Eosinophils preserve parasitic nematode larvae by regulating local immunity

Eosinophils play important roles in regulation of cellular responses under conditions of homeostasis or infection. Intestinal infection with the parasitic nematode, Trichinella spiralis, induces a pronounced eosinophilia that coincides with establishment of larval stages in skeletal muscle. We have shown previously that in mouse strains in which the eosinophil lineage is ablated, large numbers of T. spiralis larvae are killed by NO, implicating the eosinophil as an immune regulator. In this report, we show that parasite death in eosinophil-ablated mice correlates with reduced recruitment of IL-4(+) T cells and enhanced recruitment of inducible NO synthase (iNOS)-producing neutrophils to infected muscle, as well as increased iNOS in local F4/80(+)CD11b(+)Ly6C(+) macrophages. Actively growing T. spiralis larvae were susceptible to killing by NO in vitro, whereas mature larvae were highly resistant. Growth of larvae was impaired in eosinophil-ablated mice, potentially extending the period of susceptibility to the effects of NO and enhancing parasite clearance. Transfer of eosinophils into eosinophil-ablated ΔdblGATA mice restored larval growth and survival. Regulation of immunity was not dependent upon eosinophil peroxidase or major basic protein 1 and did not correlate with activity of the IDO pathway. Our results suggest that eosinophils support parasite growth and survival by promoting accumulation of Th2 cells and preventing induction of iNOS in macrophages and neutrophils. These findings begin to define the cellular interactions that occur at an extraintestinal site of nematode infection in which the eosinophil functions as a pivotal regulator of immunity.     

Critical role for IgM in host protection in experimental filarial infection

We have previously shown that B cells (in particular B1 cells) are important in host protection against brugian infections in a murine i.p. model. In this study, we show that mice deficient in circulating IgM (secIgM-/-), but otherwise normal in their humoral responses, manifest a significant impairment in worm elimination, suggesting that one critical B cell function is the production of Ag-specific IgM. Efficient elimination of larvae is IgM dependent for both primary and challenge infections. The ability to eliminate worms is restored in secIgM-/- mice by administering sera from primed mice. We corroborated these in vivo studies with in vitro observations which show that IgM is the only isotype that reacts strongly with the surface of Brugia L3. Furthermore, activated peritoneal exudate cells adhere to L3 only in the presence of filaria-specific sera or IgM purified from them. This attachment is not reduced by heat inactivation of the serum, suggesting complement independent activity. Peritoneal exudate cells from primed mice, especially activated macrophages, carry high levels of IgM on their surfaces. Our observations suggest that an IgM-mediated reaction initiates the formation of host-protective granulomas.     

Effect of testosterone on the susceptibility of C57BL/6 mice to infection with Brugia pahangi with reference to inflammatory cell response

Effects of testosterone on the susceptibility and inflammatory cell responses of C57BL/6 mice infected intraperitoneally with Brugia pahangi larvae were examined. On day 15 postinfection, female mice showed significantly greater resistance than did males, and peritoneal cell responses (lymphocytes, macrophages, and eosinophils) were great in females. Castration of highly susceptible male mice increased their resistance and peritoneal cell responses to the level of female mice; whereas, castration of female mice did not affect the susceptibility and cell responses. Furthermore, testosterone treatment at a physiological dose in the castrated male mice or a pharmacological dose in female mice suppressed resistance and inflammatory cell responses. These results suggest that male sex hormone, testosterone, but not female sex hormone has a regulatory role in the susceptibility and cellular response of C57BL/6 mice to infection with B. pahangi, and it causes differences between sexes in susceptibility.     

