£563 (“WIHHHHIIHIUIHHIHHHIHWHIHJIHIIHHIHMHI THERIQ LIBRARY Michigan Sm: University This is to certify that the thesis entitled EFFECTS OF NEUTROPHIL ADHERENCE TO MICROPORE FILTERS ON MIGRATION ASSESSMENTS IN VITRO presented by Thomas L. York has been accepted towards fulfillment of the requirements for MS degree in £aihoLog¥_ a fiajor professor Date Nov. 10, 1978 0-7639 OVERDUE FINES ARE 25¢ PER DAY PER ITEM Return to book drop to remove this checkout from your record. ABSTRACT EFFECTS OF HUMAN NEUTROPHIL ADHERENCE T0 MICROPORE FILTERS 0N MIGRATION ASSESSMENTS IN VITRO By Thomas L. York The degree of neutrophil adhesiveness to a surface may in- fluence locomotion. To investigate this relationship jfl_vitrg, ad- herence and motility were assessed by a new modification of the Boy— den assay. Decreasing adhesiveness to the filter surface by coating the filters with albumin was necessary for random and chemotactic migration of neutrOphils jn_gjtrg, Neutrophils preincubated with high concentrations of fMetPhe or a chemotactic factor from activated serum (CSa), reduced their locomotive responsiveness and signifi- cantly enhanced adherence to albumin coated filters. If the adhesive- ness reduced, then the locomotive responsiveness returned to control levels. Other chemotactic solutions affected neutrophil adhesiveness differently when used to coat the filter or when placed with the cells. Apparently, agents existed in these solutions that affected both the filter surface (decreasing adherence like albumin), and the neutro- phils (increasing adherence like C5a and fMetPhe). ACKNOWLEDGMENTS I wish to express my sincere appreciation to Dr. C. Wayne Smith, the chairman of my committee, for continued encouragement and assis- tance in my graduate program. I particularly appreciate his guidance in developing concepts of experimental design and the communication of experimental data. I would also like to thank Mr. James C. Hollers for technical assistance and, more importantly, creating an exciting and enjoyable . laboratory atmosphere, stimulating creativity. I am also grateful to Dr. Richard A. Patrick, Mrs. Martha T. Thomas, Mrs. Kathryn D. Colando, and Dr. Stuart 0. Sleight for serving on my guidance committee. My deepest appreciation goes to my wife, Jane York, for support in all areas of this program. ii TABLE OF CONTENTS Page LIST OF TABLES ........................ iv LIST OF FIGURES ....................... v INTRODUCTION ......................... 1 REVIEW OF THE LITERATURE ................... 3 MATERIALS AND METHODS .................... 16 RESULTS ........................... 27 DISCUSSION .......................... 53 LITERATURE CITED ....................... 59 iii LIST OF TABLES Table Page l. The effects of albumin in a gradient on neutrophil migration ........................ 28 2. Effects of casein in a gradient on neutrOphil migra- tion .......................... 29 3. Effects of albumin or casein on neutrOphil shape change ......................... 29 4. The effects of albumin on neutrophil adherence to glass .......................... 37 5. The effects of pretreating micropore filters with albumin on the chemotactic response of neutrophils to fMetPhe ....................... 38 6. The effects of pretreating micrOpore filters with chemotactic solutions on neutrophil adherence and migration ........................ 45 7. The effects of chemotactic solutions in a gradient on neutrophil adherence and migration .......... 49 8. The effects of heating serum or the addition of heparin to serum on neutrophil adherence and migration ..... 50 iv Figure 1. LIST OF FIGURES a. Vertical cross section of a blind well chamber . . . b. Vertical cross section of a slide chamber ..... The morphological alterations on neutrOphils when exposed to various solutions .............. The effects of pretreating micropore filters with solutions of albumin on neutrophil adherence and random migration .................... The effects of pretreating micropore filters with solutions of albumin on neutrophil distribution . . . . The effects of albumin in solution on neutrophil adher- ence and migration ................... The effects of pretreating micropore filters with albumin on the adherence and random migration of deactivated neutrophils ................ The effects of pretreating micropore filters with albumin on the chemotactic migration of deactivated neutrophils to fMetPhe ................. The effects of pretreating micropore filters with solutions of HSA or serum on Neutrophils adherence and migration ..................... The distribution of neutrophils responding to serum or zymosan activated serum (ZAS) in the stimulus compart- ment of the blind well chambers ............. Page . 19 . l9 . 24 . 3T . 33 . 35 , 40 . 42 . 46 . 51 INTRODUCTION »Leukocyte migration to inflammatory sites is a fundamental event in mammalian homeostasis. The mechanisms of cell locomotion have been studied extensively in the past. Since Boyden developed a technique to assess leukocyte migration through micropore filters, much data has accumulated about neutrophil locomotion in response to 5 a gradient of various biological agents jn_vitro. Chemokinetic fac- tors are agents that influence the rate of migration and chemotactic factors are agents that influence the rate and direction of migra- tion. Chemotactic and chemokinetic factors have been shown to affect nany cellular mechanisms, but the mechanism controlling locomotion re- main unclear. Surface adhesiveness has been shown to influence the rate and 7,18 direction of locomotion of certain tissue cells ig_vitro. Several observations made in the past indicate that adherence of neutrophils to a surface may influence locomotion as well. l) Albumin has a chemokinetic effect in the Boyden assay and has been found by many in- vestigators to be important in obtaining an effective chemotactiC' 3 4 response. ’ 19’30 Albumin also decreased neutrophil adherence to glass. 2) Chemotactic factors have a proven effect on neutrOphil migration jg_ vitro and have been shown to increase neutrophil adherence to protein 26,30 coated glass and plastic. 3) Certain drugs which increase intra- cellular cyclic adenosine monophosphate decrease neutrophil migration 2 2 6 and adherence to glass.’ 4) Deficiency of magnesium ions in the cul— ture medium significantly reduced neutrophil migration in the Boyden 3,12 ,assay and adhesiveness to protein coated glass. 