LIBRARY Mlchlgan State Unlversity PLACE IN RETURN BOX to roman this chockout from your «cord. TO AVOID FINES Mum on or him date duo. _‘—————-———_————j DATE DUE DATE DUE DATE DUE Jl a :Jl __ ”WL___ bar—:1 MSUIcAnAMnndivoAcuorVEqudOppommltylmwon W m1 ASPECTS OF EOSINOPHIL BIOLOGY IN THE AUGUST RAT INCLUDING A MODEL OF NEMATODE INFECTION WITH NIPPOSTRONG YL US BRASILIENSIS By Robert Rosback Eversole A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pathology 1996 ABSTRACT ASPECTS OF EOSINOPHIL BIOLOGY IN THE AUGUST RAT INCLUDING A MODEL OF NEMATODE INFECTION WITH NIPPOSTRONG YL US BRASILIENSIS By Robert Rosback Eversole Various novel paradigms of eosinophil involvement in the adaptive immune response have been described in the literature over the last two decades. The fundamental objective of this thesis was the elucidation of active eosinophil involvement during the primary immune response to the enteric phase of a nematode infection in rats. Initially we tested the hypothesis that the previously observed eosinophil accumulation in the peritoneal cavity of the August (AUG) strain of rat is primarily the consequence of phenotype. This characterization of the strain would also facilitate the next phase of the project, which was the comparison of eosinophil activity in the AUG rat during the intestinal phase of primary infection by the nematode parasite Nippostrongylus brasiliensis (Nb). The investigations presented here utilized primarily morphological methods, including a new photoreactive fluorescent technique using Biebrich scarlet (BS) for the detection of eosinophil specific granules. The eosinophil accumulation in the peritoneal cavity of AUG rats was determined to be principally due to the phenotypic expression of heritable trait(s) in this inbred strain. Consistent peritoneal lavage harvests of large numbers (6.9 i 0.5 x 106/animal) of eosinOphils from AUG rats of both sexes provided a ready source of these cells that is not present as a rule in other rat strains. In contrast, eosinophil numbers in the peripheral blood (113 i 22 cells/pl) and in the jejunal mucosa (25.9 i 1.1 cells/villus—crypt (VC) unit) of these rats were within the standard range observed in other rat strains. A significant eosinophilic response to adult stage Nb occurred in the lamina propria of the jejunum (72.9 i 5.2 cells/VC unit). These cells showed altered morphological profiles including cytoplasmic dispersion of specific granules, nuclear change and cytoplasmic vacuolization. Additionally, eosinOphils were observed migrating through the basement membrane of the gut wall and found within the epithelial layer. The peritoneal cavity showed a similar reactivity with a two-fold increase in eosinophil number and increased level of leukotriene C4. These responses occurred at time points relevant to the rejection of the worms by the gut and were thus consistent with the active involvement of eosinophils in the primary immune response to adult stage Nb. To my parents, Charles and Virginia Eversole, by their examples of love, strength and courage, taught me that all things are possible... iv ACKNOWLEDGMENTS I would like to take a moment to thank the members of my graduate committee, Drs. Jon Patterson, Patricia Senagore, Kathryn Lovell and Charles Mackenzie. I am grateful for their contributions of time and effort in order that I might fulfill a lifelong dream in a manner which brings with it the respect of their validation. Also, the early committee contributions by Dr. Jeff Williams provided direction that is much appreciated. Of course, I am particularly indebted to Dr. Charles Mackenzie for the guidance and patience he has shown me as my academic and research advisor. I simply cannot thank him enough for all of the special meetings (fi'om the “Flame” to the many times at his home) required to help me through this process in the midst of his already crushing schedule. I am grateful for his kind mentoring and look forward to our future activities in science and life. On the Kalamazoo front, I. must thank Dr. George Conder and Sandra Johnson at The Upjohn Company (now Pharmacia/Upjohn), who provided support and guidance in their laboratory. They graciously provided the lab space, research animals and Nippostrongylus brasiliensis cultures in the early stages of the research. I must also extend my gratitude to all my friends in the Biological Sciences department at Western Michigan University who provided moral support while I worked at both my job there, and my graduate training. This is especially true for Dr. Leonard Beuving, my friend and mentor since I began my graduate work in science. He has been particularly helpful and understanding in my times of frustration at trying to balance the combined responsibilities of my job and graduate program. Without his collaboration and support, much of the research in this thesis would not have been possible. Also, a special thanks to Dr. Karim Essani for the hybridoma experience and Dr. John Jellies for assistance in developing the Biebrich scarlet story. I would be remiss to forget Betty Chamberlain, without whom Dr. Mackenzie and I would never have arranged all of those meetings; and Kathy Campbell, who provided all of the slides that I needed yesterday. I am grateful for your help and support. To my wife Laura, goes my deepest gratitude for all of the sacrifices that she has made in support of yet another hurdle in my life. The extra love and understanding that she, my son Caleb and daughter Kiley have shown to me during this process are greatly appreciated. vi TABLE OF CONTENTS LIST OF FIGURES ................................................................................................... x KEY TO ABBREVIATIONS ................................................................................. xv CHAPTER 1 REVIEW OF LITERATURE Introduction ...................................................................................................... 1 Eosinophil Morphology .................................................................................... 2 Cell Biology of Eosinophils ............................................................................. 4 Physical Heterogeneity of Eosinophils .......................................................... 12 Immunobiology of Eosinophils ...................................................................... 17 Animal Models of Peritoneal Eosinophilia .................................................... 26 Nippostrongylus brasiliensis ......................................................................... 32 References ...................................................................................................... 38 CHAPTER 2 PERITONEAL EOSINOPHILIA IN THE IN BRED AUGUST RAT ................... 56 Introduction .................................................................................................... 56 Methods and Materials ................................................................................... 57 Experimental Design ................................................................................... 57 Animals ....................................................................................................... 58 Tissue Collections ................................................................................... ,....58 Sample Preparation and Counting Methods ............................................... 59 Statistics ...................................................................................................... 61 Results ............................................................................................................ 62 Peritoneal Cavity ......................................................................................... 62 Peripheral Blood ......................................................................................... 68 Villus—Crypt Units ....................................................................................... 68 Ultrastructure .............................................................................................. 73 Discussion ...................................................................................................... 76 References ...................................................................................................... 79 vii CHAPTER 3 PRELIMINARY INVESTIGATIONS .................................................................... 82 A: Biebrich Scarlet: An Eosinophil Specific Granule Fluorochrome ................... 82 Introduction .................................................................................................... 82 Methods and Materials ................................................................................... 84 Sample Preparation ..................................................................................... 84 Fluorescence Microscopy ........................................................................... 85 Laser Scanning Confocal Microscopy ........................................................ 85 Results ............................................................................................................ 86 Fluorescence Microscopy ........................................................................... 86 Laser Scanning Confocal Microsc0py ........................................................ 91 Discussion ...................................................................................................... 91 References ...................................................................................................... 94 B: The Eosinophilic Response to Primary Infection with Nippostrongylus brasiliensis in the August Rat ................................................................................. 95 Introduction .................................................................................................... 95 Methods and Materials ................................................................................... 96 Experimental Design ................................................................................... 96 Animals ....................................................................................................... 97 Nb Cultures ................................................................................................. 97 Sample Collection ....................................................................................... 98 Sample Preparation ..................................................................................... 99 Results ......................................................................................................... 100 Discussion ................................................................................................... 104 References ................................................................................................... 107 CHAPTER 4 CHANGES IN EOSINOPHILS IN THE INTESTINAL PHASE OF THE NEMATODE INFECTION NIPPOSTRONGYL US BRASILIENSIS ................. 109 Introduction ................................................................................................. 109 Methods and Materials ................................................................................ 112 Experimental Design ................................................................................ 112 Nb Cultures .............................................................................................. 112 Animals .................................................................................................... 113 Tissue Collections .................................................................................... 1 13 Sample Preparation and Counting Methods ............................................ 114 Microscopy ............................................................................................... l 15 Statistics ................................................................................................... l 16 Results ......................................................................................................... 117 Villus—Crypt Unit Counts ......................................................................... 1 l7 Villus—Crypt Change ................................................................................ l 19 Eosinophil Morphological Changes ........................................................ 1 19 viii Eosinophil Ultrastructural Changes ......................................................... 126 Intraepithelial Eosinophil Migration ........................................................ 126 Discussion ................................................................................................... 1 32 References ................................................................................................... l 36 CHAPTER 5 THE PERITONEAL RESPONSE TO NIPPOSTRONG YL US BRASILIENSIS IN THE AUGUST RAT ....................................................................................... 141 Introduction ................................................................................................. 141 Methods and Materials ................................................................................ 143 Experimental Design ................................................................................ 143 Reagents ................................................................................................... 143 Nb Cultures .............................................................................................. 143 Animals .................................................................................................... 144 Peripheral Blood Samples ........................................................................ 144 Peritoneal Lavage ..................................................................................... 145 Omental Samples ..................................................................................... 146 Statistics ................................................................................................... 147 Results ......................................................................................................... 147 Peripheral Blood Counts .......................................................................... 147 Peritoneal Eosinophilia ............................................................................ 149 Peritoneal LTC4 Levels ............................................................................ 152 Omental Change ....................................................................................... 152 Discussion ................................................................................................... 1 56 References ................................................................................................... 160 CHAPTER 6 CONCLUSIONS .................................................................................................. 163 BIBLIOGRAPHY ................................................................................................. 169 ix Figure 2.1 Figure 2.2 Figure 2.3 Figure 2.4 Figure 2.5 Figure 2.6 Figure 2.7 Figure 2.8 LIST OF FIGURES Cytospin preparations (cytoprep) and ultrastructure of the resident cells obtained by peritoneal lavage of the AUG rat. They are: mononuclear cells (M), eosinophils (E), small lymphocytes (SL) and mast cells (MC). Bar = 5.0 pm. Total cell harvest from the peritoneal cavity of AUG rats. The (*) indicates significance over 2wk group and (**) indicates significance over all three younger groups. Differential count on the cells harvested from the peritoneal cavity of AUG rats. Eosinophils in the peritoneal cavity of AUG rats. The (*) indicates significant difference from younger age groups. Differential white blood cell counts in the AUG rat. Eosinophils in the peripheral blood of AUG rats. Eosinophils in the proximal jejunum of AUG rats (DIC optics). Bar = 20.0 um. Eosinophils in the proximal jejunum of AUG rats. The (*) represents significant difference fi'om 2wk group. X 64 65 66 67 69 7O 72 Figure 2.9 Figure 3A.] Figure 3A.2 Figure 3A3 Figure 3A4 Figure 3A.5 The ultrastructural profiles of blood- and peritoneal- derived eosinophils in the AUG rat. A different dispersion profile of specific granules (9) is evident. Small granules (it) are prominent in the cytoplasmic zones unoccupied by specfic granules in the blood- derived eosinophil. Note the vesicular cytoplasm (*) common to the peritoneal-derived eosinophils. The chemical structure of Biebrich scarlet. Micrographs of the fluorescent emissions from the BS- protein complexes in NBF-fixed jejunal lamina propria (A) and unfixed cytospin preparations (B). The lablel includes: eosinophil specific granules (<5), cytoplasmic granules of LGL (9) and red blood cells (*). Bar = 10.0 um. Micrographs of a cytospin preparation showing the specific granules of eosinophils labelled with BS fluorescing under the LSCM. Bar = 5.0 pm. Digital micrograph of a computer-aided 3-dimensional reconstruction of the BS-eosinophil specific granule complex fluorescence (=9) against the remaining tissues of the lamina propria as detected by LSCM. The (II!) are red blood cells. Bar = 10.0 um. Variation in fluorescence emission by different BS- protein complexes as detected by the LSCM. Eosinophil specific granules (=5) and LGL granules (O) were differentiated while the red blood cells (*) emitted at a similar wavelength as the eosiniophil granules. Bar = 10.0 um. xi 75 83 87 88 89 90 Figure 38.1 Figure 33.2 Figure 33.3 Figure 4.1 Figure 4.2 Figure 4.3 Figure 4.4 Eosinophil differential counts of catheter lavage obtained from uninfected (U) and Nb infected (1) AUG rats (Phase I). Eosinophil differential counts of terminal lavage obtained fiom uninfected (U) and Nb infected (1) AUG rats (Phase II). Eosinophils in the proximal jejunum of uninfected (U) and Nb infected (1) AUG rats. Eosinophils in the proximal jejunum of uninfected and Nb infected AUG rats. The (*) denotes significant difference from uninfected level and (lid!) as significant over both uninfected and the 2WK PI groups. Eosinophils (=9) stained by Luna’s method in the lamina propria of villi from the proximal jejunum of uninfected (A) and Nb infected (B) AUG rats. Specific granule dispersion in the infected group impedes eosinophil identification (DIC optics). Bar = 10.0 um. Eosinophils (=9) stained with BS in the lamina propria of villi from the proximal jejunum of uninfected (A) and Nb infected (B) AUG rats. Even the greatly dispersed specific granules in the infected group are easiliy identified under epi-fluorescence microscopy. Red blood cells (it) are the only other cells visible in these micrographs. Bar = 10.0 um. Computer reconstruction of optical sections obtained on the LSCM gives a clear 3-dimensional view of specific granule dispersion profiles in eosinophils (=9) from the lamina propria an Nb infected animal. * = Red blood cells. Bar = 10.0 um. xii 101 102 103 118 120 121 122 Figure 4.5 Figure 4.6 Figure 4.7 Figure 4.8 Figure 4.9 Figure 5.1 Eosinophils (=9) in the lamina propria of the VC units from the infected animals showed cytoplasmic vacuolization (*) not seen in cells from the uninfected group. Bar = 10.0 mm. Dual fluorescence label provides positive eosinophil (BS-red) identification while showing the difference in nuclear (Hoechst 33258-blue) profiles observed between these cells in the uninfected (A) and infected (B) lamina propria. Bar = 10.0 um. Ultrastructure (TEM) of eosinophils (0) in the lamina propria of the VC units confirmed the morphological findings of increased specific granule dispersion within eosinophils from the infected (B) group when compared to the uninfected (A) tissue. Note that specific granules remain confined within cell plasma membranes in both groups. Bar = 2.0 pm. Eosinophils (Q) line the basement membrane of the mucosa] epithelium by 3 weeks PI (A). In the same time frame many of these cells are found within the epithelial layer (B). Bar = 25.0 pm. (A) Transmission electron micrograph of eosinophil (O) migration from the lamina propria (lp) through the basement membrane (bm) and into the epithelium (e) of the jejunal mucosa at 3 weeks PI. (B) Lower magnification from the same experimental group showing an eosinophil in the epithelium wedged between enterocytes. Bar = 1.0 pm. Eosinophils in the peripheral blood of Nb infected and uninfected AUG rats. A significant (*) increase was observed in the 2wk PI animals when compared to the uninfected animals. xiii 124 125 127 129 131 148 Figure 5.2 Figure 5.3. Figure 5.4 Figure 5.5 Total cell harvest from the peritoneal cavities of Nb infected and uninfected AUG rats. Total cells harvested in both infected groups were significantly (*) increased over the uninfected levels. Eosinophils in the peritoneal cavities of Nb infected and uninfected AUG rats. The infected animals had significantly (*) higher numbers of eosinophils than the uninfected animals. Leukotriene C4 levels in the peritoneal exudate of Nb infected and uninfected AUG rats. Only the 3 week PI animals had significantly (*) increased LTC4 in their peritoneal cavities when compared to the uninfected group. Aggregates of cells around the “milky spot” of the omentum (as seen in the uninfected (A) animals), showed an increased cellularity which included eosinophils (O) in the Nb infected (B) animals. Bar = 15.0 um. xiv 150 151 153 155 ANOVA AUG BS EC P EDN EDTA EPO FcR F MLP GM-CSF HBSS HEPES IACUC IFN KEY TO ABBREVIATIONS analysis of variance August strain Biebrich scarlet complement eosinophil cationic protein eosinophil-derived neurotoxin ethylene diamine tetra-acetic acid eosinophil peroxidase cell receptor for antibody n-fomiyl-methionyl-leucyl-phenylalanine granulocyte-macrophage colony stimulating factor Hank’s balanced salt solution hydroxyethyl-piperazine-ethane sulfonic acid human leukocyte antigen institutional animal care and use committee interferon immunoglobulin XV IHES IL LFA LSCM LT MBP MHC Nb NBF PAF PECAM PI RANTES RT SD SEM SNK SOZ TEM idiopathic hypereosinophilic syndrome interleukin third stage Nb larvae fourth stage Nb larvae leukocyte functional antigen laser scanning confocal microscopy leukotriene major basic protein major histocompatibility complex Nippostrongylus brasiliensis neutral buffered formalin platelet activating factor platelet endothelial cell adhesion molecule post infection regulated and normal T-cell expressed and secreted protein room temperature Spraque-Dawley strain standard error of the mean student Newman-Kuels serum opsonized zymosan transmission electron microscopy xvi Th VC VLA WBC thymocyte helper (cell) tumor necrosis factor villus-crypt very late antigen white blood cell xvii Chapter 1 REVIEW OF LITERATURE Introduction In 1879 Paul Ehrlich discovered that the cytoplasmic granules of certain blood cells had a strong affinity for the negatively charged stain, eosin. These bright orange-red staining cells he classified as a distinct group of leukocytes and aptly named them eosinophils. Later it was shown that the proteins found in the compact specific granules of eosinophils were highly cationic thus explaining their affinity for the bright red, brominated fluorescein derivative; eosin. This histologic feature remains perhaps the most widely known characteristic of eosinophil granulocytes. Armed with the ability to detect eosinophils in blood and tissue, Ehrlich began the first investigations linking these cells with specific pathologic changes in certain disease states of humans. He proposed that eosinophils originated in the bone marrow, demonstrated the transient nature of their levels in blood during disease and hypothesized that eosinophils “exerted their fimction in tissues” (Ehrlich & Lazarus, 1900). The evidence produced in these early investigations is, for the most part, still the accepted dogma in the field today. In the same time flame, physicians described the accumulation of eosinophils in tissues during 1 2 asthma (Gollasch, 1889), allergic skin diseases (Canon, 1892) and endoparasitic infections (Brown, 1898). These clinical conditions remain the primary areas of