OVERDUE FINES: 25¢ per 40' per item RETUMI" LIBRARY MTERIALS: Place in book return to mauve charge from circulation records IMMUNOGLOBULIN-CONTAINING CELL POPULATIONS IN TISSUES OF RATS INFECTED WITH TAENIA TAENIAEFORMIS BY Martha C. Lindsay A THESIS Submitted to , Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1981 k? ABSTRACT IMMUNOGLOBULIN-CONTAINING CELL POPULATIONS IN TISSUES OF RATS INFECTED WITH TAENIA TAENIAEFORMIS BY Martha C. Lindsay Immunoglobulin-containing inflammatory cells appear in murine tissues infected with migrating and sessile helminths. The purpose of this study was to characterize the appearance of IgE antibody-containing cells during the course of infection with the ceStode, Taenia taeniae- formis. We used immunofluorescence techniques and demonstra- ted that IgE-containing cell populations increased both in the intestinal mucosa and surrounding metacestodes in the liver. However, not all of these were plasma cells and many corresponded morphologically, histochemically and in their responses ig_zivg to dexamethasone and com- pound 48/80 to mucosal mast cells (Enerback, Acta Path et Microbiol. Scandinav., 66:303, 1966). Mayrhofer, et al. (Eur. J. Immunol., 6:537, 1976) first described IgE—containing mucosal mast cells in the intestinal mucosae of rats infected with Nippostrongylus brasiliensis. Our results now extend these findings to a different hosteparasite system and demonstrate the appearance of IgEvpositive mucosal mast cells in the liver of T. taeniaeformis—infected rats. The origin and possible function(s) of theSe cells are discussed. To my parents and my husband ii ACKNOWLEDGEMENTS My deepest gratitude goes to my advisor, Dr. Jeffrey F. Williams. I have learned much from his advice, pa- tience and foresight which have guided me through my graduate and veterinary school years. I could not have completed this work without his understanding and con- certed efforts on my behalf. I owe my thanks to my fellow workers, Dr. Anthony Musoke, Dr. Roger Cook, John Picone, Steven Hustead, Anne Zajac, Dr. George Conder, Dr. Tjaart Schillhorn van Veen, Dr. John Kaneene, Chuck Munsell, David Blaies and Myrnice Ravitch. They all offered advice and their ' friendship over the past 5 years. Ms. Marla Signs and Ms. Alma Shearer provided expert technical support for this work and I greatly appreciate the excellent secre- tarial assistance of Mrs. Margaret Toomey and Mrs. Kate Baird. Thanks to them all - my door will always be open. iii TABLE OF CONTENTS LIST OF TABLES . . . . . . . . . . . . . . . . LIST OF FIGURES. . . . . . . . . . . . . . . . INTRODUCTION . . . . . . . . . . . . . . . . . LITERATURE REVIEW. . . . . . . . . . . . . . . Humoral Immunity to Taeniid Infection . Circulating proteCtive antibodies . . Immunoglobulin-producing cells and gut immunity. . . . . . . . . . . . Immunoglobulin E (IgE). . . . . . . . Cellular Immunity to Taeniid Infection. Histopathology of‘Taenia taeniaefOrmis Infection . . . . . . . . . . . . . . Hepatic cellular responses. . . . . . Intestinal cellular responses . . . . LIST OF REFERENCES . . . . . . . . . . . . . . ARTICLE 1 - IMMUNOGLOBULIN E-CONTAINING CELLS IN INTESTINAL AND LYMPHATIC TISSUES OF RATS INFECTED WITH TAENIA ‘ TAENIAEFORMI S O O O C O O O O I O O ARTICLE 2 - MAST CELLS IN RATS INFECTED WITH TAENIA TAENIAEFORMIS . . . . . . . ARTICLE 3 - A SUPERIOR FIXATIVE FOR THE DETECTION OF IMMUNOGLOBULIN E- CONTAINING MAST CELLS IN IMMUNO- FLUORESCENCE STUDIES . . . . . . . iv 29 69 102 Table LIST OF TABLES Page ARTICLE 1 Total IgE-containing cell counts and fluorescent MMC counts in rats at in- tervals after infection with Taenia taeniaeformis and in age-matched controls. . . . . . . . . . . . . . . . 48 ARTICLE 2 Effect of treatment with Compound 48/80, Dexamethasone or saline on mucosal mast cells in rats infected with Taenia taeniaeformis . . . . . . . 82 Figure LIST OF FIGURES Page ARTICLE 1 Fluorescence micrographs illustrating trends in IgE-positive cell popula- tions in duodenal mucosae of infected rats. . . . . . . . . . . . . . . . . . . 42 IgE-containing MMC in the duodenal mucosa of a Taenia taeniaeformis-infected rat 0 I O O O O O O O O O O O O O I O O O 45 IgE-positive fluorescent cells in the duodenal mucosa of rats infected with Taenia taeniaeformis and in age-matched controls. . . . . . . . . . . 50 Cells containing IgE in the duodenal mucosa of a Nippostrongylus brasiliensis- infected rat. . . . . . . . . . . . . . . 52 ARTICLE 2 Comparison of mast cell responses in rats infected with g. taeniaeformis and treated with Compound 48/80 or dexamethasone on days 27-32 after exposure. . . . . . . . . . . . . . . . . 84 Fluorescence micrographs demonstrating the decline in the IgE—positive hepatic cell population around metacestodes of Taenia taeniaeformis. . . . . . . . . . . 90 ARTICLE 3 Fluorescence micrographs of duodenal villi. . . . . . . . . . . . . . 107 vi INTRODUCTION Cysticercosis and hydatidosis were considered for- merly to be of significant public health and economic importance in underdeveloped countries, but they are now becoming of increasingly greater concern to indus- trialized nations. A number of recent reviews of the literature on taeniid infections are available which provide a comprehensive coverage of the biology, epidem- iology, and immunology of these parasites and which address the current public health and economic aspects of tapeworm infections in man and domestic animals [Gemmell and Johnstone 1977; Matossian et a1., 1977; Hird and Pullen, 1979]. The life cycles of all these cestodes involve inter- mediate hosts (e.g., cattle, sheep, swine) in the mus- culature or visceral organs of which the larval parasites establish and grow. Their presence in food animals often results in partial or total carcass condemnation and in man they may cause serious diseases. The definitive hosts (e.g., man, dogs) ingest parasitized tissues and the cystic larvae develop into adults in the intestine. Zoonotic cestode infections such as these pose special problems for experimental work, but Taenia taeniaeformis is transmitted only between cats and ro— dents, and it therefore serves as a convenient and safe laboratory model for studies of immunity to cestode infec- tion. Infective eggs from the cat are ingested by the rat, and hatch in the small intestine.. The liberated activated oncospheres penetrate the intestinal wall and migrate to the liver. Metacestodes can survive in the liver for prolonged periods despite the early onset of marked inflammatory cellular reactions and protective antibody production by the host. This thesis concerns the characterization of mast cell reactions in experimental taeniiasis in rats, and the findings have important implications for understanding the hosteparasite relationship in other cestode infec— tions. The literature review therefore summarizes the most salient characteristics of humoral and cellular immunity in taeniid parasitism, with emphasis on the protective responses of animals infected with T. taeniae- formis. Where no specific information is available for T.‘taeniaeformis infections, examples are drawn from studies of other intestinal or hepatic helminthiases. which illustrate phenomena relevant to the discussion of mast cells and their function and role in cestodiasis. Comprehensive reviews of the immune responses to cestode infection in_general have been compiled (Weinmann, 1966, 1970; Gemmell and MacNamara, 1972; Williams, 1979), and Leid (1977) recently reviewed the status of.our know- ledge of immunity in'Taenia taeniaeformis infection. The following two sections focus upon those aspects of the immune responses to taeniid infection which are related to protection against challenge, and which.form an im- portant part of the background to the work reported be- low. The third section deals with literature on histo— pathological responses in rodent taeniiasis with particu— lar emphasis on the participation of mast cells and their relationship to immunoglobulins. LITERATURE REVIEW Humoral Immunity to Taeniid Infection Circulating_protective antibodies There is substantial evidence for the development of active immunity to taeniid metacestodes (Urquhart, 1961; Blundell et al., 1968; Gemmell et al., 1969) and many workers have demonstrated passive transfer of re- sistance (e.g., Taenia hydatigena [Blundell et al., 1968, Gemmell et al., 1969); Taenia ovis (Rickard and Arundel, 1974), Taenia pisiformis and Taenia taeniae- formis (Miller and Gardiner, 1932, 1934; Campbell, 1938]). Leid and Williams (1974) confirmed and extended the studies on T. taeniaeformis infections, and showed that 7852a is the major protective antibody produced in in- fected rats by 14 days after infection (DAI). Although colostral IgA can protect weanling rats (Musoke et al., 1975) and intestinal IgA was found to be protective a- gainst a T. taeniaeformis challenge infection in mice (Lloyd and Soulsby, 1978), IgA is not essential for pro- tection in rats (Leid and Williams, 1974; Leid, 1977). Many factors have been found to influence the class of protective antibodies produced. Firstly, susceptible host species vary in the type of antibody produced. Mice 4 develop protective-7S;1 antibody to T. taeniaeformis, while infected rats produceprotective‘7sx2a (Musoke and Williams, 1975a). SeCondly, the route of infection is important because when rats are immunized by the intra- peritoneal implantation of T. taeniaeformis, the pro- tective antibodies produced are of the classes 7851 and IgM (Musoke and Williams, 1975b). Thirdly, the age of the parasites is a significant factor because other classes of protective antibody appear as infection pro- gresses (Musoke and Williams, 1975a). Varela-Diaz et al. (1972) postulated that antigenic changes occur on the surfaces of taeniid metacestodes during their development and suggested that this may be one way in which parasites survive in infection. Damian (1964) had proposed that the parasites may, in fact, pro- duce surface host-like antigenic determinants. These kinds of antigenic changes may be responsible for the induction of different antibody classes over time, though there is little direct evidence for this at the moment. Rickard (1974) proposed that blocking antibodies may adhere to the parasites and mask previously exposed antigens, thereby protecting the parasites from lethal effects of attack mechanisms. However, again, direct eVidence for this is lacking. In addition to altering surface antigens, metacestodes may protect themselves from antibody damage in other ways. Following the study by Musoke and Williams (1975a) demonstrating that com- plement-fixing antibodies were involved in resistance to T. taeniaeformis, Hammerberg et a1. (1976) showed that metacestodes release a complement-depleting sub- stance which may act locally to prevent complement-de- pendent antibody-mediated parasite destruction (Musoke and Williams, 1975a). This may also be true for other taeniids (Kassis and Tanner, 1976; Herd, 1976; Rickard et al. 1977c). The concept of parasite interference with inflammatory mechanisms such as complement and co- agulation cascades has been discussed recently in detail by Leid and Williams (1979). Immunoglobulin-producing cells and gut immunity The intestine and its associated lymphoid tissues are likely to be especially important sites for immune responses to cestodes, but few studies have been done on these systems. Increases in immunoglobulin-producing cell populations occur within lymphatic tissues of ani- mals infected with intestinal nematodes such as Ascaris snug (Khoury and Soulsby, 1977; Crandall and Crandall, 1971), Trichinella spiralis (Ruitenberg and Duyzings, 1972; Crandall et a1. 