred when the concentrations of fMP were near 5 x 10’6M. Figure 17. 141 This diagram illustrates a tissue culture chamber with 10 isolated wells affixed on top of a single glass microsc0pe slide. ' Approximately 0.3 m1 of solutions contain— ing protein or isolated neutrophils could be injected into each of the isolated compartments. The chambers were washed by removing the fluids from each well by suction. Cells injected into the chambers settled onto the glass slide by gravity and unattached cells removed by inverting the chamber and removing the compartments. The compartments were easily detached yet fixed well enough to the glass to with- stand the rigors of most experiments. 142 4 - TOP [r l l ‘ l r came we r _] : GLASS SLIDE [115110113113 I DIDIDICJ E] 143 Table 17. Neutrophil Adherence to Protein Coated Glass * Glass Pretreatment ** % Adherence (a) 1 PBS 100% (7) Human C3 (1000 U/ml) 5 i 2% (7) Gelatin (1 mg/ml) 4 i 1% (4) Human Collagen (1 mg/ml) 6 i 1% (4) Heat Inactivated Serum (50%) 3 i 1% (3) Human Fibrinogen (5 mg/ml) 6 i 2% (4) * *‘k I NeutrOphils (2 X 105 cells/0.3 ml of PBS) were injected into the tissue culture wells and incubated for 10 minutes at 37°C. Within 2 minutes all cells were previously shown to have settled onto the bottoms of these wells (i.e., glass slide). Following a 10- minute incubation, the chamber was inverted to allow unattached cells to fall off the glass slide. The tissue culture wells were then removed and the slide rinsed. The cells attached to the glass slide were fixed, stained and counted. The numbers of cells attached to the PBS pretreated glass was allowed to represent the maximum number of cells able to adhere under the conditions of the experiment (i.e., 100%). The numbers of cells adhering to the protein coated glass surface were compared to the numbers of cells adhering to PBS and expressed as a %. 0.3 m1 of PBS or a variety of human and non-human proteins suspended in PBS were injected into individual wells of a tissue culture chamber and incubated for 10 minutes at 37°C. The solutions were then removed by suction and the wells washed X3 with PBS. n = the number of experiments. ax’ir Figure 18. 144 Neutrophils (2 x 105 cells) were pretreated in PBS or various concentrations of fMP for 10 minutes at 22°C. The cells (0.3 ml) were injected into tissue culture wells and incubated for 10 minutes at 37°C. These wells were coated with human C , gelatin, human collagen, human fibrinogen, or heat inactivated human serum prior to the experi- ments. The numbers of cells adhering to the PBS or protein coated surfaces were then counted. The number of cells adhering to a PBS pretreated surface was allowed to represent the maximum number of cells able to adhere (i.e., 100% adherence). The number of cells adhering to the other surfaces were compared to the number adhering to the PBS pretreated surface and expressed as a percentage. The brackets indicate the value of the SEM. All experiments were performed with n=4. 145 - ”A ‘1 100V GELATI N HUMAN C3 COLLIGEN sat P PERCENT 3 1r 6 g ADHERENCE ‘00., r "CRIME" SERUM 501 D 1 ,““” 5 i 3 3 FUEI Figure 18. The Effects of Chemoattractants on Neutrophil Adherence to Protein Coated Glass ”Q T 3‘,- ~ w' 1'" f 146 NeutrOphils were next pretreated (10 minutes at 22°C) with a concentration of fMP previously shown to cause a thirty percent or greater increase in neutrophil adhesiveness to C3, gelatin, collagen, fibrinogen or serum coated glass. These cells were then split into two aliquots. One aliquot was centrifuged, the supernatant removed, and the cells washed in PBS. The washed and unwashed cells were then injected into wells pretreated as previously deScribed and the adhering cells counted. In addition, a second chamber was also pre- treated with human C collagen, serum, fibrinogen or gelatin, 3: and washed. The supernatant from the fMP pretreated cells was then injected into the wells, incubated for 10 minutes at 37°C, the fluids removed, and each well washed in PBS. NeutrOphils pretreated in PBS were then injected into these wells and adhering cells also counted. The data in Table 18 demon- strates again that PBS pretreated cells adhered very poorly to protein coated glass and fMP enhanced this adherence. This data also demonstrates that some factor within the fMP pretreated cell supernatant was apparently augmenting neutro- phil adherence to C3, gelatin, and collagen coated glass. Washing the cells dramatically lowered the numbers of cells adhering to these surfaces. Chemoattractant induced neutro— phil adherence to fibrinogen and serum coated glass did not require such a factor since the washed cells adhered well. Pretreating the C3, gelatin, and collagen (but not the fibrinogen and serum coated glass) with the fMP cell super- natants also greatly enhanced neutrophil adherence to these surfaces (Table 18). Table 18. * ** § 147 Neutrophils (5 X 105 cells/0.3ml) were incubated in PBS or various