H'HESIS \ LIBRARY Michigan State University wr—v This is to certify that the dissertation entitled IN VITRO STUDY OF DOG NEUTROPHIL CHEMOTAXIS: TECHNIQUE DEVELOPMENT AND STUDY OF DOGS WI'IH DIABETES MELLITUS presented by Julia Evans Stickle has been accepted towards fulfillment of the requirements for Philosophy degree in Pathology Harold W. Tvedten, D.V.M., Ph.D. Major professor Date OCtOber 22. 1984 MS U is an Aflinmm've Action/Equal Opportunity Institution 0-12771 lVlESI_l BEIURNING MATERIALS: Place in book drop to “saunas remove this checkout from ”In. your record. FINES will be charged if book is returned after the date stamped below. IN VITRO STUDY OF DOG NEUTROPHIL CHEMOTAXIS: TECHNIQUE DEVELOPMENT AND STUDY OF DOGS WITH DIABETES MELLITUS BY Julia Evans Stickle A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pathology 1984 ABSTRACT IN VITRO STUDY OF DOG NEUTROPHIL CHEMOTAXIS: TECHNIQUE DEVELOPEMENT AND STUDY OF DOGS WITH DIABETES MELLITUS BY Julia Evans Stickle This research achieved several goals; assays of neutro- phil function in other species were adapted for use with canine cells, canine cells were found unresponsive to formyl peptides, and the neutrophil responsiveness of diabetic dogs was evaluated. Neutrophil response to stimulation was assessed by shape alteration and found to deviate from human cell re- sponse by a time dependent return to spherical in the pre— sence of stimulant. Utilizing this technique it was found that dog neutrophils did not respond to formyl-L-methionyl- L-leucyl-L-phenylalanine (fMLP). Binding studies with radio- labelled fMLP confirmed a lack of receptors for this com- pound on dog neutrophils. Neutrophil adherence was evaluated by flow and gravity techniques and optimal test conditions were determined for whole blood enui isolated granulocyte preparations. Assess- ment of neutrophil adherence in whole blood was found de- pendent on platelet activation by heparin at room tempera— ture. Platelet activation was determined by increasing Julia Evans Stickle particle counts (platelet clumps) and visual examination of the blood film. Neutrophils behavior from dogs with juvenile onset diabetes mellitus which were well regulated with insulin was compared to cells from control dogs. Neutrophils from dia- betic dogs changed shape readily but the return to spherical was inconsistently more rapid then normal dog cells. The source of this abnormality was not identified. Diabetic neutrophils produced increased amounts of thromboxane when stimulated. Neutrophil adherence in whole blood decreased with increased serum glucose concentration but was not dif— ferent from normal cell adherence when isolated cells were evaluated. The decreased adherence in whole blood was con- sidered the result of media factors and not dependent on altered neutrophil function. Cell migration (unstimulated and stimulated) was measured by passage through a filter and was not different from normal cell behavior. Science becomes dangerous only when it imagines that it has reached its goal. George Bernard Shaw ii ACKNOWLEDGEMENTS Many individuals have aided in this thesis and my develope- ment. The graduate committee has been most supportive and helpful by sharing their expertise, experience, and time generously. More specifically Harold TWedten has tirelessly reviewed manuscripts, advised, and given me the time to pursue and develope this re- search. Wayne Smith generously provided laboratory space and supplies allowing bench work start-up long before funding had been secured. But more significantly he was a major source of informa- tion and direction while fostering independent thought. Numerous faculty and staff from Michigan State University have aided in my graduate studies. It is not possible to mention all individuals but this limitation does not deminish my apprecia- tion for their assistence. The Department of Pathology provided laboratory space and some equipment which allowed work to continue after Dr. Smith's departure. Jimmy Hollers shared invaluable advise on laboratory techniques and research experience. Everyone associated with the clinical pathology section has been most supportive and helpful. On the personnel front, "Aunt" Pat Virtue has provided con- sistent, loving, and dependable child care as well as giving me a genuine appreciation for the complications associated with dia- betes mellitus. Last, but not least, I owe appreciation to Russ and Sara who have tolerated me during this difficult period. They have had to endure my mental and/or physical abscence and have done it pa- tiently and with good humor. Russ' loving support was also crutial in the completion of this work. iii TABLE OF CONTENTS Page LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . vi LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . vii INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . 1 CHAPTER 1 NEUTROPHIL CHEMOTAXIS: A REVIEW OF THE LITERATURE . . HISTORICAL PERSPECTIVE . . . . . . . . . . . . . . . CELL SHAPE AND ORIEN'PATION O O O O O C O C O O O O O ADHERENCE O O I C O O O O O O O O O O O O O O O O O \IU'I-b U CHAPTER 2 NEUTROPHIL FUNCTION IN THE DOG: SHAPE CHANGE AND RESPONSE TO SYNTHETIC TRIPEPTIDE . . . . . . . . . 10 LITERATURE REVIEW . . . . . . . . . . . . . . . . 11 MATERIALS AND METHODS . . . . . . . . . . . . . . 13 Animals . . . . . . . . . . . . . . . . . . . . 13 Neutrophil Isolation . . . . . . . . . . . . . l3 Chemotactic Factors . . . . . . . . . . . . . . 14 Human Neutrophil Isolation . . . . . . . . . . 15 Neutrophil Shape Evaluation . . . . . . . . . . 15 Binding Studies . . . . . . . . . . . . . . . . 16 RESULTS . . . . . . . . . . . . . . . . . . 16 Neutr0phil Shape Change . . . . . . . . . . . . 16 Binding Studies . . . . . . . . . . . . . . . . 17 DISCUSSION . . . . . . . . . . . . . . . . . . . . 23 CHAPTER 3 TECHNIQUES FOR THE STUDY OF DOG NEUTROPHIL ADHERENCE 25 INTRODUCTION . . . . . . . . . . . . . . . . . . . 26 MATERIALS AND METHODS . . . . . . . . . . . . . . 27 Animals . . . . . . . . . . . . . . . . . . . . 27 Sample Collection and Preparation . . . . . . . 27 Isolation of Granulocytes . . . . . . . . 27 Flow Assessment of Neutrophil Adherence . . . . 28 Gravitational Assessment of Neutrophil Adherence O O O O O O O I O O I O O O O O O O 29 iv Page RESULTS . . . . . . . . . . . . . . . . . . . . . 30 Flow Assessment of Neutrophil Adherence, Whole Blood . . . . . . . . . . . . . . . . 30 Flow Assessment of Neutrophil Adherence, Isolated Granulocytes . . . . . . . 33 Gravitational Assessment of Neutrophil Adherence . . . . . . . . . . . . . . . . . . 35 DISCUSSION . . . . . . . . . . . . . . . . . . . . 35 CHAPTER 4 CANINE DIABETES MELLITUS: IN VITRO STUDY OF NEUTROPHIL CHEMOTAXIS O O C O O O C O O C O O C O O 39 LITERATURE REVIEW . . MATERIALS AND METHODS Animals . . . . . . Blood Samples . . . Neutrophil Isolation . . . Preparation of Neutrophil Stim Y o o o o e o o o o o o o o o o o o o o o o o o o o o o o o o o e o o o o o o o e e o o o e U'| N ulants 52 Neutrophil Shape Change Assa . . . . 52 Neutrophil Adherence Assay . . . . 52 Chemotaxis Assay . . . . . . . 53 Glucose, Triglycerides, & Cholesterol Determination . . . . . . . . . . . . . . . . 55 Cortisol Determination . . . . . . . . . . . . 55 Prostacycline and Thromboxane Determination . . 56 Glycosylated Hemoglobin Assay . . . . . . . . . 56 EXPERIMENT I . . . . . . . . . . . . . . . . . . . 56 Introduction . . . . . . . . . . . . . . . . . 56 Experimental Design . . . . . . . . . . . . . . 57 Results . . . . . . . . . . . . . . . . . . . . 58 Discussion . . . . . . . . . . . . . . . . . . 61 EXPERIMENT II . . . . . . . . . . . . . . . . . . 63 Introduction . . . . . . . . . . . . . . . . . 63 Experimental Design . . . . . . . . . . . . . . 64 Results . . . . . . . . . . . . . . . . . . . . 67 Discussion . . . . . . . . . . . . . . . . . . 