1967) and Nippostrongylus brasilen- sis (Mayrhofer et a1. 1976). In general, the changes in antibody—containing cell populations parallel the appear- ance of the corresponding immunoglobulins in the circula— tion. Moreover, antibody—producing cell increases appear to occur primarily in lymphoid organs draining parasi- tized tissues. Leonard and Leonard (1941) suggested that the in- testinal wall acts as a barrier to the migration of T. pisiformis in rabbits because few oncospheres reach the liver in immune animals. Although no data are avail- able on cell types that respond to T. taeniaeformis in the lamina propria, a marked influx of lymphocytes and eosinophils occurs in the intestinal mucosae of animals infected with other helminths such as T, spiralis (Larsh and Race, 1954; Zaiman and Villaverde, 1964), Tricho- "strongylus colubriformis (RothWell and Dineen, 1972, Rothwell and Love, 1974; Dineen et a1. 1968) and g, brasiliensis (Love and Ogilvie, 1977). It seems possible that antibody may be produced locally in response to T, 'taeniaeformis antigens and contribute to mucosal resis- tance to reinfection. The observations of Crandall et a1. (1967) are especially relevant here because they demon- strated that a primary 3. spiralis infection in rabbits resulted in an increase in IgM-producing plasma cells in the intestinal mucosa, followed by a relative increase in IgG-containing cells. This pattern paralleled the appear- ance of the corresponding antibodies in the serum. Immunoglobulin E (IgE) The production of circulating reaginic (IgE) anti- body is a remarkably consistent host response to para- sitic infection (Ishizaka et a1. 1976). Ogilvie (1964) first demonstrated reagin in sera of rats infected with g. brasiliensis using passive cutaneous anaphylaxis (PCA) assays. IgE sensitizes mast cells in vitro for antigen-mediated histamine release, and may remain in skin sites of the rat for weeks after intradermal admin— istration. In the rat, reagins are first detectable by 19 days after infection (DAI) with T. taeniaeformis, peaking at 32 days and thereafter declining (Leid and Williams, 1974). IgE antibodies are also detectable in rabbits infected with T. pisiformis (Leid and Williams, 1976) and in rats infected with other hepatic parasites such as T. hepatica (Day et al., 1978) and Schistosoma mansoni (Ogilvie et al., 1966; Rousseaux-Prevost et al., 1977). The function of IgE in resistance to parasitic in- festation is not yet known. It has been proposed for some time that IgE sensitizes mast cells for antigen-in- duced amine release, thereby increasing vascular permea- bility and facilitating the influx of circulating pro- tective antibody and immune cells (Murray, 1971; Murrell et al., 1975; Steinberg et al., 1974; Rousseaux-Prevost et al., 1977). Leid et a1. (1975) demonstrated an antigen-induced release of histamine Tn vitro by peri- toneal cells from rats infected with T. taeniaeformis. In addition to influencing local vascular permeability, amines may have direct inhibitory effects on parasite sur- vival as demonstrated in Tn_!TE£g studies with T. colubri- formis (Rothwell et al., 1974) and Tn_gizg_experiments in‘ which amines were infused into occluded loops of small in- testine containing activated T. taeniaeformis oncospheres (Musoke et al., 1978). Although IgE is not essential for protection against T. taeniaeformis, it may enhance para- site destruction by protective 7332a globulin (Musoke et al., 1978), or may function to potentiate 7832a-mediated antigen-induced amine release (Leid and Williams, 1974). IgE may also participate more directly in resistance to reinfection. Capron et a1. (1975, 1977), have demon- strated an IgE-mediated cytotoxic macrophage adherence to schistosomulae iE.XiE£2° Also, Li Hsfi et al. (1979) showed recently that IgE was fixed to the tegument of Schistosoma japonicum in the skin of challenged rhesus monkeys. Earlier, Ogilvie et a1. (1966) had demonstrated that an intradermal injection of IgE antibodies in rats prevented schistosome cercarial development at that site - a resistance that was not attributable to increased vas- cular permeability. The origin of the circulating reagin in T. taeniae— formis—infected rats has not been studied. IgE-producing 10 plasma cells are normally located in the respiratory and gastrointestinal mucosae and in the regional lymphatic organs (Tada and Ishizaka, 1969). Reagin production may be a regional lymphoid response to parasitism because large numbers of IgE-positive cells are found in the mesenteric lymph nodes of rats infected with intestinal parasites, such as g. brasiliensis (Ishizaka et al., 1976; Mayrhofer et a1, 1976), and they are also found in the axial and bronchial lymph nodes draining the tissues af- fected by migrating g. brasiliensis larvae (Mayrhofer et al., 1976). Cellular Immunity_to Taeniid Infection Cellular immune (CMI) responses are said to play im- portant secondary roles in immunity to the intestinal parasites, T. brasiliensis (Wells, 1962; Ogilvie and Jones,1961), Ascaris suum (Taffs, 1968) and T. spiralis (Larsh, 1953; Markell and Lewis, 1957) and the hepatic parasite, T. hepatica (Lang et al., 1967). Although there is no direct evidence for cell mediated protection in larval taeniid infections, CMI responses have been demon- strated to occur in natural and experimental infections with taeniid larvae when Tn 2TE£g_or Tn 1122 skin tests were performed (Kagan et al., 1966; Rickard and Outteridge, 1974; Kwa and Liew, 1977). Delayed type hypersensitivity skin responses were also elicited in rats vaccinated with ll taeniid antigens (Kwa and Liew, 1977), and these were adoptively transferred to recipients with peritoneal cells from the vaccinated rats. Friedberg et al., (1967) were able to transfer some resistance to the cestode Hymenolepis nana with immune spleen cells:n1mice, and Cook (1979) found that immune lymphocyte populations exert a partial but significant protective effect during the first twelve hours of T; taeniaeformis infection in the rat. In addition, an oral infection of T. taeniaeformis oncospheres appears to trigger cell defense mechanisms against cysticerci im- planted in an abnormal site (Musoke and Williams, 1976). These reactions are at least partially non-specific be- cause similar cellular responses are stimulated by com- plete Freund's adjuvant (Musoke and Williams, 1976). Despite the considerable indirect support for pro- tective cellular immunity, many workers have been less than successful in demonstrating cell-mediated responses to T. pisiformis in rabbits (Nemeth, 1970), and Mitchell et a1. (1977) were unable to protect "nude" (athymic) mice against T. taeniaeformis with purified T cells from in- fected animals. In addition, Blundell et a1. (1969) were unsuccessful in attempts to transfer immunity to‘Taenia ovis or Taenia hydatigena in sheep with cells from in- fected donors. The significance of cellular immunity in resistance to taeniid infections may be underestimated 12 because it can be difficult to correlate Tn_vitro or ‘Tn vivo CMI reSponses with actual cell-mediated resis- tance (Cook, 1979). For eXample, Rickard and Katiyar (1976) found that the antigens of T. pisiformis that were responsible for protedtion were thOse that induced CMI reactions. 'Histopathology of Taenia taeniaeformis InfeCtion Hepatic cellular responses The maximum cellular reaction occurs around hepatic metacestodes by 15-20 DAI when levels of humoral pro- tective antibody are also rising to a peak at 28 DAI (Cook, 1979; Leid and Williams, 1974). The host capsules surrounding the parasites contain many cell types includ- ing numerous eosinophils, lymphocytes, plasma cells and fibroblasts (Singh and Rao, 1967; Ansari and Williams, 1976; Cook, 1979). Considerable numbers of mast cells have been noted in the fibrous capsule which results from chronic infections (Coleman and DeSilva, 1963; Varute, 1971; Cook, 1979). The mast cell is a common component of chronic inflammatory reactions in the rat (Smith et al., 1972) but there are relatively few accounts of their preSence in local reactions to hepatic parasites (Cheever, 1965; Andrade and Barka, 1962; Rahko, 1973; Shirai et al., 1976). It is possible that under similar conditions different hosts respond to any given organism with a 13 distinct pattern of cellular infiltration, and.that mast cells do not participate to any great extent in most cases. However, an alternative explanation for the pau- city of data on mast cell involvement in hepatic helmin- thiases is that routine histopathological techniques (e.g., formalin fixation followed by haematoxylin—eosin stain- ing) do not reveal mast cells reliably (Enerback, 1966). Intestinal cellular resPonses Cook (1979) recently described villar hyperplasia in the small intestines of rats infected with T. taeniae— formis with a concomitant increase in the mucosal mast cell (MMC) population. The reason for this MMC rise is as yet unknown and immunoglobulin«labelling assays have not been performed to determine whether or not they are coated with IgE. Increases in MMC numbers have been shown to occur in animals infected with the intestinal helminths, T. brasiliensis (Befus et al., 1979; Jarrett et al., 1967; Kelly and Ogilvie, 1972; Miller and Jarrett, 1971), T. colubriformis (Rothwell and Dineen, 1972) and T. spiralis (Tronchin et al., 1979). Of special impor- tance are the observations of Mayrhofer et a1. (1976) who demonstrated IgE on the surfaces and within the cytoplasm of MMC in g. brasiliensissinfected rats. Since metacestodes of T. taeniaeformis reside in the liver, it is clear that the physical presence of parasites 14 in the intestine may not be the determining factor in the stimulation of mucosal mast cells. Mucosal mast cell pre- cursors may reside in the lamina propria of the intestine and become stimulated locally to divide or they may be triggered to migrate to the mucosa by some parasitic stimu- lus much as lymphocytes preferentially migrate from the mesenteric lymph node to the small intestine (Parrott and Ferguson, 1974; Guy-Grand et al., 1974; McWilliams et al., 1974; Rose et al., 1976). A second hypothesis for the increase of MMC