investigation in the current eosinophil-related literature. Eosinophil Morphology Morphology remains the single most reliable method for determining the presence and activity of eosinophils within a given tissue compartment (Owen, 1993). Mature eosinophils range in size from 8-17 pm with a bilobed nucleus in humans and a multilobed donut-shaped nucleus in rats (Hamada et al., 1992). Their characteristic specific or secondary granules allow rapid identification at the light and electron microscopic levels. As mentioned previously, the arginine-rich basic proteins in eosinophil specific granules have a high affinity for acid dyes such as eosin (Ehrlich, 1879), chromotrope 2R (Lendrum, 1944) and Biebrich scarlet (Spicer & Lillie, 1961), all of which give these granules a distinct orange- red appearance under bright-field light microscopy. The ultrastructural profile of eosinophil specific granules is a distinctly unique ellipsoid shape with a bimodal packaging scheme where one or more axial, crystalline cores are surrounded by an outer matrix, all packaged in a double-unit membrane. The cores are comprised entirely of major basic protein (MBP) and the matrix is a composite of eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN) and eosinophil peroxidase (EPO) in humans (Egesten et al., 1986). Rat eosinophils contain the enzymes described in human cells (Pimenta et al., 1987) and also have 3 peroxisomal enzymes in their crystalloid granules (Yokota et al., 1984). The relative electron—densities of these granule compartments varies with staining methodology but the profile remains relatively consistent in normal tissue eosinophils (Faller, 1966). The number of eosinophil specific granules has been shown to vary in normal eosinophils for both humans and rats with 20—40/cell being the typical range. The volume of individual specific granules varies within cells as well as total granule volume/cell (Elmalek & Hammel, 1987', Caulfield et al., 1990). The ratio of total specific granule volume to total cytoplasmic volume in tissue-dwelling eosinophils is markedly decreased compared to the majority of blood- and bone marrow-derived eosinophils (Owen, 1993). These specific granule characteristics contribute to the physical heterogeneity observed in eosinophils in vivo and will be discussed in detail later in this review. A second type of granule common in eosinophils are primary granules. Primary granules are the principal type in early promyelocyte eosinophils but are also observed in tissue dwelling cells where specific granules are the predominant granule type. Primary granules vary in size, often larger than specific granules and are spherical with a homogeneous electron-dense ultrastructural profile (Zucker- Franklin, 1980). Mature eosinophils have a third population of granules which are round and much smaller in size than the specific or primary granules at 0.1-0.5 um diameter and which possess acid phosphatase and arylsulphatase activity (Parmley & Spicer, 1974). The greater number of the small granules in tissue dwelling 4 eosinophils compared to blood and bone marrow eosinophils has led to the hypothesis that these small granules may be derived from the specific granules since both have acid phosphatase; this enzyme is only detectable, however, in specific granules of degranulating eosinophils which have lost their central crystalloids (Enomoto & Kitani, 1966). Additionally, the small granules have been shown to accumulate microendocytosed gold particles demonstrating involvement in heterophagic activity in peritoneal-derived eosinophils (Fukuda et al., 1985). The eosinophil cytoplasm contains a highly developed cytoskeletal microtrabecular lattice which is capable of orchestrating rapid cellular and granular shape changes (Pryzwansky, 1987). Microvesicular and vacuolar cistemae of the smooth endoplasmic reticulum are a common feature of eosinophils and extensive membrane-bound, labyrinthine, tubulovesicular structures have been observed in peritoneal—derived eosinophils (Fukuda et al., 1985). Golgi apparatus, mitochondria and rough endoplasmic reticulum are also found in both blood and tissue eosinophils (Spry, 1988). A prominent cytoplasmic feature of tissue eosinophils involved in disease states are non-membrane bound cytoplasmic lipid bodies (Weller & Dvorak, 1985). Cell Biology of Eosinophils Migratory populations of immune regulatory cells, of which eosinophil leukocytes are one type, require complex signal transduction pathways and communication molecules to accomplish a vast array of functions in a multitude of 5 tissue microenvironments. In order to perform some of these functions, cell surface receptors are known to serve as initiation sites for many communication pathways. Some of the dynamic cell surface receptors that have been identified on eosinophils include those for soluble serum-derived mediators, cytokine mediators, immunoglobulin (Fc) receptors and adhesion receptors for both cell-cell and cell- matrix interactions (Gleich et al., 1992; Giembycz & Barnes, 1993; Wardlaw, 1994). Known complement (C) receptors include Clq, C3b/C4b (CR1), iC3b (CR3) and C5a and eosinophils will respond to C3a. Cytokine receptors have been described on eosinophils for: interleukins (IL-2,-3,-5), granulocyte, macrophage— colony stimulating factor (GM-CSF), regulated and normal T-cell expressed and secreted protein (RANT ES), and are presumed for tumor necrosis factor (TNF-a) and interferon (IFN-y). Additionally, the experimental peptide n-formyl-methionyl- leucyl-phenylalanine (FMLP) has been shown to bind to eosinophils via membrane receptors. Receptors for highly potent lipid mediators of eosinophil function include platelet activating factor (PAF) and leukotriene B4 (LTB4). Immunoglobulin (lg) binding receptors for the IgG, IgA and IgE classes are expressed on the surface of eosinophils. There are three receptors for IgG: high affinity FcyRI and the lower affinity FcyRII and FcyIII. Multiple receptors for the IgE class are also known. The first discovered was the low affinity FceRIl but a recent finding by Capron and colleagues (1995) indicated that the high affinity 6 FcaRI is also expressed on eosinophils in vivo. Adhesion receptors for eosinophils include: the integrins, VLA-4, VLA-6, LFA-l and Mac-l (CR3); Immunoglobulin- like PECAM; the L-selectin moiety and ligands for P-selectin and E-selectin. It is important to note that VLA-4 is the only member of the integrin family known for both cell-cell adhesion and binding to the extracellular matrix molecule, fibronectin (Anwar et al., 1993; Anwar et al., 1994) suggesting a versatile cellular migration mechanism in tissues. Eosinophils have the ability to secrete many types of mediators in the chemical web of the inflammatory response. These include relatively large stores of the highly alkaline preformed protein mediators found in the specific granules (Gleich et al., 1992). MBP with a pI of 10.9 contains 17 arginine residues contributing to its cationic status. It is synthesized and packaged as an acidic proprotein (p1 6.2) which may serve to protect the cell itself from its highly cytotoxic constituent (Barker et al., 1988). MBP has distinctive helminth-killing capability which has been shown to be inhibited by mast cell-derived or endogenous heparin by an unknown mechanism (Kierszenbaum etal., 1982). The strongly positive charge of MBP has been implicated in toxicity to respiratory epithelium at low concentrations. MBP can cause the release of histamine from mast cells and both MBP and EPO have been shown to increase arachidonic acid metabolism and granule secretion in platelets (Rohrbach et al., 1990). The activity of the heme containing protein EPO has been shown to be more potent than neutrophil 7 myeloperoxidase when utilizing bromine in the halide-H202 killing mechanism (Wardlaw, 1994). EPO is also a cationic toxin with a p1 of 10.8 that can mediate the killing of parasite and mammalian cells. The remaining highly cytotoxic granule protein found in eosinophils is ECP which also has a pl of 10.8 and possesses some ribonuclease activity. ECP shares a strong homology to eosinophil-derived neurotoxin (EDN) but EDN has a much higher ribonuclease activity and is only weakly cytotoxic. A number of other preformed granule enzymes have been isolated from eosinophils but a clear functional role for these has yet to be established (Spry, 1988). These include: acid phosphatase (large amounts located in the small granules), lysophospholipase, alkaline phosphatase (in rats), collagenase, arylsulphatase B, histaminase, phospholipase D, catalase and non-specific esterases. The primary lipid mediators of inflammation produced by eosinophils are eicosanoids and platelet activating factor (PAF) (Weller, 1993). Arachidonic acid metabolism in mammalian eosinophils has been shown to act primarily through the lipoxygenase pathway with 15-hydroxyeicosatetraenoic acid as a major product (Turk et al., 1983). Additionally, the late phase sulfidopeptide leukotrienes of anaphylaxis, LTC4 and LTD4 are produced with relatively large amounts of LTC4 (up to 70 ng/106 cells) being a unique eosinophil capability (Weller et al., 1983). In fact the levels of LTC4 generated by eosinophils in many experimental models far and away exceeds other inflammatory cell populations’ capacity to produce this 8 sulfidopeptide leukotriene (personal communication, Dr. F. Sun). Interestingly, evidence suggests that LTC4 concentrations are lowered in the presence of EPO as a possible self-regulatory scheme by eosinophils in hypersensitivity reactions (Goetzl, 1982). PAF also can elicit LTC4 from eosinophils (Bruynzeel et al., 1986) which indicates another autocrine mechanism since eosinophils are also known to produce PAF. However, when eosinophils produce PAF more than 90% of it remains cell-associated (Cromwell et al., 1990). The investigations on stimulus-response coupling in eosinophil function began with in vitro models of helminth parasite killing. The finding that the eosinophil leukocyte could kill the schistosomula of Schistosoma mansom‘ in vitro provided the first bioassay for eosinophil-mediated cytotoxicity (Butterworth et al., 1974). The mechanism was shown to be a multi-step process of reversible adherence to the complement and antibody opsonized schistosomula followed by the eosinophils actively degranulating, at which time, binding became irreversible and killing ensued. This mechanism was confirmed for rat and human eosinophils (Mackenzie et al., 1977', McLaren et al., 1977; Ramalho-Pinto et al., 1978). Additionally, models including trematodes, cestodes and other nematodes also demonstrated eosinophil-mediated killing mechanisms (Butterworth & Thome, 1993). The released granule contents may serve as ligand or “glue” in this last stage (Butterworth et al., 1979) as well as their known cytotoxic activities. The opsonizing antibody was shown to be IgG and, therefore, could be inhibited by 9 protein A which binds the Fc portion of this antibody class (Mackenzie et al., 1977). Additionally, complement or antibody were shown to be equally effective in eliciting eosinophil adherence and degranulation but neutrophils and mast cells required antibody for adherence (Ottesen et al., 1977; Mackenzie et al., 1981). Some variations in eosinophil adherence were observed and ascribed to immunologic variation in the nematode surfaces that are either stage- or species- specific (Mackenzie et al., 1978). The in vivo and in vitro correlates of these mechanisms of eosinophil adherence were described in the killing of microfilariae in human onchocerciasis (Mackenzie et al., 1985; Williams et al., 1987). Eosinophil binding and degranulation on antibody opsonized targets is known to be regulated by the FcyRII, which is the only constitutively expressed IgG receptor on eosinophils. This receptor regulates many other eosinophil functions including phagocytosis and the production and release of LTC4 and PAF (Wardlaw, 1994). Recent evidence has demonstrated the upregulation of the high- affinity FcaRI on human eosinophils in vivo and the involvement of this receptor in eosinophil-mediated cytotoxicity in vitro (Gounni et al., 1994a). Prior in vitro evidence has linked the low affinity FcaRII to some eosinophil effector functions in rats and humans via binding assays (Gleich et al., 1992). The release of eosinophil granule proteins has been shown to be differentially controlled depending on the stimulus of a given immunoglobulin receptor class. In one study, IgG complexes 10 caused release of ECP but not EPO but IgE complexes induced the opposite secretory profile (Khalife etal., 1986). The aforementioned studies clearly tie complement receptors on eosinophils to cytotoxic effector functions. A strong synergism was noted when both antibody and complement were added to the in vitro cytotoxicity assays (Ottesen et al., 1977; Mackenzie et al., 1981). The binding of complement receptors CR1 and CR3 has been identified as potent stimulus for eosinophil degranulation in vitro (Spry, 1988). These receptors are differentially up—regulated by histamine, FMLP and LTB4 (CR1) (Anwar & Kay, 1977; Kay et al., 1979; Nagy et al., 1982) and IL- 5 (CR3) (Vadas et al., 1986). Another study has linked the CR3 receptor to the respiratory burst in eosinophils stimulated with serum-opsonized-zymosan (802). This phenomenon was further enhanced by PAF exposure (Koenderman et al., 1990). Possible roles for C5a and Clq have been suggested by in vitro investigations but definitive studies remain to be done. Several cytokines have been shown to produce marked effects on eosinophil function (Walsh et al., 1993). These include short-term effects where response is immediate and long-term effects where protein synthesis is required such as in increased receptor expression. TNF was shown to increase eosinophil cytotoxicity to helminth and mammalian cells (Silberstein & David, 1986; Sluggard et al., 1990). IL-3, IL-5 and GM-CSF can affect mature eosinophil function in regard to enhanced cytotoxicity, prolonged survival, enhanced adhesion to vascular 11 endothelium and delayed apoptosis (Silberstein et al., 1986; Owen, Jr. et al., 1987; Rothenberg et al., 1987; Rothenberg et al., 1988; Tai et al., 1991; Lopez et al., 1996). Eosinophils stimulated with GM-CSF and IL—3 synthesized and expressed CD4 protein which allowed entry of the human immunodeficiency virus (Lucey et al., 1989). Recent work by Weller and colleagues (1993) has produced evidence for the role of GM-CSF stimulated eosinophils in HLA-DR dependent, MHC- restricted antigen-presentation. The discovery that pediatric dialysis patients presented with peritoneal eosinophilia and the eosinophils obtained expressed HLA-DR proteins (Roberts et al., 1990) led to further in vitro study. When eosinophils were co-cultured with fibroblasts and GM-CSF they produced HLA- DR proteins and IL~10L indicating plausible antigen-presentation and T-cell stimulation capability. The significance of these cytokines to eosinophil accumulation and activity has been supported by their detection in vivo under various eosinophilic conditions (Gleich et al., 1992). Early studies on eosinophil chemotaxis produced an enormous list of potential chemotactic agents for eosinophils in vitro (Spry, 1988). Further investigations narrowed the list with the most potent chemotactic agents in viva being PAF, LTB4, C5a and RANTES. Recent evidence indicates that T-cell and platelet- derived RANTES is a long acting eosinophil-specific chemotaxin (Kameyoshi et al., 1992). The production of PAF by mast cells and LTB4 by neutrophils may be the most relevant sources of these lipid mediators in the early stages of eosinophil- 12 related inflammation. While LTB4 is known for potent eosinophil chemotaxis in guinea pigs it is weak in human systems and has not been shown to stimulate degranulation in eosinophils (Wardlaw et al., 1986). The pro-inflammatory capabilities of PAF are beyond the scope of this review, however, it is probably the most biologically relevant chemotactic and secretory stimulus for eosinophils in vivo (Giembycz & Barnes, 1993). PAF selectively enhances eosinophil migration over neutrophils in vitro (Wardlaw et al., 1986) and produces an eosinophil-rich infiltrate when given intradermally (Henocq & Vargafiig, 1988). Virtually all lipid mediators in the eosinophil repertoire are secreted in response to PAF. Helminth— killing capability is enhanced by a PAF mediated up-regulation of FcaRI and cytophilic IgE binding producing increased degranulation of both specific and small granules of eosinophils (Capron et al., 1984; Capron et al., 1985). In general, functional stimulation of eosinophils has been demonstrated by PAF both with and without degranulation occurring. Physical Heterogeneity of Eosinophils In the previous section the functional heterogeneity of eosinophils was demonstrated. The vast majority of these studies examined peripheral blood eosinophils of humans purified by density gradient centrifugation. Various types of discontinuous gradients and cushions were used including bovine serum albumin, Metrizamide and Percoll (Alexander & Spriggs, 1962; Day, 1970; Gleich & Loegering, 1972). These early purification protocols designed to provide l3 segregated eosinophils for in vitro bench investigations led to the observation that peripheral blood eosinophils had a wide range of functional capabilities. In an attempt to find clinical correlates for the peripheral blood eosinophilia associated with helminth infections, comparative studies of healthy and infected donors were run on density-centrifiigation gradients. The observation was made that the eosinophilic donors had more prominent centrifugation bands of less-dense eosinophils than the non-eosinophilic donors (Vadas et al., 1979; Prin et al., 1983; Prin et al., 1984). Similar studies in asthma (Fukuda et al., 1985; Kauffman et al., 1987) and tryptophan-induced eosinophil myalgia syndrome (Owen, Jr. et al., 1990) produced the same prominent “hypodense” bands; whereas normal subjects usually had blood eosinophils that are 90% “nonnodense”. These investigations clarified the concept of physical heterogeneity in blood eosinophils and demonstrated at least an increase in population of certain physical phenotypes in eosinophilic disorders. Unfortunately, attempts were made to class all “hypodense” and “nonnodense” eosinophils into singular functional categories. This approach has proved to be too simplistic. It became apparent that the various gradient materials produced multiple standards of sedimentation coefficients for defining “hypodense” eosinophils. In fact, these standards proved to be overlapping with “nonnodense” standards depending on the eosinophilic disorder that was being investigated, the location the cells were harvested from and the gradient that was chosen for the l4 separation (Owen, 1993). Also, an overlap in functional capacity was demonstrated when “normodense” eosinophils from idiopathic hypereosinophilia syndrome (IHES) patients elicited a three-fold increase in LTC4 production over healthy donors (Owen et al., 1989). Regardless of these discrepencies, it has been shown that peripheral blood eosinophils of lesser centrifugation density generally have increased capacity when assayed for antibody-dependent helminth cytotoxicity and LTC4 generation in vitro (Owen, Jr. et al.,. 1987b). This correlative evidence for physical and functional heterogeneity led researchers to hypothesize that certain pathogenic stimuli gave rise to the “hypodense” phenotype from the ‘normodense” which was the the phenotype of the majority of eosinophils in the circulation. Multiple investigations were performed in vitro which demonstrated the conversion (Owen, 1993). Acute conversions of eosinophils were accomplished by PAF, F-MLP, LTB4, calcium ionophore (A23187) and 802 after 15 minute exposure (Kloprogge et al., 1987; Fukuda & Gleich, 1989). Long-term culture (7 days) -which can only be accomplished in the presence of GM-CSF, IL-3 and IL-5- produced the “hypodense” from the “normodense” phenotype in 90% of the cells (Owen, Jr. et al., 1987a; Rothenberg et al., 1988a; Rothenberg et al., 1989a). These investigations demonstrated in vitro the production of physically heterogeneous eosinophil populations by the same cytokines known for inflammatory regulation and specific eosinophilopoiesis in viva. 15 Evidence that eosinophils obtained from human tissues (bronchial and pleural lavage) were mainly “hypodense,” while there was a relative paucity of these cells in the peripheral circulation, provided evidence for the hypothesis that lower density eosinophils may be the majority tissue phenotype (Prin et al., 1984; Prin et al., 1986). This was also confirmed in rat peritoneal lavage where the eosinophil majority in control animals were “hypodense” and these populations were fithher increased by T. spiralis infection (Hamada et al., 1992). Strong in vitro evidence supporting this showed that co-culture of a homogeneously dense eosinophil population with 3T3 fibroblasts alone could produce the physical and functional heterogeneity described above (Rothenberg et al., 1989; Owen, Jr. et al., 1991). Further work on describing the morphological heterogeneity of tissue eosinophils in various fiinctional and pathogenic settings in vivo would provide much needed information toward delineating the roles that eosinophils play in mammalian immunity. It is of interest, that neither the in vivo nor in vitro-derived “hypodense” eosinophils possessed a singular morphologic profile consistent with the lighter densitometric profile. In fact, the in vitro conversion described previously (Owen, Jr. et al., 1987a) produced 90% “hypodense” eosinophils but none had the specific morphological granule changes usually seen in “hypodense” cells taken from the blood of hypereosinophilic patients. Therefore, it is not surprising that several morphometric and ultrastructural profiles have been observed in the eosinophils 16 taken from the less dense bands of centrifugation gradients performed on peripheral blood leukocytes in humans and rats. These included various combinations of nuclear hyper-segmentation, alterations in specific granules, increased cytoplasmic lipid content, increased cytoplasmic vacuolization and cell swelling (Owen, 1993). Increased nuclear segmentation would be consistent with long-lived fully'differentiated cells which most likely occurs in eosinophilia (see Immunobiology of Eosinophilia). The increased cytoplasmic lipid stores may serve as a source of arachidonic acid metabolites (Weller & Dvorak, 1985) and would definitely affect buoyant density. Cell swelling would be consistent with the increased metabolic activity demonstrated in “hypodense” eosinophils. A variety of alterations in ultrastructure have been observed in disease states, producing a heterogeneous specific granule appearance within and among eosinophils involved in pathogenesis in vivo (Dvorak et al., 1982; Peters et al., 1988; Torpier et al., 1988; Caulfield et al., 1990; Colombel et al., 1992). Important correlations between hypodensity, function and ultrastructure were made utilizing peripheral blood eosinophils from human subjects with IHES (Peters et al., 1988; Caulfield et al., 1990). The relative volume of specific granules Within the cytoplasm decreased in eosinophils of lower density. This was due primarily to a decrease in granule size and enhanced by cell swelling as indicated by an increase in measured cell diameter (Caulfield et al., 1990). The specific granule matrix and crystalloid cores showed heterogeneous clearing and loss of electron density. Cellular MBP 17 content was shown to be 41% of control cells and plasma MBP levels were eight times higher in IHES patients (Peters et al., 1988). Similar ultrastructural evidence for MBP release by eosinophils from the lamina propria of the small intestine have been described in gastroenteritis and adult coeliac disease (Torpier et al., 1988; Colombel et al., 1992). Unfortunately, the morphologic profiles of eosinophils during most eosinophil-related immune responses in vivo remain poorly characterized, especially for