Vasoactive intestinal peptide stimulates T lymphocytes to release IL-5 in murine schistosomiasis mansoni infection.

In murine schistosomiasis, granulomas form around ova deposited in the liver and intestines of infected mice. The granulomas have eosinophils that produce vasoactive intestinal peptide (VIP) and T cells that display VIP receptors. IL-5 is a lymphokine important for the development and maturation of eosinophils. It seemed plausible that VIP, released from eosinophils, may interact with lymphocyte VIP receptors and modulate IL-5 production as part of a feedback regulatory circuit. Thus, we determined whether granuloma T cells make IL-5 and whether VIP modulates IL-5 production. Isolated granuloma cells enriched for T lymphocytes spontaneously released IL-5. Culture of these cells in the presence of VIP increased IL-5 secretion. Spleen cells were also studied. Spleen cells from infected mice did not spontaneously release IL-5 or express IL-5 mRNA and VIP did not stimulate these resting spleen cells to produce this IL. However, these cells did express IL-5 mRNA and secreted IL-5 in response to Con A or soluble egg Ag. VIP could not appreciably modulate IL-5 release when cells were cultured with VIP and the Ag or mitogen. Spleen cells washed free of Con A ceased IL-5 secretion within 24 h. These preactivated splenic T cells resumed vigorous IL-5 secretion in response to either Con A or VIP. Yet only Con A prominently induced IL-5 mRNA expression. VIP was an effective stimulus at concentrations equal to or above the kDa of the VIP receptor on both splenic and granuloma T cells (10(-8) M). It is concluded that, in murine schistosomiasis, VIP invokes IL-5 release from activated T cells that are not undergoing immediate TCR stimulation.     

Release of substance P by granuloma eosinophils in response to secretagogues in murine schistosomiasis mansoni.

In murine schistosomiasis, granulomas form around parasite ova which lodge in the liver and intestines. The granulomas contain eosinophils which produce substance P. In order to demonstrate substance P release by individual granuloma eosinophils and mechanisms regulating this release, a reverse hemolytic plaque assay was developed. Release of substance P was demonstrated by plaque formation around granuloma eosinophils only when a specific substance P antiserum was used. Few cells released substance P in the basal state. However, eosinophils produced plaques in the presence of calcium ionophore A23187 or histamine. Plaque size and number were dependent upon secretagogue concentration. It is thus concluded that granuloma eosinophils can release substance P in response to both pharmacological and physiological agents.     

Granulomas in murine schistosomiasis mansoni contain vasoactive intestinal peptide-responsive lymphocytes.

Granulomas develop around schistosome ova in murine Schistosoma mansoni. These granulomas have eosinophils that produce VIP. It is possible that VIP participates in immunoregulation. VIP-mediated effects usually operate through a cAMP-dependent mechanism. To identify VIP-responsive inflammatory cells in murine schistosomiasis, inflammatory cells were exposed to VIP and assessed for adenylate cyclase activation and VIP binding. VIP increased adenylate cyclase activity in splenic lymphocytes from both normal and infected mice. In each case, the half-maximal stimulation was at about 5 x 10(-8) M. [125I]VIP bound to splenic lymphocytes specifically, with a Kd of 10(-8) M. This suggested that maximal adenylate cyclase activation requires full receptor occupancy. The receptor was highly specific for VIP. Hormone analogs, that are VIP receptor antagonists in some tissues, were only weak agonists of the lymphocyte VIP receptor. Granuloma cells also bound VIP and responded with adenylate cyclase activation in a manner similar to that of spleen cells. Both splenic T and B lymphocytes responded to VIP. Deletion experiments, using anti-Thy 1.2, suggested that most of the responsive granuloma cells were T lymphocytes. Thus, VIP alters cAMP metabolism in granuloma T cells through a receptor-coupled mechanism similar to that observed for spleen cells. Binding studies on mouse intestinal epithelial cells suggested that their VIP receptor is functionally and possibly structurally different from the VIP receptor on mouse lymphocytes. Additional experiments suggested that VIP and other neuropeptides are unlikely to alter the granulomatous response through a primary interaction with the granuloma macrophages.     