concentrations of fMP for 10 minutes at 22°C. The fMP pretreated cells were split into two aliquots. Both aliquots were centrifuged and the supernatants removed from one and saved. This aliquot was diluted X50 with PBS, washed again, and reconstituted to its original concentration with PBS. The remaining aliquot was left suspended in its original supernatant. Individual wells within a tissue culture chamber were injected with PBS or various protein solutions (0.3ml) and incubated for 10 minutes at 37 C. These solutions were then removed by suction and washed with PBS. After washing, several chambers were reinjected with 0.3ml of various super- natants, incubated for an additional 10 minutes at 37°C, and washed in PBS before use. 5 X 105 neutrophils were injected into the pretreated wells of a tissue culture chamber and incubated for 10 minutes at 37°C. The chamber was then inverted, the wells removed and the glass slide rinsed in PBS. The slide was then immersed in abso- lute ethanol, attached cells stained and counted using an image analyzer (10 fields, 6X objective). A percent adherence was obtained by comparing the number of cells adhering to those adhering to untreated glass using the same experimental conditions. n = the number of experiments. 148 Ame wm u we uceumcwmmcm Hamo is OH x ml mam + elecm 1Ha\caac cecaaeou mme Ame we H a ias\asac ceaseeou leesmmac axe swuoa x m Ame we H mm “Hs\msac cmmmaaoo lemcmmzecc ash amuse x m Ame we H m 1He\mawc cmmmaaou mme Ame wm I ma uceeacwmecm Have is (OH x my use + e can las\msac caeewwc mma Ame we H NH AasxmeHc caumemu leashesc ash Sense x m Awe ww H mm leaxmaac seeeemc leechesccc ash zeuoe x m rec wH H e “Hs\msac caemHmo mme rec wm . we .ucmwmcwwene Heme lseyce.x.mc.mse + paw Aas\o oooHc no cases mme lei we H ea 1Hs\s cecal mu cease leechesc ash zeuce x m rec wm H ew Aaexc oocec mu sees: lememmzcsc axe zenoa x m lac wm H w Aaaxo cocwc mu cease mme mace + mocmumsefi w we ucmfiummuumhm mumuquSm « ucmfiumwuumnm HHsmouusmz + mmmHo emumou chuoum ou mocmuwnem HHnmouusmz so usefiummnumnm ASH Ho ucwpmcnwmsm HHwU wan Ho muommmm one .eH oHame 149 Amy wm u HH acmemcwwdsm Heme A: oHc mzH + cam: Awomc aswmm cmem>HuomcH been mmm lee wm H mm chmc snwmm cmum>aeomcH seem Acmemmzc eze shuoa lee we H oe lwomv sshmm emem>HuomcH ummm Acmcmmscsc axe spice lav wm H OH Awomv sawmm emum>HuomcH ummm mmm Amy wm . ea hememchmesm Heme is ch axe + Unml AHE\mEmV ammocHHme cmesm mmm Ami we H we Aas\msmv cmmocHwnHm cease Aememmzc axe spice lee we H me Aaaxmemc ammocwhnam cease emcmmzcec ash sauce rec wm H He Aeaxmamc smmosaweae amass mma Ace H mocwuwzpm w ee pcwEummHuwum mamuumnsm e ucwfiummuwmum HHnmouusmz A.U.ucoov mH mHQma 150 Several authors have shown that fMP binds to protein coated glass 115’ 116. However, solutions containing fMP (lo-SM) were incubated in wells pretreated with all of the proteins used in the above experiments and this pretreatment failed to significantly enhance neutrophil attachment to these surfaces (p :> 0.90, n=3). NeutrOphils contain a number of trypsin and Chymotrypsin- 1ike enzymes within their lysosomal granules 80. Wells within tissue culture chambers were pretreated with human C3, collagen, fibrinogen, heat inactivated serum and gelatin as previously described. To these wells, 0.3 ml of PBS, trypsin (100 U/ml of PBS) or Chymotrypsin (100 U/ml of PBS) were injected and incubated for 1 minute at 37°C. The solutions were then removed using suction and the wells washed X3 in PBS. Neutrophils (2 X 105 cells, 0.3ml of PBS) were injected into each well and incubated for 10 minutes at 37°C. The chamber was then inverted, the wells removed, and the attached cells fixed, stained and counted as previously described. The data in Table 19 demonstrates that exposing the protein coated glass to either of the enzyme solutions enhanced neutr0phil adherence to those surfaces. Yet, the surfaces coated with human C3, collagen, and gelatin were most affected by trypsin and those coated with human fibrinogen or serum by Chymotrypsin (Table 19). Neutrophils were then preincubated with fMP (5 X lO-GM) for 10 minutes at 22°C. A specific trypsin inhibitor (TLCK, 5 X 10_4M) was then added to one aliquot of the Table 19. * ** 151 Individual wells were injected with 0.3 ml of a variety of protein solutions. The solutions were incubated for 10 minutes at 37°C and washed with PBS. Solutions containing either trypsin or Chymotrypsin were then added to the wells and incubated for 5 minutes at 37°C. These solutions were removed by suction and washed in PBS X3. Neutrophils (2 X 105 cells/0.3m1 PBS) were injected into the wells and incubated for 10 minutes at 37°C. The chamber was then inverted, the wells removed, and the adherent cells counted using an image analyzer (10 fields, 6X objective). A percentage was derived by comparing the number of adherent cells for each experiment to the number of cells adhering to untreated glass and multiplying by 100. § n = the number of experiments. 