77 SUMMARY C O O O O O O O O O O O O O O O O O O O O O O O 81 APPENDIX A O O O O O O O O O O O O O O O O O O O O O O O 83 BIBLIOGRAPHY O O O O I O O O O O O O O O O O O O O O O O 90 Table LIST OF TABLES Page Dog neutrophil bipolar shape formation with various stimulants; cells were incubated for 10 minutes prior to fixation . . . . . . . . . . 21 Statistical significance between normal and diabetic dog neutrOphil shape change kinetics for several groups of data . . . . . . . . . . . 68 Filter migration distance ix: microns of unstimulated and stimulated neutrophils from normal and diabetic dogs . .. . .. . .. . .. 69 Serum concentrations of glucose, triglycerides, cholesterol, cortisol, and glycosylated hemoglobin from normal and diabetic dogs . . . . . . . . . . . . . . . . . 71 Serum glucose and neutrOphil adherence in whole blood and isolated granulocytes from normal and diabetic Golden Retrievers . . . . . 73 vi Figure LIST OF FIGURES Stimulated and unstimulated neutrophil shapes: Unstimulated cells are spherical (A), stimulated cells appear ruffled (B), bipolar (C), or bipolar with urOpod (D) depending on time and degree of stimulation . PrOportion of neutrophil :hn bipolar conformation for 180 min. after stimulation, (-1,- ) mean of 13 samples stimulated with 1% ZAP, ( --- ) mean of 9 samples stimulated with 0.5% ZAP . . . . . . . . . . . . . . . . Proportion of neutrOphils in bipolar conformation for 120 min. after stimulation with 2.5% pooled dog serum . . . . . . . . . NeutrOphil bound activity of fML[3H]P. Mean of cells with 3 dogs ( -—— ) and after the addition of excess unlabeled fMLP ( --- ). Mean of cells from 2 human beings (O) and after the addition of excess unlabeled fMLP Ll) . . . . . . . . . . . . . . . . . . . . Whole blood nylon wool adherence assay: Effect of nylon wool weight and anticoagulant on neutrOphil adherence . . . . . . . . . . . Whole blood nylon wool adherence assay: 5 samples from one dog incubated at room temperature for various times. Increased neutrophil adherence was accompanied by increased WBC count . . . . . . . . . . . . . Isolated neutrophil nylon wool adherence assay: Effect of column pretreatment with albumin 0 O O O O O O O O O O O O O O O O O O Gravity adherence assay: Effect of cover glass pretreatment with serum .. . .. . .. vii Page Figure 10 ll 12 13 14 15 16 17 18 19 Experiment I: Experiment I: Experiment I: dogs tested . . . . . Neutrophil shape change kinetics of normal and diabetic Golden Retrievers Serum glucose concentration and whole blood neutrophil adherence for normal and diabetic Golden Retrievers . . . Neutrophil shape change kinetics; cells from Data from normal and diabetic dogs in PBS. study of media effects on shape change reaponse Effect of variable media isotonic glucose concentration on shape change kinetics for normal and diabetic dogs Effect of variable medial hypertonic glucose concentration on shape change kinetics for normal and diabetic dogs Effect of variable media insulin concentration on shape change kinetics for normal and diabetic dogs . . . . . Effect of repeated stimulation on neutrophil shape change response for normal and diabetic dogs Residual stimulatory activity after incubation with normal or diabetic dogs' cells at 37° for 10, 30, 60 or 90 minutes viii Neutrophil shape change kinetics, data from all dogs tested . Neutrophil shape change kinetics, data from Golden Retrievers Glycosylated hemoglobins concerzrations from all normal and diabetic Page . 74 . 84 INTRODUCTION This work deals with thelaboratory evaluation of neutrophil chemotaxis. This area of study is just beginning to appear in the veterinary literature and holds great interest for the author. However, before worthy evaluation of neutrophil function in disease is possible it is mandatory for the testing techniques to be fully evaluated and understood for the species to be studied. The initial 2 experimental chapters therefore, represent technique developement and modification for the dog. The later chapter applies those techniques and a general assessment of chemotaxis (which had also been evaluated for use with dog cells) to a specific disease. A brief general literature review will be followed by chapters dealing with specific areas of inquiry, each will contain a more specific litera— ture review and detailed discussion. It.is the intent of this author that each of the later chapters stand as in- dividual papers. Before embarking on this work it is appropriate to review definitions of words and terms which will appear in this thesis. The following definitions are taken from Brecher (1976). Locomotion: movement with displacement of the entire cell. Motility: capacity to move actively. Migration: active or passive movement of cells (usually a group or class of cells) from one location or organ to another. Taxis: process of cell reacting to a stimulus by locomotion. Positive: in direction of the stimulus. Negative: away from stimulus. Chemotaxis: taxis induced by chemical substance. Phagocytosis: process by which extracellular solid particles are interiorized. Capping: movement of selected portion of cell surface toward one pole. Spreading: process of cell extending over increased surface of its support or another cell. Protopod: front end of moving cell. Uropod: tail end of moving cell. Microvilli: short, fingerlike surface extensions. Filopod: fine, long extensions. Attachment filaments: cytoplasmic extension attached to support surface or other cells as a result of initial attachment and subsequent locomotion of cell, re- traction of veil, etc. Also referred to as retraction filaments. CHAPTER 1 NEUTROPHIL CHEMOTAXIS: A REVIEW OF THE LITERATURE NEUTROPHIL CHEMOTAXIS: A REVIEW OF THE LITERATURE HISTORICAL PERSPECTIVE The role of phagocyte mobilization in the inflammatory response was first reported in 1888 by Theodore Leber. The initial study by this ophthamologist involved movement of leukocytes into rabbit corneas. Metchnikoff (1887) expanded the appreciation of phagocytes by demonstrating their role in protecting the body against pyogenic infections. The theory of phagocytes as the sole defenders was soon modified as the interaction and cooperation of cellular and noncellu- lar defense systems became apparent (Wright & Douglas, 1904). The interest in this discovery was intense and extended into the literature of the day as a major theme in George Bernard Shaw's play The Doctor's Dilemma (1911). After this auspicious beginning, study of phagocytes waned with most studies searching for the source(s) of stimulatory factors. As recently as 1960, Harris summarized the search for stimulatory factors by stating the only confirmed source was from bacteria. Progress was limited by a lack of in vitro techniques to study leukocyte migration and difficulty in obtaining relatively pure populations of cells. A major technical advance in 1962 was the developement by Boyden of a reliable method to quantitate cell movement. This technique utilized a double chamber system separated by a filter with small pores. Cell migration was quantitated by movement through the filter. Modifications of the original proceedure are still widely used in evaluating cell mobility. Neutrophil isolation techniques were also improving with separation of botal leukocytes by sedimenting agents (Boyum, 1964). Isolates of greater purity were possible with density gradient centrifugation (Boyum, 1968). This combination of advances has permitted a rapid expansion in the study of neutrophil function in the last two decades. CELL SHAPE AND ORIENTATION Neutrophil movement was first described as involving altered cell morphology by Ramsey (1972b) and latter by Zigmond & Hirsch (1973). Generalized cell ruffling was identified (Gallin & Rosentha1,l974) and Ramsey described these as lamellipodia projecting in all directions with cell movement occurring only toward the lamellipodia nearest the stimulant. Retraction fibers at the rear of the cell were identified and attributed to lamellipodia which