The substance P receptor is necessary for a normal granulomatous response in murine schistosomiasis mansoni

Immune cells within the granulomas of murine schistosomiasis mansoni make the neuropeptide substance P (SP) and express neurokine 1 receptor, which is the specific receptor for substance P (SPr). It was determined if mice with deletion of the SPr (SPr-/-) would develop a normal granulomatous response to schistosome ova during the course of natural infection. Mean liver granuloma size was smaller in SPr-/- mice compared with that of wild-type control animals. Although flow analysis revealed little difference in the cellular composition of the granulomas, both splenocytes and granuloma cells from SPr-/- mice produced much less IFN-gamma and IgG2a and less IgE. The expression of Th2 cytokines (IL-4/IL-5) and IgG1 was comparable to the wild-type control. The mouse with targeted disruption of its SPr had the nonmammalian gene encoding the enzyme beta-galactosidase inserted in exon 1 of the SPr gene. There was beta-galactosidase activity in many mononuclear cells scattered throughout the schistosome granulomas of SPr-/- mice. Also, a granuloma T cell line derived from this transgenic mouse produced beta-galactosidase. These results provide further evidence that in murine schistosomiasis SPr is displayed commonly on granuloma inflammatory cells and is important for granuloma development and expression of IFN-gamma circuitry in this natural infection.     

Modulation of granulomatous inflammation in murine schistosomiasis mansoni by enteric exposure to schistosome ova: in vitro characterization of a regulatory mechanism within the granuloma

Enteric immunization with schistosome ova results in a diminished granulomatous response. This study explored a mechanism by which enteric immunization may decrease granuloma size. Granulomas from livers of acutely infected mice were dissociated and the dispersed cells were depleted of macrophages. As defined by a direct in vitro migration inhibition factor (MIF) assay, the macrophage-depleted cells, composed of lymphocytes and eosinophils, inhibited the migration of normal peritoneal exudate cells when exposed to soluble egg antigens. Anti-Thy 1.2 or -Lyt 1.1, but not -Lyt 2.1, treatment of these cells abrogated MIF activity. Next, mice were exposed enterically to eggs 4 weeks prior to sacrifice. Cells from granulomas isolated from these animals demonstrated no MIF activity unless treated with anti-Lyt 2.1. When granuloma cells from enterically immunized mice were mixed with those from unimmunized animals, MIF activity by the latter was abrogated. Treatment of cells from immunized mice with anti-Lyt 2.1 or -Thy 1.2, but not -Lyt 1.1 prior to mixing once again permitted MIF activity. These results suggest that the diminished granulomatous response induced by enteric immunization could be mediated by Lyt 2+ suppressor T cells. These suppressor cells may regulate the MIF activity of Lyt 1+ T lymphocytes residing within these lesions.     

A model of T-cell unresponsiveness using the male-specific antigen, H-Y

Granuloma formation in schistosomiasis japonica differs in several respects from those observed in Schistosoma mansoni infections. We have utilized the lung granuloma model in mice sensitized with subcutaneous injection of Schistosoma japonicum eggs to study the kinetics and mechanisms of this response. Animals injected subcutaneously with a range of 50–50,000 S. japonicum eggs elicited a significant pulmonary granulomatous response around ova subsequently injected intravenously. The pulmonary granulomas were formed of macrophages, lymphocytes, and eosinophils. Both antithymocyte globulin and antieosinophil sera reduced significantly the size of the granulomas and depleted the corresponding cell. Nude athymic mice developed markedly reduced pulmonary granulomas as did mice treated with niridazole or hydrocortisone. Sensitization to the egg antigens was demonstrable as both immediate and arthus-type footpad responses. Our data show that cell-mediated pulmonary granulomas can form around S. japonicum eggs in animals previously sensitized by the subcutaneous route. This model may provide further insights into the pathogenesis of S. japonicum granuloma.     