152 Ame we H em Awomv Ennmm pmum>HuomcH ummm Aev wb H om AHE\mEmV ammocHHnHm cmfism Ame wm H Hm las\m5wc cwuaemo Amy wm H mm AHE\mEHV cmmmHHou Ame we H em Aaa\a ocec camesweosseo 1Hs\c cecal mo cease Amy we H on Awomv Ennmm pmum>HuomcH uwmm Aev wm H Hm AHE\mE my cmmOCHunHm cmEnm Aev wm H me AHE\mEHV GHHMku Amv wm H mm AHE\OEHV cmmMHHoo Rec we H mm las\o coac cammswe Aes\s cccec mo cease m Acv H wocmnmspd w ee ucwEumee mamucm e.ucmaummnuwnm mumuumnom mmMHw pmumoo CHmuonm o» mocmumnpa HHsmonuomz co mmewucm mo muommmm was .mH mHnme 153 pretreated cell suspension. Individual wells were then pre- treated with human C3, collagen, or gelatin and washed as previously described. Neutrophils suspended in fMP with and without TLCK were then injected into the wells, incubated for 10 minutes at 37°C and neutr0phil adherence evaluated as pre- viously described. That data in Table 20 demonstrates that TLCK effectively impaired the ability of fMP pretreated cells to adhere to C3, collagen, and gelatin coated glass. Neutrophils were next pretreated with 10-7M fMP for 10 minutes at 22°C and PBS, TLCK (5 X 10-4M), or TPCK (a Chymo- trypsin inhibitor, 10°6M) added to two aliquots of the pre- treated cells. Individual wells of a tissue culture chamber were then pretreated with human fibrinogen or heat inactivated serum as previously deScribed. The pretreated cells, with or without TPCK or TLCK, were then injected into the wells, incubated, and neutrophil adherence evaluated as previously described. The data in Table 21 demonstrates that TLCK did not markedly alter fMP pretreated neutr0phil adhesiveness to fibrinogen or serum coated glass. This adherence was, however, markedly depressed by TPCK. The Chymotrypsin inhibitor was not killing the cells as determined by eosin exclusion (95% viability, n=2). This inhibitor also did not appear to be damaging neutrophil adhesion mechanisms since the majority of the cells (85 i 8%, n=3) would adhere to fibrinogen coated glass that had been pretreated with Chymo— trypsin as previously described. Table 20. 'k ** 154 Individual wells of a tissue culture chamber were injected with a variety of protein containing solutions. The solu- tions were incubated within these wells for 10 minutes at 37°C, then removed by suction and washed X3 with PBS. Neutrophils were pretreated with fMP for 10 minutes at 22°C. A trypsin inhibitor (TLCK) was then added to one aliquot of the pretreated cells. Neutrophils (2 X 105 cells/0.3ml PBS) were injected into the pretreated wells of a tissue culture chamber and incubated for 10 minutes at 37°C. The chamber was then inverted, the wells removed and the glass slide washed in PBS. Cells adhering to the slide were then counted using an image analyzer (10 fields, 6X objective). A percentage was obtained by comparing the numbers of cells adhering to the protein coated glass to those adhering to untreated ‘glass. § n = the number of experiments performed. 155 we em 12 (ca x me xoqa + e paw AEeIOH x me mse we H we Azeuca x me Haw AHs\meHc cHueHmo wm H eH Azeuoa x my Huge paw Azeuca x we axe we H mm ASeIOH x my mam AHE\mEHV cmmMHHoo :mEom we H Hm is (ca x my sons e paw Azenoa x my mam we H we Azeuca x we age AHE\D oooec mo cease mavncv + mocmuwnpm w + ee ucprmmHuwum HHnmouusmz e ucmfiummuumnm mumuumnsm ckuonm ou mocmnmnew HoananH GHmmwue m(Nn mmMHo emumoo HHnmonusmz emoseaH ucmuomnuumoamcu m Ho :oHanHscH was .om mHnme Table 21. * ** § 156 Neutrophils were incubated with fMP for 10 minutes at 22°C. Either TLCK or TPCK were added to aliquots of the cells. Individual wells of a tissue culture chamber were filled with solutions con— taining human fibrinogen or serum and incubated for 10 minutes at 37°C. The solutions were removed by suction and the wells washed X3 in PBS. NeutrOphils were injected (2 X 105 cells) into the tissue culture chamber wells and incubated for 10 minutes at 37°C. The chamber was inverted, the wells removed, and the glass slide rinsed in PBS. Cells adhering to the glass slide were fixed, stained and counted using an image analyzer (10 fields, 6X objective). A % of adherence was calculated by comparing the numbers of cells adhering to the protein coated glass to those adhering to untreated glass, and multiplying by 100. n = the number of experiments. 157 we I e As (OHV Home + e paw lseuowc mew we I em Azeuoe x ec mode + paw Azeuoav axe chec sawmm we H em cmesm emum>HuoecH ummm ASHIOHV mam we 1 a is (OHV some + paw Assuoac age we H we ASeuoe x ec acne can fishncav mam we H ee Awe\msec cemocewnae steam Azeucwc eze mAwflfiv H GUCQHmflmu/N w ee ucmEummHumHm mumumeSm e usmEummuumum HHamouuswz muoquanH chmmHHOENnO paw chmmHe ma mmMHo pmumou chuoum ou mocmnmnem HHnmouquz pmosecH HGMHUMHHHMOEan Ho coHuHQanH wse .Hm wame DISCUSSION I. Neutrophil Binding of Protein Coated Particles Neutrophil binding to protein coated substances is important for its locomotion and phagocytosis. A variety of neutrophil functions have been shown to be altered by the drug colchicine and various chemotactic factors. Of most importance to this report are the following; 1) these agents alter neutrophil adhesiveness to a number of protein coated 35. 