the cell had failed to fill (Ramsey, 1972b). Lichtman, et.a1. (1976) later observed this alteration of shape to occur in suspend- ed cells. The role ofxnicrotubules.in this shape alteration was suggested by inhibition of migration by agents which reduce microtubule formation (Olmsted & Borisy, 1973). These findings, however, were variable depending on concentration and conditions (Klebanoff & Clark, 1978). Direct observa- tion of microtubule assembly by electron microscopy has been reported (Goldstein, et.al., 1973 and Gallin & Rosenthal, 1974). Later electron microscopic studies demon- strated microtubules radiating from a prominent centriole and also mentioned prominent membrane associated microfila- ments (Gallin, et.al., 1978). The role of microfilaments in the shape change response is primarily indirect (Klebanoff & Clark, 1978) butldefective neutrophil chemotaxis has been described in a patient with dysfunctional leukocyte acto- myosin (Boxer, et.al., 1974). The shape of the cell is related to mobility with more elongate cells demonstrating faster movement (Keller, 1982). But bipolar cells are still capable of extending pseudopods in any direction when stimulated and indeed can reverse directions before a new lamellipodia has formed (Gerisch & Keller, 1981). This reorientation of the cell allows for modification and refinement of the neutrophil directional movement after initial activation (Zigmond, 1977). Previous- ly oriented cells return to spherical when the stimulus is removed and the former polarity is not retained with re- stimulation (Zigmond, 1981). The site of directional control is unknown but does not reside in the nucleus (Keller & Bessis, 1975). Initial study of individual cell locomotion reported wide variations in the average speed of individual cells (Ramsey, 1972a). Later work, however, described fairly consistent movement of individual cells with alterations of population movement due to variable numbers of active cells (Keller, 1982L. The effect of individual cell turning and reorientation was evaluated by Zigmond (1981). In this study, unstimulated cell turns generally deviated less then 20° from the original course. When cells were maximally stimulated the interval between turns was prolonged with a resultant increase in the migratory rate observed. ADHERENCE The initial movement of neutrophils out of circulation and to an inflammatory site is dependent upon adherence to the vascular endothelial cells and substratum. The need for cell adherence for emigration was demonstrated by Ackerman, et.al. (1982) who showed that treatments which decrease neutrophil adherence also decreased cell accumulation into an inflammatory site. Excessive adherence, however, is also detrimental to cell mobility as Fehr and co—workers (1979) demonstrated. High doses of fMLP greatly enhanced adhesion with an associated decrease in cell mobility; it was pro- posed that this phenomena was the basis for neutrophil deactivation and also may be instrumental in cell trapping 8 at an inflammatory site. The role of adherence in neutrophil deactivation may also be related to the irreversible nature of in vitro stimulated adherence (Smith, Rollers, et.al., 1979).Keller and co-workers (1981b) confirmed enhanced neu- trophil adherence will decreased migration. Enhanced neu- trophil adherence has been associated with circulating neu- tropenia followed by a rebound neutrophilia (O'Flaherty, et.al". 1978) but circulating neutrophil numbers are influ— enced by many factors in addition to margination. The pattern of surface adherence may also relate to cell locomo— tion which can be influenced by surface treatment with various proteins (Keller, et.al.,1979). Recent reports of patients with defective adherence have added greatly to the understanding of the crucial role of this function (Hayward, et.al., 1979, Crowley, et.al., 1980, Abramson, et.al., 1981 and Anderson, et.al., 1984). These similar works describe children with recurrent infections without neutrophil accumulation despite persistent leukocytosis. Defective neutrophil adherence and cell spreading has been documented in these children. Neu- trophils responded to stimulation by changing shape but could not orient properly inaagradient or migrate toward the stimulus. A specific membrane protein (molecular weight around 127,000 daltons) was deficient in these patients. Recent studies of neutrophil movement through a 3 dimensionalgel introduced a new perspective on the study of cell function as cells do not adhere well tomaterials in which they are very capable of advancing through (Lackie & Brown, 1982). This finding is consistent with studies of compounds which interfere with surface adherence but do not influence migration through a filter (Ackerman, et.alu 1982). This cell behavior may represent migration through the tissues while surface adherence and subsequent movement may represent the initial emigration from the vessel. CHAPTER 2 NEUTROPHIL FUNCTION IN THE DOG: SHAPE CHANGE AND RESPONSE TO SYNTHETIC TRIPEPTIDE NEUTROPHIL FUNCTION IN THE DOG: SHAPE CHANGE AND RESPONSE TO SYNTHETIC TRIPEPTIDE LITERATURE REVIEW Neutrophil chemotaxis is, in part, dependent upon cell motility (Ramsey, 1974), which requires altered neutrophil shape (Bessis, 1973) in addition to other cell activities. The human neutrophil elongates and orients with the broad irregular surface toward the chemotactic stimulus and the narrower uropod at the posterior edge (Zigmond, 1977). Initial studies suggested that neutrophil polarity required surface contact (Ramsey, 1974); however, later work confirmed that cell shape was possible in solution (Lichtman, et.al., 1976). Assessment of cell shape has since been used to indicate cell responsiveness to stimuli without the requirement for other cell functions (Smith, et.al., 1979). The present work utilized this response in assessing the effect of N-formyl-methionyl-leucyl-phenylala- nine (fMLP) on dog neutrOphils. Neutrophil chemotactic activity has been recovered from bacterial cultures (Keller & Sorkin, 1967 and Ward, et.al., 1968) with potent activity generated by Escherichia coli (Schiffman, et.al., 1974). Chemoattractant activity has also been defined for several N-formylmethionyl peptides 11 12 (Schiffman, Corcoran & Wahl, 1975L. These compounds were later equated to chemotactic factors liberated from bacterial cultures (Marasco & Becker, 1982b) and fMLP determined as the most potent stimuli (Marasco, etuaL” 1982a). The advantages of fMLP over other chemotactic agents include easier and more precise quantitation, easier preparation and availability. Species, however, vary in responsiveness to fMLP. The presence of that response must be established for each species. Cell responsiveness to fMLP may be determined by binding of radiolabeled fMLP to neutrophils or by various measures of neutrophil function. Studies of receptor numbers and kinetics utilizing tritiated fMLP (fML[3H]P) have been completed on cells from human beings (Williams, e¢.al”. 