IL-4 inhibits vasoactive intestinal peptide production by macrophages

In schistosomiasis, eggs induce granulomas that have a vasoactive intestinal peptide (VIP) immunoregulatory circuit. This study explored the regulation of VIP production at sites of inflammation. Splenocytes from uninfected C57BL/6 mice expressed VIP mRNA and protein, which stopped following egg deposition. Eggs induce a Th2 response, suggesting that Th2 cytokines like interleukin (IL)-4 can regulate VIP. To address this issue, splenocytes from uninfected mice were incubated for 4 h with or without recombinant IL-4. IL-4 inhibited VIP mRNA expression. F4/80+ macrophages were the source of constitutively expressed VIP, subject to IL-4 regulation. In IL-4 knockout mice, splenic VIP production did not downmodulate during schistosome infection, suggesting that IL-4 is a critical cytokine regulating VIP production in wild-type mouse spleen. IL-4-producing granulomas in schistosomiasis made VIP. Experiments showed that granuloma VIP derived from F4/80- (nonmacrophage) cell populations, explaining this paradox. Granuloma F4/80+ cells from IL-4 knockout mice expressed VIP. Thus macrophages can make VIP, which is subject to IL-4 regulation. However, in the Th2 granulomas, other cell types produce VIP, which compensates for loss of macrophages as a source of this molecule.     

Changes in collagen and albumin mRNA in liver tissue of mice infected with Schistosoma mansoni as determined by in situ hybridization

We have employed in situ hybridization to evaluate the molecular mechanisms responsible for hypoalbuminemia and increased liver collagen content in murine schistosomiasis. Results were compared using a simplified method of hybridizing isolated hepatocytes from Schistosoma mansoni-infected and normal mouse liver with mouse albumin (pmalb-2) and chick pro-alpha 2(l) collagen (pCg45) probes. Whereas hepatocytes from infected mice showed significantly less albumin mRNA than hepatocytes from control, there were more grains of procollagen mRNA in hepatocytes from infected as compared with control liver. Hybridization of infected liver tissue sections with the collagen probe showed more grains per field in granulomas than in liver regions, whereas with the albumin probe there was more hybridization in liver tissue than in granulomas. These results suggest that in murine schistosomiasis a reduction in albumin mRNA sequence content may be associated with decreased albumin synthesis and ultimately leads to hypoalbuminemia. In addition, although the granuloma seems to be the primary source of type I collagen synthesis, hepatocytes are also capable of synthesizing collagen, especially under fibrogenic stimulation.     

CD4+ TCR repertoire heterogeneity in Schistosoma mansoni-induced granulomas

The hallmark of Schistosoma mansoni infection is the formation of liver granulomas around deposited ova. The initiation of granuloma formation is T cell-dependent since granulomas are not formed in their absence. We investigated whether a few T cells arrive to initiate the inflammatory lesion and subsequently expand locally, or whether a large repertoire of systemically activated T cells home to the delayed type hypersensitivity reaction induced by the ova. The TCR repertoire of single granulomas from the same liver were analyzed by PCR using Vbeta-specific primers and CDR3 analysis. Each granuloma has a very diverse TCR repertoire indicating that most of the T cells recruited to these lesions are activated systemically. At the same time, sequence analysis of individually sized CDR3 products from single granuloma indicate that a fraction of T cells expand locally at the lesion site. Using TCR transgenic mice containing a pigeon cytochrome c-specific T cell population or lymphocytic choriomeningitis virus infection tracked with lymphocytic choriomeningitis virus-specific tetramers, we demonstrated that nonspecific T cells home to the granuloma if they are activated. However, recombinase-activating gene 2(-/-) pigeon cytochrome c-specific TCR transgenic mice fail to form granulomas in response to S. mansoni ova even after T cell activation, suggesting a requirement for egg-specific T cells in the initiation of these inflammatory lesions. Understanding the mechanism of T cell recruitment into granulomas has important implications for the rational design of immunotherapies for granulomatous diseases.     