Using eggs from Schistosoma mansoni as an in vivo model of helminth-induced lung inflammation

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide. In chronic infection with Schistosoma, the severe pathology, including liver fibrosis and splenomegaly, is caused by the immune response to the parasite eggs rather than the parasite itself. Parasite eggs induce a Th2 response characterized by the production of IL-4, IL-5 and IL-13, the alternative activation of macrophages and the recruitment of eosinophils. Here, we describe injection of Schistosoma mansoni eggs as a model to examine parasite-specific Th2 cytokine responses in the lung and draining lymph nodes, the formation of pulmonary granulomas surrounding the egg, and airway inflammation. Following intraperitoneal sensitization and intravenous challenge, S. mansoni eggs are transported to the lung via the pulmonary arteries where they are trapped within the lung parenchyma by granulomas composed of lymphocytes, eosinophils and alternatively activated macrophages. Associated with granuloma formation, inflammation in the broncho-alveolar spaces, expansion of the draining lymph nodes and CD4 T cell activation can be observed. Here we detail the protocol for isolating Schistosoma mansoni eggs from infected livers (modified from), sensitizing and challenging mice, and recovering the organs (broncho-alveolar lavage (BAL), lung and draining lymph nodes) for analysis. We also include representative histologic and immunologic data and suggestions for additional immunologic analysis. Overall, this method provides an in vivo model to investigate helminth-induced immunologic responses in the lung, which is broadly applicable to the study of Th2 inflammatory diseases including helminth infection, fibrotic diseases, allergic inflammation and asthma. Advantages of this model for the study of type 2 inflammation in the lung include the reproducibility of a potent Th2 inflammatory response in the lung and draining lymph nodes, the ease of assessment of inflammation by histologic examination of the granulomas surrounding the egg, and the potential for long-term storage of the parasite eggs.     

Leishmania tropica: pathogenicity and in vitro macrophage function in strains of inbred mice.

Of seven strains of inbred mice and one hybrid that were infected intracutaneously with 5, 10, or 20 × 10⁶ active promastigotes of Leishmania tropica major, two strains (CBA/Ca and C₃H/He) recovered from the infection and their lesions healed within 3 to 5 months. The other strains, with the possible exception of C57B1/6 animals, remained infected, carrying large cutaneous ulcers throughout their lives. These included DBA/2, A/Jax, Balb/c, athymic nude mice of Balb/c origin (nu/nu) and the heterozygote Balb/c (nu⁺). The responses of C57B1/6 animals were of intermediate type with a tendency toward nonhealing at higher doses of the parasite. The cutaneous infection of athymic nude mice invariably gave rise to fulminating visceral infections and death. This condition was never observed in the other strains tested. Concanavalin A (Con A)-stimulated syngeneic or allogeneic lymphocytes of intact mice activated peritoneal macrophages of both healer and nonhealer mice, resulting in complete destruction of phagocytosed L. enriettii within 24 to 48 hr. The destruction of ingested L. tropica was confined to macrophages of healer mice and required 72 to 96 hr to reach completion. However, removal of Con A-stimulated lymphocytes from macrophage cultures and regular pulsing of the cells with a lymphokine-rich supernatant produced a state of sustained activation, resulting in destruction of L. tropica inside macrophages of both healer and nonhealer mice. The ability of Con A-stimulated lymphocytes of nonhealer animals to induce effective levels of activation in healer macrophages on one hand, and eventual destruction of L. tropica in macrophages of nonhealer mice under condition of sustained activation on the other, had indicated that so far as the in vitro situation is concerned, there is no inherent defect in lymphocytes or macrophages of nonhealer animals, although the threshold of activation necessary for killing of the parasite seems to be higher for cells of nonhealer origin.     

The lymphatic pathology of Brugia pahangi in nude (athymic) and thymic mice C3H/HeN