1977) and rabbits (Asawankumar, e¢4a1., 1977). Cells from guinea pigs also responded chemotactically to N—formylmethionyl oligopeptides (Chenoweth, etualu.1980). Equine neutrophils have a low number of receptors for N-formylmethionyl peptides and these stimulants induce lysosome release, but do not initiate chemotaxis (Snyderman & Pike, 1980). Although one study did report horse neutrophil chemotactic activity induced by high concentrations of fMLP (Zinkl & Brown, 1982), this finding may be secondary to liberation of leukocyte derived chemotactic factors (Klebanoff & Clark, 1978). Studies of the pig (Chenoweth, et.al., 1980) and cow (Gray, etxal., 13 1982) did not identify receptors for N-formylmethionyl peptides on neutrophils from these Species. The present work evaluated dog neutrOphils for receptors to N- formylmethionyl peptides utilizing fML[3H]P. MATERIALS AND METHODS Animals Two healthy adult Golden Retrievers, 1 male and 1 female, and 1 male Samoyed were evaluated. The Samoyed, recovering from an E; 9213': septicemia (positive blood culture 4 days prior to sampling), was included because it may have altered receptors to fMLP. Therapy consisted of furosemide and IV penicillin in lactated Ringer‘s solution. Neutrophil Isolation Citrate anticoagulated blood (5 ml) was diluted 2-fold with calcium free Dulbecco's phosphate-buffered saline solution (Ca-free PBSS), carefully layered over 4 m1 Ficoll- diatrizoate sodium (Sigma Chemical Co., St. Louis, MO) (sp gr = 1.077) and centrifuged at 400 x g for 30 min. The upper layer of platelets and mononuclear cells and Ficoll- diatrizoate sodium were discarded and the remaining granulocytes and RBC resuspended in Ca-free PBSS. The granulocyte-RBC suspension was combined with lf7nfl.of 6% hetastarch (McGraw Laboratories, Irvine, CA) to enhance l4 gravity sedimentation of the RBC, which required 45 to 60 min. The granulocyte-rich supernatant was removed, washed with Ca-free PBSS, and centrifuged at 200 x g for 15 min. The resulting pellet was resuspended in Dulbecco's phosphate-buffered saline (P885) to a final concentration of 107 cells/ml for shape-change assay or 5 x 107 cells/m1 for binding studies. Visual examination with a phase microscope confirmed the presence of (5% nongranulocytic nucleated cells and no more than 10 RBC/granulocyte. Platelets were rarely encountered. Eosin exclusion determined cell viability between 95% and 98%. Chemotactic Factors Pooled normal dog serum was used as a known Chemoattractant stimulus for the dog (Latimer, Crane & Prasse, 1931). A stock solution of 10‘3M fMLP (Sigma Chemical Co., St. Louis, MO) in dimethyl sulfoxide was prepared every 30 days and aliquots for daily use were maintained at -70 C. Final concentration (10'6 to 10‘9M) of fMLP was attained by further dilution with PBSS. Zymozan activated plasma (ZAP) was prepared by incubating 10 mg of zymozan/ml (ICN Nutritional Biochemicals Division, International Chemical and Nuclear Corp., Cleveland, OH) of fresh heparinized plasma (25 units/ml) at 37(2for Srmhn Zymozan was removed before aliquots were 15 stored at -70(L. Final stimulatory dose was 1% ZAP in the cell suspension. Labeled fMLP (New England Nuclear, Boston, MA) at 0.167mM with 60 Ci/mM specific activity was stored at -70 C until diluted with P385 to the final concentrations of 51c 10'3, 10'7, 1.5 x 107 and 3 x 107M. Human neutrophil isolation As a control, citrated blood from 2 healthy female human volunteers was processed similar to dog samples for neutrOphil isolation, however, RBC sedimentation required 30 to 40 min. The final concentration of cell suspension was 5 x 107 cells/m1. Neutrophil shape evaluation Suspensions of 0.5 to 1.0 x 106 cells in PBSS were incubated with pooled serum, ZAP or fMLP at 37 C. Cells were fixed for at least 1 hour with an equal volume of refrigerated 2% buffered glutaraldehyde (Sigma Chemical Co., St. Louis, MO) after 5, 10, 15, 30, 60, 90, 120 and 180 min. of incubation. Wet mounts of fixed cells were prepared with 100 cells classified according to shape with 100 X phase- contrast objective. 16 Binding studies Formylmethionyl-peptide binding to dog neutrophils was assessed with fML[3H]P. Ten million cells HLZ ml of 5 x 107 cells/m1 suspension) were incubated for 10 min. at room temperature with 20 1 of fML[3H]P in the final concentration (5 x 107 cells/ml). Additional binding was terminated by the addition of 2 ml of cold PBSS. Neutrophils were collected by rapid filtration glass filters (Whatman, Inc., Clifton, NJ) and washed twice with 5 ml cold PBSS. Filters were dried for 24 hours at room temperature and suspended in 10 rml of insta-gel scintillation cocktail (Packard Instruments Co., Inc., Downers Grove, IL). Radioactivity was determined with scintillation counter (Beckman L57000) with a counting efficiency of 45%. After cells had been incubated with labeled fMLP for 10 min., nonspecific binding was determined by addition of 20 l of 10'4M unlabeled fMLP. Fifteen min. after this addition, the reaction was halted and samples processed as stated previously. Specific binding was defined as total cpm minus nonspecific cpm. RESULTS Neutrophil shape change Glutaraldehyde-fixed dog neutrophils in suspension had various cell shapes observed in the humans, such as 17 spherical, ruffled, and bipolar with or without a urOpod (Fig.1J. Studies with ZAP-stimulated dog cells (Fig. 2) demonstrated a dose response with more cells in the bipolar form for a longer time at the higher level of activation. Cell response to pooled healthy dog serum (Fig. 3) documented the rapid conformational change and gradual return to spherical, which was observed with dog neutrophils. Comparison of stimuli (Table 1) indicated that unstimulated neutrophils consistently maintained the spherical configuration and cells exposed to a known chemotactic factor (pooled dog serum) readily elongated to the bipolar shape. Exposure to fMLP did not induce bipolar neutrOphil shape after 10 min. of incubation: cells were also examined at 5 and 15 min. incubation with similar results. The Samoyed, recovering from septicemia (positive blood culture for E; 2311), did not have appreciably different results from the nonsepticemic dogs tested. Binding studies Human control cells had high binding of labeled fMLP (9,498 and 18,847 counts per minute); this binding activity was greatly reduced in the presence of excess unlabeled fMLP (3,755 and 3,613 counts per minute) and indicated binding specificity and reversibility (Fig. 4). l8 .GOAUMHDEHum mo mmummc can mafia so mcfipcomop AG. coach: Spas Hmaomfln Ho .AU. umfiomfln .Amv Umamusu Hmmmmm mHHmo omumHDEflum .Athe bottom surface of the menbrane. Neutrophil adherence has'been studied but all reports to date have used whole blood as the sample with adherence assessed by passage through a nylon wool column (see chapter 45 3 for discussion of adherence techniques). Streptozotocin induced diabetic rats were first found to have decreased adherence relative to normal controls (Stecher, et. al., 1977). Human diabetics were evaluated and adherence was decreased during poor regulation with reevaluation after more intensive insulin therapy demonstratimg improved ad- herence, although normal levels were not obtained. The adherence level in this study was correlated with serum glucose concentrations and incubation of the samples with additional glucose induced decreased neutrophil adherence (Bagdade, Stewart & Walters, 1978). Study of noninsulin dependent diabetics also demonstrated an adherence defect. Patients successfully treated with tolazamide demonstrated improved neutrophil adherence while neutrophil adherence continued to decline in patients who did not respond to this therapy regime. The ability of serum from diabetic patients to generate chemotactic activity has also been examined. Balch, etmal. (1963) reported increased hemolytic complement levels in serum from human patients with diabetes mellitus with no apparent correlation to clinical condition or infection. However, serum activation by antigen-antibody complexes was found deficient in juvenile diabetics by Miller & Baker (1972). Fikrig, etaaLJl977) found that the ability of sera from diabetic humans to generate chemotactic activity from zymosan activation of complement was not impaired. The level 46 of chemotactic activity generated was not correlated to duration of disease, treatment, or ketoacidosis. These findings were later confirmed by Manouchehr-Pour (1981). This discrepency of results may be related to mechanism of activation; antigen-antibody complex activation is via the classical cascade whereas zymosan stimulates the alternate pathway of complement. Opsonic activity has also been examined with Bybee and Rogers (1964) reporting no phagocytic defect observed when normal cells were tested with serum from diabetic patients. This finding was later confirmed