at a site distant from resident parasites is that a parasite-derived factor may trigger a hormonal and/or cellular reaction sequence which ultimately stimu- lates MMC preCursors. The intestinal mastocytosis is limited to the MMC, and these differ from serosal or connective tissue mast cell (CTMC) populations histochemically (Enerback, 1966, a, b, c; Miller and Walshaw, 1972), ultrastructurally (Miller, 1971) and in their responses to compound 48/80 (8011 et al., 1979; Veilleux, 1973; Heap and Kier- nan, 1973; Enerback and Lowhagen, 1979; Enerback, 1966) and glucocorticoids (Rasanen, 1960; Heap and Kiernan, 1973). Therefore, it seems likely that CTMC and MMC represent separate or divergent cell lines (Ruitenberg and Elgersma, 1976). There has been a_great deal of speculation as to the origin of the intestinal MMC that respond to 15 parasitism. Currently, the most accepted proposal is that they derive from lymphoid cell lines (Burnet, 1965; Miller, 1971; Burnet, 1977) but whether the precursor cells are T or B lymphocyte—derived or are regulated by some thymic factor is not yet clear. Thymectomized mice and thymectomized T cell—depleted rats (Ruitenberg and Elgersma, 1976, 1979; Mayrhofer, 1979) do not respond with an intestinal mastocytosis to T. spiralis or E. brasiliensis infection, respectively, and the MMC response can be restored by reconstituting nude mice with thymic, fragments (Ruitenberg anthlgersma, 1976). Although there is no direct evidence of an immunological basis for the MMC response, mast cells that appear in cultures of immune lymph node cells show a greater response to specific antigenic stimulation than do normal mast cells (Ginsburg et al., 1978). In addition, there is a heightened MMC response in rats secondarily infected with g. brasiliensis (Mayrhofer, 1979). An intestinal mastocytosis could benefit both.the host and the resident parasites if it contributed to resistance to superinfection. Because of the difficul- ties in isolating MMC, hypotheses on their functions are. based upon extrapolations made from Tn vitro and Tn_vivo studies of CTMC responses. These extrapolations are questionable because MMC and CTMC differ in many struc- tural and staining characteristics, and it seems likely 16 that they may differ considerably in function. Mucosal mast cells, like CTMC, may serve as sites for antigen- antibody-mediated amine release and thus facilitate antibody access to the parasites. CTMC release chemo- tactic factors such as ECF-A which enhance inflammatory cell migrations and thereby potentiate inflammatory re- actions, but whether this is true of MMC remains to be shown. Recently, Czarnetski et a1. (1979) found that heparin, a constituent of CTMC, will inhibit chemotactic factors in XiEEE; Therefore, it is possible that tem- pering the inflammatory reaction is a function of the mast cells described in the liver in chronic infections of T.Ataeniaeformis. A most interesting phenomenon from an immunological viewpoint is the presence of IgE within the cytoplasm? of CTMC in the skin of parasitized and atopic individ- uals (Halliwell, 1973, 1975; Feltkamp-Vroom et al., 1975; Li Hsu et al., 1979; Schopfer et al., 1979) and within MMC in the intestinal mucosa of E. brasiliensis- infected rats (Mayrhofer et al., 1976). Specific IgE may play a significant role in resistance to challenge infections and MMC might serve at sites of parasitic invasion to absorb and store and/or synthesize reagin (Halliwell, 1975). Since plasma cells are of lymphocyte origin and MMC are suspected to be of shmilar lineage then it seems likely that these cells may share certain l7 characteristics, even to the extent of synthesizing im- munoglobulin. Further in vitro and in vivo studies will be necessary to determine which of the functions of CTMC are shared by MMC and which of these contribute to in— testinal resistance to parasitic invasion. The observa- tions presented in this thesis open up several avenues by which to explore these questions experimentally. LIST OF REFERENCES LIST OF REFERENCES Andrade, Z.A., Barka, T. 1962- Histochemical observations on experimental schistosomiasis of mouse. Am. J. Trop. Med. Hyg., 11: 12«16. Ansari, A., Williams, J.F., 1976. The eosinophilic re- sponse of the rat to infection with Taenia taeniae- formis. J. Parasitol., 62: 728—736. Befus, A.D., Johnston, N., Bienenstock, J. 1979. 'Ni“o- ‘strongylus‘brasiliensis: Mast cells and histamine levels in tiésues 5f infected and normal rats. Exper. Parasitol., 48: 1'8. BlundelleHassell, S.K., Gemmell, M.A., MacNamara, F.N. 1968. Immunological reSponses of the mammalian host against tapeworm infections: VI. Demonstration of humoral immunity in sheep induced by the activated embryos of T.‘hydatigena and T. ovis. Exper. Para- sitol., 23: 78+82. - Blundell, S.K-, Gemmell, M.A., MacNamara, F.N. 1969. 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Para51tol., 12: 82-101. Williams, J.F. 1979. Recent advances in the immunology of cestode infections. J. Parasitol., 65: 337-349. Zaiman, H., Villaverde, H. 1964. Studies on the eosin- ophilic response of parabiotic rats infected with Trichinella spiralis. Exper. Parasitol., 15: 14-31. ARTICLE 1 IMMUNOGLOBULIN E-CONTAINING CELLS IN INTESTINAL AND LYMPHATIC TISSUES OF RATS INFECTED WITH TAENIA TAENIAEFORMIS by Martha C. Lindsay and Dr. Jeffrey F. Williams 29 SUMMARY Immunglobulin-bearing cell populations in rats in- fected with Taenia taenia"” direct immunofluoresence assays. Cells positive for IgE in the intestinal mucosa increased to maximum numbers at 60 days after infection (DAI) but had fallen by 90 DAI. This increase was due largely to IgE-positive mucosal mast cells, many of which showed evidence of specific fluorescence intracytoplasmically as well as on their membranes. 196 -containing cell numbers also rose in 2c infected rats when compared to uninfected controls, but to a lesser degree than those stained for IgE. In lymphatic tissues, cells producing all classes of immunoglobulins had increased by 6 DAI. Small numbers of IgE-positive cells were found in Peyer's patches throughout the infection in all rats, however, IgE- positive cells were often detected in clusters in in- fected rat spleens through 60 DAI. By 6 DAI, mast cells had accumulated near fluorescent plasma cell clusters along splenic vessels, and by 21 DAI many showed specific fluorescence with IgE. In addition, Inga-containing cells increased in infected rat spleens until 21 DAI, but fewer were detectable at 28 days. No comparable trends occurred in spleens of age-matched control animals. More IgE-containing cells (including fluorescent mast 30 31 cells) were observed in infected rat mesenteric lymph nodes, until 60 DAI after which time they decreased to the levels seen in uninfected controls. The relationship between mucosal mast cells and IgE and their possible function(s) in immune responses to T. taeniaeformis are discussed. INTRODU CT ION Rats infected with Taenia taeniaeformis become im- mune to secondary challenge and it has been known for many years that this resistance can be transferred with antibody by both natural and artificial means (1-3). The protective antibodies implicated in recent studies (4) are of the‘Inga type, and the resistance manifested in challenged animals has been shown to be complement-de- pendent (5). Enhancement of immune attack on early stages of the parasite occurs in animals sensitized with IgE-containing serum (6). However, the sequence of events in challenged animals is not clear, and the cellu- lar participants in protection have not been identified. There is a prolonged increase in mast cell numbers in the intestinal mucosa of rats infected with Taenia taeniaeformis, even though parasites are no longer present in the gut after the first few hours post infection (7). It seems likely that these cells contribute to resistance to reinfection, but their relationship to the pattern of antibody production has not been investigated. Studies of immunoglobulin-containing cell populations have pro- vided some important insights into immune responses to other helminthiases (11), and recent work has surfaced some new cell-immunoglobulin associations in relation to IgE (12-14). The present study was therefore undertaken 32 33 to investigate these relationships in the intestine and associated lymphatic tissues in rats infected with T; taeniaeformis. Evidence is presented for the appearance of IgE-containing gut mast cells, similar to those de- tected in parallel groups infected with Nippostrongylus brasiliensis. MATERIALS AND METHODS Parasites The strain of Taenia taeniaeformis used in these ex- periments was obtained originally from Mr. C.E. Claggett in the Laboratory of Parasitic Diseases, National Insti- tutes of Health, Bethesda, Maryland. The parasite has been maintained in our laboratory as described by Leid and Williams (4). Third stage larvae of Nippostrongylus brasiliensis were kindly supplied by Dr. Paul Weinstein, Notre Dame University, South Bend, Indiana. Experimental infections Infections with T. taeniaeformis were established in 28 day old female Sprague-Dawley-derived rats (Spb:SD) purchased from Spartan Research Animals, Haslett, Michi- gan. Animals were infected by gastric intubation under light ether anesthesia, and with a suspension containing 1000 eggs. Immunofluorescence and histochemical observa- tions were made on a total of 37 infected rats, and on an equal number of age-matched control animals. Six 15 week old female Wistar rats (Charles River Breeding Laboratories, Wilmington, Massachusetts) each received 2000 N. brasiliensis third stage larvae sub- cutaneously in the dorsum of the neck. Six age-matched 34 35 uninfected rats of the same strain served as controls. Histology At specified time intervals post infection with T; taeniaeformis, groups of 3 to 7 rats were killed by ex- posure to carbon dioxide vapor and exsanguinated. Unin- fected control groups of matching sizes were treated similarly. Samples of the spleen, mesenteric lymph node, the proximal 2-3 cm of duodenum and Peyer's patches were removed from each rat. During the sampling procedure, an estimation was made of the numbers of metacestodes in each liver to ensure that control rats had not been ac- cidentally infected and that infected livers carried ex- pected parasite burdens. Approximately 20% of the in- fective eggs gave rise to viable hepatic parasites in this experiment. Tissue specimens were fixed for 24 hr at 4° C in lead subacetate-ethanol-acetic acid (15), washed in 70% ethanol for 12 hr, dehydrated, embedded in Paraplast Plus (Scien- tific Products, Romulus, Michigan) and stored at 4° C. Four micron thick sections were cut, mounted on gelatin- coated glass slides, incubated at 56° C for 10 min and stored at 4° C until used in immunofluorescence or histo- chemical studies. Fifteen and 20 days after receiving N. brasiliensis larvae, groups of 3 rats each were killed and 36 exsanguinated. Samples of the duodenum.were removed from infected and control rats, fixed and processed as described above. 