5) Chemotactic solutions of casein bound to micropore filters increase neutrophil mi- gration in the Boyden assay.10 This paper describes an investigation of the possible relation- ship between neutrophil adherence and migration. Neutrophils, or the substratum on which they migrate, were exposed to chemotactic or chem- okinetic solutions and the effects of these solutions on adherence and motility were assessed by a new modification of the Boyden assay. Al- though the techniques used ot assess migration by Boyden assays have been used extensively in the past, they were modified slightly to investigate the possibility that alterations to the filter surface may influence migration. The adherence technique was unique and designed to assess adherence in the blind well chamber. This technique allows for comparison of neutrophil adherence with motility since assessments were made using the same experimental conditions. Adherence to glass was also measured. In addition, cellular responses to various agents were measured by incubation of neutrophils with the agents. fixing the cells in glutaraldehyde and observing fbr morphological alterations. Our results indicate that conditions which affect neutrophil ad- herence significantly affect neutrophil migration into and through micropore filters. This was observed when adherence was altered either by treating the cells or the substratum with various chemotactic and chemokinetic factors. Neutrophil migration in response to these agents jn_yjtrg_may reflect changes in adherence as well as changes in other cellular mechanisms of locomotion. REVIEW OF THE LITERATURE Chemotaxis, a phenomenon commonly described as the directional movement of cells towards a gradient of specific chemical sub- 2’11’13’34 has fascinated biologists for over 100 years. Chem- stances otaxis is of obvious interest, since it may play a role in nutrition, the development of cellular organization, reproduction, the recogni- tion of noxious agents, and inflammation. Several types of motile cells exhibit chemotaxis, but due to its possible involvement in the inflammatory response, neutrOphil chemotaxis has received much atten- tion. Neutrophil mobility appears essential for the migration of cells from the vasculature to sites of inflamaation. Adequate numbers of neutrOphils, functional locomotor mechanisms, directed cell locomotion, ‘3 Abnormal adherence and adherence appear necessary for host defense. has been reported in patients with recurrent bacterial infections and after injection of corticosteroids. Intrinsic defects of cellular locomotion have been observed with recurrent infections in patients described as having the “lazy leukocyte" syndrome. Numerous reports have described disease conditions with defects of directed locomotion such as Job's Syndrome, diabetes, neoplasms, rheumatoid arthritis, viral infections, and various acute bacterial and yeast infections. Techniques Used to Assess NeutrOphil Chemotaxis Historical Development Chemotactic investigations have been limited by inadequate tech- niques, and only in the past 20 years have the technical abilities de- veloped to answer questions asked over 100 years ago. In 1882, 1] He observed Cohnheim studied the vasculature during inflammation. neutrophils emigrating through the endothelial cells that lined the vessel wall, and their subsequent migration to inflammed tissues. In 1884, Pfeiffer introduced the term chemotaxis and in 1888, Leber per- formed the first jfl_yjyg_leukocyte chemotaxis experiment, by injecting noxious agents into the cornea of rabbits and observing neutrophils ‘1 Metchnikoff and co- accumulating in the surrounding capillaries. workers found, in 1890, that leukocytes were attracted to living or dead bacteria that had been injected into the peritoneal cavity. Later, Metchnikoff theorized that chemical signals were produced during inflamation and leukocytes had the ability to "sense" these signals. The observations and theories of Metchnikoff on leukocyte pagocytosis and chemotaxis, printed in the late 1800's, are the foundation of chem- otactic research today.25 Few significant jn_vjyg_techniques were developed after Leber's initial experiment. Clark and Clark, in 1920, did use a unique animal model, the tadpole.8 Substances were injected into the tail of the tadpole, and neutrophil margination, emigration, and accumulation were easily seen. Some have questioned the validity of this type of tech- nique, citing that cellular damage and trauma resulting from the in- jections may influence the results. Another jg_vivo technique, the skin window, developed by Rebuck and collegues in 1955, has been used extensively.29 A circular abrasion was made on the forearm of a human subject and test substances added to the abrasion. The site was covered with a sterile glass coverslip and the accumulation of leukocytes in response to the trauma or test substances was observed. The skin window is still a popular technique used by a number of groups in clinical studies. The establishment of a chemotactic gradient has never been de- monstrated and no technique has demonstrated that neutrophils rec- ognize, orient, or directionally migrate to chemotactic agents in 3139, Cell accumulation may be a direct result of test substances yet whether these substances selectively attract the neutrophil remains to be determined. Most of the information about chemotaxis was determined by in_vitro techniques. Although jg_vjtrg_chemotactic experiments were documented as early as 1899, the techniques were crude and unreliable. In 1954, Harris reviewed and dismissed all prior data.17 Today 2 basic systems exist to measure chemotaxis and most of the evidence for chemotaxis has been derived from these techniques. The first deals with the movement of individual cells on a flat surface. Harris layered neutrophils, in autologous serum, on glass slides and photographed migration in response to various stimuli. Lengthening exposure times or overlapping the time lapse exposures on a single negative allowed for visualization of the path of migration or "tracks" of neutro- phils. Neutrophils turned frequently and randomly in the absence of suitable stimuli. When bacteria were added, the neutrophils migrated directionally towards them. In 1972, Ramsey modified this technique 28 to observe neutrophil locomotion on a surface. Recently, Zigmond modified Harris's technique to evaluate the frequency in which neu- trophils turn, their rates of migration, and orientation towards suitable stimuii.35 Modifications of Harris's technique have produced a great deal of information about neutrophil mobility on glass. The development of the Boyden technique in 1962 produced a sim- 5 Vir— ple, accurate, and reproducible method of evaluating chemotaxis. tually all of the recent work on chemotaxis stems from Boyden's orig- inal paper which has been universally accepted among workers. This was the first technique allowing for the assessment of soluble chemotactic substances. Boyden used a chamber that consisted of an upper and lower half separated by a porous filter. Like the endothelial wall of the vessel, the diameter of the openings in the filter are smaller than the diameter of the cell. A stimulus was placed below the filter and, as the stimulus diffused into the filter, a chemical concentration gradient was established (later verified by Keller and Sorkin).H Neutrophils placed above the filter migrated into the filter in re- Sponse to the stimulus. Migration was evaluated by counting the num- ber of cells on the bottom surface of the filter (i.e., migrated com- pletely through the filter). in 1966, Cornely demonstrated that neutrophils migrated towards increasing concentrations of chemotactic factors and reversing the gradient reversed the direction of migra- tion.9 Boyden's technique has undergone many modifications, most of which were designed to eliminate errors in evaluating neutrophil mi- grational responsiveness. Keller demonstrated that as many as 50% of the cells getting through the filter detached from the filter sur- l3 face. He developed a double filter technique to prevent neutrophils from falling off. Gallin and coworkers labelled the cells