by Miller and Baker (1972) in their study of juvenile diabetics. However, this feature has not been consistent as decreased opsonic activity has been reported by Rayfield, et.al. (1978). Neutrophil phagocytic activity has been examined by many groups using a variety of methods. Bybee and Rogers (1964) reported decreased ingestion of pathogenic Staph- ylococci by leukocytes from patients with diabetes and ketoacidosis. The observed defect was reversed with cor- rection of the acidotic state. Study of peritoneal exudate neutrophils from rats with alloxan induced diabetes did not identify abnormal phagocytic activity (Briscoe and Allison, 1965). However, these animals were not ketoacidotic at the time of evaluation. Crosby and Allison (1966) found normal phagocytic activity of neutrophils from human diabetics without ketoacidosis, verylmhfliglucose concentrations in 47 the media did not influence phagocytic activity. The influence of environmental conditions on phago- cytosis has been addressed by Walters, Lessler and Stevenson (1971). These workers found no difference between normal and diabetic neutrophil phagocytic activity but when cells from the diabetic were incubated in hypertonic media the phagocytic activity decreased. This finding was disputed by a study of neutrophils from juvenile diabetics which demon- strated normal phagocytic function. The phagocytic function was unaffected by increased glucose concentration in the media. Decreased phagocytic activity of neutrophils derived from poorly controlled diabetic patients was correlated to serum glucose concentration in a study by Bagdade, Nielson and Bulger (1972). This defect was reversed when these patients were treated. Bagdade et.al.(1974) also found that serum from poorly controlled diabetic patientsimpaired phagocytic activity of normal cells but incubation of diabetic cells with normal serum did not fully correct the defect. Decreased phagocytic activity of neutrophils from nonketoacidodtic diabetics was reported by Tan, et.al. (1975), but these workers found no correlation of the phagocytic activity and diabetic serum (n: glucose concentration. When phagocytic activity was evaluated at various incubation time intervals, Nolan, Beaty and Bagdade (1978) found decreased activity bylfluacells from diabetics at 20 48 minutes of incubation but normal response after 60 minutes. A recent study (Dziatkowiak, et.al., 1982) also reported no decrease in the phagocytic function of neutrophils from diabetic children but the incubation time was not indicated. The cause of a possible phagocytic defect was addressed by Subbaiah and Bagdade (1982) when they reported decreased conversion of lysolecithin to lecithin by stimulated diabetic neutrophils; increased lecithin is required for formation of the phagocytic vacuole with conversion depen- dent on the presense of insulin; the conversion defect was partially reversed by the addition of insulin. Studies of neutrophil killing ability have primarily utilized bactericidal activity to assess this cell function. The initial report by Balch, et.al. (1963) described de- creased killing activity of neutrophils against Staphylococcus albus and Escherichia coli for 33 and 18% of diabetic patients respectively. No correlation with ketone or glucose concentration was observed. Cells suspended in saline (no report of calcium or magnesium addition) were found to have no difference in killing ability between normal and diabetic humans (Briscoe and Allison, 1965). Further study by Crosby and Allison (1966) also failed to detect a difference in killing ability between neutrOphils from normal and nonketoacidotic diabetic donors. Study of cells from "poorly controlled" but nonketotic diabetics disclosed decreased killing activity which was further 49 localized to serum from diabetics; normal cells incubated with diabetic serum had decreased killing function although evaluation of cells from diabetics with normal serum demon- strated only partial correction of the defect (Bagdade, et.alu. 1974). Differentiation between abnormal killing and delayed phagocytosis has been attempted. Bagdade, Nielson & Bulger (1972) reported delayed killing of type 25 pneumo- coccus by whole blood from diabetics to be due to delayed phagocytosis rather than altered intracellular killing mechanisms. Tan, et.al. (1975) studied 31 diabetics and reported 3 with deficient killing and 3 with defective killing and phagocytosis. The patients with only defective killing did not have a history of increased infection but the 3 patients with the combined defect all had severe bacterial infections at the time of testing; 2 patients reverted to normal function with resolution of the infec- tion. The separation of phagocytic and killing function was also considered by Dziatkowiak, Kowalska and Denys (1982) who reported normal engulfment but deficient killing by neutrophils from juvenile diabetics. The relationship of improved control of the diabetes and neutrophil dysfunction has been examined. Rayfield, et.al. (1978) reported decreased bactericidal activity of whole blood from diabetic donors with less killing activity in samples from "poorly controlled" juvenile diabetics relative to "well controlled" diabetic patients. Neutrophils from 50 adult diabetics were evaluated for killing activity by Nolan, Beaty and Bagdade (1978) and found deficient with improved function noted after more successful treatment of the diabetes. Evaluation of nitroblue tetrazolium dye reduction by neutrophils from patients with diabetes has been conflicting. Hill, et.al.(1974) studied juvenile diabetics and found no difference between activity of cells from normal and diabetic donors. However, Walters, Lessler & Stevenson (1971) reported decreased dye reduction by neutro- phils from diabetic patients despite a very wide range for normal cells. Oxygen uptake was also examined in this study and'diabetic neutrophils tended to have greater oxygen up- take with stimulated cells demonstrating a sharp early rise in oxygen consumption with an abnormally rapid decrease to nonphagocytic levels. MATER IALS AND METHODS Animals The dogs were adult Golden Retrievers. Nine dogs with diabetes mellitus were 5 intact females and 4 males. These animals had juvenile onset insulin dependent diabetes and were well controlled at the time of study and were housed with the "Animal Models of Human Disease" colony at the Veterinary Clinical Center(VCC), Michigan State University (MSU). Insulin was administered twice daily at a schedule 51 appropriate for control of the individual animal. Control dogs for this study were male and female Golden Retrievers. Some of these dogs were also housed in the Veterinary Clinical Center while other animals derived from the colony lines were housed in private homes. Some female dogs from private homes had been neutered. Blood Samples Venous blood samples were collected from the jugular vein between 8:30 and 9:00 A.M. before the diabetics had received their morning insulin and food. Samples for neutrophil isolation were collected into a syringe with an 18 gauge needle and sufficient 3.8% sodium citrate (Haemonetics Corpu,Braintree, MA 85301)imithe syringe to yield a final dilution of 1 part anticoagulant to 9 parts blood. Samples for whole blood evaluation of neutrophil adherence were taken with syringe and needle, placed in heparinized tubes (Vacutainers, Becton-Dickinson Co., Rutherford, NJ 07070), and immediately placed on ice to inhibit platelet activation. Samples for glucose, triglyceride, and cholesterol determination were drawn into Vacutainers(Becton-Dickinson & Co., Rutherford, NJ 07070) without aniticoagulant. These samples were allowed to clot and the serum removed promptly and frozen for later evaluation. Samples for cortisol determination were drawn into evacuated blood collection 52 tubes (Becton-Dickinson Co., Rutherford, NJ 07070) which contained EDTA as the anticoagulant. These