tissue—dwelling eosinophils. Immunobiology of Eosinophilia It is generally accepted that eosinophils are a well differentiated leukocyte population that arises from myeloid progenitor cells in the bone marrow, traverses the vasculature and lymph channels, and comes to reside predominately in submucosal tissues. In a restricted number of disease states, eosinophil production is up—regulated and these cells accumulate in certain tissues, a condition described in the literature as eosinophilia. Some specific criteria of eosinophilia have been established for blood in humans and rats, where total eosinophil counts above 0.65 x 109/1 are considered eosinophilic (Krause & Boggs, 1987; Wolford et al., 1987). Unfortunately, blood counts cannot account for the differentially marginated pools of eosinophils likely in the vascular compartment under eosinophilic conditions (Spry, 1988). When considering blood counts as a measure of overall eosinophil activity one must keep in mind that the blood to tissue eosinophil ratio is 1/300. The major tissue compartments of eosinophil residence are bone marrow, skin and 18 the gastrointestinal tract (Rytomaa, 1960). The eosinophilic response in most tissues relies on cell counts from tissue sections which may underestimate eosinophil involvement through sampling error and/or the poor staining characteristics of degranulated eosinophils. Therefore, assessment of eosinophil accumulation in most tissues (blood included) remains comparative and semi- quantitative and requires careful interpretation. In contrast to this, there are models of peritoneal eosinophilia where lavage provides a relatively efficient cell harvest and a simplified quantitative assessment of resident cell populations. These models of peritoneal eosinophil accumulation will be covered later in some detail. Interest lies in the nature of the conditions which induce eosinophilia, indicating immune system discretion of these particular disease states fiom other types where eosinophils are not prevalent. Eosinophil production is often strongly up—regulated in these disease states compared to other populations of immune effector cells. These features point to a mechanism of control on eosinophil production and migration that has a unique specificity. In the western world, the primary focus of pharmacological research involving pathologic conditions associated with eosinophilia has been on the pulmonary eosinophilia associated with asthma in a relatively small atopic segment of the human population. It remains evident, however, that the morbidity and mortality of asthma, though not trivial, is vastly overshadowed by the pandemic observed in helminth parasite infections in humans throughout the world. This fact, coupled with the diversity 19 and availability of experimental animal models of helminth-associated eosinophilia (see Animal Models of Peritoneal Eosinophilia), has produced the majority of the evidence in the literature defining the mechanisms of eosinophil production and accumulation in tissues. Basten and Beeson (1970) produced the first definitive evidence for the role of thymus-derived lymphocytes in the mechanism of eosinophilia. This comprehensive work showed that immunologically competent T-lymphocytes are active participants in the production of eosinophils in response to Trichinella spiralis in the rat (Basten & Beeson, 1970; Basten et al., 1970; Boyer et al., 1971). They demonstrated a highly consistent increased intensity of the vascular eosinophilic response when live muscle-stage larvae were given intravenously as opposed to an oral challenge; this effect also could not be achieved through subcutaneous, intramuscular or intraperitoneal challenge. Additionally, only intact live muscle-stage larvae produced the eosinophilia; larval homogenates were ineffective. Thymus-dependence was clearly shown, as neonatal thymectomy, anti-lymphocytic serum, lymphotoxic irradiation and chronic thoracic duct drainage performed singularly or in combination abolished the eosinophilic response. Adoptive transfer experiments demonstrated lymphocyte memory with reconstitution of irradiated rats with lymphocytes, but not serum, from primary challenged rats, giving a characteristic secondary response in the recipient animals upon challenge with T richinella spiralis for the first time. Similar work with 20 Trichinella spiralis in mice (Walls et al., 1971), Ascaris suum (Nielson et al., 1974), Ascaris Iumbricoides (Walls, 1976), Schistosoma mansoni (Colley, 1972), Taenia taeniaformis (Davis & Hammerberg, 1990) and an in vitro derived T-cell line stimulated by Mesocestoides corti (Lammas et al., 1990) have all supported a thymus—dependent mechanism of eosinophilia. The combinations of antigen condition and route of exposure in the eosinophilic response has led to some generally accepted understanding, as well as controversy. Work by Walls and Beeson (1972b) demonstrated the need for vascular transit and/or local tissue reactions to intact T. spiralis larvae for the response; a life cycle/migration pattern common to almost all helminth parasites. Yet the larval homogenates produced no eosinophilic response by either route, proving the parasite antigen alone is not enough. In contrast, certain non-parasite antigens have been shown to produce an eosinophilia, especially when the antigenic particles were of sufficient size to become lodged in the pulmonary vasculature. The intravenous injection of Sephadex beads (dextran polymers) in rats produced a distinct primary and secondary eosinophilic response whereas inert polystyrene beads of the same size did not (Walls & Beeson, 1972a). Schriber and Zucker—Franklin (1974) used latex beads adsorbed with gamma-globulin intravenously injected which also produced blood eosinophilia. A contradictory finding using the same gamma-globulin adsorbed latex beads, produced pulmonary and blood eosinophilia in athymic (nude) rats (Pritchard & Eady, 1981) when 21 Nippostrongvlus brasiliensis (Nb) infection would not (Ogilvie et al., 1980). Nevertheless, in the vast majority of investigations, a central theme of large, insoluble antigenic particles traversing tissue and vascular compartments has emerged from the literature on mechanisms of eosinophilia. Adoptive transfer experiments conducted with cell-exclusion chambers provided definitive evidence that the lymphocyte-derived mediators of eosinophilia were diffusable and did not require cell-cell contact (Basten & Beeson, 1970; Miller & McGarry, 1976). These data led to the in vitro search for eosinophilopoietic cytokines. The earliest eosinophil colony stimulating factor identified was GM-CSF. However, as the name implies it is not specific to eosinophil production alone (Metcalf et al., 1974). Sanderson and colleagues (1985) discovered an eosinophilia-specfic soluble factor in mouse spleen cells isolated fi’om animals infected with Mesocestoides corti. This factor was later identified as IL-5 (Kinashi et al., 1986). IL-5 has been shown to induce eosinophilia in vivo (Kings et al., 1990) and helminth-induced eosinophilia is attenuated by administration of anti-IL-S antibodies (Coffman et al., 1990; Sher et al., 1990). Additionally, transgenic animals with constitutive IL-5 production presented with eosinophilia (Yamaguchi et al., 1990) and patients with a clinical presentation of hypereosinophilic syndrome demonstrated measurable levels of IL- 5 in serum (Owen et al., 1989). The evidence clearly shows IL-5 as the only currently defined eosinophilia-specific cytokine. Other evidence from in vitro 22 investigations has shown that Multi-CSF, now called Interleukin-3 (IL-3) works in concert with IL-5 to produce eosinophilopoiesis in bone marrow cultures (Clutterbuck & Sanderson, 1990). Endothelial cells were also found to produce GM-CSF and therefore extend eosinophil viability in vitro (Rothenberg et al., 1987). Macrophages also are known to produce GM-CSF in host-defense (Adams & Hamilton, 1992). The elicitation of this cytokine by these cell populations and the antigen exposure experiments described earlier provides a strong mechanistic link to eosinophil production in viva. The definition of a distinctive cytokine profile in eosinophilia has shed some light upon the unique nature of the eosinophil response in a multitude of thymus- dependent immune reactions. The discovery that CD4+ T-helper (Th) lymphocyte clones have cytokine profiles with two main subclassifications and that their regulatory mechanisms are differentially coordinated, sharpened the focus on immunologic specificity in helminth infections. Distinct T-cell clones observed in a murine model of Schistasama mansam’ were given the designations Th1, which selectively produced interferon-y (IFN) and IL-2, and Th2, which selectively produced IL-4 and IL-5 (Mosmann et al., 1986). Some cytokines, such as GM- CSF, IL-3 and TNF, are produced by both Th1 and Th2 subclasses. Other lymphokine profiles have also been observed indicative of additional Th subsets (Firestein et al., 1989). These specific cytokine production profiles by clonal Th subsets help explain some of the diverse immunologic phenomena associated with 23 eosinophilia. Generally, Th1 cells regulate local inflammatory reactions in delayed-type hypersensitivity reactions and macrophage activation, whereas Th2 cells produce IL-4 and IL-5 which regulate antibody production of the IgE and IgA classes and eosinophilia, respectively (Street & Mosmann, 1991 ). Through investigations in a mouse model of Schistasama mansani, the specificity to antigen by the Th2 response was clearly defined (Grzych et al., 1991). The initial reaction to acute infection by cercariae was the Thl response; at 8 weeks post-infection, egg-laying commenced and the Thl response was down- regulated and then the Th2 response predominated (Pearce et al., 1991). Unisex infections (i.e., no eggs laid) did not produce a Th2 response and mice infected with irradiated cercariae (sterile) produced only Th1 responses and remained strongly immune to reinfection, implicating the Thl response as a primary immune mechanism in murine schistosomiasis. Conversely, in mice infected with Nb where an intestinal eosinophilia and expulsion phenomenon are hallmarks, the Th2 subclass predominates (Street et al., 1990). Rapid nematode expulsion has been shown to actually be dependent on the Th2 response in Trichuris muris infection where Th2—deficient mouse strains present with no intestinal eosinophilia and slow expulsion (Else & Grencis, 1991). The preferential production of IgE antibody often accompanies tissue eosinophilia in humans and rats infected with helminth parasites, raising serum levels as much as 25-fold in the IgE class (Ogilvie, 1964; Johansson et al., 1968). 24 This potentiation of IgE antibody production has been shown in Nb infection in the rat to be both antigen-specific and polyclonal (Jarrett & Bazin, 1974). As stated previously, the mechanism is T—cell mediated (Jarrett & Ferguson, 1974) and specifically enhanced by the Th2 subclass. Two mechanisms have been demonstrated for the selective induction of IgE class antibodies. The first involves IgE-binding factors released by T-cells stimulated in Nb infection which increase the production by B cells already committed to the IgE antibody class (Ishizaka, 1988). The second involves antibody class-switching promoted by the synergistic effects of lL-4 and IL-5 on plasma cell precursors (Pene et al., 1988). Capron and colleagues (1994b) have demonstrated the presence of high affinity E-class antibody receptors (FcaRI) on human eosinophils, providing impetus for antigen- specific IgE in the arming of resident tissue eosinophils, as well as mast cells in Type I hypersensitivity and cytotoxic immune reactions. Additional studies have showed that FcaRI activation led to the production and secretion of GM-CSF, IL—3, IL-4, and IL-5 by mast cells (Burd et al., 1989; Plaut et al., 1989; Wodnar- Filipowicz et al., 1989). Another study stimulated blood and tissue eosinophils with IgE-antigen complexes and detected ultrastructurally both intracellular and secreted IL-5 (Dubucquoi et al., 1994). These studies clearly implicate IgE in site- specific eosinophilia. 25 The development of localized tissue eosinophilia denotes a mechanism for cell-specific recruitment by the vasculature in the affected tissue. Cell adhesion in inflammation has been shown to be a multi-step process where leukocytes are first weakly bound to vascular endothelium by selectin receptors to its carbohydrate ligand. This produces a rolling of blood-bome leukocytes at the affected site. Rolling is followed by the leukocyte integrin tightly binding to the immunoglobulin-like ligand on the vascular endothelium and extravasation proceeds. Eosinophils and lymphocytes but not neutrophils are known to constitutively express the integrin VLA-4 which binds to VCAM-l on endothelial cells (Walsh et al., 1993). IL-4 has been shown to specifically up—regulate VCAM- 1 on endothelial cells (Masinovsky et al., 1990). Eosinophil adherence to endothelium is inhibited by antisera to VLA-4 in human systems (Walsh et al., 1991) and lymphocyte migration is blocked by anti-VLA-4 in viva in rats (Issekutz, 1991). The combined emigration of these two types of cells was demonstrated in late-phase cutaneous reactions from atopic human patients where CD4+ lymphocytes and eosinophils were identified (Frew & Kay, 1988). These studies implicate the VLA-4NCAM—l adhesion mechanism as specific for eosinophil recruitment in the production of eosinophilia. A second role for VLA-4 in localized eosinophilia involves the cell-matrix interaction of this receptor and fibronectin. Recent studies indicate that VLA-4 mediated binding of fibronectin 26 prolongs eosinophil survival and promotes the tissue phenotype (Anwar et al., 1993; Anwar et al., 1994). A complete understanding of the mechanisms underlying the eosinophilic response in tissues is still needed. It is evident that multiple pathways and sources exist for the release of cytokines necessary for the production and/or accumulation of eosinophils. While T-cell control remains as a primary mechanism for the development of eosinophilia, alternative mechanisms have been proposed in the literature. Reference was already given to data supporting non-thymus dependent eosinophilia in nude rats challenged with antibody-coated latex beads. A role for suppressor T-cell function in eosinophilia was implicated in cyclophosphamide- induced eosinophilia in rats (Thomson et al., 1987). Non-antigen driven T-cell involvement in an eosinophilic response to aluminum containing adjuvants (Walls, 1977) and toxin induced eosinophilia from Spanish cooking oil are other examples with unknown mechanisms in the literature. In fact, an array of hypereosinophilic syndromes has been described, most with unknown mechanisms (Spry, 1988). Animal Models of Peritoneal Eosinophilia Many experimental models of eosinophilia have been discussed in the previous sections. An obvious preponderance of work on peripheral blood eosinophilia exists in the literature. In contrast, the peritoneal cavity of rodents has been shown to be an accumulation site for eosinophils under certain experimental conditions. While the peritoneum offers ease of access to eosinophils under the specific 27 immunologic conditions of the enteric tissue microenvironment, relatively few studies have exploited this condition. Eosinophil interactions with peritoneal macrophages, lymphocytes, connective tissue mast cells, extracellular matrix, etc. remain poorly characterized. The focus of this section will be the models described in the literature and their mechanistic correlates. Over 50 years ago, eosinophilia was elicited in the guinea pig by intraperitoneal injections of insoluble antigen extracts of Ascaris suum (Campbell, 1942). Various other agents were also shown to elicit an eosinophilic response in the peritoneal cavity in subsequent studies. Single injections of bovine albumin, horse serum, keratin or Ascaris extract all produced a two- to three-fold increase in peritoneal eosinophils in mice after 48 hours (Speirs & Dreisbach, 1956). Repeated injections over several weeks of antigenic agents such as horse serum, hemocyanin and human serum albumin, as well as repeated saline lavage was shown to produce eosinophil-rich peritoneal exudates in guinea pigs (Litt, 1960; Gleich & Loegering, 1972). These agents obviously initiated an immune response from resident cell populations of mast cells, macrophages, neutrophils, lymphocytes and eosinophils through various pathways. G.T. Archer (1973) published an experimental model of relatively rapid peritoneal eosinophilia in Long Evans rats implicating the mast cell in a regulatory role. The observation that hyperplasia and degranulation of this cell accompanied Ascaris antigen-induced eosinophilia (Archer & Binet, 1971) led to experiments 28 on mast cell activation in the peritoneal cavity. The known mast cell granule constituents of heparin and amines were hypothesized to complex in viva. Therefore, protamine-heparin complexes were administered intraperitoneally as an immunologic catalyst to mimic mast cell activity. The result after 24 hours was a two-fold increase in peritoneal eosinophils (15.8 x 106 cells). This was a selective response without neutrophilia or peripheral blood eosinophilia. There was morphological evidence of phagocytosis of the protamine-heparin complexes by macrophages and eosinophils. The eosinophils were heavily degranulated and there was cytochemical and ultrastructural evidence of macrophage uptake of eosinophil granule material. A similar study was done with Ascaris suum or Echinacaccus granulasus phospholipid preparations which produced peritoneal eosinophilia and mast cell hyperplasia. Eosinophils increased IO-fold (23.0 x 106 cells/animal) over levels in control rats 24 hours after injection of the parasite phospholipid alone (Archer et al., 1977). In a continuation of the mast cell hypothesis, the role of histamine in the peritoneal eosinophilic response was examined in the guinea pig model (Pincus, 1978). This work utilized an 8-week treatment with the antibiotic polymyxin B or compound 48-80, known mucosa] mast cell degranulating agents, which elicited a strong eosinophilic response when injected into the peritoneum. This response could be blocked by the antihistamine, diphenhydramine, but could not be elicited by exogenous histamine. It is of interest, that the largest average number (52.0 x 106) of eosinophils was obtained 29 in the polymyxin B treated animals but a standard deviation of i 32.0 x 106 was due to highly inconsistent response between animals. Another study demonstrated a primary antigen sensitization protocol for the production of anaphylactic antibodies (IgG; and IgE), which upon secondary peritoneal challenge, released histamine and peptidoleukotrienes from the resident mast cells in rats. Peritoneal eosinophilia peaked at 4.0 x 106 cells/ animal 24 hours later (Spicer et al., 1985). The dependence on T-cells for the production of peritoneal eosinophilia in mice and rats has been shown in different models. Sensitization of mice to alum- precipitated tetanus toxoid with pertussis vaccine and then challenged by peritoneal injection of tetanus toxoid and alum produced a localized eosinophilia (McGarry et al., 1971). Adoptive cell transfer experiments demonstrated T-cell dependency and recipients showed a pronounced eosinophilia independent of antigen-specific antibody production. A similar T-cell dependent murine model elicited a reduced and transient eosinophilic response to alum adjuvants alone (Walls, 1977). Antigen-driven peritoneal eosinophilia in mice was elicited by various pollen extract sensitizations (Speirs & Dreisbach, 1956; Spicer et al., 1986). Antigen challenge in these models would increase peritoneal eosinophils three- to 10-fold and persist for 5 to 10 days. Athymic mice and adoptive transfer experiments were used to establish T-cell control on the eosinophilic response to the peritoneal-dwelling cestode Mesacestaides carti (Johnson et al., 1979). The infections are similar in rats and mice, and produce approximately 25.0 x 106 3O eosinophils/animal after 4 to 5 weeks of infection (Barton et al., 1984; Cook et al., 1987). It is important to note that the functional capacities of these cells from infected rats have been shown to be altered, with increased cytotoxic capacity (Chemin & McLaren, 1983) and reduced FcyRc (Cook et al., 1987). Some investigations have reported how multiple mechanisms can Operate to affect the eosinophilic response in the peritoneal cavity. A murine model, utilizing various preparations ofAscaris suum without infection, demonstrated comparative results in alternate pathways (Kano et al., 1989). This study administered saline lavage, viable and killed Ascaris eggs and Ascaris antigen to normal and athymic mice. All four stimuli produced peritoneal eosinophilia in normal mice, with viable Ascaris eggs eliciting the greatest response (9.3 x 106 cells/mouse) by the second day. Repeated saline lavage alone produced the same ( 4.4 x 105 cells/mouse) peritoneal eosinophilia by the 5th week in normal and athymic mice. Viable eggs, but not killed eggs or Ascaris antigen, produced localized peritoneal eosinophilia (1.6 x 106 cells/mouse) in the athymic animals. However, this response took 5 weeks to reach peak levels, still clearly demonstrating both T-cell dependent and T-cell independent mechanisms occur with live Ascaris suum eggs. A report on the repeated saline lavage protocol has implicated a role for potassium ions in the eosinophilic response. Adding potassium chloride to the saline lavage prevented the peritoneal eosinophilia (Oliveira et al., 1994). A human correlate to this mechanism was reported in dialysis patients who 31 developed peritoneal eosinophilia from dialysate with heparin (Santoro et al., 1985). Pediatric dialysis patients have also been reported to produce 1.4 x 109 eosinophils/L of dialysate (Roberts et al., 1990). Both of these mechanisms probably involve cell activation through receptor mechanisms and/or ion channels but experimental data currently does not exist. A model of murine Taxacara canis produces a partial T—cell dependent and mast cell dependent eosinophilia in the peripheral blood and peritoneal cavity. These conclusions were drawn from experiments with mast cell deficient mice and cyclophosphamide treatment (Sugane & Oshima, 1985', Nawa et al., 1987). A unique model of enhanced peritoneal eosinophilia was devised by Sugane and Oshima (1980) utilizing Taxacara cam's in mice. Peripheral blood eosinophilia was monitored in the course of the infection and peritoneal challenge with warm extracts was timed with maximal blood levels producing a pronounced peritoneal eosinophilia (1.0 x 108 eosinophils/mouse) in 48 hours. No data was given on the morphological appearance or firnctional status of these cells. In every case, the aforementioned models of peritoneal eosinophil accumulation rely on inflammatory stimuli which alter the functional status of the resident peritoneal cell populations from the resting state. Capron and colleagues (1984a) have shown that peritoneal-derived cells from Schistasama mansani infected donors can kill cercariae when both are placed in the skin of naive recipients whereas resident cells cannot. Regardless, most of these studies have 32 not provided information on the condition of the cells harvested and those that have showed clear evidence of functional alterations. The ability to harvest relatively large numbers of resting state tissue eosinophils by lavage has been reported in two rat models: the Am-l(2)/T or rat and the August rat, both of which have been reported to have spontaneously high levels of peritoneal eosinophils (Mackenzie et al., 1981; Pimenta & De Souza, 1982). Few studies using Am-l(2)/T or rats have been published. Those available are ultrastructural investigations on the eosinophils, as well as interactions in Leishmaniasis (Pimenta et al., 1980; Pimenta et al., 1987). The August rat has been used extensively at the National Institute for Medical Research in London with emphasis