with radioactive chromium, enabling more effective counting I3 Zigmond and Hirsh shortened the in- with a scintillation counter. cubation times, preventing the neutrophils from migrating compeltely through the filter, and measured the distance of penetration into the filter (“leading front").36 The importance of results derived from jn_vitro techniques may not apply to neutrophil locomotion jn_vjyg,22 The substrates used, either glass or micropore filters, are foreign to the neutrophil. Studies are currently underway assessing neturophil responsiveness on endothelial cells. Identification and Recognition of Chemotactic and Chemokinetic Factors The development of the Boyden assay enabled workers to identify many chemotactic substances. With few exceptions, the components producing chemotactic activity have not been isolated or chemically identified. The majority of substances assessed for chemotactic activ- ity have been implicated in inflammatory responses, but their effects were demonstrated jn_vjtrg, usually by the Boyden assay, and their importance j__ijg_is unproven. Certain substances appear to affect neutrophil chemotaxis directly, whereas other substances generate chemotactic activity in serum, plasma, or other biological fluids by activating enzymes in the complement, coagulation, fibrinolytic and I] The activated proteolytic enzymes cleave kinin generating systems. substrates, yielding chemotactic fragments. While most of the frag- ments are poorly characterized, a small molecular weight, heat stable fragment cleaved from the 5th component of the complement system was 8 partially purified by Ward, Gallin, and Snyderman.]2’13 This fragment was designated as C5a. Synderman gt_al, have contended that C5a was important for jn_vjyg_chemotaxis since neutrophil accumulation, occur- ring in response to inflammatory stimuli, was not effectively demon- 31 strated in C5a deficient mice. However, the mice were not prone to infection. Becker and Ward have suggested that other complement com- ponents are chemotactic for neutrophils (i.e., C3a and C567 complex,2’25 although the evidence presented has not been universally accepted.36 The "activation" of serum or plasma to produce chemotactic activ- ity, presumably by activating enzymes of complement or other systems, has been performed by numerous techniques. The addition of yeast cell walls or zymosan, antigen-antibody complexes, endotoxin, pro- teolytic enzymes (i.e., plasmin, cobra venom factor, trypsin, and kallikrein), or damaged tissues to serum or plasma generates chemo— tactic activity. Numerous investigators have found bacteria to be chemotactic. In addition to secretions that may activate complement (i.e., endo- toxin, antigen-antibody reactions), other soluble substances released 25 Bacteria from bacteria directly affect neutrophil chemotaxis. initiate protein synthesis with formylated methionine, unlike humans, and Shiffmann has synthesized several fermylated peptides that are 35 The formylated peptides and C5a are chemotactic for neutorphils. the only relatively purified chemotactic components available for chemotaxis experiments. Wilkinson and coworkers have demonstrated that denatured pro- teins and casein, a major milk protein, are also chemotactic.13 Additional reports have indicated that fibrin Split products, damaged cells, enzyme digested collagen, prostaglandins, lipids, and cyclic- 2,6,13 adenosine monophosphate are chemotactic. In addition, Zigmond demonstrated that soluble substances release from neutrophils acti- 35 The vated by chemotactic factors or phagocytosis are chemotactic. diversity of the chemotactic factors is immense, yet most are pro- teins, and the isolation and identification of common chemical se- quences is an active field of investigation. Chemotactic activity has been evaluated primarily by Boyden assays. Since conception of the assay, serum or albumin have been required in the cell medium to observe enhanced chemotactic migra- tion, although an explantion of this requirement is lacking.20 Early investigations of substances enhancing neutrOphil migration did not account for agents producing chemokinetic affects and many substances were probably misinterpreted as chemotactic. In 1973, Zigmong used the leading front technique with varying concentrations of test sub- stances above and below the filter to differentiate chemotaxis from 36 In the chemokinesis. These data are often presented as a "grid." absence of a test substance, neutrophils penetrated randomly into the filter and the distances of migration were similar to results de- rived with equations used to predict random diffusion of molecules from a front. Chemokinetic factors stimulated motility and the distances of migration were greater than predicted by the diffusion theory. This occurred equally well when the test substances were above or be- low the filter. Chemotactic factors also stimulated motility above or below the filter, but the distances migrated were significantly increased when the concentration of the chemotactic factor was greater below the filter. Apparently only chemotactic factors are 10 chemoattractants, and orient migration towards increasing concentra- tion gradients. High concentrations of chemotactic solutions inhib- ited cell migration, a phenomenon not produced by chemokinetic fac- tors. All chemotactic factors were chemokinetic and enhanced the rate of neutrophil migration. However, all chemokinetic substances were not chemotactic, since orientation and direction of migration were not affected. Thus, there appears to be 2 types of chemical messages presented to the cell, one influences orientation and another the motile mechanism. Using Zigmond's definitions, Wilkinson demon- strated that human and boving serum albumin were chemokinetic, but the requirement for chemokinetic factors in the medium to observe chemotaxis in Boyden assays remains unexplained.]9’34’35 The mechanism by which chemotactic or chemokinetic factors in- teract with the neutrophils remains undetermined and a fertile field of investigation. Numerous investigators have unsuccessfully attempted to demonstrate the presence of chemotactic receptors on the cell mem- brane of meutrophils. Ward and Becker found when neutrophils were incubated with serine esterase inhibitors, they were unable to give a 32 They concluded that chemotactic response to chemotactic factors. there were serine esterase receptors on the neutrophil that may be receptors for the chemotactic factors. One such identified serine esterase was termed the "activatable esterase." Saturation of acti- vatable esterase receptors with high concentrations of chemotactic factors appeared to "exhaust" the cell and produce a state of "de- activation." Deactivated neutrophils were less motile than untreated neutrophils and were unresponsive to other chemotactic factors. Multiple receptors may exist for various chemotactic factors. Zigmond 11 reviewed this concept, noting that C5a did not compete with form- ylated peptide binding.35 Therefore, there may be at least 2 types of chemotactic receptors. Zigmond also contended that neutrophils sense chemotactic receptors across their dimensions, thus enabling the cell to orient to a gradient of chemotactic factors. Therefore, the cell membrane must contain numerous receptors