56 and, 2) neutrophils form a bipolar configu- surfaces 7' ration with distinct front and tail ends when exposed to these agents, and 3) chemotactic factors promote the activation or release of neutrophil enzymes into their environment 91' 97' 10°. The first goal of this report was to determine whether colchi- cine or chemotactic factors would influence neutrophil binding of protein coated particles and bacteria. Particular emphasis was given to the effects of these agents on the topographical arrangements of neutrophil binding sites for the particles. The protein coated particles chosen for these studies were selected because of the considerable information that has accumulated about neutr0phil binding of these particles. 158 159 A. Neutrophil Binding of Immunoglobulin and/or Albumin Coated Spheres The coating of latex spheres with 5% HSA almost totally prevented neutrophil binding of the spheres. Similarly pre- treated spheres were agglutinated by immunoglobulin specific for human albumin and immunoglobulin could be detected anti- genically on their surfaces weeks after preparation. The immunoglobulin coated spheres were uniform, easily observed microscopically and preservable for weeks at 4°C. Unlike neutr0phil attachment of albumin coated spheres, immunoglobulin coated spheres were avidly bound by human neutrophils. It was assumed that this binding was a result of the spheres' inter- actions with the neutrophil Fc surface receptors. As previously reported 91, human neutrophils failed to bind spheres pretreated with goat immunoglobulins, apparently because of a difference between the Fc component of goat immunoglobulins and other animal immunoglobulins. Neither chemotactic factors nor colchicine enhanced or impaired neutrophil binding of immunoglobulin coated spheres. The spheres did adhere to neutr0phils in an asymetic manner. Neutrophils isolated from whole blood and suspended in PBS or pretreated with 10-8 M fMP were spherical. Immunoglobulin coated spheres were observed binding to the majority of these spherical neutrophils at one or two localized regions. The regions were usually ruffled in appearance, and often quite extensive. The probability of additional contacts within this region was perhaps greater than the remaining smaller spherical portion of the cell. Since the cells were 160 spherical, it was impossible to determine if sphere binding occurred at a region common to all cells. Walter, et al have reported that immunoglobulin coated erythrocytes adhere to PBS pretreated neutrophils in a "more or less random fashion" 10. Perhaps until additional data is obtained, this is the best description for spherical neutrophil binding of immunoglobulin coated spheres. The majority of the isolated neutr0phils were observed to form a bipolar configuration when exposed to colchicine or polarizing concentrations of fMP (e g.. lO-GM). Walter, et al have reported that neutrophils bind immunoglobulin coated erythrocytes only at their front ends when exposed to a gradient of chemotactic factors 10. We also report that neutr0phils bind immunoglobulin coated spheres almost exclusively at their front ends when exposed to multiple con- 8-10-°M). However, neutrophils pre— centrations of fMP (10- treated with a single polarizing concentration of fMP or colchicine also bound spheres at their front ends. Therefore, the frontal binding of immunoglobulin coated spheres does not appear to be a preferential response to a gradient of chemo- tactic factors. The only common fact known about these cells is that they all have formed a "motile configuration". The front end of the cells was ruffled in appearance before binding of the spheres occurred. It is unlikely these cells bind spheres at any region other than their front ends, since regardless of the sphere concentration or incubation time with the spheres, sphere binding at sites other than 161 their front ends was rarely observed. In agreement with others, the binding of immunoglobulin coated particles on polarized neutrophils seems to occur at the most logical site 10. The cells crawl in the direction their front end points. Collisions with particles would seem to occur most frequently at their front or "business" end. NeutrOphils rapidly ingested immunoglobulin coated spheres and did effectively remove them from their surface enabling further attachments to occur. The ingestion of spheres appeared to occur at or near the attachment site. When the cells were spherical, spheres were ingested within 1-3 minutes and they tended to fill all available internal spaces with no observable pattern. When polarized, the spheres were ingested at about the same rate, but were observed being swept away internally towards the tail