The nude (congenitally athymic) mouse, C3H/HeN is highly susceptible to infection with Brugia pahangi (Nematoda: Filarioidea). Normal, hairy mice show a strong thymus-dependent resistance and usually terminate the infection in the larval stages. The present study examined chronological histopathologic changes in the lumbar lymph nodes and adjacent lymphatic vessels of both hosts. In thymic mice, lymphangitis and perilymphangitis reached a maximum 14 to 17 days PI, about the time of disappearance of live worms. The infiltrate showed characteristics of both acute and chronic inflammation: eosinophils, neutrophils, eosinophilic precipitates, and sometimes necrotizing lymphangitis, as well as macrophages and plasma cells. The cellular infiltrate in nude mice was weaker and developed more slowly. Inflammatory responses to identifiable dead worms were seen in both types of hosts but appeared more frequently in thymic mice. Although variable in both models, the granulomas of thymic mice generally showed more tendency to cavitation, greater macrophage or epithelioid cell infiltration, more granulocytes, and appeared to be more destructive than the foreign body responses of nude mice. Whereas lymphangiectasis was generally progressive in nude mice, it was arrested before the end of the third week in thymic mice. In thymic mice, at maximum lumbar lymph node size (17 days), there were large areas of lymphocyte hyperplasia and heavy infiltration of plasma cells. Most nodes returned to normal mean size by the end of the second month. Little or no reactivity was seen in athymic mouse nodes. Our results suggest that some lesions of lymphatic filariasis are potentially thymus-independent: lymphatic fibrosis, lymphangiectasis, accumulations of macrophages and giant cells around disintegrating worms, calcification of worms, intralymphatic thrombosis, and moderate vascular infiltrates including eosinophils.     

The role of macrophages on the expression of sex difference in the susceptibility to Brugia pahangi infection in C57BL/6 mice

The role of macrophages or eosinophils on the expression of sex difference in the susceptibility to a primary Brugia pahangi infection in C57BL/6 mice was investigated by using a macrophage blockade technique (carbon treatment) or a histamine type 1 (H1) receptor antagonist (promethazine). Carbon treatment remarkably inhibited macrophage exudation, reduced the resistance of female mice, and completely abolished sex difference in the susceptibility to B. pahangi infection. Although promethazine treatment inhibited eosinophil exudation, it caused only a little increase (not significant) in the recovery rate of worms. These results suggest that macrophages have more important role(s) than do eosinophils on the expression of sex difference in the susceptibility to a primary B. pahangi infection in C57BL/6 mice.     

Extracellular traps are associated with human and mouse neutrophil and macrophage mediated killing of larval Strongyloides stercoralis

Neutrophils are multifaceted cells that are often the immune system's first line of defense. Human and murine cells release extracellular DNA traps (ETs) in response to several pathogens and diseases. Neutrophil extracellular trap (NET) formation is crucial to trapping and killing extracellular pathogens. Aside from neutrophils, macrophages and eosinophils also release ETs. We hypothesized that ETs serve as a mechanism of ensnaring the large and highly motile helminth parasite Strongyloides stercoralis thereby providing a static target for the immune response. We demonstrated that S. stercoralis larvae trigger the release of ETs by human neutrophils and macrophages. Analysis of NETs revealed that NETs trapped but did not kill larvae. Induction of NETs was essential for larval killing by human but not murine neutrophils and macrophages in vitro. In mice, extracellular traps were induced following infection with S. stercoralis larvae and were present in the microenvironment of worms being killed in vivo. These findings demonstrate that NETs ensnare the parasite facilitating larval killing by cells of the immune system.     

Pathology of pulmonary parasitic migration: morphological and bronchoalveolar cellular responses following Nippostrongylus brasiliensis infection in rats

Nippostrongylus brasiliensis has an obligatory migratory phase through the lungs during its development in rats. This migration is associated with marked tissue damage and pronounced cellular reaction. Given that cells from the lower respiratory tract, especially alveolar macrophages, can adhere to and kill larvae of N. brasiliensis in vitro, we studied the time course of morphological changes associated with parasitic migration. Compared to a primary infection, a secondary infection resulted in significant changes in the pulmonary tissue characterized by an early acute inflammation leading to granulomatous reaction in the parenchyma and a leucocytosis in the bronchoalveolar lavage fluids with an anamnestic increase in absolute numbers of neutrophils, alveolar macrophages, eosinophils, and lymphocytes. Scanning electron microscopy showed that inflammatory cells, especially alveolar macrophages, granulocytes, lymphocytes, erythrocytes, and platelets, adhered to the larvae following secondary infection and this adhesion was associated with disruption of cuticular surface in some larvae. Secondary infection also resulted in retention of larvae in granulomatous lesions in the lungs even up to 21 days postinfection. There was mast cell and type II pneumocyte hyperplasia and these cells appeared to be activated. Thus, the histopathological changes in lungs correlated with the bronchoalveolar cellular responses and further document the inflammatory and immunological reactions during the migration of N. brasiliensis larvae.     