samples were centrifuged and the plasma removed within 15 minutes of sampling; plasma was then frozen for later study. Neutrophil Isolation Neutrophil isolation was accomplished as described in chapter 2. Preparation 2; Neutrophil Stimulants The stimulant was zymosan activated plasma (ZAP) pre- pared as previously described (chapter 2). Each experiment was performed using the same batch of ZAP which had been stored at -20°C. until individual alliquots were used. Neutrophil Shape Change Assay Neutrophil shape change was assessed as described in chapter 2. The initial concentration of neutrophils used was 5 x 106 cells/m1. with the final test containing 5 x 105 cells/ml. Fixation of cells occurred after 5 & 15 or 10 minutes and after 30, 60 & 90 minutes of incubation. Neutrophil Adherence Assay The nylon wool adherence assay is described in chapter Whole blood adherence was evaluated with a column con- 53 taining 80mg. of nylon fiber packed to a final volume of (L4m1. These columns were pretreated with lml. of complete phosphate buffered saline (PBS) to hydrate the column and produce a similar flow rate between the whole blood sample and isolated neutrophil preparation. Isolated neutrophil adherence was evaluated with the same column size but the columns were pretreated with 0.5m1. of 0.01gm./dl. bovine serum albumin (BSA) (Fraction V, Sigma Chemical Co., St. Louis, MO 63178). This treatment was followed by lml. PBS to remove residual protien from the fluid phase. Sample volume was 0.8ml. for both assays and the time required for sample movement through the column noted to detect major differences in the sample flow rate. Assay of both whole blood and isolated neutrophils was done in tri- plicate with the mean adherence value entered into the data. Chemotaxis Assay Neutrophil locomotion was evaluated by movement of cells through a fine pore filter. This method is a modifi- cation of the technique originally described by Boyden (1962). Two plexiglass plates containing 30 wells were used to form double chambers seperatedby membranes between the 2 plates. Each well was 6mm. in diameter with the bottom chamber volume of 212ul. The bottom chamber was filled with 54 fluid, either 10% ZAP as the stimulant and 1% BSA in PBS as the control. A membrane with pore diameter of 3 microns (Schleicher and Schuell, Inc., Keene, NH 03431) was care- fully layered over the fluid in the lower well without trapping air bubbles at the interface. The chamber assembled and the upper well then filled with 250 ul. of cell suspension consisting of 2 x 106 cells/ml. in 1% BSA. The plates were then incubated in 100% humidity at 37°C. for one hour. The apparatus was then cooled to 4°C. to halt cell migration until further processing was possible. Membrane processing consisted of first fixing the mem- brane in absolute isopropanol (Aldrich Chemical Co., Milwaukee, IVI 53233). Staining 2h) hematoxalin (Gill's Formulation #3, Fisher Scientific Co., Fair Lawn, NJ 07410) was followed by destaining in a solution of 70% isopropanol and 1% hydrochloric acid. Stain color wasintensified by leaving the membranes in hard tap water over night. The stained membranes were then dehydrated before clearing with double baths of xylenes (Sigma Chemical Co., St. Louis, MO 63178). Cleared membranes were mounted on microscope slides with Permount (Fisher Scientific Co., Fair Lawn, NJ 07410). Cell migration was determined with a 100x oil hnmersion lens on a Zeiss Photomicroscope III. The plan of focus for the top of the membrane was determined and the fine focus calibration recorded. The plane of focus for the furthest 55 field into the membrane containing at least two nuclei in focus was then established and the difference used as the total migration distance into the membrane. Glucose, Triglycerides, g Cholesterol Determination Serum values .flmr glucose, triglycerides, and cholesterol were determined on a Flexigem Centrifigal Analyzer (Electro-Nucleonics, Inc., Fairfield, NJ 07006). All samples for a test were evaluated as a group to diminish possible variation. Glucose was measured by an endpoint reaction utilizimg hexokinase specific degradation of glucose (Beckman Instruments, Inc., Fullerton, CA 92634). Triglycerides were also measured by an endpoint reaction initiated by glycerol liberation (Electro-Nucleonics, Inc., Fairfield, NJ 07006). Cholesterol determination was based on specific hydrolysis and oxidation of cholesterol esters (Electro-Nucleonics, Inc., Fairfield, NJ 07006). Cortisol Determination Plasma cortisol levels were determined on all plasma samples at the same time. Methodology utilized was a commercially available radioimmunoassay kit (GammaCoat [1251] Cortisol Radioimmunoassay Kit, Clinical Assays, Travenol Laboratories, Inc., Cambridge, MA 02139). This technique is routinely applied to dog samples in the Veteri- nary Clinical Endocrinology Laboratoryu Michigan State 56 University. Prostacycline and Thromboxane Determination Prostacycline and thromboxane levels were determined in the laboratory of Dr. Scott Walsh, Department of Physiology, Michigan State University (Walsh, eteflu, 1984a and Welsh & Fenner, 1984b). Methodology employed was radioimmunoassay using dog specific antibodies (Seragen, Inc., Boston, MA) labelled with [3H] (New England Nuclear, Boston, MA). Glycosylated Hemoglobin Assay Samples for glycosylated hemoglobin determination were drawn 4 to 5 weeks prior to the study of neutrophil function and assayed at the University of New Mexico (Standefer & Eaton, 1983). EXPERIMENT I Introduction In the spring of 1983 a diabetic dog from the MSU colony of diabetic dogs developed multiple infectious pro- cesses. Neutrophils from this dog were isolated and tested for shape change, adherence on a nylon wool column, and migration in a modified Boyden filter system. The results were compared with cell functions of a control dog from this colony run at the same time. Adherence and chemotactic 57 activity between the 2 dogs was essentially the same but the neutrophil shape change over time appeared quite different between the 2 animals. Cells from the diabetic dog respond- ed differently than expected from previous experience. The diabetic dog cells had adopted the elongated conformation to the same extent as cells from normal dogs but had reverted to spherical shape much more quickly than expected. To determine if this alteration was consistently observed in neutrophils from diabetic dogs several dogs from both the MSU colony and client animals from the MSU veterinary Clinical Center were evaluated over the remainder of the summer 0 Experimental Design Diabetic dogs studied were from two sources; residents of the MSU colony of diabetic dogs were well regulated with insulin treatments at the time of evaluation and client dogs. The client owned dogs were not well regulated at the time of sampling either they had just been diagnosed as having diabetes or their current medical management was insufficient to control serum glucose concentration. Con- trol animals were from the Animal Models of Human Disease kennel, dogs derived from the Golden Retrievers diabetic dogs and returned for breeding, or dogs housed in the VCC as teaching animals or blood donors. The shape change assays were completed with the same 58 batch of ZAP which had been alliquotted into individual vials and frozen at -70°C. for later use. Isolated neutro- phils at a standard comcentration of 5.2-5.3 x 106cells/m1. were incubated with a final concentration of 0.5 & 1% ZAP at 37°C. for 5, 10, 15, 30, 60, s 90 minutes before fixation with 2% neutral buffered glutaraldehyde (Sigma Chemical Co., St. Louis, MO 63178). After fixation for at least 1 hour 100 cells were classified as to shape using a 100x oil immersion phase contrast lens (American Optical, Buffalo, NY 14215). Sixteen diabetic dogs were evaluated, 6 male and 4 female Golden Retreivers, and 6 female dogs of various types. Nine normal dogs were used as controls, 2 male and 4 female Golden Retreivers, 2 female English Pointers, and 1 male Boxer. Glycosylated hemoglobin levels were also determined on all dogs as a long term indicator of the level of control of the diabetes mellitus attained in each animal. Results Comparison of the data from the controls and diabetic dogs using split plot factorial analysis of variation failed to define a statistically significant difference in shape change between the populations (Figure 9%. No difference was noted between control dogs and "poorly regulated" dogs when cell shape change was examined. But examination of data from only the Golden Retreiver colony of dogs (Figure 59 IOO ” Iv. ZAP 90 _. NORMAL n-8 \ DIABETIC n=l5 — —— 80 I— . . 