end of the cell. This appeared to be a tremendously efficient method of removing particles enabling additional attachments and unencumbered ingestion to occur. The mechanisms by which polarized neutrophils selectively bind and ingest individual particles at different sites remain unknown. The means by which internalized spheres are swept rearward also are unknown. The presence or absence of microtubules appear unimportant for these proceSses since chemotactic factors have been reported to promote microtubule 103 within neutrophils while colchicine causes 93. polymerization microtubule depolymerization 162 In the beginning of this investigation, we also verified earlier reports by Smith and Hollers 7. NeutrOphils did bind spheres coated with only 1.0% human albumin. Pretreating 8M fMP enhanced their ability to bind neutrophils with 10— spheres, but did not cause shape change. Increasing the con- centrations of fMP used to pretreat the cells further enhanced sphere binding and caused the cells to form a bipolar con- figuration. Pretreating neutrophils with colchicine somewhat diminished their ability to bind the spheres yet caused shape change. Of particular intereSt were the binding patterns for albumin coated spheres on bipolar neutr0phils pretreated with colchicine; single or multiple concentrations of fMP (i.e., 6 8 6 10- M, and 10- -10— M, respeCtively). As reported 7, neutro- phils bound albumin coated spheres in a random fashion when 8M, or lO-GM fMP. Yet, spheres were pretreated with PBS, 10- only observed bound to the tail process of cells pretreated with colchicine or multiple doses of fMP. Smith and Hollers offered this and other evidence that pretreating neutr0phils with colchicine and multiple concentrations of fMP may have altered their binding site topography. In agreement with two independent laboratories, human neutrophils appear to selectively bind two different particles (i.e., albumin and immunoglobulin coated particles) at two different locations 7' 10. We proved that this can occur simultaneously on neutrophils. NeutrOphils exposed to colchicine or multiple increasing concentrations of fMP were shown to bind immunoglobulin coated particles at their front 163 ends while also selectively binding albumin coated spheres at their tail ends. Spherical neutrophils or cells only exposed to a single chemotactic stimulus also bound immunoglobulin coated spheres in a localized fashion and at the same time bound albumin coated spheres randomly. B. Neutrophil Adhesion and Surface Rearrangements of Membrane Bound Lectins and Bacteria Concanavalin A binding to leukocytes and its subsequent surface redistribution on colchicine pretreated cells have been extensively documented in the past 6'98' 103. We did not quantitate the binding of ConA to neutr0phils but did evaluate the topographical rearrangements of binding sites for' ConA on neutrophils pretreated with chemotactic factors or colchicine. Chemotactic factors have been reported to promote the redistribution of tetravalent ConA adhesion sites to the front ends of neutrophils 8. We were unable to document this localized adhesion pattern of fMP pretreated cells in suspen- 4, 100 sion. Like others , we observed that ConA was bound randomly on all pretreated cells. However, we do report a selective redistribution of surface bound ConA on fMP stimulated cells. The distribution of ConA on the surface of 8 neutr0phils exposed to a single dose of fMP (10- M or 10-6M) did not appear to be altered with time. Yet, when cells were 8-10'6ml a pretreated with multiple doses of fMP (10- rearrangement of surface bound ConA was observed. Most of the surface bound lectin was observed to flow to the tail 164 process on each of the cells or "cap". This capping process was indistinguishable from that previously reported on col— chicine pretreated cells. We also observed ConA redistribtur- ing to the tail ends of colchicine pretreated cells. The redistribution of ConA on colchicine or chemotactic factor pretreated cells apparently required lectin crosslinking of receptors since bivalent ConA did not redistribute rearward on similarly pretreated cells. The redistribution of surface bound ConA to the tail ends of colchicine and chemotactic factor pretreated cells required 10 minutes at 37°C for completion. The addition of ConA specific immunoglobulin to polarized cells with ConA previously attached promoted a much. faster redistribution of surface bound ConA. This rapid redistribution (within 1 minute) occurred on all pretreated cells (e.g., 10-6M fMP, 10.8-10-6 M fMP or colchicine). The addition of immunoglobulin did more than accelerate the redistribution of ConA, since there were no restraints on the redistribution of ConA on cells pretreated with a single polarizing dose of fMP. Salmonella typhimurium have been reported to possess ConA-like lectins on their surface pili 1' 68. As previously 1, 68 reported by others , human neutrophils