Infection-induced respiratory burst in BALB/c macrophages kills Leishmania guyanensis amastigotes through apoptosis: possible involvement in resistance to cutaneous leishmaniasis

The immune mechanisms that underlie resistance and susceptibility to leishmaniasis are not completely understood for all species of Leishmania. It is becoming clear that the immune response, the parasite elimination by the host and, as a result, the outcome of the disease depend both on the host and on the species of the infecting Leishmania. Here, we analyzed the outcome of the infection of BALB/c mice with L. guyanensis in vivo and in vitro. We showed that BALB/c mice, which are a prototype of susceptible host for most species of Leishmania, dying from these infections, develop insignificant or no cutaneous lesions and eliminate the parasite when infected with promastigotes of L. guyanensis. In vitro, we found that thioglycollate-elicited BALB/c peritoneal macrophages, which are unable to eliminate L. amazonensis without previous activation with cytokines or lipopolysaccharide, can kill L. guyanensis amastigotes. This is the first report showing that infection of peritoneal macrophages with stationary phase promastigotes efficiently triggers innate microbicidal mechanisms that are effective in eliminating the amastigotes, without exogenous activation. We demonstrated that L. guyanensis amastigotes die inside the macrophages through an apoptotic process that is independent of nitric oxide and is mediated by reactive oxygen intermediates generated in the host cell during infection. This innate killing mechanism of macrophages may account for the resistance of BALB/c mice to infection by L. guyanensis.     

Ultrastructural aspects of the host cellular immune response to Taenia crassiceps metacestodes.

When three Taenia crassiceps metacestodes were injected intraperitoneally into C3H mice primed by previous subcutaneous inoculation of metacestodes, larvae which were resistant to early immune damage by the humoral response were encapsulated by host cells and rejected. Initially, normal larvae were encapsulated primarily by eosinophils and macrophages. In the early stages of encapsulation, both cell types showed severe degenerative changes and disruption of cell membranes, but there was no evidence of tegumental damage to the encapsulated larvae. Later, mast cells appeared in the capsules surrounding the larvae. After mast cells became common, all of the cell types present were normal, and damage to the larval Tegument became apparent. Ultimately, interaction of eosinophils, mast cells, macrophages, and lymphocytes resulted in death of the encapsulated larvae. These results suggest that larvae may secrete substances toxic to host cells, and that mast cells are necessary for rejection of larvae.     

Taenia taeniaeformis: evasion of complement-mediated lysis by early larval stages following activation of the alternative pathway

Activation of the alternative pathway of complement by T. taeniaeformis oncospheres and early stage metacestodes, although a factor in host defense against primary infection, does not directly lead to the killing of the parasite larvae observed prior to day 6 post-infection in innately resistant BALB/cByJ inbred mice. Immunogold labelling techniques clearly demonstrated tegument-associated C3 on in vitro-activated oncospheres incubated with non-immune mouse sera. However, C5, a protease necessary for the assembly of the membrane attack complex, was not detected. Early stage larvae cultured from in vitro-activated oncospheres escaped membrane damage and survived incubation in non-immune sera from both BALB/cByJ and taeniid-susceptible C3H/HeDub mice. Comparisons of cobra venom factor-treated and untreated C5-deficient B10.D2osn mice revealed no significant differences in parasite burden and local eosinophil infiltration at 6 days post-infection, suggesting that the terminal arm of the complement system is necessary for the previously reported role of complement in resistance to primary infection in BALB/cByJ and C3H/HeDub mice. An in vivo test of chemotaxis indicated that although both complement-intact mouse strains examined responded to intraperitoneal injections of inulin, there were lower numbers of eosinophils in C3H/HeDub mice than in BALB/cByJ mice, perhaps pointing to possible mouse strain differences in C5a generation/catabolism or eosinophil ability to respond to C5a. Lectin-binding studies showed an affinity of PNA for the exposed surface of taeniid oncospheres and 4-day post-infection metacestodes; however, binding of lectin to the carbohydrate moiety did not inhibit complement activation.     