'Antisera Sheep anti-rat IgE (Miles Laboratories, Elkhart, Indiana) was tested in immunoelectrophoresis against both normal rat serum and IgE myeloma protein. A known speci- fic Goat anti-rat myeloma IgE antiserum was used in order to establish specificity. The myeloma protein and mono- specific anti-IgE-myeloma serum.were made available through the generosity of Dr. E.E.E. Jarrett, University of Glasgow, Glasgow, Scotland. Sheep anti-rat IgA was prepared in our laboratory according to the methods described by Leid and Williams (4). Goat anti-rat IgGZa' Rabbit anti-goat IgG (FITC), Goat anti-rabbit IgG (FITC), and Rabbit anti-sheep IgG (FITC) were purchased from Miles Laboratories, Elkhart, Indiana. Rabbit anti-rat IgM, Rabbit anti-rat IgG2b' Goat were purchased from anti-rat IgG and Sheep anti-rat IgG 2c 1 Pel-Freez Biological, Rogers, Arizona. All antisera were teSted in immunoelectrophoresis before use in immunofluor- escence‘assays. Prior to use, antisera were diluted at least 1:10 with phOsphate-buffered saline (PBS), pH 7.4, and absorbed with 10 mg of rat liver acetone powder (Sigma Chemical Company, St. Louis, Missouri) per 1 mg of protein for 1 37 hour at 25° C with constant gentle agitation. The sus- pension was centrifuged at 5000 X g for 10 minutes. The supernatant was then filtered through .22 micron Millipore filters (Millipore Corporation, Bedford, Massa- chusetts) and aliquots stored at -70° C. Final dilutions ‘ were prepared immediately prior to use in assays. Immunofluorescent'and histological staining Sections were dewaxed in xylene, rehydrated and stained for 30 min with 0.1% Alcian blue (Alcian blue 8GX, Gurr, London), pH 1.0, to stain mucosal mast cell mucopolysaccharides (12). After a 10 min wash in PBS, indirect immunofluorescence assays were begun. Sections were incubated with a primary antiserum for 30 min at 25° C with occasional gentle agitation and then washed in 3 l-liter changes of PBS for a total of 30 min. They were incubated further with the corresponding FITC-con- jugate for 30 min at 25° C and washed in 4 1-1iter changes of PBS for a total of 60 min. Stained sections were mounted in 50% PBS/50% glycerol, pH 9.0, for exami- nation of fluorescence. ‘Specificity of antisera for tissue immunoglobulins The following teats.were performed to determine the specificity of the f1uore3cent reactions observed with each antiserum. A series of control slides was prepared in which either normal serum was substituted for each 38 prflmary antiserum, the primary antiserum was omitted, or an unconjugated secondary antiserum was applied prior to the corresponding FITC-labelled antiserum in order to block binding of the conjugate. Counting method The numbers of fluorescent cells were counted in 5 duodenal villus crypt units (16-17) in each of 2 separate tissue sections giving a total count for 10 villus crypt units (VCU). The arithmetic mean was cal- culated, expressed as the.number of fluorescent cells/VCU and served as the cell count for each rat in subsequent statistical analyses. Statistical analysis and rationale The total fluorescent cell numbers in infected and control rats werecompared using a one—way analysis of variance method for data collected at 6, 42, 60 and 90 DAI. We then wished to determine which cell populations were responsible for any differences. The fluorescent mast cell count was subtracted from the total fluorescent cell number for each rat and the remaining cells were designated the "E" population. Since fluorescent mast cell counts were zero in all control groups, the total IgE-positive cell counts were equal to the E populations in these animals. A comparison was made between the E 39 values of control and infected groups at each time inter- val, again using a one-way analysis of variance with the ex level set at 0.01. If a difference was found between the total fluorescent cell counts of infected and control groups but their E levels were the same, then the dif- ference was attributed to MMC in the parasitized rats. Microscopy and Photography Sections were examined for immunofluorescence with a Zeiss Photomicroscope III (Carl Zeiss, Oberkocken, West Germany) by dark field illumination with light from a halogen quartz lamp. A 36-38 3mm red suppressor filter placed over the FITC exciter filter and a 530 nm barrier filter were used for photography. Fluorescence photo- graphs were taken with Kodak Ektachrome 400 ASA daylight film. Bright field photographs were taken on Kodachrome 64 ASA daylight film using the same microscope. RESULTS Duodenum The specificity of the Sheep anti-rat IgE prepara- tion was confirmed prior to use in immunofluorescence studies. This antiserum produced one precipitin arc in immunoelectrophoresis against normal rat serum, and recognized rat myeloma IgE protein. The arc was identical to that formed with monospecific goat anti-IgE. When normal sheep serum was substituted for sheep anti-IgE in immunofluorescence tests or if the primary antiserum was omitted, all fluorescent staining was lost. In ad- dition, unconjugated Rabbit anti-sheep IgG blocked fluorescent staining by the FITC-conjugate. Sheep anti- IgE was the only reagent that bound to mucosal mast cells (MMC) in this study. We chose to sample at 6, 13, 21, 28, 42, 60 and 90 DAI because MMC numbers are significantly elevated by 21 DAI and circulating IgE is detectable from day 19 to 50 DAI (18). Figure 1 illustrates the changes in IgE- positive cell populations in the duodenum through 90 DAI. From 6 through 28 DAI (Figure 1A), fluorescent cells occupied the lower half of each villus. Many were plasma cells with eccentric nuclei and homogeneously fluorescent cytoplasm, but there were also a few 40 FIGURE 1 Fluorescence micrographs illustrating trends in IgE- positive cell populations in duodenal mucosae of infected rats (X 50). A, 13 days after infection (DAI) showing positive cells located in the lower half of each villus. B, 42 DAI. Cell numbers have increased and they are now distributed in the lower two-thirds of most villi. C, 60 DAI. Maximum numbers of cells are visible at this stage, and they extend to the tips of the villi. D, By 90 DAI, fewer cells are present and they are no longer detectable at the tips of villi. 41 43 unidentifiable round cells, some with granular cytoplasm, which were coated with IgE, and occasional Alcian blue- positive MMC with labelled surfaces. IgE-positive lympho- cytes and granulocytes were difficult to identify, either because their morphological characteristics were not distinguishable or because they showed autofluor- escence, respectively. However, both types could have contributed to the population of Alcian blue-negative cells whose surfaces appeared to bind anti-IgE. At 42 DAI (Figure 1B), increased numbers of fluor- escent cells were localized in the lower two-thirds of each villus and up to 50% of MMC either contained or were coated with IgE. Mast cells containing IgE were very similar to those described by Mayrhofer et a1. (12) and Halliwell (19). The cytoplasm was brightly fluores- cent around the unstained granules which appeared as dark inclusion bodies. IgE-positive cell numbers peaked at 60 DAI (Figure 1C) and occupied the entire lamina propria of most villi. Approximately 90% of MMC became labelled with anti-IgE, and fluorescence was almost exclusively confined to cell surfaces. The fluorescent cell numbers declined by 90 DAI (Figure 1D) and IgE-binding cells were seldom found in the tips of villi. Only 50% of the MMC population be- came coated with anti-IgE at this time. Figure ZB illus- trates the different fluorescent cell types found in the FIGURE 2 IgE-containing MMC in the duodenal mucosa of a Taenia taeniaeformis-infected rat (X125). A. Light micrograph showing Alcian blue-stained MMC with arrows (1) pointing to cells that contain IgE. B. Fluorescence micrograph of the same field demon- strating IgE-positive cells with arrows (2) pointing to the two mast cells selected in A. 44 33"“ . at . TABLE 1 Total IgE-containing cell counts and fluorescent MMC counts in rats at intervals after infection with Taenia taeniaeformis and in age-matched controls. 47 .cmma owuoaouwum .DO> mom maaoo mo Hogans coma caumanuflum on» ma unsoo comm M o m~.om m~-o , sn-s~ +.. o ,. ms.may o sm.ma o ,Nm N . on. o. mm x o as 0 mm 0 en o mm o mm o «a 0 mm o as o ma o om o em o as o NH 0 mm o mm o «H 0 so aomezoo o me o HN o so o on 8 RN 0 NN o as o NH ms.sm . «H.mm ,a~.am sm.sm sm.sa .So-mm... o _.nqa.~a mm om _ m‘ Hm‘ .Hm. as o m mm mm as ea ma mm o as as no em as «a «N o as we as as mm am an 0 NH ms ms mm as m - o no amaomazH mm as mm was ma mm o «H mm mm as mos ms mm o «as masmo, mHHmo .maamo maamo ..maamo.tmaamo....masmo mHHmo one: Hopes you: dance , .umoz Houoe .ummz.. Hmuoa. Om . , Om . _ . . a . A .Nv. . . 7 v . . 0 Bzaafiafi mafia . ZOHHUMAZH . who. mZHB. 48 FIGURE 3 IgE-positive fluorescent cells in the duodenal mucosae of rats infected with Taenia taeinaeformis and in age- matched controls. Coarsely-stippled columns represent total fluorescent cells in infected rats and white columns illus- trate those which are mast cells. Finely-stippled columns correspond to total fluorescent cell numbers in control ani- mals, and black columns represent those which are fluor- escent mast cells. Vertical bars indicate standard error. 49 r_. _ uouoewl 190d Mag 317 09 Mean Number Of Cells Per VCU l l FIGURE 4 Cells containing IgE in the duodenal mucosa of a Nippostrongylus brasiliensis-infected rat (X 125). A. Alcian blue-positive MMC with arrows (l) pointing to cells that contain IgE. B. Fluorescence micrograph of the same field demon- strating MMC (2) and plasma cells (3) containing reagin. 51 53 taeniaeformis-infected animals, Alcian blue-positive cells showing bright intracytoplasmic fluorescence with anti- IgE were commonly seen. All other antisera produced one precipitin arc in immunoelectrophoresis when tested against normal rat serum, with the exception of Goat anti-rat IgGZa‘ Batches of this reagent always produced two arcs, one of which corresponded to IgG23 and the other to Ingb or I9G2c' However, the results of immunofluorescence assays for Goat anti-rat Inga were distinctly different from those for the monospecific Goat anti-rat IgGZC and Rabbit anti-rat IgGZb. Control procedures in the indirect immuno- fluorescence tests confirmed the specificity of binding by antisera. Cells containing IgGZa' IgG2b and IgM were detected in small numbers in the duodenal mucosae of infected and control rats throughout the observation period. IgG - and IgA-positive cell numbers increased steadily in 1 all animals with age and counts were not elevated notice- ably in infected rats. MMC showed no fluorescence with any of these reagents. Cells containing intracytoplasmic Ingc increased in number with time, and by 60 DAI there were greater numbers in infected rats than in non-infected controls,_but the magnitude of these differences was much lower than for IgE-containing cell populations. 