failed to bind the non-piliated strain of this bacteria but did form mannose sensitive attachments with the piliated strains. Chemotactic factors enhanced neutrophil binding of the piliated bacteria and colchicine pretreatment somewhat impaired the ability of the neutrophil to bind piliated 165 bacteria. Neutrophil adhesion and redistributions of surface bound S. typhimurium were indistinguishable from those described for ConA. If the bacteria extensively cross-linked neutrophil binding sites, then this process did not accelerate their redistribution on the neutr0phil's surface or release the restraints generated by a single chemotactic stimulus. Several other piliated micro-organisms have been reported to be bound by neutrophils independently of serum opsonins. Some of them are apparently ingested after attach- ment, others are not. Bacteria bound but not ingested by neutr0phils have been observed to redistribute to the tail processes of motile neutr0phils after attachment. These bacteria were not internalized and remained viable 128. The conditions affecting the selective internalization of these bacteria are unknown.” S. typhimurium bound by neutr0phils were also observed to redistribute rearward across the neutr0phil's surface, but only after the cells were pre- treated with colchicine or multiple doses of chemotactic factors. Bacteria that accumulated at the tail processes of these cells were not ingested 30 minutes after attachment. Yet, externally bound S. typhimurium were rarely observed on the surface of neutrophils exposed to a single dose of fMP. The ability of neutrophils to remove surface bound piliated bacteria by ingestion or redistribution may reside in the number of chemotactic stimuli received by the cell. We also report that redistribution of surface bound particles does not interfere with the localized adhesion of 166 latex bound immunoglobulin on polarized neutrophils. S. typhimurium were bound by neutrophils pretreated with col- chicine or multiple doses of fMP and, in 10 minutes, the cells redistributed the bacteria to their tail ends. Following this surface redistribution, neutrophils were still able to bind and ingest immunoglobulin coated spheres at their front ends. The redistribution of the binding sites with attached bacteria apparently did not sweep the immunoglobulin binding sites from the front end of the cell. C. Preparation and Neutrophil Binding of C,b Coated Latex Spheres J Two techniques were used to attach C to latex spheres. 3 Bacteria or spheres were incubated with solutions containing serum or purified human C3. Whether fresh serum or solutions containing C3b were used for sphere pretreatment, sphere attachments were enhanced by 20-50X as compared to neutrophil binding of spheres pretreated with heat inactivated serum or human C3. The following evidence indicates this enhanced binding was a result of C3b absorption to the spheres. First, the conditions used have been used in the past to fix C3b to a variety of particles 79’ 84' 91. Second, C3b was demon- strated to be antigenically present on the treated spheres. Third, immunoglobulins specific for C3b (e.g., goat or rabbit anti-human C3c) blocked (i.e., goat anti-C3c) or modified (e.g., goat or rabbit anti-human C3c) neutrophil binding of the treated spheres. Last, physical (i.e., sonification) or 167 enzymatic (i.e., trypsin) removal or latex absorbed proteins to the sphere surface were not influencing our results. The assumption was made that neutrophil binding of these particles was a result of particle bound C b and the neutrophil C b 3 3 receptor interactions. Although bacteria and erythrocyte bound C3b have been used in the past 5’ 84' 97, the advantages for using C3b coated spheres were as previously described for immunoglobulin coated spheres. Several groups have reported that macrophages bind C3b coated particles randomly across their surfaces 5' 91. Bianco, et al have also shown that "activated" macrophages ingest C3b coated particles 129. We also observed spherical PBS pretreated neutrophils binding latex bound C3b randomly 8M fMP did not on their surfaces. Exposing neutr0phils to 10- morphologically alter the cells or attachment patterns for latex bound C b. Yet, unlike the control cells, (i.e., PBS 3 pretreated) neutr0phils exposed to even low doses of fMP ingested C3b coated spheres. Unlike previous reports 10, col— chicine pretreated neutrophils did not initially bind latex bound C3b randomly on their surface. The initial particle attachments occurred almost exclusively at the front ends of these polarized cells. Seconds (5 seconds) later, the attached spheres traveled rearward and appeared randomly distributed. By 90 seconds, the attached spheres were almost exclusively at the cell's tail end. This was an extremely rapid clearance as compared to the surface redistribution of ConA on neutrophils. All observations of this redistribution 168 implied that the rearrangements