encompassing the neutrophil. Chemotactic factors specifically activate several neutrophil activities other than enhancing locomotion. These include: promoting lysosomal enzyme release, altering the cell's morphology, assembly of microtubules and microfilaments, increasing metabolic activities, and cellular adhesivenessI3’25’30 The involvement of these activities with locomotion remain unexplained, and obscure interpretations of measurements of cellular responses to chemotactic factors such as cation fluxes, net surface charges, membrane potential, and the con- centration of cyclic nucleotides. Control of Chemotactic Response So far, only mechanisms that tend to increase the chemotactic response, with the exception of deactivation, have been discussed. In 1974, Ward reviewed the clinical importance of chemotactic inactiva- tions for turning off the inflammatory process.13 Again, most of the evidence for inhibiting neutrophil locomotion was derived fron jn_ vitrg_observations and scattered clinical abnormalities. Inhibition may be accomplished by affecting cellular mechanisms of locomotion or by preventing the activation of chemotactic factors. As suggested by Ward and Becker, deactivation by incubating neutrophils with high 12 concentrations of chemotactic factors inhibits the ability of the 32,35 cell to detect and respond to other chemotactic factors. This nay be a biological control, preventing cells from leaving the site of' inflammation. Goetzel and Austen described a neutrOphil immobilizing factor released after phagocytosis which may inhibit the ability of 15 the cell to migrate. Gallin observed the same effect with lyso- 13 They suggested the possibility of internal somal enzyme release. cell controls for directed cell locomotion. Others have described patients with a variety of disorders having serum inhibitors that directly impair neutrophil locomotion, but the nature of these in- 11’25 Other researchers have used various hibitors remains unclear. chemicals to inhibit neutrophil locomotion. The specificity (If these chemicals on specific components within the cell are questionable, but may provide evidence for the mechanisms of cell locomotion. Mal- awiste, although not supported by others, has reported that colchi- cine, which prevented microtuble assembly, inhibited random locomo- 25 Becker reported that cytochalasin B, which prevented micro- tion. filament assemble, inhibited random and chemotactic neutrophil locomo- tion.2 Various chemical inhibitors of metabolism, membrane ATPase activity, and protein synthesis also inhibit neutrophil locomotion.]3’25 Several inhibitors of chemotactic factor activation may be an important control of chemotaxis. C5 inactivator, Cl inhibitor, and alpha-2- macroglobulin inhibit the chemotactic activity generated by C5, C1, and kallikrein or plasminogen activator respectively 1n_vitro.]1’]3’25 13 Relationshipgof Neutrophil Adherence to Motility In the early 1800's, Dutrochet described leukocyte margination 1‘ Numerous investigators have focused on near inflammatory sites. investigations of neutrophil adhesiveness since adhesion to the endo- thelial lining of the vessel may be a critical event in the inflam- matory response. Other investigators have noted the importance of cell attachment to a surface on which these cells crawl. In 1972, 28 As observed in Boyden assays, Ramsey reviewed locomotion on glass. serum or albumin were required for neutrophil locomotion on glass, since the cells just flattened out on the glass surface without serum or albumin. In a protein environment, neutrophils attach to the surface, sending out pseudopods which also attach to the glass. The intracellular contents were observed flowing from the tail pro- cess or ur0pod to the pseudopods. The uropod was then released, or borken off, drawing the cell to the attached pseudopod.4 DeBruyn, in 1946, showed neutrophils to be more adherent at the pseudopod 11 than the uropod. Machesi noted neutrophils migrating through the vasculature also sent pseudopods between the endothelial cells and the cell contents flowed from the uropod to the pseudopod.H It ap- pears that adhesion provides the frictional forces required for trans- location and is a vital characteristic of neutrophils. Techniques Assessing Neutrophil Adherence Ig_yiyg_assessments of neutrophil adherence have relied upon direct observations of marginated neutrophils or upon counting the numbers of circulating neutrophils. The assumption was made that factors decreasing the numbers of circulating neutrophils increased 14 the numbers of neutrophils in the marginal pool due to increased neutrophil adhesiveness. Several jn_vitro techniques have developed to assess neutrophil adherence to glass and nylon.13 Most of the in_ yitrg_techniques allow for neutrophil attachment to the surface and the numbers of cells that detach as external forces were applied reflect neutrophil adhesiveness. The forces used to detach the cells include centrifugation, elution off a column, shaking, and rotation. The external forces may promote cell shearing, destruction and aggre- gation, thereby confusing evaluations of adhesiveness. Smith gt_al, have recently described a technique to assess neutrophil adherence to 30 O'Flaherty and Coworkers have glass without using external forces. developed a technique to assess cell to cell adhesion or aggregation by counting the numbers of cells passing through a sizing aperture 26’27 The experimental conditions or by using a platelet aggregometer. of the adherence techniques are extremely different from the conditions used to evaluate neutrophil motility (i.e., substrate, cell distribu- tion, external forces, chemotactic gradient). Therefore, correlations of effects on adherence to motility may have little meaning. Control of Adherence Mechanisms Very little is understood about the mechanism of adherence or agents that influence this mechanism. The nature of the binding site to glass also remains speculative. Carter (1965) and Harris (1973) demonstrated that substratum adhesiveness was a determining factor 7,18 for translocation. Grant and Epstein (1974) damaged the vascular endothelium with laser beams and produced margination, suggesting "11 that the damaged endothelial cells became "sticky. However, 15 several workers have implied that chemotactic factors increased neu- 26,27,30 trophil adhesiveness. Smith gt_gl, (1978) have demonstrated that incubation of neutrophils with high concentrations of chemotactic factors irreversibly increased cellular adherence.30 Keller and Smith demonstrated that albumin reduced neutrophil adherence to ‘9’30 McGregor has shown that anti-inflammatory drugs decrease 13.23 glass. adherence and a plasma factor augments adherence. Bryant gt_ 1. have shown that c-AMP and prostaglandins decreased neutrophil ad- .herence.6 Kvarstein and Smith have demonstrated that neutrophil ad- herence was dependent on magnesium ions.2]’30 It appears that the substrate or cellular adhesiveness may be affected by various agents. Not surprising is the fact that many of the substances shown to influence adherence also affected neutrophil translocation. It is surprising, however, that no technique exists to assess neutrophil adhesiveness to micropore filters when the bulk of information about neutrophil locomotion has been