54 Peyer's patches Cells containing all classes and subclasses of im- munoglobulins were found in small numbers beneath the domes by 6 DAI. No consistent population trends were observed throughout the study, and no one immunoglubulin predominated. No remarkable differences were noted be- tween infected and control rats. At 6 DAI, a few Alcian blue-staining cells appeared in the domes but none was positive for IgE. Occasional IgE-positive mast cells were found at the periphery of the Peyer's patches by 42 DAI and they were no longer detectable at 60 days. Mast cells were found rarely in controls and none bound anti-IgE. Spleen All classes of immunoglobulins were represented in clusters of splenic cells by 6 DAI, and thereafter through- out the observation period in both control and infected rats. No single immunoglobulin-containing cell popula- tion was predominant, but IgA-positive cells were present in least numbers in all rats through 90 DAI. IgGZa'con' taining cells in spleens of infected rats peaked at 21 DAI and there were noticeably fewer by 28 DAI. There were many IgE-positive cell clusters through 60 DAI, but by 90 DAI they had declined. These patterns did not occur in control animals. 55 In spleens of control rats few mast cells were seen until 13 DAI, when scattered clusters were found through- out the parenchyma. By 42 DAI, and during the remainder of the study, mast cells were found rarely. The pattern was markedly different in infected rat spleens. By 6 DAI, many more Alcian blue-staining cells were found sin- ‘gly and in clusters of 2 or more cells than had been de- tected in control spleens at any time. Mast cells were often present near IgE-positive cell clusters along small splenic vessels throughout the parenchyma. By 21 DAI, many individual IgE-positive mast cells appeared and clusters of more than 2 to 3 Alcian blue cells seldom fluoresced. Positive cells either contained or were coated with IgE, and the population increased until 28 DAI, before declining. By 60 DAI only occasional fluor- escent mast cells were detected although clusters of IgE-positive Alcian blue-negative cells were plentiful. Mast cells were rarely seen at 90 DAI and none was posi- tive for IgE. Mesenteric lymph node Trends in each immunoglobulin-containing cell popula- tion were established by estimating the proportion of lymph node parenchyma containing positive cells at each time interval. Cells positive for each class of immunoglobu- lin were detected at 6 DAI in control and infected animals. 56 Approximately one-third of mesenteric lymph node parenchyma in infected rats showed IgE-positive cells (including occasional fluorescent mast cells) by 13 DAI. This situation persisted through 42 DAI, then decreased to control levels (less than 10%) by 60 DAI. Cell popula- tions containing IgGZC, IgGZa' IgG1 and IgM increased in all animals until 21 DAI, then decreased, whereas IgGZb- positive cells were present to their greatest extent at 27 DAI, then declined. Except for IgE-staining cells, the trends did not differ in infected and control rats. DISCUSSION Mucosal mast cells increase in numbers in the lamina propria of rats infected with the intestinal parasites N. brasiliensis (20-21) and Trichinella'spiralis (22), and also with the hepatic metacestode T. taeniaeformis (7). Our results show that the marked increase in IgE- positive cells in the intestines of T. taeniaeformis- ‘infected rats was largely attributable to these MMC. Not only were MMC coated with IgE, but many also showed specific extragranular cytoplasmic fluorescence, indica- tive of intracellular IgE. There are precedents for this interpretation, because mast cells with cytoplasmic IgE have now been described in the dermis and intestinal mucosa of both atopic and parasitized individuals (23-26). It is unlikely that chance tangential mast cell section- ing had any effect on our observations (24), because the proportion of MMC showing fluorescent cytoplasm changed with time even though all tissues were processed and sectioned identically. ‘In view of the significance of this finding in terms of the relationship between MMC and IgE, we felt it im- portant to validate our procedures by applying them to samples of duodenum from rats infected with N. brasiliensis (12). IgE-containing MMC have been described in the gut 57 58 at 15-21 DAI and we were able to confirm this in Charles 'River Wistar rats. However, we also found many IgE- producing plasma cells in the intestinal mucosae of rats infected with either N.‘brasiliensis or T.'taeniaeformis. This represents a marked discrepancy between our results and those of Mayrhofer et a1. (12) who observed very few plasma cells. It does not seem likely that the host strain contributed to this difference because both studies were done with Wistar rats from the same commercial sup- plier. A further point of difference from the situation in nippostrongylosis was that MMC in T.taeniaeformis-in- fected animals were not subepithelial or epithelial in location. The distribution of IgE-positive MMC under- went some remarkable changes during the course of taenii- asis, perhaps related to the selectivity of location of precursors at different sites in the villi, or due to loss of IgE from established MMC population in a con- sistent pattern within each villus. Quantitative studies comparing total MMC and IgE-positive MMC within the same villi would be necessary to explore these possibilities experimentally. Our results certainly providerx>evidence that IgE-positive MMC migrate to the intestinal lumen in primary infections, although.this is generally believed to be their fate in other helminthiases (27, 28). The re- sponse of MMC in T.‘taeniaeformis-infected rats exposed to 59 secondary challenge would be particularly interesting in light of the recent observation that epithelial migration of mast cells occurs following re-exposure of rats after primary nippostrongylosis (29). The increase in IgE-positive cells in the gut was preceded by a rise in similar populations in the spleen and mesenteric lymph nodes. There is considerable evi- dence for the preferential migration of mesenteric lympho- blasts to the intestinal mucosa (30-33), and our findings are consistent with this notion. Presumably some inducing factor, perhaps parasite-derived, could affect the local lymphatic tissues and stimulate plasma cell precursors to product IgE and MMC precursors to migrate to the in- testine. In addition to plasma cells, the spleen and Peyer‘s patches also showed increased numbers of IgE- positive Alcian blue-staining cells. Antigen uptake by mast cells in lymphatic tissues has been described and it is possible that they increase in numbers in response to an infection in order to facilitate the processing of parasite antigens. It is tempting to consider all these changes as part of an effort to develop a highly sensitized cell population in the gut lamina propria specifically prepared to respond to secondary challenge organisms as they migrate through the villar epithelium. Although no direct evidence exists, it has often been suggeSted that an intestinal immune 60 barrier is mounted to circumvent larval migration via this route (35) and the MMC may be an important component, even thOugh their main contribution might be to enhance accessibility of protective'Inga antibodies (6). They could also accelerate the local accumulation of immune effector cells through the release of chemotactic fac- tors after parasite antigen-induced triggering. The de- velopment of resistance to challenge infection and pre- vention of potentially debilitating superinfections could be advantageous both to the host and established para- sites. Results of immunofluoreScence with other anti-immuno- globulin sera indicate that there is no marked increase in either IgG2a or IgA-containing cells in the gut of infected rats. Antibodies of both these types have been implicated in acquired resistance in rodents (4, 36, 37). IgGZa' in particular, is formed very rapidly after infec- tion (4) and there was evidence in our study of increases in IgGZa-forming cells in lymphoid tissues. The observa- tion that lamina propria increases do not occur rein- forces our view that mast cell modulation of vascular permeability in the intestine may be an integral part of serum antibody-mediated parasite destruction in challenged animals. These proposals do not take into account other pos- sible roles for IgE which.have surfaced recently from 61 studies on anti-parasite immunity. IgE adheres to the surface of schistosomulae Tn_yTyg (25), and can sensi- tize macrophages Tn yTE£g_for the destruction of g. man- soni (13). If IgE antibodies were released from intra- cytoplasmic sites in MMC after stimulation by T. taeniae- formis, these reports suggest that they could have a direct role in resistance effector mechanisms. It has also been suggested that IgE accumulated within mast cells represents a storage system (23) and this would be compatible with a protective role at mucosal or epithelial sites. Secretory cells do not generally store macromolecular products of other cell lines, and the possibility that certain MMC may have IgE-synthe- sizing capabilities cannot be discarded entirely in at- tempting to account for its presence within the cytoplasm. MMC are believed to be derived from a lymphoid cell lineage (38, 39, 40), and a subpopulation with functions similar to those of plasma cells could exist. However, the re- cent demonstration of peroxidase-labelled IgE within rat macrophages (41) establishes a convincing precedent for the hypothesis that preformed antibody can be selec- tively absorbed and retained for future use. Our results shed no light on the question of how hepatic parasites influence the proliferation and/or accumulation of MMC at gut sites, but they do raise the possibility of using an additional discriminatory 62 parameter (IgE binding) in pursuing the characteristics of mastocytosis in this infection. Elsewhere we have shown that MMC increases are probably mediated via some circulating factor, and that prolonged hypergastrinemia accompanies chronic infection (42), although its role in gut-related pathologic changes is far from clear. More definitive accounts of the functions of MMC must await demonstration of their sensitivity to antigen and the nature of secretory responsiveness. In the meantime, hypotheses can only be developed based on the assumption that the findings for connective tissue mast cells also hold true for MMC. In fact, the two cell types differ morphologically, histochemically and in their responses to pharmacologic agents (40, 43, 44, 45, 46) and some divergences in function seem likely to emerge. The isolation of MMC from gut tissues (43) opens the way to exploration of these differences, but the hazards of in vitro characterization of enzymatically dispersed cells require that continued efforts be made to assess their roles in vivo. The situation in T. taeniaeformis in which reactivity of intestinal MMC can be inVestigated without the complicating presence of parasites in the gut offers unique advantages for research towards this goal. ACKNOWLEDGMENTS We are especially grateful to Alma Shearer for tech- nical assistance in many aspects of this work, and to Dr. John Kaneene for advice on statistical analysis. These studies were supported by NIH grant AI-10842 and NIH training grant AI-07203-01. This is Journal Article No. from the Michigan Agricultural Experiment Station. 