were occurring on the cell surface. As previously reported for colchicine pretreated macrophages 10, we also report that neutrophil ingestion by colchicine pretreated cells was undetectable 30 minutes after the sphere-neutrophil attachment. Adhesion of latex bound C3b also occurred at the front ends of neutrophils pretreated with single or multiple polar- izing doses of fMP. Following attachments, two neutrophil mechanisms were, however, activated for removing C3b coated particles from the front ends of these polarized cells, and the meChanisms inseparable when the cells were examined using light microsc0py. First, fMP polarized neutrophils appeared to ingest approximately half of the frontally attached spheres within 60 seconds of attachment. Following internalization, spheres traveled relatively slowly to the rear of the cells much like those spheres coated with immunoglobulin. Spheres not ingested by the fMP polarized neutrophils were redistributed rearward by a second, much faster, mechanism which occurred on the cell surface. Surface bound spheres traveled from front to tail on neutrophils in approximately 60 seconds. The different rates of rearward flow for internalized and externally bound spheres often created the impression, using light microscopy, that the spheres were randomly distributed 1510 minutes after attachment. Each redistribution event had to be studied using light, electron, and immunochemical microscopic techniques to separate externally bound spheres from ingested spheres. As observed 169 on colchicine pretreated neutrophils, spheres observed attached on the tail process of fMP stimulated cells remained exteriorly bound for 30 minutes. Neutrophils avidly bound C3b coated spheres or bacteria. As previously reported 124, polarizing concentrations of fMP almost doubled the binding of C3b coated spheres. Low concen- trations of fMP (i.e., 10-8 M) did not significantly enhance sphere binding and ingestion of the spheres was relatively slow (10-20 minutes). The inability of the cells to clear spheres from the cell surface rapidly may have accounted for their lack of enhanced sphere binding. However, spheres were cleared rapidly from their attachment sites on colchicine pre- treated cells, and these cells failed to bind more spheres than control cells. Only polarized cells ingesting spheres and redistributing spheres rearward on their surfaces bound significantly more spheres than the control cells. Their rate of sphere ingestion was much quicker than cells pre- treated with 10-8 M fMP. Ingestion occurred at about the same rate as for immunoglobulin coated spheres (1—3 minutes). The rapid sphere ingestion by the cells may have exposed hidden or new binding sites for C3b coated particles on the neutrophil surface 109. Neutrophil binding, ingestion and redistribution of surface bound particles was again shown to be highly selective. Although neutrophils bound C3b and immunoglobulin, coated spheres simultaneously at their front ends, their fates after binding were as predicted. The immunoglobulin 170 coated spheres were rapidly ingested at or near their attach- ment sites. The C3b coated particles were swept rearward on the same cell (e.g., colchicine pretreated) or ingested and 8 -6 swept rearward on the cell surface (e.g., 10-6M or 10- -10 M fMP pretreated). II. The Effects of Chemoattractants on ‘ Substrate Adhesiveness There were two conceivable means of altering the ability of a cell to adhere to a surface. First, alter cellular adhesiveness and, second, alter the adhesiveness of the attachment surface. Our research demonstrated an example of each. Chemotactic factors were shown to enhance neutrophil binding of bacteria and spheres coated with 2% HSA or C3b. The CF appears to have altered the neutr0phil's adhesiveness for these particles. The second example was neutrophil binding of spheres coated with human C3 before and after trypsinization. Enzymatic alteration of this protein dramatically altered particle adhesiveness. Chemotactic factors have also been shown to cause neutrophils to release trypsin and Chymotrypsin-like enzymes into their environment, and may also activate similar enzymes on the neutr0phi1 membrane 12’ 73' 99. The second goal of this report was to determine whether enzymes could influence the adhesiveness of protein coated surfaces. To perform such experiments, another adherence assay technique 171 was needed that provided a surface which could be coated with proteins yet afforded a surface onto which neutr0phils could settle and act on. The assay technique chosen (i.e., the tissue culture chamber) proved an accurate, reliable, simple and inexpensive technique for evaluating the ability of neutr0phils to attach to a surface over an extended period of time. This technique could also be modified for utilizing automated methods of counting surface bound cells improving its accuracy and precision. A. Neutrophil Adherence to Protein