derived from Boy- den assays. MATERIALS AND METHODS Reagents All reagents were reagent grade and adjusted to pH 7.3. Ficoll, glutaraldehyde, cacodylic acid, sodium heparin sulfate, casein, human serum albumin (HSA), crystallized 1 time and bovine serum albumin (BSA) crystallized 4 times were obtained from Sigma Chemical Company, St. Louis, Missouri. Hypaque was purchased from Winthrop Laboratories, New York, New York. The blind well chambers were purchased from Neuroprobe Incorporated, Bethesda, Maryland. The micropore filters and Swinny adaptors (18mm. diameter) were obtained from Millipore Corporation, Bedford, Massachusetts. Zymosan was obtained from Nutri- tional Biochemicals Corporation, Cleveland, Ohio, and fMetPhe from Andeulis Research Corporation, Bethesda, Maryland. Hanks balanced salt solution (HBSS) was obtained from Gibco, Grand Island, New York. The C5a was a generous gift from Dr. Richard A. Patrick, prepared by Clare Hassett in the Department of Microbiology and Public Health, Michigan State University, East Lansing, Michigan. Preparation of Chemotactic and Chemokinetic Solutions Zymosan activated serum (ZAS) was prepared by incubating 10 mg of zymosan with 1 m1 of fresh human serum for 30 minutes at 27C and then diluting this mixturel()fold with HBSS. The zymosan particles were removed by centrifugation. The formylated peptides were dissolved in H858 to a concentration 16 17 of 10'3 M and diluted further with HBSS to desired concentrations. Casein was dissolved in 1N NaOH, diluted to desired concentra- tions with HBSS, and adjusted to pH 7.3 with HCl. A low molecular weight chemotactic factor was prepared from activated human serum by the method of Gallin, gt_al,12 Briefly, human serum was incubated with 0.1 mg/ml E, gglj_1ipopolysaccharide at 37C for 1 hour. The serum was then heated at 56C for 30 minutes. After rapid cooling, the activated serum was layered on a Sephadex G-75 column (90 x 5 mm). The column was equlibrated with calcium and magnesium free HBSS by descending flow at 4C. The column was cal- ibrated with the following substances: blue dextran, molecular weight (MW) 2 x 105; cytochrome c, nw 12,384; ribonuclease A, MW 13,700; chymotrypsinogen, MW 25,000; and ovalbumin, MW 45,000. Frac- tions in the 20,000 to 10,000 MW range were assayed in modified Boy- den chambers and those showing chemotactic activity were pooled and frozen at -70C. The pool containing chemotactic activity had 40 pg of protein/ml. This reagent will be referred to as C5a. Human serum albumin and BSA were dissolved in HBSS and all solutions were adjusted to pH 7.3 with NaOH. Isolation of Human Neutrophils Blood samples were obtained from 6 healthy adult volunteers (3 men and 3 women). Blood was collected in plastic syringes and placed in heparinized (15 units/m1 blood) tubes. Dextran (6%) was mixed with the samples to enhance red blood cell sedimentation. After approximately 45 minutes at room temperature, the leukocyte rich plasma was removed and placed in 17 mm siliconized tubes. The 18 leukocyte rich plasma was diluted with an equal volume of HBSS, cen- tringed at 500 G, and the plasma and HBSS removed by suction. The cell button was resuspended in 4 m1 of HBSS and centrifuged at 800 G for 30 minutes on a Ficoll-Hypaque solution (4 ml) consisting of 10 parts of 33.9% Hypaque and 24 parts of 9% Ficoll. This solution pro— vided a density gradient to separate granulocytes from platelets and other leukocytes in the cell suspension. When the Ficoll-Hypaque solution and HBSS were removed by suction, the cells in the cell button were resuspended in HBSS and differentially counted. This cell suspension contained greater than 98% polymorphonuclear leuko- cytes of which approximately 95% were neutrOphils. No platelets were seen in the preparations and the red blood cell to neutrophil ratio was consistently less than 2:1. Neutrophil viability was greater than 98% as determined by eosin exclusion. Assessment of Neutrophil Motility Neutrophil motility was tested by a modified Boyden technique using blind well chambers (Figure la). The chambers were prepared by 4 2 of exposed filter) suspended placing cells (2 X 10 neutrophils/mm in various reagents in the cell or upper compartment. The cells then settled onto a micropore filter (3 pm pore size) which separated the cell compartment from the stimulus compartment. Various reagents were placed into the stimulus compartment, and the concentration dif- ferences of reagents in the two compartments established a concentra- tion gradient in the filter. After the chambers were prepared, they were incubated at 37C in an atmosphere of 5% CO2 and high humidity. Incubation times were controlled for each experiment such that cells 19 Figure la: Vertical cross section of a blind well chamber. Magni- fication 5X. Figure lb: Vertical cross section of a slide chamber. Magnifica- tion 12X. 20 SCHEMATIC CROSS SECTION OF A BLIND WELL CHAMBER. CAP CELL COMPART MENT Fl LTER C STNULUS I CWNRMNT Figure 1a. I SCHEMATIC cnoss secnow or A 1 SUDECHAMBER. F r Vlswlwc PO RT fli/I/I/I/I/I/I/I/I/Ill/I/I/I/I/I/I/I/l‘. — F| LT E R — GASKE T NECTION PORT — CELL If/I/I/I/l/I/l/I/l/I/ll/I/I/l/l/I/I/I/I; COMPARTMENT Figure 1b- 21 migrated into or completely through the filters. (Migration from the top of the filter to the bottom of the filter). These time inter- vals varied and are specified for each experiment. The experiment was terminated after the incubation period by removing the filter, rinsing it in absolute methanol to fix the cells present and staining it with hematoxylin. Hematoxylin stained the cells, but not the filter. The filters were then soaked in xylene to make them transparent. Migra- tional behavior in the filters was assessed as follows. The depth of migration was determined microscopically. When neutrophils migrated completely through the filters, the cells in 10 randomly chosen 40X microscopic fields on the bottom surface of the filter were counted. Each experiment was duplicated and counts averaged. When neutrophils did not migrate completely through the filter, migration was assessed by 2 methods as previously described by Zigmond and Hirsch.35’36 1) Briefly, the distance of neutrophil penetration was determined by measuring the distance (pm) from the cell origin (top of the filter) to which only 2 cells remained in focus ("leading front"). In each experiment, the leading front was determined by averaging 5 measure- ments for each of duplicate filters. 2) The distribution of cells in the filter was determined by counting the number of cells/40X micro- scopic field at 20 um intervals through the filter. At least 3 deter- minations were made for each of duplicate filters and averaged for each experiment. NeutrOphil migration was assessed when varying concentrations of chemotactic or chemokinetic solutions were placed above and below the filter or used to pretreat the neutrOphils or filters. 22 Assessment of Neutrophil Adhesiveness The interaction of neutrophils with 2 surfaces, glass and micro- pore filters, were evaluated using 2 techniques. 