CD8+ T lymphocytes are not required for murine resistance to human filarial parasites.

Mice are resistant to the establishment of infection with the nematode parasite Brugia malayi, an etiologic agent of human lymphatic filariasis. We have recently shown that T and B lymphocyte-deficient C.B.-17 scid/scid mice are permissive for infection with this parasite, whereas coisogenic C.B.-17+/+ mice are resistant. This observation suggests that T and B lymphocytes that comprise the antigen-specific immune system orchestrate murine resistance to B. malayi. In order to define the component of the antigen-specific immune response that is responsible for this resistance, we have tested the susceptibility of beta 2M-/- mice to infection with B. malayi L3 larvae. These mice are homozygous for insertional disruption of their B2m genes, which encode beta 2-microglobulin, the small subunit of the major histocompatibility (MHC) antigens. They do not express beta 2-microglobulin and, as a consequence, fail to express the class I major histocompatibility antigens, and they do not develop the CD8+ class I MHC-restricted cytotoxic T cell subset. We find that these mice are completely resistant to B. malayi, indicating that the CD8+ T lymphocyte subset is not an obligate requirement for murine resistance to human filarial parasites.     

Lack of correlation between in vitro and in vivo activation of complement in mice by infective Trichinella spiralis larvae

Very little is known about the role played by complement in vivo during Trichinella spiralis infections, although previous reports indicate that it binds readily to the surfaces of muscle stages of the parasite in vitro. In order to study the binding of complement to muscle-stage larvae in vivo, larvae were recovered from BALB-c inbred, NFR/N inbred, and Swiss white outbred mice from 20 to 95 days postinfection. The presence of C3 was examined by direct immunofluorescence and leucocyte- and erythrocyte-adherence tests. Complement was found on a few larvae from the outbred strain and only rarely on larvae from the 2 inbred strains. Histological sections prepared from inbred strains and used in immunofluorescence tests to study in situ complement activation and binding were negative. Larvae from all 3 mouse strains bound complement 100% of the time when it was added to the worms in vitro. The results indicate that extrapolation from in vitro to in vivo activation and binding of complement to T. spiralis larvae may not be valid.     

The granulomatous inflammatory response in jirds, Meriones unguiculatus, to Brugia pahangi: an ultrastructural and histochemical comparison of the reaction in the lymphatics and peritoneal cavity

A granulomatous inflammatory response develops in jirds with lymphatic or intraperitoneal infections of Brugia pahangi. Light, histochemical, and ultrastructural microscopy were used for comparative studies of the reactions in these 2 locations. The reactions observed were categorized into 3 types: (1) an initial response in which lymphocytes, monocytes, macrophages, and eosinophils were present; (2) an intermediate one which consisted of macrophages, epithelioid cells, lymphocytes, eosinophils, collagen, and mesothelial/endothelial cells with central areas of necrosis; and (3) a terminal reaction consisting of degenerating, necrotic cells. Microfilariae and adult worms were associated with these reactions. Macrophages were the predominant cell type in the lesion and were often found attached to the surface of the parasite. The inflammatory responses to B. pahangi in the lymphatics and in the peritoneal cavity appear to be similar, and thus, the peritoneal cavity may be useful in studying specific cell-parasite interactions to further define the pathogenesis of filarial disease.     