63 10. LIST OF REFERENCES Miller, B.M., and M.L. Gardiner. 1932. Passive immunity to infection with a metazoan parasite Tys- ticercus fasciolaris in the albino rat. J. Prev. Med. 67749. Miller, H.M., and M.L. Gardiner. 1934. Further studies on passive immunity to a metazoan parasite, ”Cysticercus fasciolaris.- Am. J. Hyg. 20:424. Campbell, D.H. 1938. 'The specific property of serum from rats infected with CysticerCus crassi- ‘collis. J. Immunol. 35:195. Leid, R.W., and J.F. Williams. 1974. -Immunologica1 response cf the rat to infection with Taenia taeniae- formis. I. Immunoglobulin classes involved ififipas- sive transfer of resistance. 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Scandinav. 66:289. 41. 42. 43. 44. 45. 46. 47. 68 Dessaint, J.F., Torpier, G., Capron, M., Bazin, H., and Capron, A. 1979. Cytophilic binding of IgE to the macrophage. I. Binding characteristics of IgE on the surface of macrophages in the rat. Cell. Im- munol. 46:12. Cook, R.W., J.F. Williams, and L.M. Litchtenberger. Hyperplastic gastropathy in the rat due to Taenia ‘taeniaeformis infection: Parabiotic transfer and hy- pergastrinemia. Gastroenterology. (submitted for publication). Enerback, L. 1966. Mast cells in rat gastrointest- inal mucosa. 3. Reactivity towards compound 48/80. Acta. path. et microbiol. Scandinav. 66:313. Veilleux, R. 1973. Staining properties of duodenal mast cells in 48/80etreated rats. Histochemie. 34:157. Heap, E.J., and J.A. Kiernan. 1973. Histological, histochemical and pharmacological observations on mast cells in the stomach of the rat. J. Anat. 115:315. Enerback, L. 1979. Long term increase of mucosal mast cells in the rat induced by administration of compound 48/80. Cell. Tissue. Res. 198:209. Befus, A.D., Pearce, F.L., Gauldie, J., Horsewood, P., Goodacre, R.L., Cole, P., Heatley, R.V., and Bienenstock, J. 1979. In the Mast Cell. Edited by J. Pepys and A.M. Edwards. Pitman Medical Publishing Co., Ltd. Tunbridge Wells. p. 702. ARTICLE 2 MAST CELLS IN RATS INFECTED WITH TAENIA TAENIAEFORMIS by Martha C. Lindsay and Dr. Jeffrey F. Williams 69 ABSTRACT Mast cells appeared in the liver around metacestodes of Taenia taeniaeformis by 13 days after infection (DAI). The population increased until 28 DAI, then gradually de- clined. These hepatic mast cells (HMC) were compared to intestinal mucosal mast cells (MMC) and connective tissue mast cells (CTMC) histochemically, morphologically and in their responses in vivo to compound 48/80 and dexa- methasone. HMC were similar to MMC in that they stained positively with Astra blue, could not be demonstrated with .005% toluidine blue, and disappeared after 5 days of treatment with dexamethasone, but were unaffected by 48/80. Immunoglobulin—containing cells in the liver were characterized using immunofluorescence assays. IgGZa-, IgGZc- and IgE-positive cell populations surrounding the parasites increased until 28 DAI then declined. Of these three populations, changes in anti-IgE-labelled cells were the most marked. Many IgE-positive cells were found to be HMC, and there was frequent evidence of intra-cyto- plasmic IgE. The possible origin of these IgE-containing HMC and their potential role(s) in the local immune re- actions to g. taeniaeformis are discussed. 70 71 KEY WORDS: mast cells, IgE, immunofluorescence, Taenia taeniaeformis, cestodes, immunoglobulins, liver. INTRODUCTION Experimental cysticercosis in the rats provides a useful laboratory model for study of host-parasite rela- tionships and the persistence of tissue helminths in im- mune animals (38). In the early stages of infection with Taenia taeniaeformis there are intense inflammatory changes around the organisms (33). Eventually, however, the host response subsides and the growing foreign mass is ac- commodated remarkably well in the liver. Histopathological accounts of inflammatory lesions around taeniid cestodes and other hepatic helminths sel- dom mention the participation of mast cells, (19, 23, 36), and in the few instances where they have been de— scribed their nature and possible function have not been investigated (2, 5, 6, 7, 33, 37). In view of the central role played by these cells in inflammatory processes and their known interactions with immunoglobulin E, we attempted to characterize the appearance of mast cells around de- veloping larvae of g. taeniaeformis in the liver, and their relationship to IgE in the encapsulating response. The results provide evidence for the proliferation of a popula- tion of mast cells, similar in morphology and drug re- sponsiveness to the mucosal mast cells (MMC) of the 72 73 intestine. Furthermore, these cells appear to exhibit not only surface sensitization by IgE,_but also the capacity for intracytOplasmic accumulation of this im— munoglobulin, described recently for intestinal MMC in the parasitized rat (22). 'MATERIALS'AND’METHODS PARASITE The strain of Taenia Taeniaeformis used in this study was maintained as described by Leid and Williams (20). EXPERIMENTAL INFECTION Twenty—eight day old female Sprague—Dawley-derived rats (Spb: [SD]) were purchased from Spartan Research Animals, Haslett, Michigan. Animals were infected orally with 1000 eggs under light ether anesthesia. COMPOUND 48/80 AND DEXAMETHASONE ADMINISTRATION Groups of 6 rats infected for 27 days received intra- peritoneal injections twice daily for 5 consecutive days of 0.1 mg compound 48/80 (Sigma Chemical Company, St. Louis, Missouri)per 100 gm body weight in 0.1 ml sterile saline (increased daily by increments of 0.1 mg/lOO gm), 0.1 mg dexamethasone (Azium, Schering Corporation, Kenil- worth, New Jersey) in .05 ml, or equivalent volumes of isotonic saline. The protocol described by Enerback (10) was followed for 48/80 treatments, and the regimen used by Heap and Kiernan (15) for dexamethasone administration. 74 J- 75 HISTOLOGY AND HISTOCHEMISTRY Animals which received compound 48/80, dexamethasone or saline were killed 4 hours after the first injection on day 5 by exposure to carbon dioxide vapor. Samples of tongue, skin, liver, duodenum and stomach (sampled ac- cording to the method of Heap and Kiernan, (15))were placed in cold lead subacetatevethanol—acetic acid fix- ative (26) for 24 hours. Groups of 3 rats each were killed at 6, 13, 21, 28, 42, 65 and 90 days of infection. Groups of age- matched uninfected animals served as controls. Samples of the liver were taken from each rat and fixed for 36 hours at 4°C in either lead subacetate-ethanol-acetic acid or 10% neutral-buffered formalin. After fixation, samples were treated for 12 hours in absolute ethanol before dehydration, embedding in Para- plast Plus (Scientific Products, Romulus, Michigan) and storage at 4°C. Sections were cut at 4“, mounted on gela- tine—coated glass slides, incubated at 56°C for 10 minutes and stored at 4°C until used in immunofluorescence or histochemical studies. Tissue sections from dexamethasone-,48/80-, or sa- line—treated rats were dewaxed in xylene, rehydrated, stained for 30 minutes either with 0.5% Alcian blue (Al- cian blue 8GX, Gurr, London), pH 1.0, and counter-stained with alcoholic acid fuchsin for 2 minutes, or with 0.5% 76 Astra blue (Astrablau FM, Roboz Surgical Instruments Com- pany, Inc., Washington, D.C.), pH 1.0, and counterstained with 0.5% safranin (Safranin, Fisher Scientific Company, Fair Lawn, New Jersey) for 1 minute. Tissues were then dehydrated and mounted in Pro—Tex mounting medium (Sci- entific Products, McGaw Park, Illinois). Other sections were stained with .005% toluidine blue (Toluidine Blue 0, Central Scientific Company, New York, New York), pH 0.5, to detect differences between affinities of connective tissue, mucosal or hepatic mast cells for thiazanine dye (16). In order to determine the identity of immunoglobulin- containing cells, serial sections paired with those ex- amined in immunofluorescence assays were dewaxed, re- hydrated and stained with giemsa (May-Grunwald Giemsa method) or with an alcoholic Astra blue/acid fuchsin technique (Blaies and Williams, submitted for publica- tion). Stained tissue sections were then dehydrated and mounted. ANTISERA All antisera were tested for specificity in immuno- electrophoresis prior to use in immunofluorescence assays. Sheep antivrat IgE (Miles Laboratories, Elkhart, Indiana) were tested against both normal rat serum and IgE myeloma protein in parallel with a known specific goat anti-rat myeloma IgE. The monospecific antivmyeloma antiserum and the myeloma protein were generously provided by Dr. E.E.E. 77 Jarrett, University of Glasgow, Glasgow, Scotland. Sheep anti-rat IgA was prepared in our laboratory ac- cording to the methods described by Leid and Williams (20). Goat anti-rat IgG .Rabbit anti-goat IgG (FITC), 2a' Goat anti-rabbit IgG (FITC) and Rabbit anti-sheep IgG (FITC) were purchased from Miles Laboratories, Elkhart, Indiana. Rabbit anti-rat IgM, Rabbit anti-rat IgGZb' Goat anti-rat IgG c and Sheep anti-rat IgG were pur- 2 l chased from Pel-Freez Biologicals, Rogers, Arizona. Prior to use in immunofluorescence studies, anti- sera were absorbed with rat liver acetone powder (Sigma Chemical Company, St._Louis, Missouri) as described pre- viously (Lindsay and Williams, submitted for publication). IMMUNOFLUORESCENCE ASSAY Tissue sections were dewaxed in xylene, hydrated to distilled water than washed for 10 minutes in phosphate- buffered saline (PBS), pH 7.4. The slides were treated with a 0.01% solution of trypsin 1:250 (Difco Laborato- ries, Detroit, Michigan) in 0.1 M CaCl , pH 7.8, for 30 2 minutes at 37°C. They were then washed for 10 minutes in two changes of PBS prior to antiserum incubations. Indi- rect immunofluorescence assays.were conducted according to the procedure described by Lindsay and Williams (sub- mitted for publication). 