1) Slide chambers (Figure lb) similar to those reported by Lichtman et_al,24 were filled with suspensions of neutrophils (l X 106/ml in various reagents). The chambers were immediately placed on the stage of an inverted phase contrast microscope and cells settling onto the glass surface were observed with a 50X oil immersion objective at room temperature. The numbers of neutrophils on the surface were counted in 5 randomly selected 50X microsc0pic fields 350 to 500 seconds after injecting cells into the chamber. The chamber was then inverted and the un- attached cells allowed to fall off the surface. After 1000 seconds, cells remaining attached to the glass surface were again counted. Each experiment was perfbrmed in duplicate. The comparison of counts of cells remaining attached to the glass surface with the initial cell count was expressed as percent adherent cells. Adherence was assessed when reagents were in the cell suspension, used to pretreat the cells, or used to pretreat the glass’coverslips. 2) Blind well chambers were filled with 0.5 ml of cell suspension (5 X 105 neutro- phils/ml of various reagents) in the cell compartment and HBSS or various reagents in the stimulus compartment. The chambers were in- cubated for 5 minutes at room temperature as the neutrophils settled onto cell impermeable micropore filters (0.22 um pore size). Hanks' balanced salt solution was gently added over the cell suspension until the cell compartment was completely filled. An 18 mm glass coverslip was placed over the cell suspension (insuring that no air bubbles were introduced) to seal the cell compartment. After an additional 15 23 minute incubation at room temperature, the chambers were inverted, ‘allowing the unattached cells to fall off the filter. The filter was removed after 20 minutes, and the cells attached to the surface counted. The cells in five 50X oil objective fields were counted on each of duplicate filters for each experiment. The counts of cells remaining attached under various conditions were compared to counts in HBSS remaining attached to untreated filters. Since virtually 100% of the neutrophils remained attached to untreated filters when in HBSS, this comparison was expressed as the percentage of adherent cells. Adherence was assessed when reagents were in a gradient, used to pretreat the cells, or used to pretreat the filters. Assessment of Changes in Neutrophil Shape A modification of the method of Lichtman gt_al,24 was used. Neutrophils (106 neutrophils/ml of reagent) were exposed to various reagents. The pH was adjusted to 7.3 using 5% C02 in air. After a 30 minute incubation time with these reagents, the cells were added dropwise to 10 ml of cold (4C) glutaraldehyde (1%) in 0.1M cacodylic acid. The glutaraldehyde solution was mixed constantly while the rcells were being added. After remaining in the cold glutaraldehyde solution for 1 hour, the cells were washed and resuspended in 0.1 ml of distilled water. The neutrophils were examined by using a 100x phase contrast objective and they were classified either as round or motile (Figure 2). Pretreatment of MicrOpgre Filters and Glass Coverslips Glass coverslips used in the slide chamber were pretreated with various agents. Unless otherwise specified, the coverslips were 24 Figure 2: The morphological alterations of neutrophils when exposed to various solution. Magnification 1000X. Cells similar to those in picture (a) were classified as round, while those similar to the neutrophil in picture (b) as motile. 25 Figure 2(a). Figure 2(b). 26 incubated in the pretreated solution for 2 minutes, removed and washed in 3 changes of HBSS. If 2 solutions were used in pretreatment, the coverslips were washed after each incubation. Filters used in the blind well chamber were also pretreated. The filters were incubated in various solutions for 4 minutes (2 minutes/side), placed in a Swinny adaptor, and washed with 20 ml of HBSS. If 2 pretreatment solutions were used, the filters were rinsed in HBSS after the initial incuba- tion and washed after the second incubation. Pretreatment of Neutrophils with Chemotactic Factors Neutrophil suspensions were placed in glass tubes coated with serum and centrifuged at 500 G fOr 10 minutes. The supernate was re- moved by suction. Chemotactic or control solutions were added to the cell button, mixed, and subsequently incubated at 37C for 30 minutes. After incubation, the treated neutrophils were washed 3 times with 5 ml HBSS and resuspended in HBSS to the desired working concentration. The concentrations of chemotactic factors used to treat the neutrophils 5 6 were 0.1 ml of 10' M fMetPhe or 40 ug of protein/ml of C5a per 10 neutrophils. Presentation and Analysis of Data The data are expressed in terms of the mean t standard error of the mean; n represents the number of duplicate experiments. Students' t test was used to assess significance. Zigmond and Hirsch have presented leading front data in the form of a "grid.“ This presentation was used to differentiate random chemotactic, and chemokinetic migration, as defined by them.35’36 RESULTS Agents Assessed for Chemokinetic and Chemotactic Activity Chemotactic and chemokinetic activity was assessed by placing varying concentrations of the reagents in the cell and stimulus com- partment of the blind well chamber and assessing neutrophil migration by grid analysis of the leading front data. Albumin solutions (BSA and HSA) were chemokinetic, since the albumin solutions enhanced migra- tion to the same extent whether above or below the filter (Table 1). Zymosan activated serum, C5a, and fMetPhe were chemotactic, producing results similar to those presented fer solutions of casein (Table 2). Enhanced migration was more pronounced when casein was below the filter. Smith, gt 31, have reported that neutrophils form motile config- urations after exposure to chemotactic factors (C5a, ZAS, or fMetPhe), 30 Human serum but do not do so after exposure to chemokinetic BSA. albumin did not significantly affect the cellular morphology whereas solutions of casein did (Table 3). Effect of Albumin on Neutrophil Adherence and Migration Albumin is often required in the medium while assessing random or chemotactic neutrophil migration in Boyden assays. The reason(s) for this requirement are unknown. The following experiments were de- signed to determine the effects generated by the addition of albumin 27 28 Table l. The effects of bovine or human serum albumin (BSA or HSA respectively) in a gradient on neutrophil migration HSA concentration in the cell compartment (mg/ml) HSA concentration in the stimulus compartment (mg/ml) 0 3.5 35 140 O 26* 32 42 76 3.5 33 35 35 67 80 140 88 90 BSA concentration in the cell compartment (mg/ml) BSA concentration in the stimulus compartment (mg/ml) O 10 30 50 0 41* 61 88 115 10 75 100 110 30 87 120 119 50 106 128 *Numbers represent the distances of migration (pm) as determined by the leading front technique with 60 or 90 minute incubations fer HSA or BSA respectively. 29 Table 2. Effects of casein in a gradient on neutrophil migration Casein concentration in the Casein concentration in the stimulus compartment (mg/ml) cell compartment (mg/m1) O 0.1 1 5 10 O 25* 29 56 110 120 0.1 33 32 1 56 65 5 7O 85 10 6O 79 *Numbers represent distances of migration (mp) as determined by the leading front technique; incubation 45 minutes. Table 3. Alterations of neutrophil morphology when the cells were suspended in various reagents* . . . Round Motile Solution in Cell Suspen51on I’: SEM E's SEM n p Hanks' Buffer 90 i 3 10 t 3 3 Human Serum Albumin (35 mg/ml) 88 i 5 12 i 5 3 > 0.5 Casein (1 mg/ml) 13 i 5 87 i 5 3 < 0.01 *The morphology of the neutrophile in suspension was determined microscopically (50X oil objective) after fixing the cells with glutaraldehyde; cells were classified as round or, when pseudopids and uropods present, as motile. 