Genetic control of murine immune responses to larval Dirofilaria immitis

Previous studies have demonstrated that BALB/c mice, immunized against infection with Dirofilaria immitis, were capable of killing a significant percentage of challenge larvae found within diffusion chambers. The percentage of larvae killed by immunized mice was, however, less than in immunized dogs and unlike immunized dogs, mice were unable to retard the development of the surviving larvae. The objective of the present study was to test 3 inbred strains of mice to determine whether a higher level of protective immunity would develop in these hosts and if larval growth retardation would occur. DBA/2J and C57BL/6J mice and their F1 hybrids B6D2F1/J were used in these studies; it was determined that there were differences in susceptibility among the 3 strains but no difference in ability to eliminate larvae from challenge infections. Growth retardation was seen in larvae recovered from immunized DBA/2J and C57BL/6J mice but not in B6D2F1/J. No difference was noted between immune and control mice in the cell types found in the diffusion chambers. The predominant cell types seen were mononuclear macrophages, multinucleate syncytial cells, and neutrophils. Antibody responses to soluble third- and fourth-stage larval antigens and larval excretory/secretory antigens were measured. Although antibodies to all 3 antigen groups were found in higher concentrations in immunized mice than in their respective controls, only antibody responses to soluble L-3 antigens provided a clear correlation with protective immunity.     

TNF regulates chemokine induction essential for cell recruitment, granuloma formation, and clearance of mycobacterial infection

Host immunity to mycobacterial infection is dependent on the activation of T lymphocytes and their recruitment with monocytes to form granulomas. These discrete foci of activated macrophages and lymphocytes provide a microenvironment for containing the infection. The cytokine, TNF, is essential for the formation and maintenance of granulomas, but the mechanisms by which TNF regulates these processes are unclear. We have compared the responses of TNF-deficient (TNF(-/-)) and wild-type C57BL/6 mice to infection with Mycobacterium smegmatis, a potent inducer of TNF, and virulent Mycobacterium tuberculosis to delineate the TNF-dependent and -independent components of the process. The initial clearance of M. smegmatis was TNF independent, but TNF was required for the early expression of mRNA encoding C-C and C-X-C chemokines and the initial recruitment of CD11b(+) macrophages and CD4(+) T cells to the liver during the second week of infection. Late chemokine expression and cell recruitment developed in TNF(-/-) mice associated with enhanced Th1-like T cell responses and mycobacterial clearance, but recruited leukocytes did not form tight granulomas. Infection of TNF(-/-) mice with M. tuberculosis also resulted in an initial delay in chemokine induction and cellular recruitment to the liver. Subsequently, increased mRNA expression was evident in TNF(-/-) mice, but the loosely associated lymphocytes and macrophages failed to form granulomas and prevent progressive infection. Therefore, TNF orchestrates early induction of chemokines and initial leukocyte recruitment, but has an additional role in the aggregation of leukocytes into functional granulomas capable of controlling virulent mycobacterial infection.     

Macrophages as effector cells of protective immunity in murine schistosomiasis

Mice infected with Schistosoma mansoni develop a dramatic (five- to eightfold) increase in numbers of peritoneal leukocytes, and approximately 65% of these cells are macrophages. By several biochemical and cytochemical criteria, these cells were comparable to resident peritoneal macrophages of normal mice. However, macrophages from schistosome-infected mice exhibited significant nonspecific tumoricidal activity in vitro, a function associated with immunologically activated cells. The time course for development of activated macrophages in the peritoneal cavity was dependent upon the route of infection. Cytotoxic cells were present in the peritoneal cavity by 3 weeks after intraperitoneal infection, but were not evident until several weeks later in animals infected percutaneously, subcutaneously, or intravenously. However, by 3 weeks after subcutaneous infection, tumoricidal macrophages appeared in the peritoneal cavity after intraperitoneal challenge with soluble schistosome antigens. Macrophage activation was independent of the development of egg granulomas, since tumoricidal cells could be found prior to the onset of egg production and were also present in mice infected with only male worms. Development of activated macrophages in these instances is thus consistent with previous observations on induction of T lymphocyte reactivity toward schistosomula. Since other manipulations known to activate macrophages have been shown to induce partial resistance to schistosome infection, the finding that macrophage activation results from primary S. mansoni infection itself suggests that these cells may play a major role in acquired immunity to this parasite.