78 SPECIFICITY OF IMMUNOFLUORESCENT REACTIONS The following tests were performed to determine the specificity of the fluorescent reactions observed with each antiserum. A series of control slides was prepared in which either normal serum was substituted for each pri- mary antiserum, the primary antiserum was omitted, or an unconjugated secondary antiserum was applied prior to the corresponding FITC-labelled anti—IgG in order to block the binding of the conjugate. COUNTING METHOD The numbers of duodenal mast cells were counted in 3 villus crypt units (VCU (17)) in eachnof two separate tis- sue sections giving a total number of 6 villus crypt units. The arithmetic mean was calculated, expressed as the num- ber of mast cells/VCU and served as the cell count for each rat in subsequent statistical analyses. STATISTICAL ANALYSIS Mast cell counts of rats in each treatment group were analyzed using a one-way analysis of variance method, and the alpha level was set at .01 for all comparisons. MICROSCOPY AND PHOTOGRAPHYA Tissue sections were examined for fluorescence using a Zeiss Photomicroscope III (Carl Zeiss, Oberkochen, West Germany) and dark ground illumination with a halogen quartz lamp. A BG—38 3mm red suppressor filter placed .4 79 over the FITC exciter filter and a 530 nm barrier filter were used for photography. Fluorescence photographs were taken with Kodak Ektachrome 400 ASA daylight film. Bright field microscopy was performed on the same microsc0pe and photographs taken using Kodachrome 64 ASA daylight film. RESULTS HEPATIC MAST CELL POPULATIONS DURING THE COURSE OF INFECTION Round to ellipsoidal, Astra blue-positive cells (5.5-13.2um x 4.4v8.8pm) containing coarse to fine cyto- plasmic granules and round to ovoid nuclei were present in small numbers around portal vessels and in clusters within the developing host capsules around metacestodes by 13 days after infection (DAI). This population of mast cells, hereafter designated as hepatic mast cells (HMC), increased markedly up to 28 DAI, then declined so that fewer cells were visible by 42 DAI. They were de— tectable throughout the host capsules, but the majority were localized in the outer capsular layer nearest normal hepatic tissue. Large numbers of HMC were observed in all portal areas through 28 DAI, but subsequently they disappeared from these areas, even though Astra blue- staining cells were still detectable around the encapsu- lated parasites at 90 DAI. COMPOUND 48/80 - DEXAMETHASONE EXPERIMENT In order to compare the responses of HMC with mast cells of connective tissue and mucosal types, rats were treated with compound 48/80 or dexamethasone for 5 80 81 consecutive days. Connective tissue mast cells (CTMC) of the tongue and skin were unaffeCted by dexamethasone administration but very few could be found in 48/80- treated animals. Gastric and duodenal mucosal mast cells (MMC) (Figures 1A, 13, 1D, 1E) were unaffected by 48/80 treatment and they were almost undetectable in dexamethasone-treated rats. When the lamina propria mast cell numbers were recorded (Table l) and analyzed, MMC counts in the dexamethasone-treated group were sig- nificantly lower (p S .01) than counts in 48/80- or saline-treated animals. Hepatic mast cells were not detectable in the host capsules around parasites in rats that received dexameth- asone (Figure 1F). However, those in saline- and 48/80- treated animals were not noticeably different from mast cells in untreated infected rat tissues (Figure 1C). The CTMC and MMC exhibited different affinities for toluidine blue in lead subacetate-fixed tissues. Mast cells of the tongue and skin stained darkly, whereas MMC were seldom seen in the gastric or duodenal mucosae. Toluidine blue-staining mast cells were found occasionally in host capsules surrounding hepatic metacestodes and rarely around portal vessels. TABLE 1 TREATMENT COMPOUND 48/ 80 . DEXAMETHASONE ISOTONIC SALINE 18 1 15 30 2 17 19 2 14 20 2 14 18 6 17 14 0 16 MEAN : SE 19.8: 5.38 2.2 i 2.04 15.5 i 1.38 Effect of treatment with Compound 48/80, Dexamethasone or saline on mucosal mast cells in rats infected with Taenia taeniaeformis. 82 FIGURE 1 Comparison of mast cell responses in rats infected with T. taeniaeformis and treated with Compound 48/80 (A,B,C) or dexamethasone (D,E,F) on days 27-32 after exposure. Al- cian blue/acid fuchsin stain. (A) and (D), gastric mu- cosae. (B) and (E), duodenal mucosae. (C) and (F), host capsules surrounding hepatic metacestodes. Parasite mem- brane is at the top in (C) and (F) (arrows). X50. 83 85 IMMUNOGLOBULIN-CONTAINING MAST CELLS Sheep anti-rat IgE was found to be monospecific for rat IgE in immunoelectrophoresis tests. In indirect im- munofluorescent tests no fluorescence occurred when normal sheep serum was substituted for the primary anti-IgE serum. Unconjugated Rabbit antivsheep IgG blocked fluorescent staining by the FITC conjugate. All other antisera were found to be monospecific when tested in immunoelectrophoresis, with the exception of Goat antierat IgGZa‘ All batches of this antiserum exhibited two arcs, one of which corresponded to IgC2a and the other to IgGZb or IgGZc' However, the results of immunofluorescence assays for anti—I962a were dis- tinctly different from those for the monospecific anti— Ingc and antivIgGZb. No immunoglobulin—containing cells were detectable in livers at 6 DAI. However, at 13 DAI, IgE-positive cells appeared around portal vessels and by 21 DAI they were also present in host capsules around the parasites. IgEvcontaining cells were most evident at 28 DAI (Figure 2A) with fewer present by 42 DAI (Figure 2B). Some of these were plasma cells with eccentric nuclei and uni- formlyefluorescent cytoplasm, but others were round to ovoid with granular cytoplasm (5.5—13.2pm x 4.4—8.8pm). IgEvfluorescent cells were found around portal vessels through 65 DAI, and at 90 DAI a few cells positively FIGURE 2 Fluorescence micrographs demonstrating the decline in IgE— positive hepatic cell populations around metacestodes of Taenia taeniaeformis (to the right in each micrograph). (A), 28 days after infection (DAI) showing positive cells around portal vessels as well as within the host capsule. (B), 42 DAI illustrating fewer positive cells primarily within the host capsule. (C), 90 DAI when few cells are detected around parasites. X50. 86 88 labelled with anti—IgE were still evident in host cap- sules (Figure 2C). Many more cells were positive for IgE than for other immunoglobulins at each time sampled. Immunoglobulin—containing cells were detectable occasion- ally in portal areas of uninfected control livers through- out the study. Plasma cells containing IgG2a and IgG2c appeared around portal vessels near parasites by 13 DAI. At this time, a few IgGZaepositive plasma cells were also found around metacestodes within the developing host capsules. Cells positive for these and all other immunoglobulin classes were detected around parasites at 21 DAI, in- creased by 28 DAI, and declined thereafter, with few cells detected at 42 DAI. Positive plasma cells were not scattered randomly but were distributed in one or two layers within the host capsule. IDENTIFICATION OF IgE-POSITIVE CELLS The vast majority of the IgE—positive cells in in- fected livers were round to ovoid, brightly—fluorescent irregularly-outlined cells containing intracytoplasmic granules of various sizes. Most were coated with IgE (i.e. outlined with fluorescein), and a large proportion of this population contained fluorescent cytoplasm sur- rounding the darker granules (Figures 3C and 3D). In order to identify the IgE—positive granular cells, FIGURE 3 Distribution patterns of IgE-positive cells in the liver of a Taenia taeniaeformis-infected rat. (A), light micrograph showing Astra blue-positive mast cells. (B), fluorescence micrograph demonstrating IgE-positive cells in the same field. (C), IgE-containing plasma cell (top right) and four Astra blue-positive granular cells, two coated with IgE and two containing intracytoplasmic IgE. (D), two granular IgE- containing cells (top right) and two positive cells coated _ with IgE. Figures 3A and BB, X50; Figures 3C and 3D, X200. 89 91 paired sections of livers at 28 DAI were stained, one of each pair with anti-IgE and the other with either Astra blue/acid fuchsin or giemsa stain. Figures 3A and 3B illustrate the identical distribution patterns of IgE- positive cells and mast cells which were remarkably con- sistent in all infected rat livers. At higher magnifi- cations, individual granular IgE-containing cells were found to be Astra blueepositive mast cells. No other cell types in giemsa—stained sections were similarly distributed or corresponded with IgE-positive granular cells. DISCUSSION The objective of this study was to characterize the course of the mast cell responses to developing meta— cestodes of T. taeniaeformis, and to establish their re— lationship to IgE and other immunoglobulins in the local hepatic lesions. The results provide evidence for the prompt appearance and persistence of a population of mast cells around the parasites which resemble the MMC of the intestinal lamina propria in terms of their histochemical traits, their affinity for IgE, and the accumulation of intra—cytoplasmic IgE. The briskness and intensity of the hepatic mast cell reaction was unexpected. Mast cells are generally few in number in parenchymatous organs containing little connective tissue such as the liver (34). However, they have been shown to increase in certain pathological con- ditions including cirrhosis (1) and some chronic para- sitic inflammatory diseases. There is a hepatic mast cell response in schistosomiasis (2,5) and fascioliasis (29, 32), and mast cells have been described in the thick fibrous host capsules around metacestodes of g. taeniae- ’formis in long—term infeCtions (6, 7, 33, 37). The pro- portion of these increases has not been defined, and the time course of their occurrence has not been investigated 92 93 previously. Our reSults suggeSt that mature mast cells or pre- cursors of HMC may migrate from the periportal areas to the site of metacestode establishment, because they were detected around portal veSsels before appearing in the de- veloping capsule. Moreover, the accumulation of mast cells in clusters around the encapsulating fibroblastic response raises the possibility of local proliferation and/or differentiation (18). Since HMC and MMC reacted so similarly to dexamethasone and Compound 48/80, and shared affinities for copper phthalocyanine and thiaza- nine dyes which.were quite distinct from those of CTMC, we conclude that most HMC are likely to be related to the intestinal mast cells often implicated in pathologic re- sponses to gut helminths and their rejection in immune hosts (9,10,15,25). NeVertheless, the fact that a small minority of cells in T. taeniaeformis-infected livers did resemble CTMC is consistent with the possibility that representatives of both these populations respond to in- vading metacestodes. Recent evidence for the existence of specialized mast cell types in species other than the rat (16) has stimulated interest in their origin and function in a variety of pathologic states. There are observations which suggest their derivation from lymphoid cell lineage (4, 24), and the preferential migration of lymphoblasts 94 , from draining lymph nodes to sites of inflammation (3) may be important in the induction of