Brugian infections in the peritoneal cavities of laboratory mice: kinetics of infection and cellular responses

Standard, immunocompetent, inbred strains of mice are non-permissive for infection with the human filarial nematode, Brugia malayi or the closely related Brugia pahangi. This non-permissiveness allows one to address the mechanism(s) that might be used by mammalian hosts to eliminate large, multicellular, metazoan, extracellular invertebrate pathogens. We describe here the time course of intraperitoneal Brugian infections in na?ve and primed +/+ mice from two commonly used, inbred laboratory strains (C57BL/6J and BALB/cByJ). We believe that this documentation of the course of infection in normal mice will serve as a reference for future studies using mice with gene-targeted immunological deficits or which have been pharmacologically or immunologically manipulated to manifest such deficits. Our data show that even though both strains of mice eliminate the parasite before the onset of patency, there are significant differences in the time course of infection and in the fractions of input larvae that can be recovered at any time after infection. In a secondary infection, the time course of elimination is accelerated. We examined the cells in the peritoneal cavity, the site of infection, by flow microfluorimetry using forward and side scatter properties and cell surface antigen expression using fluorescent antibodies. These studies reveal a complex cellular pattern, predominated by B lymphocytes, macrophages, and eosinophils. The most notable gross morphological findings at necropsy during the phase of elimination of the parasite are nodules of tissue containing larvae, which appear viable in some cases and undergoing various stages of disintegration in others. These nodules, which are histologically granulomas, are primarily composed of macrophages and eosinophils, with few if any lymphocytes. Transmission electron micrographs reveal that eosinophils can penetrate under the cuticles of the larvae and be seen in close approximation with internal structures. These granulomas may represent an important mechanism by which worms are eliminated.     

Eosinophils from granulomas in murine schistosomiasis mansoni produce substance P

Granulomas are chronic, usually focal, tissue-destructive inflammatory reactions that usually form around slowly degradable, poorly soluble substances. They are dynamic lesions, regulated by complex immune mechanisms. Tachykinins are a family of neuropeptides characterized by the common C-terminal amino acid sequence -Phe-X-Gly-Leu-Met-NH2. One such tachykinin, substance P, has been reported to modulate immunologic responses. In this investigation, granulomas were examined for substance P. Granulomas were isolated from the livers of mice infected with murine schistosomiasis, and substance P was extracted. Immunoreactive substance P was detected by RIA. The authenticity of the molecule was confirmed by elution profile on HPLC. Immunoreactive substance P, identified by immunostaining, localized to eosinophils derived from collagenase-dispersed granulomas. Granulomas were then probed for expression of the gene for substance P (preprotachykinin). Preprotachykinin mRNA was localized to granuloma eosinophils by in situ oligonucleotide hybridization. It is concluded that substance P is present within the granuloma as a result of preprotachykinin production by eosinophils.     

Organ-dependent differences in composition and function observed in hepatic and intestinal granulomas isolated from mice with Schistosomiasis mansoni

In murine schistosomiasis mansoni, eggs deposited in the liver and intestines induce a cell-mediated granulomatous reaction. Previous studies have shown that maximal granuloma size differs in the liver and various segments of the gastrointestinal tract. The objective of this investigation was to isolate intestinal granulomas and to determine whether organ-dependent differences in cell composition and granuloma function exist. Intestinal granulomas representative of those in tissue were isolated by a combination of chemical and mechanical techniques. When dissociated by collagenase, these lesions yielded a viable heterogeneous population of inflammatory cells. Granulomas from the liver, colon, and ileum showed differences in cellular composition. Liver lesions contained the largest number of T and B lymphocytes, eosinophils, and mast cells whereas ileal granulomas comprised mostly macrophages. Immunofluorescence studies on frozen tissue sections revealed that T and B lymphocytes also displayed different patterns of distribution within granulomas from different tissues. In contrast to isolated cultured liver granulomas that produced MIF-like substances, isolated colonic and ileal granulomas had weak or no MIF activity. It thus appears that granuloma formation in various organs is influenced by local factors that could affect the ultimate resolution of the lesions.     