30 to Boyden assays. Neutrophil adherence and the distances of neutrophil migration on filters treated with albumin were assessed by the leading front technique (Figure 3). Increasing the concentrations of albumin used to pretreat the filters enhanced migration but decreased neutrophil adherence. The distribution of cells in the treated filters was also determined (Figure 4). Increasing the concentrations of albumin to treat the filters increased the numbers of neutrophils getting into the filters. The effects of adding albumin to the cell suspension on neutro- phil migration were determined previously (Table 1). In addition, albumin was placed in the cell suspension and adherence to filters was determined. Neutrophil adherence and leading front measurements were similar when albumin was used to treat the filters or placed in solution with the cells (Figure 5). The effects of albumin on neutrophil adherence to glass were determined using the slide chamber technique. Glass coverslips were pretreated with the same solutions used to pretreat the filters. Cell adherence was significantly decreased when albumin was used to treat the coverslips (Table 4). The addition of albumin to the cell sus- pension also significantly reduced adherence to untreated glass (Table 4). Albumin has been shown to be required in the cell suspension to assess neutrophil migration in response to several chemotactic solu- 34 Micropore filters (3 pm pore tions, including C5a and fMetPhe. size) were soaked in solutions of albumin, albumin then fMetPhe, or HBSS and placed in blind well chambers. Cells were placed in the cell Figure 3. 31 The effects of pretreating micropore filters with solu- tions of human or bovine serum albumin (HSA or BSA re- spectively) on neutrophil adherence and random migration. The filters were pretreated with albumin solutions or Hanks' buffer (HBSS) for 4 minutes and washed by passing 20 m1 of H855 through the filter. Top panel presents the distance of migration determined by the leading front technique with 120 or 75 minutes incubation for filters treated with BSA or HSA respectively. Lower panel pre- sents the adherence of neutrophils to filters pretreated with albumin relative to the adherence to filters pre- treated with HBSS. The number above each bar is the num- ber of duplicate determinations and the vertical line represents 1 1 SEM. All measurements within each of the 4 groups are significantly different (p < 0.02). 32 I40 3:5 35 £5 20 50 O 6 % 51w KTII+II. .bIII$II e, 5+ x , 3+ 5% m m w 10 . . I . . . . e m e e o m m m w m m 553 20.52522 wozwmwzo< .rzmommn. .m ocsm_m HSA BSA ALBUMIN (mg/m1) Figure 4: 33 The effects of pretreating micropore filters with solu- tions of albumin (BSA) on neutrophil distributions. Neutrophil distribution was assessed after neturophils had migrated into treated filters for 120 minutes. Fil- ters were incubated in solutions of albumin or HBSS for 4 minutes and washed by passing 20 ml of HBSS through the filters. The solid dots represent the mean number of neutrophils counted/40X microscopic field at the specified distances into the filters from the cell or- igin. Each dot represents the mean for 10 counts. The numbers to the left of the first count (30 pm into the filter) for each curve are the albumin concnetrations (mg/m1) used to treat the filters. CELLS / FIELD 34 1201 5C) 0 \ 200 . I3C)1 55 O 60‘ 0 40‘ o 9 c) o 20i ‘ 0 ‘\\\\\\\.~\\\\\\‘. I 1 :¥1\\T 20 4O 60 80 IOO 120 DISTANCE MIGRATED (um) Figure 4. Figure 5: 35 The effects of bovine serum albumin (BSA) on neutrophil adherence when used to treat micropore filters or when placed in the cell suspension. The dashed lines repre- sent filters pretreated with albumin solutions and the solid lines are with albumin in the cell suspension. The distances neutrophils migrated (top graph) were de- termined by the leading front technique with incubation times of 120 minutes for experiments using filters pre- treated with BSA (incubated for 4 minutes and washed by passing 20 ml of Hands' buffer through the filters) or 90 minutes when BSA was in the cell suspension. Adher- ence (bottom graph) to filters pretreated with BSA or with BAS in solution is relative to the adherence to filters in the absence of albumin. The solid dots are the means of measurements from duplicate experiments and the vertical lines are :1 SEM. 36 m mm m 1 1 m a s ASS outta—s. woz 0.5)- ****Significantly different from control values (P < 0.01). 39 in chemotactic solutions were assessed in the blind well chamber using filters pretreated with solutions of albumin. Neutrophils pretreated with fMetPhe (lO'SM) did not migrate as far as control cells prein- cubated in HBSS (Figure 6, top panel), and they were more adherent (Figure 6, bottom panel). Increasing the albumin concentration used to pretreat the filters reduced adherence and enhanced migration of cells preincubated in fMetPhe or HBSS. The adherence and leading front data derived from preincubating cells in fMetPhe and exposing them to filters treated with albumin at concentrations of 50 mg/ml were not significantly different from results obtained when cells preincubated in HBSS were exposed to filters pretreated with albumin at concentrations of 20 mg/ml. Results similar to those in Figure 4, for migration and adherence were obtained when neutrophils were pre- incubated with 40 pg of protein/ml of C5a (4315 pm and 68:4% respec- tively using filters treated with 20 mg/ml of albumin). Since random migration of neutrophils preincubated with C5a or fMetPhe was indistinguishable, the responsiveness of the preincubated cells to a second chemotactic stimulus was assessed. The formylated peptides (10'6M) were placed in the stimulus compartment and the mi- gration of cells preincubated in fMetPhe or C5a on filters treated .with albumin was assessed by the leading front technique. Unlike random migration, significant differences were noted in the chemo- tactic migration of preincubated neutrophils (Figure 7). Minimal mirgation in response to fMetPhe was observed for cells preincubated in fMetPhe. Though the response of cells preincubated in C5a was significantly increased, it was not as high as in control cells pre- incubated in HBSS. When the concentration of albumin used to pretreat Figure 6: 40 The effects of pretreating micropore filters with Bovine serum albumin (BSA) on the adherence and random migra- tion of deactivated neutrophils. Neutrophils were incu- bated in 10-5M fMetPhe (hatched bars) or Hanks' buffer (HBSS) (open bars) for 30 minutes at 37°C and washed X3 ‘with HBSS. Micropore filters were incubated with albumin or HBSS for 4 minutes and washed by passing 20 ml of HBSS through the filter. The top panel shows the distances neutrophils migrated which were determined by the leading front technique with 90 minute incubation. The bottom panel shows the adherence measurements relative to the adherence of neutrophils incubated in HBSS to filters treated with HBSS. The numbers above the bars are the numbers of duplicate experiments and the vertical lines represent :1 SEM. All measurements for each group of experiments (i.e. cells pretreated with HBSS or fMetPhe) for adherence or the distances of migration are signif- icantly different. 