0.5% ZAP ' NORMAL use _ ..... \\ . - .............. :3 7o _ ~ '\\.\ DIABETIC :1 I5 :3 \\ ° 0 5° 7 0: so <1 I .. \ a 40 '- .\'\““s Q: '2. “\\. ‘. ca 30 _ ..................... ' .13 o\° a 20 - l0 - O l l l l l J 5 Io I5 30 so 90 TIME (minutes) Figure 9. Experiment I: Neutrophil shape change kinetics, data from all dogs tested. 6O IOO P 90 - l‘lo ZAP \ NORMAL n86 80 - ' DIABETIC n-Io ----- 0.5% ZAP (3 70 - \’\. NORMAL n86 ————— - _] \\\. DIABETIC nxlo .............. . \ 8 60 ., \ \ g 50 - \ . 'J Ta \ \\. R; 4{)- Thu \\‘-.:\\\. . \ \\, \O 30" .. ‘\ ° ‘\.. 20 _ "°"°°°°°"'°-°-~-...-.., l0 '- 0 l I l l l I 5 IO IS 30 60 90 TIME (minutes) Figure 10. Experiment I: NeutrOphil shape change kinetics, data from Golden Retrievers. 61 10) demonstrated a statistically significant difference unflL005) in the shape change time curve between normal and diabetic dogs. Glycosylated hemoglobin data (Figure 11) were signifi- cantly different between all normal dogs and those with diabetes mellitus (p<0.005, Students t Test). Comparison of glycosylated hemoglobin levels with the % of cells in bi- polar shape after 60 minutes of incubation with 1% ZAP yielded r=0.3672 (not significant). As glycosylated hemo- globin values increased the percent of cells remaining in the bipolar configuration increased. Discussion Neutrophil shape change kinetics differed between normal and diabetic Golden Retreivers. The importance of this difference to cell migration was unknown as human cells when stimulated maintain the bipolar shape for 60 to 90 minutes. But, if the stimulant is removed human neutrophils will return to spherical (Smith, etaal., 1979). It was unclear why dog neutrophils gradually return to spherical and whether this characteristic is related to how the dog cells respond to a chemotactic gradient or interact with the environment. Further study was indicated and undertaken. Glycosylated hemoglobin concentration was higher in the diseased population which was consistent with previous find- ings (Standefer & Eaton, 1983). The relationship of this 62 g DNORMAI. me E 4r DIABETICS n-I4 z 3)- < 2, A .1 n w # 0 Fl l T 80 90 I00 IIO I20 I30 I40 I50 760 INTERVAL (m MS‘ HMF/M Hb) Figure 11. Experiment I: Glycosylated hemoglobins concentrations from all normal and diabetic dogs tested. 63 data to the shape change assay was not significant; it was anticipated that ifaidefect was detected in the diabetic population that that defect would either be unaffected by or increase in severity with poorer regulation of the glucose levels. EXPERIMENT II Introduction The mechanism of the altered shape change was investigated in 2 general areas, examination of the inter- action of the neutrophils and their environment and eval- uation of intrinsic neutrophil functions. Technique developement of the normal dog neutrophil shape change response suggested that the return to sherical form was accompanied byIdecreased stimulatory activity in the media. The difference in shape change response between normal and diabetic dogs then may have been related to a more rapid decrease in media chemotactic activity with incu- bation. Previous investigators suggested that serum glucose concentration (and osmolality) and/or insulin levels in- fluenced neutrophil chemotaxis (Drachman, Root & Wood, 1966, Ainsworth & Allison, 1970, Mowat & Baum, 1971, Miller & Baker, 1972 and Hill, et.al., 1974). The neutrophil's intrinsic "endurance" was evaluated by repeated stimulation; if the diabetic cells were not able to respond as long as normal cells this could relate to the 64 more rapid return to spherical form. The role of arachi- donic acid metabolites as a second mediator of cell function was examined in cells from normal and diabetic dogs. This experiment was based on a recent paper which reported decreased generation of cyclooxigenase products by stim- ulated neutrophils from diabetic humans (Qvist & Larkin, 1983) A more diversified study of chemotaxis in diabetic Golden Retriever dogs was also undertaken to identify other areas of altered behavior. The literature has been contradictory on chemotactic abnormalities. Studies of neutrophil adherence have uniformly reported decreased adherence in diabetic cells but have all used the nylon wool/whole blood test system. Experimental Design The influence of different glucose concentrations on the shape change assay was done on 5 female and 2 male diabetic and 3 female and 2 male normal Golden Retrievers. Neutrophils were isolated in the standard manner but then were incubated for 1 hour at 37°C in isotonic media con- taining 3 or 6 times the standard glucose concentration (assay of standard media glucose was 96 mg/dl). Neutrophil response in hypertonic media with 3 or 6 times the concen— tration of standard media glucose was also studied. The shape change assay was then completed with cells fixed after 65 5, 15, 30, 60, and 90 minutes of incubation. The influence of insulin in the media was evaluated on cells from 4 female and 3 male diabeticrandIS female and 2 male normal Golden Retrievers. After isolation, cells were incubated for 1 hour at 37°C in media without insulin or media with 10cyclooxigenase deriva- tives. Further studies may be useful. Increased thromboxane production has been previously reported from diabetic platelets (Ziboh, et.aln, 1979). Random platelet counts established that the platelet to 77 serum glucose values. A definite correlation of whole blood neutrophil adherence and serum glucose concentration for females was established (r=0.883, p<0.05). The correlation line slope for females was -0.093 with a Y intercept of 89.08. The correlation for data from the males was irrele- vant due to the low number of samples (n=2) but the slope for the male data was -0.072 which is similar to the female data (Y intercept for males was 45.14). No other correla- tions were detected. Discussion The inability 13) repeat the shape change kinetic alteration observed in Experiment I was unexplained. With- out a reproducible abnormality present it became impossible to identify the mechanism of the change by manipulation of the abnormality. The inability to reproduce the initial finding may be the result of an initial inappropriate con— clusion, subsequent changes in technique, or the sporatic appearence of this phenomena. The lack of significant difference between diabetic and normal dog serum glucose concentration «chemotaxis study) reflects the wide range of data obtained for the diabetic animals. Also evident from these data is that most of the diabetic dogs are well regulated with insulin therapy. Glucose concentration was significantly different for fe- males in the adherence study due to less variation in the 78 neutrophil ratio never exceded 1:5 in this study. Also human platelets did not produce thromboxane when exposed to zymosan activated serum (Goldstein, et.al., 1978). The correlation between increasing serum glucose levels and decreasing neutrophil adherence for females is consis- tent with published studies on induced diabetes in rats (Stecher,et.al., 1977) and dogs (Latimer & Mahafetty, 1984) and in humans with naturally occurring disease (Bagdade, et.alu. 1978 and Bagdade & Walters, 1980). The similar