Pleural cellular reaction to the filarial infection Litomosoides sigmodontis is determined by the moulting process, the worm alteration, and the host strain

The filarial nematode Litomosoides sigmodontis model was used to decipher the complex in vivo relationships between filariae, granulomas and leukocytes in the host's pleural cavity. The study was performed from D5 p.i.: to D47 p.i. in resistant C57BL/6 mice, to D74 p.i. in susceptible BALB/c mice, and to D420 p.i. in permissive jirds. We showed that, during the first month, leukocytes only clustered as granulomas around shed cuticles (exuviae) and with eosinophils as the major constituents. In addition, carbohydrates residues became abundant on exuviae only, suggesting a glycan-dependent mechanism of eosinophil attachment. Neutrophils were absent from the pleural cavity of all rodents and from the murine granulomas, but they made up 25% of the granuloma cell population in jirds. After the first month of infection granulomas formed around developed adult worms and morphological evidence suggested that leukocytes preferentially clustered around altered, but still motile, worms. No carbohydrates were detected on these worms and neutrophils were abundant in those granulomas. Finally, a rare third type of granuloma was observed in the resistant mice only; they contained young newly moulted adult worms; typically these granulomas were attached to the lateral lines of the worm via eosinophils; this feature correlated with the persistence of carbohydrate residues on the worms' lateral lines. Neutrophils were always in low proportion in all granulomas from resistant mice, suggesting difference in their adhesive properties in these mice. In vitro neutrophil recruitment in resistant mice was similar to that observed in susceptible mice although they expressed less cell surface CD11b.     

Substance P but not vasoactive intestinal peptide modulates immunoglobulin secretion in murine schistosomiasis.

Neuropeptides including SP and VIP modulate Ig secretion by in vitro stimulated lymphocyte cultures. It is not known whether these neuropeptides effect the B cell directly, or if they significantly alter humoral immune responses to pathogens. We have previously shown that granulomas derived from schistosome-infected mice contain immunoglobulin secreting B cells (ISC) as well as eosinophils that secrete substance P (SP) and vasoactive intestinal peptide (VIP). It therefore seemed plausible that B cells derived from infected animals might respond to these neuropeptides, and that such responses might effect immunoregulatory signals. In this study, we addressed these issues in the murine Schistosoma mansoni model, at the level of immunoglobulin secretion in single B cells. Spontaneous ISC were observed in both splenic and granuloma cell preparations. The addition of SP resulted in a dose-dependent reduction in the number and size of plaques (a 50% reduction was observed at 10(-9) M). This effect was blocked with SP antagonists. Similar results were observed in T cell-depleted cell cultures. VIP had no effect on ISC number or plaque size. We conclude that SP, but not VIP, decreases spontaneous ISC number and Ig secretion in short-term cultures of spleen and granuloma cells. SP appears to exert its effects at the level of single B cells through a receptor-mediated mechanism and may thus play an immunoregulatory role in schistosomiasis.     

In vivo molecular analysis of lymphokines involved in the murine immune response during Schistosoma mansoni infection. I. IL-4 mRNA, not IL-2 mRNA, is abundant in the granulomatous livers, mesenteric lymph nodes, and spleens of infected mice

Using Northern Blot analysis, the endogenous levels of IL-4 and IL-2 mRNA in the spleens, mesenteric lymph nodes, and granulomatous livers of male CBA/J mice in the acute phase of infection with Schistosoma mansoni have been quantified. High levels of IL-4 mRNA were detected in all three tissues from infected mice, whereas none was detected in tissues from normal, uninfected, age-matched mice. Isolation of the granulomas from the livers of infected mice and subsequent extraction of total RNA from these lesions resulted in a 70-fold enrichment of IL-4 message compared with the whole, unseparated granulomatous liver tissue. Hence, the predominant source of the IL-4 mRNA detected in livers from infected mice appears to be the schistosome egg-induced granulomas within these livers. In contrast, IL-2 mRNA was never detected in any of these tissues from either infected or normal mice. Control experiments were performed that ruled out the possibility that this inability to detect IL-2 mRNA was due to a difference in the efficacy of the IL-4 and IL-2 probes or due to a selective lability of IL-2 message. These data imply that IL-4-producing, Th2 lymphocytes are active in and possibly integral to the granulomatous, delayed-type hypersensitivity response characteristic of this infection, and directly challenges the current hypothesis that delayed-type hypersensitivity responses are exclusively mediated by Th1 lymphocytes.     