on pathogenesis in Nb infection (see Nippastrangylus brasiliensis). Nippostrangylus brasiliensis The study of the nematode Nippastrangylus brasiliensis in the laboratory rat has provided a large body of information on mammalian immunity to helminth parasites (Ogilvie & Jones, 1971). This model is relatively unique in that rats generate a strong immune response to this nematode parasite which leads to the expulsion of the adult stage and acquired immunity to reinfection (Africa, 1931; Ogilvie, 1965). This rapid expulsion phenomenon has rarely been observed in natural mammalian nematode infections where adult stage senesence or pharrnacologic intervention is the usual course. It is important to note that the expulsion phenomenon was only observed in rats given large (> 2,000 L3/animal) 33 single-dose primary challenge. If a “trickle” infection of a few larvae/day were given to mimic a more natural exposure, a threshold worm population was established for prolonged periods before being eliminated. Regardless, this animal model provides a useful tool for investigating primary aspects of enteric immunology and the possible pathogenesis of certain intestinal disease states in humans. Nb cultures are relatively easily maintained in the laboratory with a short life cycle and no intermediate hosts. Humans are not susceptible to infection and the definitive hosts in nature are rats and mice. The infection has three phases in the laboratory rat, involving the skin, the lungs and the intestines (Jarrett et al., 1968). The infective L3 larvae are injected subcutaneously (2,000-8,000/animal) where they migrate to the blood and lymph channels. The slight majority of the L3 larvae migrate via the vasculature to the lungs within 24-72 hours where they erupt into the alveolar airways and molt to the L4 stage. The remaining L3 larvae are thought to be lost to lymph drainage and/or killed by immune effector cells. The L4 larvae move through the pulmonary airways (i.e., they are coughed up and swallowed) to the esophagus reaching the proximal jejunem 3-16 days later in naive August rats and 4-9 days later in secondary infections (Mackenzie & Spry, 1983). In the intestine they molt to the L5 stage (adult) and begin feeding and egg production (1000-1500/female/day) by the 5th day of residence. Eggs are passed in the feces and hatch in the external environment to produce the infective L3 larvae thus 34 repeating the cycle. The adult nematodes do not penetrate the mucosal wall but mechanically dislodge the host villous epithelium after secreting digestive enzymes onto the intestinal surface. The host cells are then sucked into the worm’s digestive tract. This extracorporeal digestion by adult Nb leads to extensive local tissue destruction to the host. Worm adherence is thought to be aided by reduced host peristalsis caused by large amounts of acetylcholinesterase secreted by the adult nematodes (Sanderson, 1969). Observed host metabolic responses to large infestations include: decreased plasma protein concentrations, catabolism of skeletal muscle and stored fat, hypoglycemia and even death in some cases (Ovington, 1987). The immunologic events which lead to the expulsion of primary Nb infections in the rat have been described in two steps. The first step involves severe antibody-induced damage to the cells lining the worm’s alimentary tract which deleteriously affects mobility and fecundity of the adult worms. This step was shown to be irreversible, independent of complement and occurred by day 10 (Jones et al., 1970). The following expulsion step requires immunologically competent hosts and was not observed in T-cell-deprived, irradiated, young or lactating rats (Ogilvie & Love, 1974). Adult worm antigens, with particular emphasis on females, were implicated in the expulsion phase as larval stages would not elicit the response (Ogilvie, 1965). Further work identified these effector cells of expulsion as thymus-dependent, antigen-specific, immunoglobulin- 35 negative lymphocytes recovered from thoracic duct lymph and/or the peritoneal cavity by day 8 post-infection (Ogilvie et al., 1977). In conjunction with these events, other aspects of the host’s immune defenses have been implicated in Nb rejection in primary and subsequent infections. It has been well documented that monocytes, mast cells and eosinophils infiltrate all the tissue sites where Nb migration occurs in initial infections and more intensely in immune animals (Taliaferro & Sarles, 1939). Additionally, goblet cell hyperplasia was observed in the intestinal phase of Nb infection and could be adoptively transferred by immune thoracic duct lymphocytes (Miller & Nawa, 1979). Protective antibodies of the IgM and IgGLz class have been detected by day 5 in primary infections and are involved in eosinophil and neutrophil adherence to all Nb stages in vitro (Jones et al., 1970', Mackenzie et al., 1980). The potentiation of both antigen-specific and polyclonal IgE production by Nb, as well as its role in arming effector cells has been previously discussed (see Immunobiology of Eosinophilia ). The peak IgE titers were detected in the third week after primary Nb infection (Ogilvie, 1967). This IgE peak coincides with maximal eosinophil and mast cell infiltration of the proximal jejunum, all occurring after expulsion of the majority of the Nb adults (Taliaferro & Sarles, 1939; Wells, 1962; Hogaboam et al., 1991). There is evidence that the infiltration mechanism of mast cells may have both antigen-specific and non-specific components (MacDonald et al., 1980). Mackenzie and Spry (1983) demonstrated homing from the vasculature of 36 peritoneal-derived radiolabelled eosinophils from both naive and immune rats to skin, lungs and proximal jejunum, all sites of tissue migration by the various stages of Nb. Several studies have described plausible roles of the various effector cell populations but a complete understanding of the expulsion phenomenon remains elusive. Urquhart and colleagues (1965) proposed a mast cell mediated anaphylaxis in the intestinal mucosa as the primary mechanism for Nb expulsion. This was countered by Ogilvie and Jones (1971) citing passive protection in neonates who did not yet possess passive cutaneous anaphylactic capability and also cited the survival of “adapted” threshold populations in “trickle” infections and transfer experiments. Further work in mast cell deficient W/Wv mice demonstrated Nb expulsion, although it was delayed (Uber et al., 1980; Crowle & Reed, 1981). 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Austen (Eds). The eosinophil in health and disease. (pp. 43-59). New York: Grune & Stratton. Chapter 2 PERITONEAL EOSINOPHILIA IN THE INBRED AUGUST RAT Introduction The use of inbred strains of rodents which possess distinct phenotypes has proven invaluable in the quest for answers to some of the most basic of research questions regarding biological processes. Inbred animals are homozygous at virtually all genetic loci making them ideal for immunological studies in which preventing rejection or uniformity of response is critical. Additionally, inbred animals have long-tenn genetic stability making strain characteristics useful for long periods of time without genetic driff (Festing, 1979). Thus, characterization of observed traits in inbred animals has provided powerful models for scientific inquiry. The August rat sublines were first developed from unknown outbred stock by Curtis and Bullock at Columbia University in 1921. They produced five inbred lines, one of which was obtained by the Chester Beatty Institute, Pollards Woods, England. After 26 brother x sister matings the current August (AUG) inbred strain was distributed for the first time in 1951 (Wright et al., 1993). Several breeding colonies were established in Europe including one at the National Institute for Medical Research, Mill Hill, London. 56 57 Some fifteen years ago, C .D. Mackenzie and colleagues reported the isolation of eosinophil leukocytes from the peritoneal cavity of AUG rats and their use as tissue-derived eosinophils in an in vitro parasite adherence assay. Further studies both in vitro and in viva went on to describe various aspects of eosinophil involvement in helminth parasite immunology utilizing the AUG rat (McLaren et al., 1977; Ogilvie et al., 1977; Mackenzie et al., 1978; Mackenzie et al., 1980; Mackenzie et al., 1981; Mackenzie & Spry, 1983). These studies established a plausible phenotypic expression of peritoneal eosinophil accumulation in the AUG rat. The present investigation will describe and quantify, utilizing morphologic criteria, the accumulation of eosinophils in the peritoneal cavity, blood and jejunal mucosa of AUG rats of both sexes at selected ages. Methods and Materials Experimental Design Age-matched AUG rats of mixed parentage and sex were grouped (n = 6-1 1) into 2, 4, 6, 8 weeks (wk), and >1 year (yr) of age. Cell isolations were obtained from the peritoneal cavity and peripheral blood, and tissue samples were taken from the proximal jejunum. Eosinophil leukocytes were identified by morpho- histological criteria and quantified in these tissue compartments. Ultrastructural profiles of AUG rat eosinophils isolated from the peritoneal and vascular compartments also were described. A single group of 6-wk-old Sprague-Dawley (SD) rats were used for the comparisons of villus-crypt (VC) units. 58 Animals Sibling breeding pairs of August (AUG) rats were obtained from Harlan Olac Limited, Bicester, England and the National Institute for Medical Research, London. Colonies of inbred progeny were established at the Biological Sciences Department, Western Michigan University, Kalamazoo, MI and the Division of Animal Health, The Upjohn Company, Kalamazoo, MI and were the source of research animals for this study. The SD rats were obtained from Harlan Sprague Dawley, Inc. (Indianapolis, IN). All animals were housed in accordance with the Institutional Animal Care and Use Committee (IACUC) of the respective institutions. The colonies were maintained under a 12hr/12hr-light/dark cycle at 21-28°C with rodent chow #5001 (Purina Mills, St. Louis, MO) and reverse osmosis purified water provided ad Iibitum. Tissue Collections All chemical reagents were obtained fiom the SIGMA Chemical Company (St. Louis, MO) unless otherwise noted. Animals were killed prior to tissue collection by lethal carbon dioxide inhalation. Peripheral blood was taken immediately after death from tail snips and smeared between microscope slides and allowed to air dry. Additionally, an aliquot of peripheral blood was drawn into a white cell pipette (Becton-Dickinson, Cockeysville, MD) and diluted with Turk’s solution for total white blood cell (WBC) determination. Peritoneal lavage was performed utilizing calcium- and magnesium-free Hanks’ Balanced Salt Solution (HBSS) 59 with 20 mM HEPES buffer at pH 7.3 kept on wet ice. This collection was accomplished by making a small (1-3 cm) incision on the lower abdomen adjacent to the ventral midline of the animal to irrigate the peritoneal cavity with 30-50 ml of the lavage solution. The resident peritoneal cells and the lavage solution were removed within 5 minutes by syringe and pelleted by centrifugation at 200 x g for 10 minutes at 4°C in a Beckman (Fullerton, CA) centrifiige. The lavage supernatant was discarded and the cell pellet brought to 5 ml with HBSS with 20 mM HEPES and held on wet ice. Following the lavage procedure, the thoracic cavity was opened and systemic blood was drawn by cardiac puncture into 7 ml blood collection tubes with 0.07 ml of 15% EDTA (Tenno Medical Co., Elkton, MD) and held at room temperature (RT). Next, 8 cm of the proximal jejunum beginning at a point just distal to the duodenal loop adjacent to the pancreatic rests, was ligated into a 2—4 cm compartment. This was injected with cold (wet ice) 10% neutral buffered formalin (NBF). These jejunal segments were excised and placed in NBF for 15 minutes at RT and then the NBF was replaced with f‘resh fixative solution and the samples stored at RT until paraffin embedment. Sample Preparation and Counting Methods All cell counting procedures were performed on a Nikon (Tokyo, Japan) Microphot-FXA® research grade microscope. Air dried peripheral blood smears were fixed for 10 minutes in cold (-20°C) methanol and stained with Diff-Quick® 6O stain kit (Baxter Healthcare, McGraw Park, IL). Differential determinations were made upon the first 300 WBCs/animal counted (from 2 smears) and expressed as mean percents (%) of the total. The Turk’s diluted peripheral blood was counted (2x) according to the method of Brown (1984) in a Neubauer hemocytometer (American Optical, Buffalo, NY) and expressed as mean WBC/u]. The peritoneal lavage was aliquoted for counting, cytocentrifuge preparation and electron microscopy. Total cell counts were done on a Neubauer hemocytometer as stated above. Cytocentrifuge preparations were carried out with a Cytospin 3® (Shandon Scientific, Cheshire, England) and stained with the Diff- Quick® system and differential cell determinations performed as described for WBCS. The remaining peritoneal cells were again pelleted, the supernatant discarded and the cell pellet immersed in cold (wet ice) 3% glutaraldehyde in 0.1M phosphate buffer at pH 7.3 for 1 hour. The pellet was then washed (2x) in buffer and secondarily fixed in buffered 1% osmium tetroxide (Polysciences, Inc., Warrington, PA) for one hour at RT. The sample was dehydrated in a graded ethanol series and embedded in Polybed 812® (Polysciences, Inc., Warrington, PA) epoxy resin and thin sectioned for transmission electron microscopy (TEM). Sectioning was done on an LKB (Bromma, Sweden) Ultrotome Nova® and sections stained with 5% methanolic uranyl acetate and Reynold’s lead citrate (Reynolds, 1963). Microscopy was performed on a Siemens (Berlin, Germany) 61 101 Elmiskop® TEM. The EDTA treated systemic blood samples were prepared according to the method of Dykstra (1993) for the preparation of buffy coats for TEM. Briefly, the blood samples were centrifuged in Wintrobe tubes and the buffy coats fixed in situ. The glass tube was then broken and the fixed buffy coat pellet was pushed out and embedded in agar to retain the cell layering of the pellet during the embedment process for TEM. The remaining preparatory steps for TEM were as described previously. The segments of proximal jejunum were trimmed into 2-4 mm rings and embedded in paraffin. Histological sections were cut (6-10 um) and stained by Luna’s (1968) for eosinophils and Lendrum’s (1944) chromotrope 2R methods. Counting of the eosinophils in the VC units of the intestinal mucosa was done according to Miller (1971). Briefly, all cells located between two gland crypts of the villus and in the lamina propria and epithelium of the villus above were counted as a single VC unit. Cells were counted only in those villi that were longitudinally sectioned and a total of 10 VC units/animal counted. Statistics All numerical data was expressed as mean 3: SEM. Equal variance was tested by Hartley’s homogeneity of variance method. Statistical significance was determined by one-way or 2 x 2 factorial analysis of variance (ANOVA) with a 62 95% confidence limit (p = 0.05). Group-wise comparisons were made utilizing Student-Newman-Kuels a posteriori test (Ott, 1988). Results Peritoneal Cavity The resident peritoneal cell populations in adult (>1 yr) AUG rats (Figure 2.1.) consisted of: 51.7 i 1.2% mononuclear cells (mononuclear phagocytes and lymphocytes), 32.8 i 1.4% eosinophils, 9.3 i 1.0% mast cells, and 6.2 i 0.5% small lymphocytes. It should be noted here that no morphological or ultrastructural evidence for the presence of neutrophils in the AUG rat peritoneal lavage was ever observed. Total cell harvests (Figure 2.2) are 20.9 i 1.6 x 106 (range = 16.2-27.5) cells/animal. Both the total cell harvest and the percent eosinophils (Figure 2.3) increased with age in the AUG rat. The absolute number of peritoneal eosinophils/adult animal averaged 6.9 i 0.5 x 106 (range = 4.7-9.6) (Figure 2.4). The peritoneal eosinophil accumulation in 8-wk and >1 yr age groups were found to be statistically significantly greater compared to the earlier time points, while no difference was seen between 8-wk and >1 yr. Additionally, no statistically significant difference was found between peritoneal eosinophil accumulation in AUG rat males or females. 63 Figure 2.1. Cytospin preparations (cytoprep) and ultrastructure of the resident cells obtained by peritoneal lavage of the AUG rat. They are: mononuclear cells (M), eosinophils (E), small lymphocytes (SL) and mast cells (MC). Bar = 5.0 pm. ('YTOI’REI’ \].\S I' ('lil.l. Figure 2.1 65 30 ~r-W-W w—w -—»_ --—- 25 *‘k ** 20... 15 ‘fi 10 -~ CELLS In MILLIONS 2WK 4WK GWK SWK > lYR AGE Figure 2.2. Total cell harvest fiom the peritoneal cavity of AUG rats. The (*) Indicates significance over 2wk group and (**) indicates significance over all three younger groups. 66 ‘1 —+—Eos 46.}. » {U —MONOS 7o _ , ..,- MAST - -~ SL so — A so - % of TOTAL fit 9 2WK 4WK GWK SWK >1YR Figure 2.3. Differential count on the cells harvested from the peritoneal cavity of AUG rats. 67 10 Fm—mm—wm-mw @- ~—-v~-----——fi \O illinflimii] Till \I l l CELLS In MILLIONS u- ih f ‘i l j : .imillllililLiLiiimlw 1L] 1 III] 9 1 L l 2WK 4WK GWK SWK > lYR AGE Figure 2.4. Eosinophils in the peritoneal cavity of AUG rats. The (*) indicates Significant difference from younger age groups. 68 Peripheral Blood Differential cell determinations from AUG rat peripheral blood were: 63.6 i 1.2% lymphocytes, 22.3 i 0.9% monocytes, 10.] :t 0.9% neutrophils, 2.2 i 0.5 eosinophils, and 1.8 i 0.6% basophils with no significant differences between groups (Figure 2.5). Total white cell counts were 5052 i 337 (range = 3295-6500) cells/u] with peripheral blood eosinophils being 113 i 22 (range = 0-549) eosinophils/u] (Figure 2.6). There was no significant difference in peripheral blood eosinophil numbers between any of the age groups. Villus-Crypt Units Light micrographs of VC units from the 2-wk and 8-wk age groups are represented in Figure 2.7. Eosinophil numbers reached 25.9 :1: 1.1 (range = 17.2- 29.8)/VC unit by 8 weeks of age in the AUG rat (Figure 2.8). A significant difference was observed between the 2-wk and 8-wk old groups. but the three oldest groups (4-wk, 6-wk and 8-wk) were not significantly different. Additionally, the 6-wk old SD rats had VC unit eosinophil counts (17.6 i 1.6, range = 14.2-21.7) that fell within the same population range as the VC counts for AUG rats at 4, 6 and 8 weeks of age. 69 70 i] so ~i§i~ MONOS +508 mam BASOS 50 -- 111111] :1 40 4 .__,,..-_ S. LYMPH 5.. - --L~‘} —~ NEUTS O . h "1 ~ _ ~— "7 ° so ——a ° 1 1 , / ~tug1,m-m~dfi * 19‘- L?‘ j \. 1 J- J " 20 e 10 - , 31 ._ .— ,__ — _, r." __ _..- _r ..1 #_ flip-«xi " - _... J— A ' ‘ ‘ Al ' Y - 1 :5- —- — r: — — — :7 o l l l 1 Figure 2.5. Differential white blood cell counts in the AUG rat. 70 300 ---~>=r-—-ul~’—--i-- -- 7,, ~- - ----— LL._L_.J N 6 e l 100 2WK 4WK GWK SWK >1YR AGE Figure 2.6. Eosinophils in the peripheral blood of AUG rats. CELLS/MICROLITER e L l l l I 1 l ‘ 71 ] a \\'K «or: (.‘ROI'I’ Figure 2.7. Eosinophils in the proximal jejunum of AUG rats (DIC optics). Bar = 20.0 um. 72 30 .1 * ‘ l E: 1 z i i D i E ‘ l y 20 ‘ 7 ‘ 5 i 6: D 1 i a 1 A u-r . ‘ i Z ‘ . m l i-I . E i a 10 1 O l J z . 1 51‘ I 1 a i . 2 l l 1 1 l I 1 ° ‘ 1 " i " l 2WK 4WK GWK SWK AGE Figure 2.8. Eosinophils in the proximal jejunum of AUG rats. The (*) represents significant difference from 2wk group. 73 Ultrastructure The ultrastructural profiles of blood and peritoneal-derived eosinophils in the AUG rat had distinctive qualitative differences (Figure 2.9). The most prominent distinction was in the distribution of the specific granules within the cytoplasm. The clear majority of blood-derived eosinophils had relatively densely packed specific granules that were closely associated with the nucleus. In the most compact states a halo of cytoskeletal filaments was visible at the perimeter of the granule pack. This condition left distinct regions of the cytoplasm free of specific granules which had the effect of making the small granules that are normally present a prominent feature in these regions of blood-derived eosinophils. This was in sharp contrast to the peritoneal-derived eosinophils; the majority of these cells had a more generalized specific granule dispersion with an almost granule- free zone around the nuclear envelope. Many more granules were located at the cell periphery and the specific granule distribution in peritoneal-derived eosinophils had a higher degree of separation throughout the cytoplasm. Small granules were present in the peritoneal—derived eosinophils but their apparent prominence was diminished as these cells had relatively few specific granule-free zones in their cytoplasms. An additional distinctive feature of peritoneal-derived eosinophils was the prominent microvesicular appearance of the smooth endoplasmic reticulum. Virtually all of the peritoneal—derived cells showed distension of the microtrabecular cistemae of the smooth endoplasmic reticulum. 74 Figure 2.9. The ultrastructural profiles of blood- and peritoneal-derived eosinophils in the AUG rat. A different dispersion profile of specific granules (O) is evident. Small granules (=9) are prominent in the cytoplasmic zones unoccupied by specfic granules in the blood-derived eosinophil. Note the vesicular cytoplasm (*) common to the peritoneal-derived eosinophils. 75 Figure 2.9 76 This condition was less prominent or not evident in blood-derived eosinophils. Lastly, the plasma membrane of blood-derived eosinophils had a smooth appearance with very few projections. Plasma membrane projections which produced a ruffled cellular profile were common in peritoneal-derived AUG rat eosinophils. Discussion This study describes the postnatal development and consistency of an observed strain characteristic of high-level eosinophil accumulation in the peritoneal cavity of the AUG rat. This trait of 7.0 x 106 eosinophils/AUG rat is in sharp contrast to other common laboratory rats. The SD rat and the PVG hooded rat produce 2.2 x 104 and 1.5 x 105 eosinophils/animal, respectively, by peritoneal lavage (Mackenzie et al., unpublished results). The eosinophil numbers in the intestinal mucosa of the AUG rat were comparable to those found in the SD rats and the eosinophil level in the peripheral blood of the AUG rat falls within the range of normative data for all laboratory rats (Wright et al., 1993). Elevations in eosinophil numbers in one or both of these tissue compartments are well documented in rodent helminth infestations (Butterworth & Thome, 1993; Wright et al., 1993) and other eosinophil-associated diseases (Vadas et al., 1986). The eosinophil levels observed in the blood and intestinal mucosa of the AUG rat being consistent with other rat strains and well below established criteria for eosinophilia 77 provides strong evidence for phenotypic control of the accumulation of eosinophils in the peritoneal cavity. The qualitative differences in ultrastructural profiles of the blood-derived and peritoneal-derived eosinophils from the AUG rat are consistent with morphological and functional differences between blood and tissue eosinophil phenotypes reviewed by Owen (1993). While high eosinophil levels in the peripheral blood can suggest the presence of eosinophil-associated disease processes, too many studies in the past have utilized only blood-derived eosinophils for mechanistic studies. Diapedesis through the vascular wall (Walsh et al., 1993) and adhesion to the extracellular matrix of the tissue environment (Silberstein & David, 1986) are most likely to have an important impact on the functional capacities of eosinophils. This study provides further support to the concept of utilizing tissue-derived eosinophils for studies that ask questions about the firnctional roles of eosinophils. As stated in the review of eosinophil literature, virtually all other animal models of peritoneal eosinophilia rely on immunologic or inflammatory stimuli to induce the accumulation needed for an eosinophil harvest of sufficient quantity for most scientific investigations. The few studies that have examined the cells derived from these models have described functionally and morphologically altered eosinophils (Chemin & McLaren, 1983; Cook et al., 1987). Additionally, an under-reported contaminant of these peritoneal models is neutrophils, a notoriously difficult cell to separate from eosinophils by centrifugation gradients alone 78 (Stewart, 1978). Peritoneal isolations fi'om the AUG rat have produced neutrophil- free preparations of >90% resting-state tissue eosinophils by metrizamide gradient (Mackenzie et al., 1981). The potential use of this unique strain characteristic of the AUG rat as a research tool for studies on eosinophil biology is strong. 