hepatic mastocytosis. Whether specifically reactive lymphocytes can differen- tiate into mast cells or elaborate factors which stimulate the development or migration of HMC precursors remains to be seen. The parasites themselves may release chemical agents which induce differentiation or direct an accumula- tion of mature cells at sites of predilection. Both mechanisms are compatible with the observation that non- infected rats parabiotically united with those bearing larvae of T. taeniaeformis exhibit intestinal mastocyto- sis to the same degree as their infected partners (7). Local increases may be accentuated still further by the degranulation of mast cells which first arrive at the in- fection site. This has been proposed to account for mucosal cell increases induced by chemical degranulation experimentally (11). The results of immunofluorescence tests show that the marked increase in IgE—positive cells around the parasites was largely due to HMC. Many of these, in addition to beraing surface IgE, contained extragranular cyt0plasmic IgE. Plasma cells producing this and other immunoglob- ulins also accumulated locally, and they may be the source of bound antibodies in and on mast cells. There are several recent reports of IgE within mast cells in atOpic and parasitized individuals (12,13,14,21,22,30,31), and 95 MMC showing specific anti-IgE fluorescence intracellular- ly also appear in the intestines of rats with T. taeniae- formis (Lindsay and Williams, submitted for publication), observations which suggest that MMC may be antigen-reactive, but there is, as yet, no direct evidence to support this. Specific sensitization to parasite products could place local mast cells in the role of controlling the influx of other inflammatory cells through the antigen-triggered release of vasoactive amines and chemotactic factors (27, 28, 35). On the other hand, they may also be re- sponsible for modulating the intensity of the host re- sponse via the secretion of heparin, a potent inactivator of chemotactic agents (8). Such a mechanism might favor the eventual subsidence of inflammatory rejection efforts on the part of the host, and contribute to prolonged parasite survival. Clearly, further work is required to define the functional characteristics of the HMC in order to sub- stantiate hypotheses on their influence on immune evasion. Their presence in such large numbers in the infected liver provides an opportunity for such studies, either through organ perfusion, or by means of isolating viable mast cells from the lesions for in_yi3£g experimentation. In the meantime, the observation that IgGZa-producing cells, in particular, are plentiful in the immediate vicinity of the parasites is especially challenging, 96 because antibodies of this type are extremely potent pro- tective factors (20). The nature of the relationship between the limiting plasma membrane of the metacestode surface and these humoral and cellular immune effector systems is the key to cestode parasite persistence in tis- sues. The results of our study emphasize the value of the rat—T. taeniaeformis model as a tool for research on this phenomenon in the future. ACKNOWLEDGMENTS We are very grateful to Alma Shearer for her tech- nical assistance. This work was supported by NIH Grant Number AI-10842 and NIH Training Grant Number AI-07203. This is journal article No. ' from the Michigan Agri- cultural Experiment Station. 97 10. LIST'OF REFERENCES Ahlquist J: Liver mast cell counts during develop— ment of cirrhosis of the liver in rats on a low protein, high fat diet.. Acta path et microbiol Scandinav 50:10, 1960 Andrade ZA, Barka T: Histochemical observations on experimental schistosomiasis of mouse. Am J Trop Med Hyg 11:12, 1962 Asherson GL, Allwood, GG, Mayhew B: Contact Sensitiv- ity in the mouse. XI. Movement of T blasts in the draining lymph nodes to sites of inflammation. Im- munology 25:485, 1973 Befus AD, Denburg J, Bienenstock J: Mechanisms of intestinal mastocytosis. In the Mast Cell, edited by Pepys J and Edwards AM, p. 115. Tunbridge Wells, Pitman Med Pub CO Ltd, 1979 Cheever AW: A comparative study of Schistosoma man- soni infection in mice, gerbils, multimammate rats and hamsters. II. Qualitative pathological differ- ences. Am J Trop Med Hyg 14:227, 1965 Coleman EJ, DeSalva JJ: Mast cell responses to ces- tode infection. Proc Soc Exp Biol Med 112:432, 1963 Cook RW: Pathology of Taenia taeniaeformis infection in the rat. PhD thesis. Michigan State University, East Lansing: Michigan, 1979 Czarnetzki BM, Panneck W, Frosch PJ: Inhibitory ef- fect of heparin on leuocyte chemotactic factors. Clin Exp Immunol 39:526, 1980 - Enerback L: Mast cells in rat gastrointestinal mu- cosa. 2. Dye-binding and metachromatic properties. Acta path et microbiol Scandinav 66:203, 1966 Enerback L: Mast cells in rat gastrointestinal mu- cosa. 3. Reactivity towards compound 48/80. Acta path.et microbial Scandinav 66:313, 1966 98 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 99 EnerbSck L, L5whagen GB: Long term increase of mu- cosal mast cells in the rat induced by administration of compound 48/80. Cell Tissue Res 198:209, 1979 Feltkamp-Vroom TM, Stallman PJ, Aalberse RC, Reerink- Brongers EE: Immunofluorescence studies on renal tisSue, tonsils, adenoids, nasal polyps, and skin of atopic and nonatopic patients with special refer- ence to IgE. Clin Immunol Immunopathol 4:392, 1975 Halliwell REW: The localization of IgE in canine skin; An immunofluorescent study. J Immunol 110:422, 1973' Halliwell REW: The sites of production and locali- zation of IgE in canine tissues. Ann NY Acad Sci 254: 476, 1975 Heap BJ, Kiernan JA: Histological, histochemical and pharmacological observations on mast cells in the stomach of the rat. J Anat 115:315, 1973 Heatley RV, James PD, Birkinshaw M, Wenham RB, May- berry J and Rhodes J: The role of intestinal mast cells and ecsinophil cells in ulcerative protocolitis in relation to prognosis and treatment. In The Mast Cell, edited by Pepys J and Edwards AM, p. 716. Tunbridge wells, Pitman Med Pub Co Ltd, 1979 Jarrett WFH, Jarrett EEE, Miller HRP, Urquhart GM: Quantitative studies on the mechanism of self cure in Nippostrongylus brasiliensis infections. In The Re- action of the Host to Parasitism, edited by Soulsby EJL, p. 191. 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Indian J Exp Biol 9:200, 1971 Williams JF: Recent advances in the immunology of cestodes. J Parasitol 65:337, 1979 ARTICLE 3 A SUPERIOR FIXATIVE FOR THE DETECTION OF IMMUNOGLOBULIN E-CONTAINING MAST CELLS IN IMMUNOFLUORESCENCE STUDIES by Martha C. Lindsay and Dr. Jeffrey F. Williams 102 KEY WORDS : mast cells immunofluorescence fixatives immunoglobulins 103 To the editor: —— In a previous report, Dorsett and Ioachiml compared the effects of different fixatives on the detection of intracellular immunoglobulins in human tissues. There is now evidence for the existence of two populations of mast cells that appear to be active in ulcerative proctocolitis and which are analogous to the connective tissue and mucosal mast cell pOpulations ob- served in the rat intestine.3 Rat mucosal mast cells have been found recently to contain intracytoplasmic IgE antibody,4'5 and because IgEdmediated hypersensitivity reactions may be involved in ulcerative proctocolitis,3 we were interested in finding a fixative suitable for identifying IgE-containing intestinal mucosal mast cells in immunofluorescence assays. Lead subacetate-ethanol- acetic acid,6 Carnoy‘s fluid and isotonic-formaldehyde- acetic acid (IFAA) preserve muc0polysaccharides in mu- cosal mast cells such that copper phthalocyanine dyes can be used to stain these cells selectively.2 On the other hand, formalin is not a suitable fixative for the detec- tion of mucosal mast cells, although it is effective in preserving the antigenicity of tissue immunoglobulins.7 Therefore, we compared the effects of the former three fixatives with those of formalin on the intracellular immunoglobulins of mast cells and morphologic integrity 104 105 of rat duodenal mucosa. Sections 4 microns thick were deparaffinized, stained for 30 minutes with 0.1% Alcian blue (Alcian blue 8GX, Gurr, London), pH 1.0, and then incubated with sheep anti- rat IgE 1:50 and FITC-conjugated rabbit anti-sheep IgG 1:10 for 30 minutes each. Reagents were purchased from Miles Laboratories, Elkhart, Indiana and were tested in im- munoelectrophoresis for specificity before use in immuno- fluorescence assays. Prior to antiserum incubations, formalin-fixed tissue sections were treated with 0.1% trypsin 1:250 (Difco Laboratories, Detroit, Michigan) as described by Lindsay and Williams.4 All four fixatives preserved satisfactorily the anti- genicity of IgE; however, tissues fixed with lead sub- acetate showed superior mucosal morphology compared to those fixed with either Carnoy's fluid or IFAA. Figure 1 illustrates the bright fluorescence of IgE-containing cells that was achieved with both formalin and lead subacetate. Alcian blue-staining mast cells were only detected in tis- sues fixed with the latter. This result suggests that lead subacetate fixation would be particularly useful in studies designed to iden- tify and enumerate mucosal mast cells in pathological conditions of the gut, and to establish their relationship to IgE. FIGURE 1 Fluorescence micrographs of duodenal villi. (A) Lead subacetate-fixed tissue section and (B) formalin-fixed tryp- sin-treated tissue section showing specific binding with anti-IgE. X55. 106 ACKNOWLEDGEMENT This work was performed at Michigan State Univer- sity, East Lansing, Michigan, and was supported by NIH Grants AI-07203 and AI—10842. 108 LIST OF REFERENCES Dorsett BH, Ioachim HL: A method for the use of im— munofluorescence on paraffin-embedded tissues. Am J Clin Pathol 69:66—72, 1978. Enerback L: Mast cells in rat gastrointestinal mu- cosa I. Effects of fixation. Acta Path et Microbiol Scandinav 66:289v302, 1966. Heatley RV, James PD, Birkinshaw M, Wenham RB, May- berry J, Rhodes J: The role of intestinal mast cells and eosinophil cells in ulcerative protocolitis in relation to prognosis and treatment, The Mast Cell. Edited by Pepys J, Edwards AM, Tunbridge Wells, 1979, pp 716—724. Lindsay MC, William JF: Immunoglobulin E-containing cells in intestinal and lymphatic tissues of rats in- fected with‘Taenia‘taeniaeformis. Submitted for pub- lication. ' ' Mayrhofer G, Bazin H, Gowans JL: Nature of cells binding anti-IgE in rats immunized with Nippostrongy- lus brasiliensis: IgE synthesis in regional nodes and concentratiOn in mucosal mast cells. Eur J Im- munol 6:537-545, 1976. Mota I, Ferri AG, Yoneda S. The distribution of mast cells in the digestive tract of laboratory animals: Its bearings on the problem of the location of his- tamine in tissues. Quart J Micro Sci 97:251-256, 1956. Qualman SJ, Keren DF: Immunofluorescence of deparaf- finized, trypsin-treated renal tissues. Lab Invest 41:483e489, 1979. 109