Schistosoma mansoni: higher free proline levels in the livers of infected mice

The concentration of L-hydroxyproline in the liver of ICR female mice increased rapidly during the 8th to 11th weeks of Schistosoma mansoni infection. Free L-proline concentration began to increase about the 7th week and reached its maximum at the 8th to 9th weeks of the infection, when the granulomatous response to the schistosome eggs in the liver was most prominent, as indicated by the increase in liver wet weight and its deoxyribonucleic acid concentration. A significant increment in the total activity of ornithine-delta-transaminase (EC 2.6.1.13) and the decrease in the specific activity of proline oxidase (EC 1.4.3.2) became detectable in the liver homogenate of infected mice on the 8th week. However, changes in these enzymatic activities were not parallel to that of the hepatic free L-proline content. Intraperitoneal administration of S. mansoni egg granulomas or 15,000g x 30 min supernatant fluid of their extracts into uninfected, normal mice significantly increased the hepatic free L-proline content without any appreciable effect on the enzymatic activities of proline oxidase and ornithine-delta-transaminase. These findings suggest that S. mansoni egg granulomas contain a factor(s) which may be responsible for the elevation of free L-proline content in the fibrotic liver caused by experimental schistosomiasis mansoni.     

Pelvic abscess from enterobius vermicularis. Report of a case with cytologic detection of eggs and worms

BACKGROUND: Enterobius vermicularis is known to produce perianal and ischioanal abscesses and invade the peritoneal cavity via the female reproductive system, causing pelvic peritonitis. However, there are only rare case reports on the cytodiagnosis of these parasitic lesions. CASE: A 28-year-old woman was admitted with a tender left iliac fossa mass and greenish vaginal discharge. Ultrasonogram and computed tomography scan confirmed the presence of a mass lesion suggestive of a tuboovarian abscess. Cytologic examination of the pus obtained during left salpingo-oophorectomy revealed the presence of ova of E vermicularis and fragments of the adult worm in an inflammatory exudate consisting predominantly of neutrophils, eosinophils and occasional epithelioid cell granulomas. Paraffin sections of the tuboovarian mass showed necrotizing epithelioid cell granulomas, but neither ova nor any worm section was identified. Although the possibility of tuberculosis was considered histologically, Ziehl-Neelsen (Z-N) stain for acid-fast bacilli was negative. Z-N staining of the smear and mycobacterial culture of the pus also did not yield positive results. CONCLUSION: E vermicularis may cause tuboovarian abscess with necrotizing epithelioid granulomas mimicking tuberculosis. Cytologic examination of the pus is helpful in the diagnosis.     

Tachykinin production in granulomas of murine schistosomiasis mansoni

Preprotachykinins, the products of one gene, are the precursor molecules of three mammalian tachykinins called substance P (SP), substance K (SK), and neuropeptide K. An additional mammalian tachykinin, neurokinin B, has also been described. SP and possibly other tachykinins may modulate immunologic responses. Granulomas that form around parasite ova in murine schistosomiasis were examined for tachykinins. Tachykinins were extracted from granulomas by boiling or with detergent. Extracts examined by RIA and HPLC contained only immunoreactive SP. Granulomas were dispersed with collagenase and cultured in vitro for up to 4 h. Only immunoreactive SP appeared in the culture medium. SP immunoreactivity localized solely to granuloma eosinophils as demonstrated by a sensitive immunohistochemical technique. An antiserum that recognized SK, neuropeptide K, and neurokinin B, but which possessed low reactivity to SP, also stained these cells. Only prior absorption of each antiserum with the appropriate synthetic neuropeptide would abrogate the immunostaining. This suggested that tachykinins other than SP were present within these cells. However, results of in situ hybridization experiments intimated that eosinophils produced predominantly preprotachykinin mRNAs which encode SP but are devoid of the SK/neuropeptide K sequence. It is concluded that granuloma eosinophils make predominantly SP in deference to other tachykinins, and that tachykinins other than SP are unlikely to be important in the regulation of the early granulomatous response of murine schistosomiasis.     

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.