Coated Glass As observed for similar protein coated particles, neutro- phil adherence to glass coated with human C or 50% heat 3 inactivated serum was extremely poor. NeutrOphils also adhered very poorly to glass coated with human collagen, fibrinogen and gelatin. Unlike neutr0phil binding of particles, exposing neutrophils to CF greatly enhanced their adherence to glass coated with each of these proteins. The dose response curves reported for fMP stimulated adherence to glass coated with C3, gelatin or collagen were similar. Neutrophil adherence was not altered until the cells were exposed to relatively high concentrations of fMP. The CF enhanced adherence to these surfaces was dramatically impaired by the addition of a trypsin specific inhibitor to the cell suspension or by washing the fMP pretreated cells prior to their addition to the adherence chamber. We demonstrated that chemotactic factor binding to the protein coated 172 surfaces could not explain these results. Exposing human C3, collagen, or gelatin coated glass briefly to trypsin also 'greatly enhanced neutrophil adherence to the protein coated glass. These results imply l) trypsin like enzymes released from CF stimulated neutrophils may enhance neutrophil adheSiveness to surfaces coated with trypsin sensitive substrates, and 2) these enzymes may not be released until the cell is exposed to relative high concentrations of fMP. A different fMP dose response curve was observed when evaluating neutr0phil adherence of glass coated with human fibrinogen or heat inactivated serum. Significant increases in neutr0phil adherence were observed on these substrates after the cells were exposed to lower concentrations of fMP and neutrophil adherence increased proportionally with each subsequent increase of the fMP concentration. This adherence could not be diminished by washing the cells and was not inhibitable by trypsin inhibitors. Neutrophil adherence to these surfaces was greatly enhanced following a brief (1 minute) exposure of these surfaces to Chymotrypsin. The addition of a Chymotrypsin specific inhibitor to the CF stimulated cells greatly reduced their ability to adhere to these surfaces. It was concluded that 1) CF pretreatment may activate or release Chymotrypsin like enzymes that cannot be removed or inactivated by washing the cells, 2) these enzymes can alter neutr0phi1 adhesiveness on Chymotrypsin sensitive substrates, and 3) relatively low doses of fMP can stimulate this action. Several authors have documented that 173 a Chymotrypsin like enzyme is necessary for neutrophil locomotion on protein coated surfaces and that CF activated a surface bound Chymotrypsin like enzyme on neutrophils 12’ 121. S UMMARY We report that chemotactic factors and colchicine influence the ability of neutrophils to bind bacteria, and a variety of protein coated particles. A summary of their effects on neutrophil binding and redistribution of albumin coated spheres, lectins and bacteria, immunoglobulin coated spheres and C3b coated particles are illustrated in Figures 19 and 20. Only the binding of lectins or piliated bacteria occurred in a random fashion on all pretreated cells. The binding of immunoglobulin or C3b coated particles was restricted to the front ends of polarized neutr0phils. The binding of albumin coated spheres was‘restricted to the tail process of neutrophils pretreated with colchicine or multiple doses of chemotactic factors and occurred randomly on spherical cells or those exposed to a single CF stimulus. Two methods were observed for removing particles from sites of attachment on the neutrophil surface. First, neutrophils ingested particles. All neutrophils ingested immunoglobulin coated spheres and only CF stimulated cells ingested bacteria or C3b coated spheres. Ingested particles were swept rear- ward but only within polarized cells. Second, particles were swept rearward on the exterior surface of neutrophils. C3b coated particles were redistributed rapidly in this 174 175 manner on all polarized neutrophils. Similar, but much slower redistributions of lectins or bacteria occurred only on cells exposed to colchicine or multiple concentrations of CF. NeutrOphils pretreated with colchicine or CF were shown to selectively bind particles simultaneously at different regions on the same cell. We also report that neutrophils adhere poorly to glass coated with a variety of proteins and CF enhanced neutrophil adherence to these surfaces. Brief enzymatic action on surfaces coated with protein substrates also enhanced neutro- phil adherence. The adherence of CF stimulated cells to protein coated glass was impaired by the addition of enzyme inhibitors. Our results suggested trypsin and Chymotrypsin like enzymes were involved in augmenting neutr0phil adherence to protein coated surfaces after neutrophil exposure to a chemotactic factor. 1..fi.__ Colchicine wave-16511) m(1o'6n) who’s") Control Cells (PBS) Figure 19. 176 < ’k’