79 References Brown, BA. (1984). Hematology: Principles and Procedures. (4th ed.) Philadelphia: Lea &Febiger. Butterworth, A. & Thome, K. (1993). Eosinophils and parasitic diseases. In H. Smith & RM. Cook (Eds). Immunopharmacology of Eosinophils (pp. 119-150). New York: Academic Press. Chemin, J. & McLaren, DJ. (1983). The pathology induced in laboratory rats by metacestodes of Taem'a crassiceps and Mesacestaides carti (C estoda). Parasitology, 87, 279-287. Cook, R.M., Musgrove, N.R.J., & Ashworth, RF. (1987). Activity of rat peritoneal eosinophils following induction by different methods. Int Arch Allergy Appl Immunol, 83, 423—427. Dykstra, M.J. (1993). A manual of applied techniques for biological electron microscopy. New York: Plenum Press. Festing, F .W. (1979). Inbred Strains. In H]. Baker, J .R. Lindsey & S.H. Weisbroth (Eds). The Laboratory Rat (pp. 55-72). Lendrum, A. (1944). The staining of eosinophil polymorphs and enterochromaffin cells in histological sections. J Path Bact 36, 441. Luna, LG. (1968). Manual of histolpgic staining methods of the Armed Forces Institute of Pathology. (3rd ed.) New York: McGraw-Hill Book Co. Mackenzie, C.D., Ramalho-Pinto, F.J., McLaren, D.J., & Smithers, SR. (1977). Antibody-mediated adherence of rat eosinophils to schistosomula of Schistoma mansoni in vitro. Clin Exp Immunol, 3_()_, 97-104. Mackenzie, C.D., Preston, P.M., & Ogilvie, BM. (1978). Immunological properties of the surface of parasitic nematodes. Nature 27_6, 826-828. Mackenzie, C.D., Jungery, M., Taylor, P.M., & Ogilvie, BM. (1980). Activation of complement,the induction of antibodies to the surface of nematodes and the effect of these factors and cells on warm survival in vitro. Eur J Irnmunol. 10, 594- 601. 8O Mackenzie, C.D., Jungery, M., Taylor, P.M., & Ogilvie, BM. (1981). The in-vitro interaction of eosinophils, neutrophils, macrophages and mast cells with nematode surfaces in the presence of complement or antibodies. J Pathol. 133. 161-175. Mackenzie, C.D. & Spry, C.J. (1983). Selective localization of intravenously injected lllindium- labelled eosinophils in rat tissues infected with Nippastrangylus brasiliensis.. Parasite Immunol. 3, 151-163. McLaren, D.J., Mackenzie, C.D., & Ramalho-Pinto, F.J. (1977). Ultrastructural observations on the in vitro interaction between rat eosinophils and some parasitic helminths (Schistasama mansoni,Trichinella spiralis and Nippastrangylus brasiliensis). Clin Exp Immunol, 32, 105-118. Miller, H.R.P. & Jarrett, W.F.H. (1971). Intestinal mast cell response during helminth expulsion in the rat. Immunology, 22, 277-288. Ogilvie, B.M., Mackenzie, C .D., & Love, R.J. (1977). Lymphocytes and eosinophils in the immune response of rats to initial and subsequent infections with Nippastrangzlus brasiliensis. Am J Trop Med Hyg, 2_6_, 61-67. Ott, L. (1988). An introduction to statistical methods and data analysis. (third ed.) Boston: PWS-Kent Publishing Company. Owen, W.F. (1993). Eosinophil Heterogeneity. In H. Smith & RM. Cook (Eds). Immunophannacologyof Eosinophils (pp. 57-69). Reynolds, ES. (1963). The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J Cell Biol 11, 208-212.(abstract). Silberstein, D.S. & David, J .R. (1986). Turner necrosis factor enhances eosinophil toxicity to Schistasama mansoni larvae. Proc Natl Acad Sci U S A. 83, 1055-1059. Stewart, W.W. (1978). Functional connections between cells as revealed by dye- coupling with highly fluorescent naphthalimide tracer. Q_e_]1, 151, 741-759. Vadas, M.A., Lopez, A.F., Begley, C.G., Williamson, D.J., Warren, D.J., & Sanderson, C]. (1986). Activation of human eosinophils by an eosinophil specific colony-stimulating factor. Presented to the 6th International Congress of Immunology, Toronto, Canada. J Immunol (abstract). 81 Walsh, G.M., Wardlaw, A.J. & Kay, AB. (1993). Eosinophil accumulation, secretion, and activation. In H.Smith and R.M.Cook (Ed). lmmunopharrnacology of Eosinophils (pp. 73-90). New York: Academic Press. Wright, S.J., Centonze, V.E., Stricker, S.A., DeVries, P.J., Paddock, S.W. & Schatten, G. (1993). Introduction to confocal microscopy and three-dimensional reconstruction. In B. Matsumoto (Ed). Cell biological applications of confocal microscopy (pp. 2-45). New York: Academic Press. Chapter 3 PRELIMINARY INVESTIGATIONS A: Biebrich Scarlet: An Eosinophil Specific Granule F luorochrome Introduction The need for markers of eosinophil specific granule proteins in rats has provided difficulties for investigators to fully utilize this species in eosinophil- related research. After screening >1000 hybridomas for monoclonal antibodies to AUG rat eosinophils it became apparent that we would not be making any contributions to the bereft pool of antibody reagents for rat leukocytes. The only known clone to rat eosinophil peroxidase (Keeping & Lyttle, 1984) died in storage and could not be regrown in our hands. In an attempt to provide stark contrast for rapid counting of eosinophils in tissues under light microscopy, Luna’s stain for eosinophil granules (Luna, 1968) was chosen as a staining method in Chapter 2 of this thesis. Luna’s protocol utilizes Biebrich scarlet (BS), 3 chromophore with high specificity to basic proteins (Spicer & Lillie, 1961). This water soluble, deep red, anionic dye is synthesized by coupling diazotized 4-amino-l , l’-azobenzene—3, 4’-disulfonic acid to 2-naphthol (Figure 3A. 1) and is also known as acid red 66. 82 83 O Q. 0 Figure 3A. 1. The chemical structure of Biebrich scarlet. It was evident upon examination of the known structure which showed 4 aromatic hydrocarbon ring structures linked by double-bond (azo) nitrogens that there was a strong capacity for electron resonance in this molecule. Available spectra] absorbance data on this compound showed a broad (450 nm-575 nm) peak with a maxima at 505 nm. These data support a hypothesis that this compound might possess fluorescent capabilities. The advantages of fluorescent probes are many. Analysis of microscopy specimens by fluorescent markers is not limited to the interference properties of transmitted light. Fluorecent markers provide point sources of narrow band emission spectra as opposed to the transmission of full spectrum light through the entire thickness of a sample under bright field microscopy. Tissue often has a high degree of variability with respect to optical properties such as refractive index and absorption. Therefore, fluorescent markers often provide better three—dimensional imaging than bright field chromophores and produce exceptional clarity for labelled objects nearest the objective. Additionally, if a given fluorophore provides 84 enough quantum yield at wavelengths sufficiently separate from the excitation wavelength then a brighter image on a darker field will result. This improvement of signal-to-noise can provide better resolution when imaging in thick biological specimens and/or tissue sections (Inoue, 1990). Lastly, the use of laser scanning confocal microscopy (LSCM) requires fluorescent markers. The prime virtue of this technology is high resolution optical sectioning of biological specimens. Optical sectioning eliminates the structural artifacts and invasive nature of mechanical sectioning and allows for the visualization of both living and fixed cells. The shallow depth of field (0.1-0.5 pm) of better LSCMs limits the information gathered to a small section of the whole sample. This eliminates the background and scattered fluorescence produced by the rest of the specimen and improves contrast, clarity and detection (Wright et al., 1993). This investigation identifies a new fluorochrome, Biebrich scarlet, and describes some of its properties and applications in epi-fluorescent microscopy and LSCM. Methods and Materials Sample Preparation Deparaffinized histological sections (6-10 um) of proximal jejunum from the AUG rat infected with Nippastrangylus brasiliensis (Nb) fixed in 10% neutral buffered formalin, as well as cytocentrifuge preparations of peritoneal lavage, similarly fixed and unfixed, were used. Preparations were rehydrated in distilled 85 H20 at room temperature (RT) for 5 minutes The slides were then stained in an aqueous solution (pH 6.8) of 1% BS (SIGMA Chemical Co., St. Louis, MO) for 10 seconds and cleared in running HzO for 5 minutes. Slides were dehydrated in 100% ethanol (2x) and washed (2x) in xylene before being coverslipped with Pennount® (Fisher Scientific,Fair Lawn, NJ) and allowed to dry overnight. Fluorescence Microscopy Microscopy was performed on a Nikon F XA epi-fluorescent research microscope (Nikon, Tokyo, Japan) with a lOO-watt mercury arc lamp and a G-2A filter cassette (excitation filter EX510-590, dichroic mirror DM580, barrier filter BA590). Images were recorded on Kodak Ektachrome 50® slide film (Eastman Kodak Co., Rochester, NY) and transferred to Polaroid 59® prints (Polaroid Corp, Cambridge, MA) utilizing a Polaroid Daylab II®. Laser Scanning Confocal Microscopy (LSCM) The AUG rat samples were observed on the Ultima—Z-312® (Meridian Instruments Inc., Okemos, MI) LSCM with an excitation line at 514 nm and emission filters of 575 nm (short) and 605 nm (long) and on the lnSight Plus® (Meridian Instruments Inc., Okemos, MI) LSCM with an excitation at 488 nm and bandpass filter of 530 nm/30 nm and longpass filter at 605 nm. These images were recorded as TIF image files on floppy discs and converted to dye sublimation prints on a digital color printer (Tektronix, Inc., Wilsonville, OR). 86 Results Fluorescence Microscopy A deep red fluorescent label was observed in eosinophil specific granules, red blood cells and the granules of large granular lymphocytes (Figure 3A2.) within the tissues examined (fixed and unfixed). These fluorophore-tissue complexes were the source of emission spectra (>605 nm) upon photoexcitation and proved to be photoreactive, producing increased quantum emission for the first 1-10 minutes of continuous photoexcitation. This was observed qualitatively by a halving of microscope-indicated exposure times on successive micrographs from identical fields. All non-specific autofluorescence phenomena associated with these tissues were photobleached in this time frame including areas of background BS binding where photoreactive complexes were not formed (i.e., in areas of connective tissue collagen). The remaining deep red fluorescent emissions were from the various cellular constituents described above in strong contrast to the dark background. 87 Figure 3A.2. Micrographs of the fluorescent emissions from the BS-protein complexes in NBF-fixed jejunal lamina propria (A) and unfixed cytospin preparations (B). The lablel includes: eosinophil specific granules (<>), cytoplasmic granules of LGL (O) and red blood cells (It). Bar = 10.0 um. 88 Figure 3A3. Micrographs of a cytospin preparation showing the specific granules of eosinophils labelled with BS fluorescing under the LSCM. Bar = 5.0 pm. 89 Figure 3A4. Digital micrograph of a computer-aided 3-dimensional reconstruction of the BS-eosinophil specific granule complex fluorescence (=9) against the remaining tissues of the lamina propria as detected by LSCM. The (*) are red blood cells. Bar = 10.0 urn. 9O Figure 3A.5. Variation in fluorescence emission by different BS-protein complexes as detected by the LSCM. Eosinophil specific granules (=9) and LGL granules (O) were differentiated while the red blood cells (*) emitted at a similar wavelength as the eosiniophil granules. Bar = 10.0 um. 9 1 Laser Scanning Confocal Microscopy The LSCM provided exceptional resolution of individual eosinophil specific granules (Figure 3A3) through optical sectioning the BS labelled granules were clearly visualized. Additionally, 3-dimensional reconstructions of successive optical sections resolved specific granule profiles and dispersion patterns previously unobtainable from whole eosinophils in histological sections (Figure 3A4). The use of narrow excitation lines and emission filtration showed that the emission spectra varied in wavelength with some of the BS complexes (i.e., eosinophil specific granules vs. LGL granules) and were distinctly separable (Figure 3A5). Discussion Several properties are necessary for a dye to be useful in marking cells for fluorescent detection; these include spectral properties, chemical properties and specificity (Stewart, 1978). The spectral properties of the BS complexes described here are exemplary. A high quantum yield was observed at a long wavelength minimizing interference with tissue autofluorescence common at shorter spectra. The emission was photoreactive, increasing over time; this is in sharp contrast to the vast majority of fluorochromes which tend to photobleach under continuous photoexcitation. Finally, a wide separation of excitation and emission maxima of BS, coupled with the photoreactivity, provides a brighter image against a dark field. 92 Biebrich scarlet has both a known structure and synthesis making it readily available in high purity. Solubility of BS in water is essential for its use in the preparation protocols for both fixed and live cells. The ability to form covalent bonds with cellular constituents prevents dyes from redistributing during tissue preparation and analysis. Biebrich scarlet has two separate sulfonated benzene ring structures. These vinyl sulfone groups bind covalently with proteins and other compounds containing amino or sulflrydryl groups. Spicer and Lillie (1961) have described the compound’s affinity for basic proteins. Nevertheless, the difference in photoreactivity observed between BS bound to eosinophil specific granules and BS bound to collagen can not be a simple matter of fluorophore binding concentration. The fluorophore-granule complexes described in this study were clearly photoreactive, increasing their quantum emission with continuous photoexcitation, while background BS staining photobleached. Additionally, the complexes observed in the eosinophils were separable fiom the macrophages and LGL by virtue of their emission spectra under LSCM. The specific nature of these fluorescent complexes is unknown. A possible scenario may be a complex between the two vinyl sulfone groups of BS and the two amine groups of arginine, known to be in high concentration in eosinophil specific granule proteins (Yokota et al., 1984; Egesten et al., 1986; Pimenta et al., 1987). Of interest is the fact that major basic protein, a primary constituent of eosinophil specific granules, is packaged as an acidic proprotein (pl 6.2) (Barker et al., 1988). However, since 93 many aspects of the eosinophil specific granule packaging scheme are unknown, this may have no bearing on the availability of amine groups for BS binding. Regardless, the amount of complex formation or the saturation of binding by the BS vinyl sulfone groups may affect emission spectra from the observed cellular constituents. One relevant point is the fact that the fluorescent complexes were formed in both fixed and unfixed eosinophils. This alludes to the stability of the BS binding sites and holds promise as the first hurdle in utilizing BS in living eosinophils. This investigation has described a new fluorophore for the study of eosinophil specific granules in individual cells and tissues. While investigations remain to fully characterize the nature of BS-tissue complexes, a careful analysis of emission spectra and binding targets may provide new data on the nature of packaging and release of these granule proteins from their cellular compartments. The need for fluorescent markers for LSCM and the properties of BS-eosinophil specific granule complexes described here demonstrate a strong potential for the use of Biebrich scarlet in future eosinophil-related research. 94 References Barker, R.L., Gleich, G.J., & Pease, LR. (1988). Acidic precursor revealed in human eosinophil granule major basic protein cDNA [published erratum appears in J Exp Med 1989 Sep 1;l70(3):1057]. J Exp Med. 168. 1493-1498. Egesten, A., Alumets, J., Meclenberg, C., Palmegren, M., & Olsson, I. (1986). Localization of eosinophil cationic protein, major basic protein, and eosinophil peroxidase in human eosinophils by immunoelectron microsc0pic technique. 1 Histochem Cytochem, 34, 1399-1403. Inoue, S. (1990). Handbook of biolpgical confocal microscopy. (Rev. ed.) New York: Plenum. Keeping, HS. & Lyttle, CR. (1984). Monoclonal antibody to rat uterine peroxidase and its use in identification of the peroxidase as being of eosinophil origin. Biochim Biophys Acta. 802, 399-406. Luna, LG. (1968). Manual of histologic staining methods of the Armed Forces Institute of Pathology. (3rd ed.) New York: McGraw-Hill Book Co. Pimenta, P., Loures, M., & DeSouza, W. (1987). Ultrastructural localization of basic proteins in cytoplasmic granules of rat eosinophils. J Submicrosc Cfiol. 1_9_, 387-395. Spicer, S.J. & Lillie, RD. (1961). Histochemical identification of basic proteins with Biebrich Scarlet at alkaline pH. Stain Technol. 36, 365-370. Stewart, W.W. (1978). Functional connections between cells as revealed by dye- coupling with highly fluorescent naphthalimide tracer. _C_efl, 14, 741-759. Wright, S.J., Centonze, V.E., Stricker, S.A., DeVries, P.J., Paddock, SW. & Schatten, G. (1993). Introduction to confocal microscopy and three-dimensional reconstruction. In B. Matsumoto (Ed). Cell biological applications of confocal microscopy (pp. 2-45). New York: Academic Press. Yokota, S., Tsuji, H., & Kato, K. (1984). Localization of lysosomal and peroxisomal enzymes in the specific granules of rat intestinal eosinophil leukocytes. J Histochem Cytochem. 32, 267-274. 95 B: The Eosinophilic Response to Primary Infection with Nippostrangvlus brasiliensis in the August Rat Introduction A basic pattern of warm establishment and expulsion for Nb infection in rats was first quantitatively described by Jarrett, Jarrett and Urquhart (1968a). Primary infection was characterized by an initial loss of infecting larvae in the skin and lungs (loss phase 1), with the surviving worms maturing in the proximal jejunum (plateau phase) before the majority were expelled (loss phase 2) with a small residual population remaining (threshold phase). This basic pattern was shown to be generally retained with variation in the length of the plateau phase and rate of loss for phase 2 depending on host immune status, genotype and the number of the infective larvae given (Nawa & Miller, 1979; Rothwel], 1989). Eosinophils have been described in tissue sites around the migrating worms in the rat (Taliaferro & Sarles, 1939; Ogilvie et al., 1977; Mackenzie & Spry, 1983) implicating them as effector cells of immunity in these loss phases during Nb infection. Our interests for this study were in the timing of the tissue eosinophilic response during the intestinal phase of primary Nb infection in the AUG rat. Presence of parasites in the small intestine has been reported between day 6-19 for the AUG rat strain during primary Nb infection (Mackenzie & Spry, 1983). In addition, the strain-specific characteristic of peritoneal eosinophil accumulation in 96 the AUG rat was described in Chapter 2, and the known reactivity of the peritoneal compartment to other helminth infections was discussed in Chapter 1. Therefore, the specific aims of this preliminary investigation were two-fold: first, to determine the eosinophilic reactivity of the peritoneal cavity in all phases of primary Nb infection; second, to quantitate the trends of accumulation of eosinophils in the small intestine during the same time frame. This would allow determination of the plausible time points of maximal eosinOphil involvement in the immune response of the intestinal mucosa to the adult stage of Nb. Methods and Materials Experimental Design Experiments were done in two phases. The first phase involved two groups of 6 healthy 4-week-old AUG rats. In one group, rats were injected subcutaneously into the medial aspect of the right hind leg with 3000 Nb infective larvae (L3) at 6 weeks of age. Rats in the other group similarly received sterile saline and served as uninfected controls. Catheter lavage of the peritoneal cavity was performed on each animal at 4, 6, 8, 12, 16, 20, 24 and 28 weeks of age and differential cell counts performed. In the second phase there were two groups of 16 6-week-old AUG rats. One group was infected as described above and the other contained saline controls. In this phase, two animals/group were sacrificed at day 0, 4, 7, 9, 11, 15, 20 and 35 post infection (PI). Peritoneal lavage was performed to determine differential cell counts, as above. Additionally, samples of 97 proximal jejunum were taken for histological determination of eosinophil infiltration. Animals Sibling breeding pairs of August (AUG) rats were obtained from Harlan Olac Limited, Bicester, England and the National Institute for Medical Research, London. Colonies of inbred progeny were established at the Biological Sciences Department, Western Michigan University and the Division of Animal Health, The Upjohn Company, Kalamazoo, MI and were the source of research animals for this study. All animals were housed in accordance with the Institutional Animal Care and Use Committee (IACUC) of the respective institutions. The colonies were maintained under a 12h/12h-light/dark cycle at 21-28 °C with rodent chow #5001 (Purina Mills, St. Louis, MO) and reverse osmosis purified water provided ad libitum. Nb Cultures The parasites were maintained in mice by methods previously described (Westcott & Todd, 1966). Fecal pellets were obtained on day 8 and 9 PI. These were macerated and mixed with activated charcoal and cultured at 19°C. The L3 larvae were harvested from cultures between 8-20 days old in a Baennann apparatus filled with water. The concentrated larvae were counted, brought up in sterile saline and adjusted to 3000/ml for injection. 98 Sample Collection All chemical reagents were obtained from the SIGMA Chemical Company (St. Louis, MO) unless otherwise noted. Peritoneal washings were performed utilizing a cold (wet ice) lavage solution of calcium- and magnesium-free Hanks’ Balanced Salts (HBSS) with 20 mM HEPES buffer and 10 units/ml heparin (The Upjohn Co., Kalamazoo, M1) at pH 7.3. The resident peritoneal cells and the lavage solution were removed within 5 minutes and pelleted by centrifugation at 200 x g for 10 minutes at 4°C in a Beckman (Fullerton, CA) centrifuge. The lavage supernatant was discarded and the cell pellet brought to 5m] with HBSS containing 20mM HEPES and lmg/ml gelatin then held on wet ice until counting. Separate collection protocols were used in the two phases to obtain resident peritoneal cells. In the first phase repeat collections on the same animals required catheterization of the peritoneal cavity at each time point. This was accomplished by introducing 10-20 ml of the lavage solution through an 18G catheter needle (Becton-Dickinson, Cockeysville, MD) inserted adjacent to the ventral midline of the abdomen. The needle was withdrawn and after gentle massage the lavage solution recovered through the catheter sleeve into a 50 ml centrifugation tube. The catheter sleeve was then removed and the animal housed until subsequent procedures. 