slope observed in the data from the males suggests a similar relationship exists in the male dog population but at a lower level of adherence. The lack of serum glucose influ- ence on isolated neutrophil adherence suggests that the altered adherence is not on intrinsic quality of the neutro- phil but somehow associated with the whole blood media. The role of platelets in the whole blood adherence reaction has been described previously (Rasp, eteflu, 1981) with alterations in platelet function well documented in the patient with diabetes (Mustard & Packham, 1984). The in- fluence of platelet activation on whole blood neutrophil adherence has also been described in this work (Chapter 3) with increased adherence observed as platelet activation increases. In this experiment, samples for whole blood adherence assay were rapidly placed on ice to inhibit plate- let activation by the heparin (Salzman, 1980), despite this precaution various levels of platelet clumping were observed 79 on whole blood smears. The degree of platelet activation could not be quantitated but did not appear to be influenced by the presence of diabetes. As reported in chapter 3 the samples incubated at room temperature formed platelet aggre- gates of increasing size and density and with greater incu- bation were surrounded by adherent neutrophils. This fea- ture may represent aggregation enhancement (Redl, etea1., 1983b) or platelet satellitisnI(Payne, 1981, Poon, et.al., 1981 and Y00, et. al., 1982). Payne (1981) reported that rosette forming platelets were distinct from other platelets by an increased glycogen content. However, studies of leukocytes found decreased formation and storage of glycogen in cells from diabetics (Esmann, 1961, Stossel, et. al., 1970 and Goldstein & Curnow, 1980). Poon and co-workers (1981) described the presence of nonimmune glycoconjugates as the source of platelet/neutrophil interaction in their patient; perhaps this plasma factor activity is related to serum glucose concentration in the diabetic patients. Spagnuolo and co-workers (1980) described thromboxane enhanced neutrophil adherence in a whole blood media and thrombin stimulated platelet enhancement of neutrophil adhe- siveness. Platelet stimulation by thromboxane has been reported (Hamberg, et.al.,l975 and Malmsten, et.al., 1975) with a possible role for neutrophil thromboxane generation in the diabetic patient by activation of platelets in the 80 whole blood media with subsequent enhanced neutrophil adherence. Increased serum cholesterol, observed in this experi- ment, is consistent with other reports from diabetics and may influence the lipid composition of membranes (vanOost, et. al., 1982 and Morita, et. al., 1983). But removal of neutrophils to PBS for the relatively short incubation and test period should not alter membrane composition appreci- ably and the effect of diabetes should be observed in ad- herence of isolated neutrophils also. *. a SUMMARY This thesis has dealt with 2 major areas; developement and validation of techniques for the in vitro study of canine neutrophil chemotaxis, and examination of chemotaxis of neutrophils from dogs with diabetes mellitus. Major techniques that were modified for the dog were neutrophil shape change and adherence assays although other techniques required only minor modifications for application to dog cells. The shape change assay deviated from reports on human cells, dog neutrophils adopted a bipolar configu— ration, as with other species, but a return to spherical is observed in the continued presence the of stimulant. Utilizing this assay proceedure it was determined that the dog neutrophils did not respond to the synthetic tripeptide fMLP. Lack of fMLP receptors on canine neutrophils was documented by binding studies with radiolabelled fMLP. This finding is consistent with several other species (pig, cow, and horse) which also do not have a sufficient number of receptors to initiate chemotaxis. Study of neutrophil adherence techniques established optimal experimental conditions for each assay. The influence of sample type (i.e. whole blood vs. isolated granulocyte preparation) was explored with the role of 81 A bI U- -“/A -' IA. 82 platelet activation at room temperature readily apparent. The source or mechanism of this effect was not further defined. Dogs with diabetes mellitus were studied by several techniques for evaluating chemotaxis. This work was under- taken when neutrophils of a diabetic dog had an altered shape change response. The abnormal shape change kinetics was confirmed on study of Golden Retrievers from the MSU colony of dogs with juvenile onset, insulin dependent dia- betes. The altered shape kinetics were further investigated to discover the pathogenesis of this change. The initial abnormality could not be consistently reproduced. Neutro- phil migration through filters and shape change kinetics were not significantly altered in the diabetic population. Neutrophil adherence in whole blood media was decreased with increased serum glucose concentration in the diabetic dogs. This finding is cocsistent with previous reports. Since the isolated neutrophil aderence was not effected by serum glu- cose and was not different from the normal dogs this varia- tion in response may lead, directly or indirectly, to an explanation of the mechanism effecting diabetic neutrophil adherence in vivo. Also, evaluation of neutrophil produc- tion of thromboxane with stimulation were assessed with diabetic cells generating significantly greater amounts of thromboxane then normal cells. \'.0_g~‘.xy APPENDIX A EVALUATION OF NEUTROPHIL SHAPE CHANGE RESPONSE FOR NORMAL AND DIABETIC DOGS 84 90 VARIABLE GLUCOSE STUDY 8° _. --NORMAL DOGS IN pas ass 70 .. \-\ .--DIABETIC 0065 IN PBS m? 60 - \. 40'- 30" 20P °/o BIPOLAR CELLS I0— OI I I L ' 5 I5 30 60 90 o yARIABLE INSULIN STUDY —— NORMAL 0065 IN PBS use 70 r. N -— DIABETIC 0068 IN new 60" 40" “lo BIPOLAR CELLS I0- . '4 °+L——5‘OI5 6'?) 90 TIME (minutes) Figure 14. Neutrophil shape change kinetics; cells from normal and diabetic dogs in PBS. Data from study of media effects on shape change response. 85 o PIABETIC DOGS n=7 - .\0 3° \. —-PBS \ 7o .. -\\ -—--3x ISOTONIC GLUCOSE ---ex ISOTONIC GLUCOSE 60 50 4o - 2“ '-- fl...” 30 20 “lo BIPOLAR CELLS IO O so - ' . "— P93 Q\ ~-- 3:: ISOTONIC GLUCOSE 7o .. \-\ --- 6X ISOTONIC GLUCOSE 50F- 40" 30" % BIPOLAR CELLS K)- o . l I l 5 I5 30 so so TIME (minutes) . Fugue 15. Effect of variable media isotonic glucose concentration on shape change kinetics for normal and diabetic dogs. °/o BIPOLAR CELLS % BIPOLAR CELLS 90 BO 70 60 50 4O 30 20 IO ' O 90 80 70 60 50 4O 30 20 IO 86 DIABETIC DOGS n=7 "- —PBS — \\ \'\\\, . --- 3x HYPERTONIC GLUCOSE ... \\.\ --- 6x HYPERTONIC GLUCOSE \ . _J_J__I I ' 5 I5 ‘ 30 60 90 NORMAL DOGS n=5 '_ —PBS \. --- 3x HYPERTONIC GLUCOSE _ \ --- 6X HYPERTONIC GLUCOSE O I I5 TIME (minutes) Figure 16. Effect of variable media hypertonic glucose concentration on shape change kinetics for normal and diabetic dogs. 87 DIABETIC DOGS n= 8 so 80 - \\ ' . 70 - °\ \ --_--—- PBS. As ONLY ‘ IOp UNITS INSULIN/ml 3 60 — ----- IOOp UNITS INSULIN/ml ..I I.I.l 50 - 0 . a: 40 — < 55 3° - - i— “: 20 — O\ IO - O I I ' I I J 5 I5 30 so 90 NORMAL DOGS n=7 90 I- so .. ’ ----° PBS. A's ONLY - —- IO» units UNITS INSULIN/ml 7o .. "" IOO}! UNITS INSULIN/ml 60- 50- % BIPOLAR CELLS 3 l 3O '- 20 '- IO - \ o I I I I I ' 5 I5 30 60 90 TIME (minutes) Figure 17. Effect of variable media insulin concentration on shape change kinetics for normal and diabetic dogs. 88 0.... moon uriII I I I. .2 .L .maoc ofiumnmfio can HmEuoc How Omcommmu Omcmno woman Handouuaoc :o :ofiumHDEwum omummmou mo uowwwm .mH Ousmwm ON on O? on é STIBO BV'IOdIB °/o .4. .2sz: II p... 83 a. 0596 III 00 cm 00. msolid substrata, locomotion, chemokinesis and chemotaxis in Biology 2; the Chemotactic Response, J.M. Lackie and P.C. Wilkinson, eds. 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