80' 50 20 BSA (mg/m” Figure 7: 42 The effects of pretreating micrOpore filters with Bovine serum albumin (BSA) on the chemotactic migration of de- activated neutrophils towards fMetPhe. Neutrophils were incubated in fMetPhe (open bars, C5a (hatched bars), or Hanks' buffer (HBSS) (closed bars) at 37°C for 30 minutes and washed X2 with HBSS. The bars are the distances of migration as determined by the leading front technique with 45 minutes incubation. Albumin solutions were incu- bated with micropore filters for 4 minutes and then the filters were washed by passing 20 ml HBSS through the filters. The number above each bar is the number of du- plicate experiments and the vertical line is :1 SEM. All measurements for each of the 2 groups are significantly different (P < 0.01). O m m m mu mu 8 . 6 4 2 . A83 awkdmgz moz<...m.o . 50 O 2 BSA (mg / ml) 44 the filters was increased to 50 mg/ml, the distances of chemotactic migration for cells preincubated in either chemotactic factor increased. However, only for cells preincubated in C5a and placed on filters treated with 50 mg/ml of albumin were the distances of migration com- parable to control cells preincubated in HBSS on filters treated with 20 mg/ml of albumin (Figure 7). Effects of Pretreating_Micropore Filters and Glass with Chem- otactic Solutions on Neutrgphil Adherence and Migration Micropore filters were pretreated with various chemotactic sol- utions or HBSS, washed, and placed in the blind well chamber. As shown in Table 6, filters pretreated with solutions of serum, ZAS, or casein had significantly decreased numbers of attached cells. Migra- tion in these filters was also significantly increased. Only two chemotactic solutions (fMetPhe and C5a) when allowed to bind to the filter surface did not affect neutrophil adherence or migration. Pre- treatment of the filters with serum produced essentially the same re- sults as pretreating with human serum albumin (Figure 8). These effects remained when serum was heated at 56C or when heparin (20 U/ml) was added to the serum solution. Adherence to glass pretreated with chemotactic solutions was de- termined with the slide chamber technique. The adherence of neutro- phils suspended in HBSS to glass coated with solutions of serum, ZAS, and casein was significantly reduced (Table 6). The Effectsggf Chemotactic Factors in Solution on Neutrophil Adherence and Migration Pretreatment of filters or glass with chemokinetic solutions (albumin) produced similar effects on adherence and migration to 45 Table 6: The effects of pretreating micropore filters with chemo- tactic solutions on neutrophil adherence and migration pr222552;::t* adfilgzfice n aghlighce n 3:3:22235 n p**** z 4 SEM** % 1 SEM** pm s SEM*** HBSS 100 5 100 6 30 s 4 8 fMetPhe (10'6M) 100 s 2 3 97 s 2 3 29 s 3 3 C5a 30 pg protein/m1) 97 e 4 3 101 s 3 3 27 s 3 3 Serum (10%) 30 i 5 3 53 i 3 5 55 i 5 5 **** ZAS (10%) 26 e 2 3 55 e 11 4 63 e 5 4 **** Casein (lmg/ml) 33 i 7 3 31 i 8 4 63 t 7 4 **** *Glass xoverslips were incubated for 2 minutes in the specified solutions and washed X3 in HBSS; filters were incubated in these so- lutions for 4 minutes and washed by passing 20 ml of Hanks' buffer (HBSS) through the filters. Zymosan activated serum was abbrwviated as ZAS. **Neutrophil adherence to glass was determined by the slide cham- ber technique and filter adherence by the new modified Boyden assay. ***The distances of migration were determined by the leading front technique; incubation times were 60 minutes with fMetPhe or C5a, 90 minutes for serum and ZAS, and 45 minutes for casein. ****Significantly different (P < 0.01) from control values (HBSS pretreatment) for all parameters measured. Figure 8: 46 The effects of pretreating micropore filters with solutions of human serum albumin (HSA) or serum on neutrophil adher- ence and migration. The filters were incubated in solu- tions of serum, HSA, or Hanks' buffer (HBSS) for 4 minutes and washed by passing 20 ml of HBSS through the filters. The solid lines are the distances neutrophils migrated as determined by the leading front technique with incubation times of 90 or 75 minutes for experiments using filters treated with serum or HSA respectively. The dashed lines refer to the adherence of neutrophils to filters treated with serum or HSA relative to the adherence to filters treated with HBSS. The solid dots represent measurements using filters pretreated with serum (n = 3) and the open dots are measurements with filters pretreated with HSA solutions (n = 3). The concentrations of serum used to pretreat the filter were based on estimates of albumin con- centrations in normal serum. 47 0.3 m, 8 ON 0 o 3 N t; v 04 .w mesa?» 2,595 22:34 .m.m mm m 6. ON V N . 0 , 3 AVG.Mm 9 o e / / \a m / \l / / a / /O W . // cm I / CO. 48 solutions of serum, ZAS, and casein which are chemotactic. Exper- iments were designed to assess adherence and migration with the chantr- tactic factors in the cell or stimulus compartments of the blind well chamber. The first chemotactic factors tested were fMetPhe and C5a because of our observations that these agents did not affect cell behavior when used to coat the filter. Micropore filters were coated with albumin since chemotactic migration in response to C5a and fMetPhe did not occur in the absence of albumin as demonstrated in earlier experiments. In either compartment, both chemotactic factors significantly increased neutrophil migration and adherence (Table 7). Adherence and migration were greater when the chemotac- tic factor was in the stimulus compartment. The levels of adherence were not as high as observed when chemotactic factors were preincu- bated with the cells (Figure 6). The other chemotactic solutions (serum, ZAS, and casein) were tested in a similar manner with the exception that the filters were pretreated with the chemotactic factors instead of albumin. The con- centrations of chemotactic factors used to pretreat the filters were determined as sufficient to prevent additional binding of the chemo- tactic factor to the filter. The results show that all the chemotac- tic factors tested significantly increased adherence and migration when in solution (Table 7). The effects produced by serum in the stimulus compartment were particularly interesting. The migrational responses (leading front measurements) for serum as a chemotactic agent in the stimulus com- partment were similar to those produced by ZAS. However, the 49 .cowuocm_s use mucocoguo cow mo=Po> pogucou o» «consou Amo.c v av acacmcewo Npucouwcpcmvmehee .mouscwe me .cowpmnsuc_ ”mauvecumu cog» mcwummp me» No voc_sgoumc mum: copamgmwe mo moucmumvu aghast .Aon "men coup?» oz» name» on com: anemones mo meowumw>acnn< .aommm cmvzom ms» mo :owuoowewuoe 3o: m an cocw5couwu mo: mucmgogowuuo comoexN .mcoopcc ego eosocep mmm: we peom mcwmmmq an vegan: new mwuacpe e cow meowu:_0m vmwmmuoam on» no nouoaaocv use: weep—put a... m N. 5 mp. m __ a on Ape\oe_v e_ooeo mmmz e , res, m m h oN m m 5 Po mmm: ape\oe.v ecumeo . . m N 5 mm m N h Pm mmm: mmm: AFe\ost ecomeo part c n H mm c m H mm ARC—v m