99 In the second phase the animals were killed prior to tissue collection by lethal C02 inhalation. The peritoneal washings were done by making a small (1-3 cm) incision on the lower abdomen adjacent to the ventral midline of the animal to irrigate the peritoneal cavity with 30-50 ml of the lavage solution. Next, the proximal jejunum, beginning at a point approximately 10 cm from the pyloric sphincter, was ligated into a 2-4 cm compartment. This was injected with cold (wet ice) 10% neutral buffered formalin (NBF). The jejunal segments were excised and placed NBF for 15 minutes at RT and then the NBF was replaced with fresh solution and the samples stored at RT until paraffin embedment. Sample Preparation All cell counting procedures were performed on a Nikon (Tokyo, Japan) Microphot-FXA® upright microscope. Differential cell determinations were performed on all peritoneal washings. Cytocentrifuge preparations were made on a Cytospin 3® (Shandon Scientific, Cheshire, England) and stained with the Diff- Quick® system (Baxter Healthcare, McGraw Park, IL). Determinations were made on the first 300 cells from these preparations utilizing morphologic parameters previously discussed (see Chapter 2, Sample Preparation and Counting Methods) and expressed as mean percents (%) of the total. The segments of proximal jejunum were trimmed into 2-4 mm rings and embedded in paraffin. Histological sections were cut (6-10 pm) and stained by 100 Luna’s (1968) method for eosinophil specific granules. The eosinophils in the villus—crypt (VC) units of the intestinal mucosa were counted according to Miller (1971). Briefly, all cells located between two gland crypts of the villus, in the lamina propria and epithelium of the villus above were counted as a single VC unit. Cells were counted only in those villi that were longitudinally sectioned with a total of 10 VC units/animal counted. Results The results of the first phase peritoneal catheter lavages are represented in Figure 3B]. These data are expressed as mean % i SEM. The Nb infected animals (48 i 2.8%) had higher peritoneal eosinophil differential counts than the uninfected animals (30.7 i 1.4%) at 8 weeks of age (2 weeks PI). This was followed by a return to similar ranges for infected and uninfected rats at 12 weeks of age (6 weeks PI), and counts remained similar for the two groups at subsequent time points. The second phase of terminal peritoneal lavages (Figure 3B2) also profile higher eosinophil differential counts in the infected animals compared to the uninfected animals (48% vs. 37%) at day 15 PI observed in the first phase. However, the difference between the 2 groups continued to be apparent at day 20 PI (51% vs. 37%) before counts were similar for the two groups (28-39%) on day 35 PI. lOl —+—%EOS-I —I§I-%EOS-U i so ,1 4o ‘- tn 5 I i a! O z _. 5") O 30 —‘ a - 5‘ I 20 ”I 10 I I I 1 I I I y l I I I I" 0 E 10 20 30 1 WEEKS of AGE Figure 33.1. Eosinophil differential counts of catheter lavage obtained from uninfected (U) and Nb infected (1) AUG rats (Phase I). 102 —0—% EOS-I 493*- % EOS-U 60 — *— 50 ~- In ..1 H E 8 _. z 40 n U) G H b? 30 - 20 "WT—II] '1 l I l I I 1' IT] I l I] 1 i I ] TAT—WT'ITTflli—WTTF‘J 0 5 10 15 2025303540 DAYS r1 Figure 38.2. Eosinophil differential counts of tennina] lavage obtained from uninfected (U) and Nb infected (1) AUG rats (Phase H). 103 -— soslvc-r ———:u~—— EOSIVC-U I I ~I 70 60- 50 4O EOSINOPHILSIVILLUS-CRYPT UNIT 30- 20 are DAYS PI Figure 38.3. Eosinophils in the proximal jejunum of uninfected (U) and Nb infected (1) AUG rats. 104 The eosinophil numbers determined from the VC unit counts in the segments of proximal jejunum are represented in Figure 38.3. The eosinophils/V C unit in the infected animals remained in the uninfected range (24-35 cells/V C unit) through day 11 P]. On day 15 P] the infected group reached 43 eosinophils/V C unit and peaked at day 20 P1 with 69 eosinophils/V C unit. The infected animals returned to the uninfected range by day 35 PI. Discussion These studies suggest a temporal association between the peritoneum and intestinal mucosa in regards to eosinophil accumulation in response to primary infection with Nb. This type of association in enteric helminth infections has not been well characterized. Infections with the peritoneal-dwelling cestode Mesacestaides carti (Barton et al., 1984; Cook et al., 1987) remain the only reported mode] of peritoneal eosinophilia by natural infection. Virtually all other studies of this phenomenon involve the direct injection of helminth antigen into the peritoneal cavity without infection (see Chapter 1, Animal Models of Peritoneal Eosinophilia). With the knowledge from the literature that T-cells regulate eosinophil activity (Basten & Beeson, 1970; Basten et al., 1970; Boyer et al., 1971), it is assumed that the accumulation of eosinophils in the peritoneum observed in 6- week-old AUG rats (see Chapter 2) is an indicator of immunological activity. Previous studies of Nb infection in rats younger than 6 weeks of age showed 105 prolonged infections without rapid expulsion of the adult stage from the intestine (Jarrett et al., 1968b). We therefore chose to infect the animals at 6 weeks of age and take advantage of a responsive but relatively na‘r‘ve immune system of young animals. Antigen-specific T-cells recovered after day 8 P] from thoracic duct lymph and/or the peritoneal cavity were shown to confer immunity through expulsion when transferred to na'r've hosts (Ogilvie et al., 1977). It is known that lymphocytes, monocytes, mast cells and eosinophils infiltrate the tissue sites where Nb migration occurs in initial infections and more intensely in immune animals (Taliaferro & Sarles, 1939). Several studies have described plausible roles for some 'of the various effector cell populations observed but a complete understanding of the intestinal expulsion of Nb remains elusive. Urquhart and colleagues (1965) proposed a mast cell mediated anaphylaxis in the intestinal mucosa as the primary mechanism for Nb expulsion. In contrast, work in mast cell deficient W/Wv mice demonstrated Nb expulsion, although it was delayed (Uber et al., 1980; Crowle & Reed, 1981). Nawa (1994) showed goblet cell hyperplasia and changes to the terminal sugars of the mucin produced by these cells to also be under T-cel] control and specific to expulsion of the adult worms. Ogilvie and Jones (1971) proposed a more general theory that many cells provide a concerted effort in orchestrating the expulsion of Nb adults with no cell population singularly 106 capable. In this context, the current evidence helped define plausible roles for the eosinophil in the immune response to the intestinal phase of Nb. These findings implicate days 14-21 PI as the most relevant for investigating the eosinophil accumulation profiles of the peritoneal cavity and the jejunal submucosa during Nb infection in the AUG rat. However, a need for studies including absolute eosinophil numbers in the peritoneal cavity and sufficient experimental group size to ascertain biological relevance was also established. The following studies (Chapters 4 and 5) will concentrate on detailed descriptions of eosinophil involvement in immunity to the establishment of adult phase Nb. 1 07 References Barton, A.M., Washington, E.A., Stewart, A.C., & Nicholas, W.L. (1984). Mesacestaides carti in the rat: comparisons of Mesacestaides carti infections in rats and mice. Int J Parasitol, 14, 391-294. Basten, A. & Beeson, PB. (1970). Mechanisms of eosinophilia. 11. Role of the lymphocyte. J Exp Med. 131. 1288-1305. Basten, D.A., Boyer, M.H., & Beeson, PB. (1970). Mechanisms of eosinophilia. 1. Factors affecting the eosinophil response of rats to T richinella spiralis. . J Exp Med, 131, 1271-1287. Boyer, M.H., Spry, C.J., Beeson, P.B., & Sheldon, W.H. (1971). Mechanism of eosinophilia. IV. The pulmonary lesion resulting from intravenous injection of Trichinella spiralis. Yale J Biol Med, 43, 351-357. Cook, R.M., Musgrove, N.R.J., & Ashworth, RF. (1987). Activity of rat peritoneal eosinophils following induction by different methods. Int Arch Allergy Appl Immunol, 83, 423-427. Crowle, P.K. & Reed, ND. (1981). Rejection of the intestinal parasite Nippastrangylus brasiliensis by mast cell—deficient W/Wv anemic mice. Infect Immun, 3_3_, 54-58. Jarrett, E.E.E., Jarrett, W.F.H., & Urquhart, G.M. (1968a). Quantitative studies on the kinetics of establishment and expulsion of the intestinal nematode populations in susceptible and immune hosts. Nippastrangylus brasiliensis in the rat. Parasitology, 38, 625-640. Jarrett, E.E.E., Jarrett, W.F.H., & Urquhart, G.M. (1968b). Immunological unresponsiveness to helminth parasites: I. The pattern of Nippastrangylus brasiliensis in young rats. Exp Parasitol, 23, 151-160. Luna, LG. (1968). Manual of histologic stainirpg methods of the Armed Forces Institute of Pathology (3rd ed.) New York: McGraw-Hill Book Co. Mackenzie, C.D. & Spry, C.J. (1983). Selective localization of intravenously injected lllindium- labelled eosinophils in rat tissues infected with Nippastrongylus brasiliensis.. Parasite Immunol. 3, 151-163. 108 Miller? H.R.P. & Jarrett, W.F.H. (1971). Intestinal mast cell response during helminth expulsion in the rat. Immunology, 2_0, 277-288. Nawa, Y., lshikawa, N., Tsuchiya, K., Horii, Y., Abe, T., Khan, A., Itoh, B.H., Ide, H., & Uchiyama, F. (1994). Selective effector mechanisms for the expulsion of intestinal helminths. Parasite Immunol, _lp, 333-338. Nawa, Y. & Miller, H.R.P. (1979). Adoptive transfer of intestinal mast cell response in rats infected with Nippastrangylus brasiliensis. Cellular Immunology, 42,225-239. Ogilvie, B.M., Mackenzie, C.D., & Love, R.J. (1977). Lymphocytes and eosinophils in the immune response of rats to initial and subsequent infections with Nippastrangylus brasiliensis. Am J Trop Med Hyg, 26, 61-67. Ogilvie, B.M. & Jones, VB. (1971). Parasitological review. Nippastrangylus brasiliensis: a review of immunity and host-parasite relationship in the rat. Exp Parasitol, 22, 138-177. Rothwell, T.L.W. (1989). Immune expulsion of parasitic nematodes from the alimentary tract. IntJ Parasitol, 19, 139-168. Taliaferro, W.H. & Sarles, MP. (1939). The cellular reactions in the skin, lungs and intestine of normal and immune rats after infection with Nippastrangylus mum's. J Infect Dis, 6_4_, 157—192. Uber, C.L., Roth, R.L., & Levy, DA. (1980). Expulsion of Nippastangylus brasiliensis by mice deficient in mast cells. Nature, 287, 226-228. Urquhart, G.M., Mulligan, W., Eadie, R.M., & Jennings, F.W. (1965). Immunological studies on Nippastrangylus brasiliensis infection in the rat: the role of local anaphylaxis. Exp Parasitol, 1_7, 210-217. Westcott, R.B. & Todd, AC. (1966). Adaptation of Nippastrangylus brasiliensis to the mouse. J Parasitol, 32_, 233-236. Chapter 4 CHANGES IN EOSINOPHILS IN THE INTESTINAL PHASE OF THE N EMATODE INFECTION NIPPOSTRONG YL US BRASILIENSIS Introduction The presence of eosinophil leukocytes in lesions associated with endoparasitic infections of mammalian species has been documented in the literature for nearly 100 years (Brown, 1898). Localized eosinophilic responses have been described in tissues where helminth invasion or migration has occurred. An intense and often fatal eosinophilic leukocytosis of the meninges and cerebrospinal fluid has been reported in Angiastrangylus cantanensis infection in both humans (Yii, 1976) and rodents (Hua et al., 1990). Pulmonary eosinophilia was noted in tropical filarial infections (Neva & Otteson, 1978) and during the migratory lung phases of many gastrointestinal nematodes (Boyer et al., 1971; Mackenzie & Spry, 1983; Kayes et al., 1987). As well, the intestinal mucosa has also been described as a site of localized eosinophilia around migrating worms in Angiastrangylus castaricensis infection in humans (Loria Cortes & Lobo Sanahuja, 1980), Haemanchus cantartus infection in sheep (Gorrell et al., 1988) and Nippastrangylus brasiliensis infection in rats (Taliaferro & Sarles, 1939). 109 110 Distinctive patterns of eosinophilia associated with large amounts of locally released helminth antigens concomitant with host tissue destruction, as well as parasite migration, feeding and/or resulting inflammation were evident from these and other studies (Butterworth & Thome, 1993). A plausible role for eosinophils as effector cells in parasite damage was suggested by the detailed investigations of eosinophilic responses of the skin in human onchocerciasis. Mackenzie (1980) first described increased eosinophil populations in the proximity of, and adherent to, dead microfilaria in the skin. Further study showed that some heavily infected patients had neither skin lesions nor cellular infiltrates around morphologically viable larvae. This profile of apparently “adapted” responses was in direct contrast to other indivduals who often had low filarial loads in their skin but nevertheless presented with severe skin lesions. The lesions in these reactive hosts were characterized as having many dead and damaged microfilaria surrounded by heavy eosinophil infiltrates (Mackenzie et al., 1985; Mackenzie et al., 1986). These findings support a hypothesis that eosinophils serve as direct effector cells in the immune response to limit helminth infection. The study of Nb infection in the laboratory rat has provided a wealth of information about the mechanisms behind mammalian immunity to helminth parasites (Ogilvie & Jones, 1971). Rats generate a strong immune response to the infection which leads to rapid expulsion of the adult phase fi'om the small intestine and an acquired immunity to reinfection (Africa, 1931; Ogilvie, 1965). Primary 111 Nb infection has been characterized by an initial loss of the infective stage (L3) during migration in the skin and lungs. The surviving worms then home to the proximal jejunum where they mature and produce eggs. This is followed by a sharp drop in fecundity and expulsion (Jarrett et al., 1968). The expulsion of adult Nb is known to be T-cell mediated with both humoral and cellular components (Jones et al., 1970; Ogilvie & Love, 1974; Ogilvie et al., 1977), but many aspects of the mechanism remain poorly characterized. The first evidence implicating eosinophils as effector cells in the immune response by rats to Nb was published by Taliaferro and Sarles in 1939 . In this work, mast cell, monocyte and eosinophil infiltrates were noted at all sites of Nb migration in initial infections and more intensely in immune animals. Other in vitro work showed that eosinophils, under certain conditions, would adhere and degranulate on the L3 larvae and adult Nb (McLaren et al., 1977; Mackenzie et al., 1980; Mackenzie et al., 1981). Another investigation demonstrated homing of radiolabeled eosinophils from the vasculature to the small intestine of August (AUG) rats during the intestinal phase of primary Nb infection (Mackenzie & Spry, 1983). These studies allude to the probable involvement of eosinophils in the rejection of the adult stage of Nb. However, more detailed investigations are needed to provide more definitive morphological evidence of direct eosinophil involvement in the intestinal phase of adult Nb. 112 In the present study we have quantitated the eosinophil infiltrates of the intestinal mucosa at selected time points during the expulsion phase of primary Nb infection in rats. Moreover, we have analysed in detail the distinctive changes in the morphological profiles and behavior of eosinophils involved in the inflammatory response to adult stage Nb in the small intestine. This necessary detail was accomplished utilizing several morphological assessment technologies. Methods and Materials Experimental Design Six-week-old August (AUG) rats of mixed parentage and sex were placed into 4 experimental groups (n = 6-11). Two of the groups were infected with 3000 Nb infective larvae (L3) injected subcutaneously into the right hind leg. The remaining groups were similarly injected with sterile saline and served as age- matched uninfected controls. Tissues were harvested from one infected and one uninfected group at 2 weeks post infection (PI), and from the remaining 2 groups at 3 weeks PI. Determinations of eosinophil numbers were made from segments of the proximal jejunum and in addition, changes in the morphological, ultrastructural and behavioral profiles of these cells were described. Nb Cultures The parasites were maintained in mice by methods previously described (Westcott & Todd, 1966). Fecal pellets were obtained on day 8 and 9 PI. These were macerated and mixed with activated charcoal and cultured at 19°C. The L3 113 larvae were harvested from cultures between 8-20 days old in a Baerrnann apparatus filled with water. The concentrated larvae were counted, brought up in sterile saline and adjusted to 3000/ml for injection. Animals Sibling breeding pairs of AUG rats were obtained from Harlan Olac Limited, Bicester, England and the National Institute for Medical Research, London. Colonies of inbred progeny were established at the Biological Sciences Department, Western Michigan University, Kalamazoo, MI and the Division of Animal Health, The Upjohn Company, Kalamazoo, MI and were the source of research animals for this study. All animals were housed in accordance with the Institutional Animal Care and Use Committee (IACUC) of the respective institutions. The colonies were maintained under a 12hr/12hr-light/dark cycle at 21-28°C with rodent chow #500] (Purina Mills, St. Louis, MO) and reverse osmosis purified water provided ad Iibitum. Tissue Collections All chemical reagents were obtained from the SIGMA Chemical Company (St. Louis, MO) unless otherwise noted. Animals were killed prior to tissue collection by lethal carbon dioxide inhalation. Following this, an 8-cm segment of the proximal jejunum, beginning at a point just distal to the duodenal loop adjacent to the pancreatic rests, was ligated into two 2-4 cm compartments. One segment was injected with cold (wet ice) 10% neutral buffered formalin (NBF), excised and l 14 placed in NBF for 15 minutes at RT with the NBF then replaced with fresh fixative solution and the samples stored at RT until paraffin embedment. The other segment was injected with cold 3% glutaraldehyde in 0.1 M phosphate buffer at pH 7.3, excised and placed in cold fixative for 1 hour prior to preparation for transmission electron microscopy (TEM). Sample Preparation and Counting Methods The glutaraldehyde-fixed jejunal segments were sliced into 1 x 5 mm strips. The strips were then washed (2x) in buffer and secondarily fixed in buffered 1% osmium tetroxide (Polysciences, lnc., Warrington, PA) for one hour at RT. The samples were dehydrated in a graded ethanol series and embedded in Polybed 812® (Polysciences, Inc., Warrington, PA) epoxy resin and thin sectioned for transmission electron microscopy (TEM). Sectioning was done on an LKB (Bromma, Sweden) Ultrotome Nova® and sections were stained with 5% methanolic uranyl acetate and Reynold’s lead citrate (Reynolds, 1963). The NBF-fixed segments of proximal jejunum were trimmed into 24 mm rings and embedded in paraffin. Histological sections were cut (6-10 pm) and stained by Luna’s (1968) method for eosinophils. These preparations were used for counting of the eosinophils in the villus-crypt (VC) units of the intestinal mucosa according to the method of Miller (1971). Briefly, all cells located between two gland crypts of the villus and in the lamina propria and epithelium of 115 the villus above were counted as a single VC unit. Cells were counted only in those villi that were longitudinally sectioned with a total of 10 VC units/animal counted. Similar histological sections were cut from the NBF-fixed jejunal segments for fluorescence microscopy and deparaffinized in xylene (5 minutes at RT). The sections were rehydrated in distilled H20 at room temperature (RT) for 5 minutes. The slides were then stained in an aqueous solution (pH 6.8) of 1% Biebrich scarlet (BS) for 10 seconds and cleared in running HZO for 5 minutes. These preparations were utilized in two ways. The first protocol, in which fluorescence observation of BS-complexes alone was desired, were dehydrated in 100% ethanol (2x) and washed (2x) in xylene before being coverslipped with Pennount® (Fisher Scientific, Fair Lawn, NJ) and allowed to dry overnight. The second was secondarily labeled with the nucleic acid-specific fluorophore, Hoechst 33258 (Molecular Probes, Inc., Eugene, OR). These slides were incubated with Hoechst 33258 (lug/ml) in 0.1 M Tris buffer (ph 7.3) for 1 hour. The preparations were cleared to desired intensity in a graded ethanol series and coverslipped in 100% glycerol. Microscopy All cell counting procedures and bright—field photography were performed on a Nikon Microphot-FXA® upright microscope (Nikon, Inc., Tokyo, Japan). 116 Fluorescence microscopy was also performed on the Nikon-FXA epi-fluorescent research microsc0pe with a 100-watt mercury light source. A G-2A filter cassette (excitation filter EX510-590, dichroic mirror DM580, barrier filter BA590) was utilized for BS. Both the G-2A and the UV-lA filter cassette (excitation filter EX365/10, dichroic mirror DM400, barrier filter BA400) were used for the BS/Hoechst 33258 stained specimens and photographed by dual exposure. Images were recorded on Kodak Ektachrome 50® slide film (Eastman Kodak Co., Rochester, NY) and transferred to Polaroid 59® prints (Polaroid Corp, Cambridge, MA) utilizing a Polaroid Daylab II®. Laser scanning confocal microscopy (LSCM) was carried out on a Ultima-Z- 312® (Meridian Instruments Inc., Okemos, MI) LSCM with an excitation line at 514 nm and emission filters of 575 nm (short) and 605 nm (long). These images were recorded as TIF image files on floppy discs and converted to dye sublimation prints on a digital color printer (Tektronix, Inc., Wilsonville, OR). Differential interference contrast (DIC)-Nomarski optics were utilized on a Nikon Optiphot-2® microscope. Ultrastructural analysis was accomplished on a Siemens 101 Elmiskop® TEM (Siemens, Inc., Berlin, Germany). l 17 Statistics All numerical data was expressed as mean i SEM. Equal variance was tested by Hartley’s homogeneity of variance method. Statistical significance was determined by one-way analysis of variance (ANOVA) with a 95% confidence limit (p = 0.05). Group-wise comparisons were made utilizing Student-Newman- Kuels (SNK) a posteriori test (Ott, 1988). Results Villus-Crypt Unit Counts Figure 4.1 represents the eosinophil counts for the VC units from each of the experimental groups. The uninfected animals had 25 .9 :t 1.1 eosinophils/V C unit (range = 17.2-34.2). Eosinophil counts were significantly greater in the infected group at 2 weeks P1: 39.8 i 2.2 eosinophils/VC unit (range = 29.5-54.6). Statistical significance over both the uninfected animals and the 2 week PI group was determined for the infected animals at 3 weeks PI; their levels were at 72.9 i 5.2 eosinophils/V C unit (range = 49.4-87.4). 118 ** 70 lLllJJi'HiliLI 60 l ‘ J 50 40 30 20 EOSINOPHILS/VILLUS-CRYPT UNIT 10 illllilllIilLIlilillLlLIIi UNINFECTED 2WK PI 3WK PI GROUPS Figure 4.1. Eosinophils in the proximal jejunum of uninfected and Nb infected AUG rats. The (It!) denotes significant difference from uninfected level and (*4!) as significant over both uninfected and the 2WK PI groups. l 19 Villus-Crypt Change The pathology of the jejunum associated with the presence of adult stage Nb has been described (Symons, 1957). The changes observed in both infected groups of this study were consistent with the literature. They included: distension and flabbiness of the jejunum; blunted, edematous, hyperemic villi; crypt hyperplasia, with increased numbers of mucosal epithelial cells and goblet cells; increased populations of mast cells, lymphocytes, plasma cells and eosinophils in the lamina propria, as well as the appearance of large granular lymphocytes (LGL). Eosinophil Morphological Changes The morphological profiles of eosinophils in the VC units of the infected animals were distinct from those of the uninfected rats. Luna’s stained sections of jejunum indicated morphologic changes at both time points post-infection. It was apparent that in addition to the increased numbers of eosinophils there was a marked change in specific granule dispersion within these cells in the infected lamina propria (Figure 4.2). The granule dispersion was so pronounced in many of these cells that their characteristic orange-red color was difficult to detect under regular bright-field microscopy. This problem was addressed in the epi-fluorescence images of the BS-stained jejunal segments. These fluorescent preparations produced striking contrast in regard to eosinophil specific granule dispersion between the uninfected and the infected VC units (Figure 4.3). The specific granules of the eosinophils in 120 Figure 4.2. Eosinophils 09) stained by Luna’s method in the lamina propria of villi from the proximal jejunum of uninfected (A) and Nb infected (B) AUG rats. Specific granule dispersion in the infected group impedes eosinophil identification (DIC optics). Bar = 10.0 um. Figure 4.3. Eosinophils (=9) stained with BS in the lamina propria of villi from the proximal jejunum of uninfected (A) and Nb infected (B) AUG rats. Even the greatly dispersed specific granules in the infected group are easiliy identified under epi-fluorescence microscopy. Red blood cells (*) are the only other cells visible in these micrographs. Bar = 10.0 um. 122 Figure 4.4. Computer reconstruction of optical sections obtained on the LSCM gives a clear 3-dimensional view of specific granule dispersion profiles in eosinophils (=9) from the lamina propria an Nb infected animal. * = Red blood cells. Bar = 10.0 um. 123 the uninfected intestines remained densely packed around the nucleus and, coupled with lower cell numbers, they occupied very little of the volume of the lamina prOpria. In contrast, the eosinophils in the infected intestines were more numerous, and their specific granules were greatly dispersed throughout the cytoplasm; thus, the granules occupied a greater proportion of the villus. These specific granule dispersion profiles, while always evident, varied from compact to widely dispersed in the eosinophils that populated the lamina propria of the infected animals. This was best demonstrated by 3-dimensional computer reconstruction of successive optical sections obtained on the LSCM (Figure 4.4). Other morphologic alterations in the eosinophils from the infected groups included regions of expanded granule-free cytoplasm and changes in nuclear profiles. The DIC optics (Figure 4.5) revealed that the expanded areas of cytoplasm free of specific granules contained vacuoles; these areas were not observed in the eosinophils of the uninfected animals. The Hoechst 33258 fluorophore provided definitive imaging of the nuclear profiles of the cells in the lamina propria and the dual-label with BS allowed eosinophils to be distinguished from the other resident cells (Figure 4.6). Nuclear morphological heterogeneity was a hallmark of the eosinophils observed in the VC units in the infected animals with many more having nuclei that were donut-shaped and segmented rather than rounded and compact as was seen in the eosinophils in the uninfected groups. 124 Figure 4.5. Eosinophils (=9) in the lamina propria of the VC units from the infected animals showed cytoplasmic vacuolization (*) not seen in cells from the uninfected group. Bar = 10.0 urn. 125 8 Figure 4.6. Dual fluorescence label provides positive eosinophil (BS-red) identification while showing the difference in nuclear (Hoechst 33258-blue) profiles observed between these cells in the uninfected (A) and infected (B) lamina propria. Bar= 10.0 um. 126 Eosinophil Ultrastructural Changes Ultrastructural analysis of the jejunal segments taken from the experimental animals in this study confirmed the previously described morphological findings (Figure 4.7). Eosinophil specific granule dispersion throughout the cytoplasm was observed in a clear majority of the cells from the infected groups. In contrast, the eosinophils from the uninfected lamina propria showed a relatively dense granule pack in an annular ring around the nucleus. While some ultrastructural heterogeneity in specific granules was observed, no qualitative differences could be attributed to any particular experimental group. Furthermore, no ultrastructural evidence of eosinophil degranulation within the lamina propria was detected and all intact specific granules were located within the confines of plasma membranes. In no instance were eosinophil specific granules found in the extracellular compartment of the tissues examined in this study. Intraepithelial Eosinophil Migration Concomitant with the eosinophil changes described in the aforementioned results, came marked distinctions in their distribution within the VC units of the different experimental groups. In the uninfected animals most eosinophils were found centrally located within the lamina propria of the VC units. This same condition held for the infected jejunum at 2 weeks PI although eosinophil numbers 127 1 do...“ .9 I... .II Figure 4.7. Ultrastructure (TEM) of eosinophils (O) in the lamina propria of the VC units confirmed the morphological findings of increased specific granule dispersion within eosinophils from the infected (B) group when compared to the uninfected (A) tissue. Note that specific granules remain confined within cell plasma membranes in both groups. Bar 2.0 pm. 128 had increased significantly with the majority found near the base crypts of the VC units. In addition, some eosinophils (1-4 cells/VC unit) were seen within the epithelial layer near the base crypts at 2 weeks P], a condition rarely observed (1 cell/10 VC units) in the uninfected jejunum. At 3 weeks P], the eosinophils appeared adherent to the basement membrane of the mucosal epithelium in large numbers and many more (2 - 10 cells/V C unit) were observed within the epithelial layer (Figure 4.8). These intraepithelial eosinophils were in the basal region between enterocytes and not found above the epithelial cell midline. The eosinophil migration through the basement membrane into the epithelium of the jejunal mucosa was confirmed by TEM in the infected animals at 3 weeks PI (Figure 4.9). 129 Figure 4.8. Eosinophils (O) line the basement membrane of the mucosal epithelium by 3 weeks P] (A). In the same time frame many of these cells are found within the epithelial layer (B). Bar = 25.0 pm. 130 Figure 4.9. (A) Transmission electron micrograph of eosinophil (O) migration from the lamina propria (lp) through the basement membrane (bm) and into the epithelium (e) of the jejunal mucosa at 3 weeks PI. (B) Lower magnification from the same experimental group showing an eosinophil in the epithelium wedged between enterocytes. Bar = 1.0 pm. 13] .WW m D m 0 CU Figure 4.9 1 32 Discussion Several theories have been advanced on the cell-mediated events associated with the expulsion of the adult stage Nb from the intestinal tract (Ogilvie & Jones, 1971; Miller & Nawa, I979; Rothwell, 1989; Nawa et al., 1994). While their presence has been noted, (Taliaferro & Sarles, 1939) evidence for an active role for eosinophil leukocytes in warm damage and expulsion has been limited. In this study, we provide documentation of morphological and behavioral changes consistent with functional activity by eosinophils in the primary immune response of the jejunal mucosa to the adult stage of Nb. Earlier studies on mast cell involvement in nematode expulsion have been criticized for not providing evidence of morphological change along with population counts (Rothwell, 1989). Comparisons of infected and uninfected intestines in the current study show not only increased numbers of eosinophils, but also distinct morphological profiles. The eosinophils in the infected jejunum at 2 and 3 weeks PI had widely dispersed specific granules, often making granule detection difficult. The extent of this granule dispersion was best assessed by the 3-dimensional capabilities of BS fluorescent labeling detected by the LSCM with computer reconstruction of the optical sections. This profile was consistent with the dispersion and rapid motion observed by specific granules within eosinophils when exposed to adult stage Nb in vitro (Mackenzie et al., 1981). In that study, the rapid motion and specific granule dispersion preceeded adherence and degranulation of the eosinophils on 133 the surface of the worm, events which required serum (and was greatly enhanced by the addition of complement factors) from immune rats. We also have observed the same specific granule activity in AUG rat peritoneal eosinophils stimulated with calcium ionophore in vitro (unpublished data). The ultrastructural findings of this study clearly place eosinophil specific granules, though often widely dispersed in tissue sections, within the confines of their cell plasma membranes. The eosinophils also showed no ultrastructural evidence of extracellular degranulation within the infected lamina propria. Enteric conditions like adult celiac and Crohn’s disease, for which there is direct evidence of eosinophil degranulation, are characterized by erosion of the tissue architecture of the intestinal wall (Dvorak, 1980; Hallgren et al., 1989; Colombel et al., 1992). The resident cells in the lamina propria of the infected rats in this investigation showed none of the expected effects of exposure to cytotoxic levels of eosinophil granule proteins (Gleich et al., 1992; Walsh et al., 1993). The observed change in eosinophil nuclear profiles and appearance of cytoplasmic vacuoles in the infected lamina propria are consistent with previous findings in vitro as morphological indicators of exposure to cytokines. IL-3, IL-4 and IL-5 are known to specifically influence localized eosinophil accumulation and have been detected in mesenteric lymph nodes of Nb infected rats (Matsuda et al., 1995). These same cytokines, as well as GM-CSF have also been shown to regulate prolonged eosinophil survival and conversion to the “hypodense” 134 phenotype in vitro (Rothenberg et al., 1987; Rothenberg et al., 1988; Owen, Jr. et al., 1991). Moreover, these “hypodense” eosinophils have also demonstrated increased helminth-killing capability in vitro (Owen, Jr. et al., 1987). Nuclear hypersegmentation and cytoplasmic vacuolization observed in eosinophils in this study are accordant with an extended lifespan and the “hypodense” phenotype respectively (Owen, 1993). Adult stage Nb feed on the host mucosal epithelium from days 6-24 P1 in primary infections in the AUG rat, in which time they mate and produce eggs (Mackenzie & Spry, 1983). It is well known that worms established in the intestinal mucosa undergo many degenerative changes prior to expulsion. These changes include damage to the cells lining the alimentary tract of the worm, leading to a loss of fecundity and stunted growth. These changes are requisite for expulsion and once complete, even na‘r‘ve intestines can expel passively transferred damaged worms (Ogilvie & Hockley, 1968). This damage to adult Nb is thought to be mediated by both humoral and cellular components of the immune response (Jones et al., 1970; Rothwell, 1989). We show eosinophil morphology in the infected lamina propria consistent with stimulation toward increased anti- helminthic capability, without evidence of cytotoxicity to the host. These observations support eosinophil involvement in potential damage to adult Nb, as well as a stepwise paradigm of priming prior to the delivery of their cytotoxic granule proteins to the appropriate targets. 135 Our demonstration of the intraepithelial migration of eosinophils in response to the presence of adult Nb in the jejunal lumen of rats conflicts with a previous report in which only large granular lymphocytes (LGL) were reported to exhibit this behavior (Taliaferro & Sarles, 1939). Most likely, this can be attributed to the sensitivity of the detection methodology. Regardless, this finding has many important connotations. Recent work in vitro has shown that eosinophils do not migrate across intestinal epithelium without prior stimulation by GM-CSF (Resnick et al., 1995). As stated previously, the morphologic profile of the eosinophils in the infected lamina propria seen in the present study is consistent with GM-CSF priming. Furthermore, we have demonstrated increased numbers of eosinophils along the basement membrane and in the mucosal epithelium in the successive time points PI. The positioning of these cells is consistent with an active role for eosinophils in the developing gut immunity to primary Nb infection where successive worm establishment is sharply curtailed. Finally, the intraepithelial migration of eosinophils observed in this study supports their role in direct cytotoxic damage to adult stage Nb, perhaps through their ingestion by these worms. l 36 References Africa, CM. (1931). Studies on the host relations of Nippastrangylus muris with special reference to age resistance and acquired immunity. J Parasitol, 18, 1-13. Boyer, M.H., Spry, C.J., Beeson, P.B., & Sheldon, W.H. (1971). Mechanism of eosinophilia. IV. The pulmonary lesion resulting from intravenous injection of Trichinella spiralis. Yale J Biol Med. £1.31, 351-357. Brown, T. (1898). Studies on trichinosis with special reference to the increase of the eosinophilic cells in the blood and muscle, origin of these cells and their diagnostic importance. J Exp Med. 3, 315-347. Butterworth, A. & Thome, K. (1993). Eosinophils and parasitic diseases. In H. Smith & RM. Cook (Eds). Immunopharmacology of Eosinophils (pp. 119-150). New York: Academic Press. Colombel, J .F., Torpier, G., Janin, A., Klein, 0., Cortot, A., & Capron, M. (1992). Activated eosinophils in adult coeliac disease: evidence for a local release of major basic protein. 911;, 33, 1 190-1 194. Dvorak, AM. (1980). Ultrastructural evidence for release of major basic protein- containing crystalline cores of eosinophil granules in vivo: cytotoxic potential in Crohn's disease. J Immunol, 125, 460-462. Gleich, G., Adolphson, C. & Leifennan, K. (1992). Eosinophils. In J. Gallin, I. Goldstein & R. Snyderman (Eds). Inflammation: Basic Principles and Clinical Correlates (2nd ed.) (pp. 663-700). New York: Raven Press. Gorrell, M.D., Miller, H.R., & Brandon, MR. (1988). Lymphocyte phenotypes in the abomasal mucosa of sheep infected with Haemanchus cantartus.. Parasite Immunol, _1_Q, 661-674. Hallgren, R., Colombel, J.F., Dahl, R., Fredens, K., Kruse, A., Jacobsen, N.O., Venge, P., & Rambaud, J .C. (1989). Neutrophil and eosinophil involvement of the small bowel in patients with celiac disease and Crohn's disease: studies on the secretion rate and immunohistochemical localization of granulocyte granule constituents. Am J Med, 83, 56-64. Hua, X., Sugaya, H., & Yoshimura, K. (1990). Alteration in density of eosinophils in the cerebrospinal fluid of mice infected with Angiastrangylus cantanensis.. Int J Parasitol, 2, 443-445. M2 I'd/2i My 137 Jarrett, E.E.E., Jarrett, W.F.H., & Urquhart, GM. (1968). Quantitative studies on the kinetics of establishment and expulsion of the intestinal nematode populations in susceptible and immune hosts. Nippastrangylus brasiliensis in the rat. Parasitology, 38, 625-640. Jones, V.E., Edwards, A.J., & Ogilvie, BM. (1970). The circulating immunoglobulins involved in protective immunity to the intestinal stage of Nippaslrangylus brasiliensis in the rat. Immunology, 18, 621—633. Kayes, S.G., Jones, R.E., & Omholt, PE. (1987). Use of bronchoalveolar lavage to compare local pulmonary immunity with the systemic immune response of Taxacara cams-infected mice. Infect Immun, 33, 2132—2136. Loria Cortes, R. & Lobo Sanahuja, J.F. (1980). Clinical abdominal angiostrongylosis: a study of 116 children with intestinal eosinophilic granuloma caused by Angiastrangylus castaricensis.. Am J Trop Med, 2_9, 538-544. Luna, LG. (1968). Manual of histologic staining methods of the Armed Forces Institute of Pathology (3rd ed.) New York: McGraw-Hill Book Co. Mackenzie, CD. (1980). Eosinophil leucocytes in filarial infections. Trans R Soc Trop Med Hyg, 14, $51-$58. Mackenzie, C.D., Jungery, M., Taylor, P.M., & Ogilvie, BM. (1980). Activation of complement,the induction of antibodies to the surface of nematodes and the effect of these factors and cells on worm survival in vitro. Eur J Immunol, 12, 594- 601. Mackenzie, C.D., Jungery, M., Taylor, P.M., & Ogilvie, BM. (1981). The in-vitro interaction of eosinophils, neutrophils, macrophages and mast cells with nematode surfaces in the presence of complement or antibodies. J Pathol. 133, 161-175. Mackenzie, C.D., Williams, J.F., Sisley, B.M., Steward, M.W., & O'Day, J. (1985). Variations in host responses and the pathogenesis of human onchocerciasis. Eey Infect Dis, 1, 802-808. Mackenzie, C.D., Denham, D.A., & Spry, C.J. (1986). The cellular components of tissue changes in filariasis: fiinctional aspects. J Commun Dis. 18, 308-309. Mackenzie, C.D. & Spry, C.J. (1983). Selective localization of intravenously injected lllindium- labelled eosinophils in rat tissues infected with Nippastrangylus brasiliensis.. Parasite Immunol. Q, 151-163. 138 Matsuda, S., Uchikawa, R., Yamada, M., & Arizono, N. (1995). Cytokine mRNA expression profiles in rats infected with the intestinal nematode Nippastrangylus brasiliensis.. Infect Immun, 63, 4653-4660. McLaren, D.J., Mackenzie, C.D., & Ramalho-Pinto, F.J. (1977). Ultrastructural observations on the in vitro interaction between rat eosinophils and some parasitic helminths (Schistasama mansoni, Trichinella spiralis and Nippastrangylus brasiliensis). C lin Exp Immunol. 3_0, 105-118. Miller, H.R.P. & Jarrett, W.F.H. (1971). Intestinal mast cell response during helminth expulsion in the rat. Immunology, _2_0,, 277-288. Miller, H.R.P. & Nawa, Y. (1979). Nippastrangylus brasiliensis: Intestinal goblet- cell response in adaptively immunized rats. Exp Parasitol, 41, 81-90. Nawa, Y., lshikawa, N., Tsuchiya, K., Horii, Y., Abe, T., Khan, A., Itoh, B.H., Ide, H., & Uchiyama, F. (1994). Selective effector mechanisms for the expulsion of intestinal helminths. Parasite Immunol, 16, 333-338. Neva, F .A. & Otteson, EA. (1978). Tropical (filarial) eosinophilia. N Engl J Med, 228,1129-1131. Ogilvie, BM. (1965). Role of adult worms in immunity of rats to Nippastrangylus brasiliensis.. Parasitology, 33, 325-335. Ogilvie, B.M., Mackenzie, C.D., & Love, R.J. (I977). Lymphocytes and eosinophils in the immune response of rats to initial and subsequent infections with Nippastrangylus brasiliensis. Am J Trop Med Hyg, 26, 61-67. Ogilvie, B.M. & Hockley, DJ. (1968). Effects of immunity on Nippastrangylus brasiliensis adult worms: reversible and irreversible changes in infectivity, reproduction and morphology. J Parasitol, fl, 1073-1084. Ogilvie, B.M. & Jones, VB. (1971). Parasitological review. Nippastrangylus brasiliensis: a review of immunity and host-parasite relationship in the rat. Exp Parasitol 22, 138-177. Ogilvie, B.M. & Love, R.J. (1974). Cooperation between antibodies and cells in immunity to a nematode parasite. Transplant Rev, E, 147-155. Ott, L. (1988). An introduction to statistical methods and data analysis. (third ed.) Boston: PWS-Kent Publishing Company. 139 Owen, W.F. (1993). Eosinophil Heterogeneity. In H. Smith & RM. Cook (Eds). Immunopharmacolog of Eosinophils (pp. 57-69). Owen, W.F., Jr., Rothenberg, ME, Silberstein, D.S., Gasson, J.C., Stevens, R.L., Austen, K.F., & Soberman, R.J. (1987). Regulation of human eosinophil viability,density,and function by granulocyte/macrophage colony-stimulating factor in the presence of 3T3 fibroblasts. J 13g) Med, 166, 129-141. Owen, W.F., Jr., Petersen, J., & Austen, K.F. (1991). Eosinophils altered phenotypically and primed by culture with granulocyte/macrophage colony- stimulating factor and 3T3 fibroblasts generate leukotriene C4 in response to FMLP. J Clin Invest. 82, 1958-1963. Resnick, M.B., Colgan, S.P., Parkos, C.A., Delp-Archer, C., McGuirk, D., Weller, P.F., & Madara, J.L. (1995). Human eosinophils migrate across an intestinal epithelium in response to platelet-activating factor. Gastroenterology, 168, 409- 416. Reynolds, ES. (1963). The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J Cell Biol, 11, 208-2]2.(abstract). Rothenberg, M.E., Owen, W.F., Jr., Silberstein, D.S., Sobennan, R.J., Austen, K.F., & Stevens, R.L. (1987). Eosinophils cocultured with endothelial cells have increased survival and functional properties. Science, 237, 645-647. Rothenberg, M.E., Owen, W.F., Jr., Silberstein, D.S., Woods, J., Sobennan, R.J., Austen, K.F., & Stevens, R.L. (1988). Human eosinophils have prolonged survival,enhanced functional properties,and become hypodense when exposed to human interleukin 3. J Clin Invest, 81 , 1986-1992. Rothwell, T.L.W. (1989). Immune expulsion of parasitic nematodes from the alimentary tract. Int J Parasitol. 12, 139-168. Symons, LEA. (1957). Pathology of infestation of the rat with Nippastrangylus muris (Yokogawa). 1. Changes in water content, dry weight and tissues of the small intestine. Aust J Biol Sci, 16, 374-383. Taliaferro, W.H. & Sarles, MP. (1939). The cellular reactions in the skin, lungs and intestine of normal and immune rats after infection with Nippastrangylus maria. J Infect Dis, 64, 157-192. 140 Walsh, G.M., Wardlaw, A.J. & Kay, AB. (1993). Eosinophil accumulation, secretion, and activation. In H.Smith and R.M.Cook (Ed). Immunopharmacology of Eosinophils (pp. 73-90). New York: Academic Press. Westcott, R.B. & Todd, AC. (1966). Adaptation of Nippastrangylus brasiliensis to the mouse. J Parasitol, _52, 233-236. Yii, CY. (1976). Clinical observations on eosinophilic meningitis and meningoencephalitis caused by Angiastrangvlus cantanensis.. Am J Trop Med Eyg, 23, 233-249. Chapter 5 THE PERITONEAL RESPONSE TO NIPPOSTRONG YLUS BRASILIENSIS IN THE AUGUST RAT Introduction At the turn of the century Ehrlich (1900) suggested that “eosinophils exert their functions in tissues”. Subsequent research has led to a general consensus among investigators that eosinophils are primarily tissue-dwelling cells. However, due to the ease of access, the changes in eosinophil numbers in the peripheral blood during disease has often been the singular diagnostic standard. Blood eosinophil counts have been found to be most accurate during steady-state flux between bone marrow and tissues (Spry, 1993). Therefore, this measure cannot account for marginated vascular pools, localized tissue accumulations. or functional changes common to eosinophil-associated disease (Butterworth & Thome, 1993). Clearly, the utility of measuring circulating cell numbers alone as a indication of overall behavior by eosinophils in a given immune response is limited. The peritoneal cavity is the most extensive serous membrane in mammals with a surface area roughly equal to that of the skin (diZerege & Rodgers, 1992). It is well known that the serous exudate of the peritoneal cavity in man (Roberts et al., 141 142 1990) and rodents (see Chapters 1& 2) contains a mixture of migratory cells with various immunoregulatory capabilities. The cells include: mononuclear phagocytes, lymphocytes, eosinophils, connective tissue mast cells and neutrophils. Specialized folds of the peritoneal membrane form the omenta. Within the omenta, aggregates of these migratory cells called “milky spots or omental glomeruli” have been observed and are considered to be the source of the cells in the serous exudate of the peritoneum (Cranshaw & Leak, 1990). These specialized lymphoid organs of the peritoneum have been shown to be highly reactive to various inflammatory stimuli by demonstrating antigen trapping, cellular proliferation and elicitation of inflammatory mediators (Shimotsuma et al., 1993). Peritoneal reactivity to Nippastrangylus brasiliensis (Nb) was demonstrated through direct instillation of infective larvae into the peritoneal cavity of rats. This caused adherence of resident macrophages to the worms and trapping against the omentum which led to death of the immobilized larvae (Greenberg & Wertheim, 1973). The peritoneal immune responses to helminth parasites are not firlly characterized. Furthermore, most of the literature follows the approach of direct intraperitoneal antigen exposure (see Chapter 1, Animal models of peritoneal eosinophilia) with little or no data on peritoneal reactivity to the natural course of infection. In the present study we characterize aspects of the cellular and functional responses of the peritoneal exudate and omental membranes during the intestinal 143 phase of primary Nb infection in AUG rats. This will be accomplished by quantifying changes in cell populations and leukotriene C4 levels in the serous exudate of the peritoneal cavity. Histological changes in the omental tissues will be described through light microscopy. In addition, the eosinophilic response of the peripheral blood to Nb infection will be assessed and compared to the accumulation of eosinophils in the peritoneal cavity at specific time points in relation to the host immune response. Methods and Materials Experimental Design Six-week-old August (AUG) rats of mixed parentage and sex were placed into 4 experimental groups (n = 6-11); two groups were infected with 3000 Nb infective larvae (L3) injected subcutaneously into the right hind leg. The remaining groups were similarly injected with sterile saline and served as age- matched uninfected controls. Sample harvests were performed on one infected and one uninfected group at 2 weeks post infection (PI) followed by the remaining groups at 3 weeks PI. Reagents All chemical reagents were obtained from the SIGMA Chemical Company (St. Louis, MO) unless otherwise noted. l 44 Nb Cultures The parasites were maintained in mice by methods previously described (Westcott & Todd, 1966). Fecal pellets were obtained on day 8 and 9 PI. These were macerated and mixed with activated charcoal and cultured at 19°C. The L3 larvae were harvested from cultures between 8-20 days old in a Baermann apparatus filled with water. The concentrated larvae were counted, brought up in sterile saline and adjusted to 3000/ml for injection. Animals Sibling breeding pairs of August (AUG) rats were obtained from Harlan Olac Limited, Bicester, England and the National Institute for Medical Research, London. Colonies of inbred progeny were established at the Biological Sciences Department, Western Michigan University, Kalamazoo, MI and the Division of Animal Health, The Upjohn Company, Kalamazoo, MI and were the source of research animals for this study. All animals were housed in accordance with the Institutional Animal Care and Use Committee (IACUC) of the respective institutions. The colonies were maintained under a l2hr/12hr-light/dark cycle at 21-28°C with rodent chow #5001 (Purina Mills, St. Louis, MO) and reverse osmosis purified water provided ad Iibitum. Animals were killed prior to tissue collection by lethal carbon dioxide inhalation. 1 45 Peripheral Blood Samples Peripheral blood was taken from the rats immediately after euthanasia from tail snips, and was smeared between microscope slides and allowed to air dry. Additionally, an aliquot of peripheral blood was drawn into a white cell pipette (Becton-Dickinson, Cockeysville,MD) and diluted with Turk’s solution for total white blood cell (WBC) determination. Air dried peripheral blood smears were fixed for 10 minutes in cold (-20°C) methanol and stained with Diff-Quick® stain kit (Baxter Healthcare, McGraw Park, IL). Differential determinations were made upon the first 300 WBCs/animal counted (fiom 2 smears) and expressed as mean percents (%) of the total. The Turk’s diluted peripheral blood was counted (2x) according to the method of ‘ Brown (1984) in a Neubauer hemocytometer (American Optical, Buffalo, NY) and expressed as mean WBC/pl. All cell counting procedures were performed on a Nikon (Tokyo, Japan) Microphot-FXA® research grade microscope. Peritoneal Lavage Peritoneal lavage was performed utilizing calcium- and magnesium-free Hanks’ Balanced Salt Solution (HBSS) with 20 mM HEPES buffer at pH 7.3 kept on wet ice. This collection was accomplished by making a small (1-3 cm) incision on the lower abdomen adjacent to the ventral midline of the animal to irrigate the peritoneal cavity with 30-50 ml of the lavage solution. The resident peritoneal 146 cells and the lavage solution were removed within 5 minutes by syringe and pelleted by centrifugation at 200 x g for 10 minutes at 4°C in a Beckman (Fullerton, CA) centrifuge. The lavage supernatant was decanted and the cell pellet brought to 5 ml with HBSS with 20 mM HEPES and held on wet ice. Total cell counts were done on a Neubauer hemocytometer as stated above. Cytocentrifuge preparations were carried out with a Cytospin 3® (Shandon Scientific, Cheshire, England) and stained with the Diff-Quick® system and differential cell determinations performed as described for WBCs The lavage supematants were diluted (3x) with ice-cold methanol and stored at -70°C until LTC4 determinations were performed. Lavage LTC4 content was determined by an acetylcholinesterase (AChE) enzyme immunoassay kit (Caymen Chemical, Ann Arbor, MI) developed by a previously described method (Pradelles et al., 1985). Prior to determination, 4 ml samples were thawed and centrifuged (1500 x g for 10 minutes at 4°C) to remove precipitates and cell debris. The resulting supematants were dried in a heated water bath (50°C) under a continuous stream of nitrogen gas. Dried samples were then reconstituted to 1 ml in assay buffer and stored overnight at 4°C before LTC4 determinations were done in duplicate. Results were interpreted from standard curves, generated with each assay, of percent displacement of bound LTC4 AChE tracer versus LTC4 concentration (pg/ml). 147 Omental Samples Portions of the greater omentum (gastro-splenic section) were excised and placed in 10% neutral buffered formalin (NBF) for 15 minutes at RT and then the NBF was replaced with fresh fixative and the samples stored at RT until paraffin embedment. After embedment, histological sections were cut (6-10 pm) and stained by Luna’s (1968) method for eosinophils. Statistics All numerical data is expressed as mean 3: SEM. Equal variance was tested by Hartley’s homogeneity of variance method. Statistical significance was determined by one-way analysis of variance (ANOVA) with a 95% confidence limit (p = 0.05). Groupwise comparisons were made utilizing Student-Newman- Kuels (SNK) a posteriori test (Ott, 1988). Results Peripheral Blood Counts Absolute blood eosinophil numbers increased significantly in the infected animals at 2 weeks PI (Figure 5.1). Peripheral blood eosinophil counts in uninfected animals were 158 j: 49 (range = 0-492) cells/u] compared to 827 i 170 (range = 51-1 ,746) cells/pl for the 2 weeks PI experimental group. However, by 3 weeks P], the peripheral blood eosinophil count had returned to uninfected levels, at 197 :t 93 (range = 0-734) cells/pl. 148 1100 , *- 1000 -~4 900 -3 800 700 600 500 400 CELLS/MICROLITER 300 200 - 100 UNINFECTED 2WK PI 3WK PI GROUPS Figure 5.1. Eosinophils in the peripheral blood of Nb infected and uninfected AUG rats. A significant (*) increase was observed in the 2wk PI animals but returned to levels observed in the uninfected animals by the 3 wk PI time point. 149 Peritoneal Eosinophilia Significant increases in the total cell harvest by peritoneal lavage were observed in both the 2-week and 3-week Pl groups compared to the uninfected animals (Figure 5.2). The peritoneal exudate population in the uninfected group was 19.9 i 1.2 x 106 cells/animal (range = 15.0-26.9). This number increased to 35.8 i 2.2 x 106 cells/animal (range = 23.5-44.3) by 2 weeks PI. The accumulation continued in the 3 week PI group with peritoneal cell harvests reaching 40.8 i 1.9 x 106 cells/animal (range = 33.5-47.8). As described in Chapter 2, the cells found in these lavages include: mononuclear cells (macrophages and large lymphocytes), eosinophils, mast cells and small lymphocytes. This cellular constitution did not change between groups in this study but a trend (not significant) of increased percentages of eosinophils was noted in the infected groups. This trend, coupled with the overall increase in cellularity, led to significant absolute eosinophil accumulation in the infected peritoneal cavities (Figure 5.3). Uninfected levels were 6.6 i 0.4 x 106 eosinophils/animal (range = 4.0-8.9). Eosinophil numbers rose to 15.6 i 1.3 x 106 (range = 9.2-22.6) and 18.0 i 1.9 x 106 (range = 1 1.6-24.4) cells/animal by 2 and 3 weeks PI, respectively. 150 40 30 20 CELLS In MILLIONS 10 UNINFECTED 2WK PI 3WK PI GROUPS Figure 5.2. Total cell harvest from the peritoneal cavities of Nb infected and uninfected AUG rats. Total cells harvested in both infected groups were significantly (*) increased over the uninfected levels. 15] 20 10- CELLS In MILLIONS UNINFECTED 2WK PI 3WK PI GROUPS Figure 5.3. Eosinophils in the peritoneal cavities of Nb infected and uninfected AUG rats. The infected animals had significantly (*) higher numbers of eosinophils than the uninfected animals. 1 52 Peritoneal LTC4 Levels LTC4 levels in the peritoneal lavage from the experimental groups are represented in Figure 5.4. The LTC4 concentration was found to be 14.5 i 2.0 pg/ml (range = 6.2-26.0) in the lavage from uninfected animals. This level was mirrored (14.9 i 1.6 pg/ml, range = 96-218) by the 2 week PI group. However, a 3-fold increase in LTC4 was measured in the 3-week PI group, reaching 48.] i 5.9 pg/ml in the peritoneal lavage. This level in the 3-week PI group was found to be a statistically significant increase, compared to the other groups. Omental Change As a specialized fold of the mesentery, the greater omentum lies attached to the greater curvature of the stomach, extends from the spleen and drapes over the anterior portion of the small intestine. Changes were evident in the greater omentum of the Nb infected groups including hyperemia and increased cellularity of the milky spots. These conditions produced an overall increase in the surface area of the omentum, roughly doubling in size in the infected groups. Histological examination revealed increased populations of mononuclear cells (macrophages and lymphocytes) and eosinophils within the layers of omental connective tissue (Figure 5.5). 153 60 "* pglml of LTC4 UNINFECTED 2WK PI 3WK PI GROUPS Figure 5.4. Leukotriene C. levels in the peritoneal exudate of Nb infected and uninfected AUG rats. Only the 3 week PI animals had significantly (*) increased LTC4 in their peritoneal cavities when compared to the uninfected group. 154 Figure 5.5. Aggregates of cells around the “milky spot” of the omentum (as seen in the uninfected (A) animals), showed an increased cellularity which included eosinophils (O) in the Nb infected (B) animals. Bar = 15.0 um. 155 Figure 5.5 1 56 Discussion The peripheral blood data indicates a significant rise in eosinophils only in the 2-week PI group. Peripheral blood eosinophil count in the 3 week PI group returned to uninfected levels and this blood profile is consistent with a similar profile in bone marrow eosinophil counts from another study (Ogilvie et al., 1977b). However, these data are not consistent with the behavior of the tissue eosinophils in response to Nb infection in this and our previous study (see Chapter 4). In that work, eosinophils in the jejunal mucosa continued to rise through the 3 week PI time point and this was paralleled in the peritoneal cavity in the present study. Additonally, the ranges observed in peripheral blood eosinophil counts showed more variability between the infected animals than the peritoneal counts. Overall, eosinophil counts of peritoneal lavage provides a higher degree of accuracy for tracking the enteric phases of eosinophilia than peripheral blood in this model. Currently, we have provided evidence demonstrating reactivity of the peritoneal cavity to the intestinal phase of Nb infection. Previously we demonstrated the consistency of resident peritoneal cell populations in the uninfected AUG rat in Chapter 2. In this study, this consistency was altered in the course of primary Nb infection by an almost 2-fold increase in the total cellular complement of the peritoneal cavity which included a 3-fold increase in eosinophils. Additionally, the morphologic alterations in the omental milky spots 157 were consistent with work in mice which showed these specialized lymphoid organs as a source of peritoneal cells in immunologically stimulated (Cranshaw & Leak, 1990) and Schistasama mansoni infected (Weinberg et al., 1992) animals. One mechanism of cellular traffic in the peritoneum is by the exit of these migratory cells through the lymphatic portals of the diaphragm (Leak & Rahil, 1978). This trafficking of cells through the peritoneal cavity provides a paradigm within the enteric microenvironment where soluble and cellular components of the primary immune response to Nb infection can interact to produce immunocompetent effector cells for systemic redistribution. Current theory suggests that the control over which arm of the acquired immune response that predominates (Th1 vs Th2) requires specialized instructional roles by antigen- presenting cells in microenvironmental interactions with na‘r've T-cells (Fearon & Locksley, 1996). Weller and colleagues (1993) have demonstrated antigen- presentation capabilities in cytokine-stimulated eosinophils. Moreover, peritoneal- derived eosinophils in rat helminthiases have been shown to have increased cytotoxic responses in vitro (Cook et al., 1987) and can confer protective immunity to infection when passively transferred to na'r've recipients (Ogilvie et al., 1977a; Ogilvie et al., 1977a; Capron et al., 1984a). These experiments include conveyance of the ability to expel adult Nb from na'r've intestines after the passive transfer of peritoneal cells taken after day 12 PI from infected donor rats and put into the peritoneal cavity of the recipients (Ogilvie et al., 1977a). 158 Previous investigations have demonstrated the presence of LTC4 (ng/ml levels) in the intestinal mucosa and gut lumen during Nb infection (Moqbel et al., 1986; Perdue et al., 1989). These studies concentrated on the acute phase of the anaphylactic shock response in the small intestine to massive secondary challenge and implicated eosinophils and mucosal mast cells as the LTC4 sources. Our findings during primary Nb infection Show peak peritoneal levels (pg/ml) of LTC4 at 3 weeks PI. It is difficult to ascribe a similar biological significance to these findings when. compared with the previous work where LTC4 levels were found to be several hundred times higher. However, blocking of the lipoxygenase pathway in resident peritoneal cells causes an inhibition of saline-induced eosinophil accumulation in the peritoneal cavity (Oliveira et al., 1994). thus demonstrating the importance of eicosinoid metabolism by these cells in peritoneal eosinophilia. Minimally, the change in LTC4 observed in the current study is an indication of an immunologic stimulus with potential regulatory effects on all of the resident peritoneal cells during the intestinal phase of Nb infection. In conclusion, we have demonstrated peritoneal reactivity in the intestinal phase of primary Nb infection in rats. The observed eosinophilic response of the peritoneal cavity reflects a more accurate indication than blood of the worth- specific eosinophil response in the intestinal mucosa without losing the practically useful ease of access. Further work must be done to ascertain the interactions that 159 occur within the peritoneal cavity which help produce competent effector cells in the immune response to helminth parasites. l 60 References Brown, BA. (1984). Hematolggy: Principles and Procedures. (4th ed.) Philadelphia: Lea &Febiger. Butterworth, A. & Thome, K. (1993). Eosinophils and parasitic diseases. In H. Smith & RM. Cook (Eds). Immunopharmacolog of Eosinophils (pp. 119-150). New York: Academic Press. Capron, M., Nogueira-Queiroz, J.A., Papin, J .P., & Capron, A. (1984). Interactions between eosinophils and antibodies: in vivo protective role against rat schistosomiasis. Cell Immunol, 83, 60-72. Cook, R.M., Musgrove, N.R.J., & Ashworth, RF. (1987). 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Chapter 6 CONCLUSIONS This thesis has centered around investigating eosinophil function and behavior in viva with the development of tools toward this end; most of this work has relied on morphological and ultrastructural assessment methods. Despite the advanced state of molecular biology today many of the cell-cell interactions that occur in the pathophysiological processes between specific immunoregulatory cells and tissues still remain unattainable due, in part, to a scarcity of in situ probes and well characterized experimental models. Morphology based assessment has historically proved invaluable for developing new lines of evidence to broaden the knowledge of eosinophil firnction in viva, and is again utilized in this current work. The data presented in Chapter 2 supports the hypothesis that the eosinophil accumulation observed in the peritoneal cavities of AUG rats is the result of phenotypic expression of a heritable trait or traits in this inbred strain. We showed that the expression of peritoneal eosinophilia develops with time reaching maximal numbers by the 8th week of life. No differences were found between males and females with regard to peritoneal eosinophil accumulation either in timing of appearance or extent of accumulation. Further analyses of the eosinophil component of peripheral blood and the intestinal mucosa showed AUG rats to be 163 164 within the normal range of other rat strains in this respect. These findings help negate pathOphysiologicalimechanisms as the primary cause in the peritoneal accumulation of eosinophils observed in these rats. Thus, this animal model provides a readily available source of tissue-derived eosinophils in an immunologically resting state. In addition, the genetic stability of this rat through its inbred nature has provided for very consistent expression of this trait throughout all of the studies which have encompassed many generations of these animals from our own breeding colony. The characterization of this trait in the August strain of rats provided a solid base for the subsequent research of this thesis. With a paucity of eosinophil-specific antisera, the most dependable means of identifying eosinophils still revolves around the chemical staining properties of the proteins found in eosinophil specific granules. However, difficulties can arise when specific granule packaging is altered during pathophysiological responses by the cell. Histological dyes that rely solely on the property of interference of light often lose their intensity when the density or relative position of the specific granules within eosinophils are changed, such as in inflamed tissues. This can lead to under-representation of eosinophil involvement in a given condition. We addressed this problem in part, by the development of a new photoreactive fluorescent method utilizing Biebrich scarlet for the detection of eosinophil specific granules (see Chapter 3). The granule protein-Biebrich scarlet complexes 165 gave deep red fluorescent emissions which provided superior microscope imaging of eosinophil involvement in tissue even when specific granules were widely dispersed within the cells. This method provided exemplary results and holds much promise for firture work with technologies that require fluorescent probes, such as laser scanning confocal microscopy, to assist in delineating activities in eosinophil specific granules. The infection of rats with the nematode Nippostrongylus brasiliensis has been used to characterized many aspects of host-parasite relationships. While the presence of eosinophil leukocytes has been described in the responses to the intestinal phase of Nb, no specific action has been ascribed to these cells. Operating under the hypothesis that eosinophil leukocytes play an active role in the intestinal immune response to the adult stage of Nb we proceeded with the investigations described in Chapter 3 and 4 of this thesis. Our results indicated a clear difference in eosinophil accumulation in the jejunal villus-crypt units between uninfected and Nb infected animals. Initial work showed a time point between 2 week and 3 week P1 to be most appropriate to study eosinophil accumulation in the intestinal wall. These results were consistent with previous work in primary Nb infection describing the arrival of the worms in the proximal jejunum to occur between days 6-19 P1 in AUG rats. With primary expulsion expected between day 12-14 P], the timing of eosinophil accumulation 166 was compatible with these cells’ participation in the rejection of existing Nb adults- and in possibly preventing the establishment further incoming worms. More detailed work, as described in Chapter 4, confirmed these preliminary findings on eosinophil accumulation in the intestinal wall. Moreover, the morphological profiles of the eosinophils that had collected in the intestinal wall of the infected rats were distinctly different fi'om those found in uninfected animals. We carefully detailed these eosinophil changes through Nomarski (DIC) optics, as well as histological, fluorescence and ultrastructural technologies. The changes seen in eosinophils observed in Nb infected VC units included dispersion of specific granules within their cytoplasm, nuclear changes and emigration of the cells themselves from the lamina propria into the intestinal epithelium. These changes were concordant with eosinophil involvement in immune rejection of the intestinal phase of Nb from rats. Furthermore, the observed intraepithelial migration of eosinophils provides support for their role in direct cytotoxic damage to adult stage Nb. This strategic location of these cells in the epithelium allows for their ingestion by feeding Nb and the concerted or involuntary delivery of eosinophil cytotoxic proteins to the wonn’s gut. The results presented in Chapter 5 demonstrate peritoneal reactivity to primary Nb infection in the AUG rat model. As discussed in the review of literature (see Chapter 1), studies abound that demonstrate peripheral blood eosinophilia in response to infection by helminth parasites while ignoring the important tissue 167 phases. We compared eosinophil accumulation in the blood and the peritoneal cavity during the intestinal phase of Nb in rats and found the peritoneal profile to be a more accurate reflection of eosinophil involvement in the intestinal tissue. The highly variable measure of peripheral blood eosinophilia is at best an indication of bone marrow release and transit of these cells usually falling to control levels at the time that tissue activity crested. In contrast, the peritoneal accumulation of eosinophils reaches a peak in concert with eosinophil levels in the jejunal VC units. We also found an increased cellularity (including eosinophils) in the omental “milky spots” lending support for these sites as portals for eosinophil traffic in the peritoneal cavity. Our results on LTC4 levels in the peritoneal exudate are somewhat different from the studies of others. Past work involved massive secondary challenges with Nb and showed high levels (ng/ml in gut and plasma) of LTC4 in support of anaphylaxis as a mechanism of expulsion. The findings of our primary Nb challenge did not show increased LTC4 in the peritoneal exudate over uninfected animals at the 2 week Pl time interval, which would correspond to the time of warm expulsion. We did, however, measure significant increases at pg/ml levels in the 3 week PI group coinciding with the maximal eosinophil accumulation in the peritoneal cavity and the intestinal wall. This level of LTC4 exposure (and plausible release) by the cells in the peritoneal cavity of infected rats may indicate a functional priming of cells and/or the development of an immune response. At 168 the very least, this evidence supports the concept of an altered status of the peritoneal exudate in response to primary infection by Nb. In common with all scientific inquiry, this work has produced many new questions that are ripe for investigation. Of primary importance is an understanding of the mechanisms surrounding the intraepithelial migration of eosinophils in response to adult Nb in the gut lumen. In addition, the elucidation of the potential cytotoxic role that these cells play in damaging enteric parasites in viva may provide valuable insights into the roles that eosinophils play as a first line of immune defense from the extracorporeal environment. Toward this end, further development of Biebrich scarlet as an eosinophil fluorophore should be productive. 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