Role of IL-4 and IFN-gamma in Schistosoma mansoni egg-induced hypersensitivity granuloma formation. Orchestration, relative contribution, and relationship to macrophage function.

A well defined model of T cell-mediated hypersensitivity-type granulomatous inflammation induced by Schistosoma mansoni eggs was used to assess the role of IL-4 and IFN-gamma in granuloma development. Synchronized pulmonary granulomas were induced and isolated from S. mansoni-infected mice during vigorous (8 wk) and modulated (20 wk) stages of the disease. The sequential production of IL-4 and IFN was determined and related to temporal changes in granuloma macrophage production of IL-1, TNF, and superoxide anion (O2-). During the vigorous stage, IL-4 was produced on days 1 and 2 of granuloma formation, whereas IFN was released in greatest amounts on days 4 to 8. The peak of IL-4 occurred in a window between the peak of IL-1 (1 day) and maximal TNF production (8 to 16 days). Maximal O2- release tended to parallel IFN production. During the modulated stage when the inflammatory response is attenuated, IL-4 production was dramatically reduced as were levels of IL-1 and TNF, but IFN production persisted and maximum O2(-)-producing capacity was only delayed in onset. mAb specific for IL-4 and IFN were used to examine the effect of in vivo depletion of these cytokines on granuloma development. Administration of a single 1.0-mg dose of anti-IL-4 antibodies to mice with synchronously developing granulomas dramatically reduced granuloma size (40 to 50% suppression of area) during an 8-day study period, whereas antibodies to IFN had no effect on size. However, the latter treatment reduced giant cell formation. Our results indicate that granuloma development involves an orchestrated production of cytokines possibly resulting from sequential participation of different Th cell populations. Moreover, IL-4 is a pivotal cytokine in anamnestic cellular recruitment and subject to endogenous regulation.

Vaccine induced immunity to Schistosoma mansoni: spleen cell proliferative responses before and after challenge in BALB/c mice given irradiated or normal schistosomula.

Spleen cell proliferative responses in BALB/c mice were assessed at varying intervals after vaccination or primary infection and subsequent cercarial challenge. Mice were vaccinated with 500 50-Krad-irradiated Schistosoma mansoni schistosomula or infected with 20 normal schistosomula. Prior to challenge, splenic responses in the two test groups to phytohemagglutinin (PHA) declined progressively while schistosomula (SMA)-driven responses increased. After challenge, PHA responses increased in both groups on day 3 then declined to significantly lower levels compared to normal controls. On day 3 after challenge, SMA responses in vaccinated mice were vigorous, and greater than twice the responses in infected mice. Thereafter, responses in vaccinated mice declined while responses in infected mice increased on days 7 through 25 but dropped markedly by day 39. For the infected group, in vitro depletion of plastic adherent cells or Lyt 2.2+ lymphocytes resulted in augmented SMA responses 3 days post-challenge by greater than 400% and greater than 100%, respectively. Depletion of either cell population in the vaccinated group had no significant effect. Protection assessed by total worm burdens showed a reduction of 62% in vaccinated mice and 43% reduction in infected mice. The post-challenge results indicate that these two models of anti-schistosomula immunity differed in the dynamics of their splenocyte antigen-specific proliferative responses. These findings may contribute to an understanding of the mechanisms by which resistance to S. mansoni is induced.

Schistosoma mansoni tropomyosin: production and purification of the recombinant protein and studies on its immunodiagnostic potential.

A cDNA that encodes Schistosoma mansoni tropomyosin, except for 10 amino acids at the amino terminus, has been cloned into a pOTSNCO plasmid vector. Induced expression resulted in a constant level of recombinant protein production. The recombinant S. mansoni tropomyosin was purified from preparative SDS-PAGE gel and by a combination of 20% ammonium sulfate fractionation and fast protein liquid chromatography-ion-exchange chromatography. The purified recombinant S. mansoni tropomyosin was tested as an immunodiagnostic reagent in Western blot and enzyme-linked immunosorbent assays. Sera from individual patients with chronic S. mansoni infection, but not S. haematobium, S. japonicum, parasitic infections other than schistosomiasis, and without infection reacted with the recombinant tropomyosin. The species specificity of S. mansoni tropomyosin suggests that further study of its potential as an immunodiagnostic reagent is warranted.

Quantitative cellular cytotoxicity to Brugia malayi microfilariae: in vitro studies of sera from an area endemic for filariasis in Andhra Pradesh, India.

The extent of eosinophil and neutrophil cell-mediated cytotoxicity on Brugia malayi microfilariae by sera from an area endemic for bancroftian filariasis in Andhra Pradesh, India, has been studied in vitro in terms of the clinical status of the subjects. At physical examination, 66 serum samples were collected. Group "A" included patients with various disease manifestations like lymphoedema, hydrocoele, lymphangitis and elephantiasis. Group "B" had microfilaraemia ranging from 1-300/ml of blood. Subjects with no history of infection past or present (endemic normals) were studied as Group "C". Out of 38 sera tested individually with eosinophils, 14/18 of Group A, 10/11 of Group B and 7/9 of Group C promoted higher (21-97%), moderate (18-88%) and highest (51-95%) range of cytotoxicity, respectively. The age, clinical status and duration of disease among the infected subjects appeared to correlate with the microfilarial mortality. In Group B, we observed the highest microfilarial count (16-300/ml) in lower (1-20 yrs) age groups. These individuals promoted higher (77-83%) cytotoxicity compared to the older age group (21-40 or 41-60) with low (1-36/ml) microfilaraemia. Group C sera were highly toxic to microfilariae. All those that were positive promoted greater than 50% mortality. Eighteen nonendemic normal sera had no effect on microfilariae. The overlapping but differential toxicity of the sera indicates that various clinical manifestations are associated with different types of cellular and humoral responses. These studies should help focus identification of the target epitopes of various immune responses of the host.

T cell clones for antigen selection and lymphokine production in murine Schistosomiasis mansoni.

The role of T cells in immunity to murine schistosomiasis was examined through the use of T cell clones that recognize the live schistosomulum stage of Schistosoma mansoni. T cell clones of three different phenotypes were isolated and expanded into long term culture from lymph nodes of C57B1/6J mice vaccinated with irradiated S. mansoni larvae. They were characterized by surface markers, lymphokine production, and functional assays. The m.w. range of the Ag recognized by one clone was identified through nitrocellulose blotting and confirmed with a preparation of the putative protein made by immunoaffinity purification. All but one of the clones were CD4+, CD5+, Th cells. One clone, 35, produced Il-2 and IFN-gamma and was designated a TH1 clone. The others were designated TH2 clones because of Il-4 production. One clone was CD8+ and failed to show help. Clone 35 recognized live schistosomula and produced Il-2 when presented a 27-kDa protein from nitrocellulose. It proliferated in response to purified Ag. Clone 35 participated along with macrophages to induce up to 98% killing of live schistosomula in vitro. IFN-gamma and TNF-alpha were essential to the killing mechanism whereas Il-1, Il-2, and Il-4 were not required. This study has approached Ag identification for vaccine development from the point of view of T cells and showed that TH1 cells are essential to in vitro macrophage killing of schistosomula in murine schistosomiasis.

Course of infection and humoral response to Leishmania major in inbred Meriones unguiculatus.

Numerous species of Meriones have been incriminated as natural reservoir hosts of Leishmania major in Mongolia, Soviet Asia, Afghanistan, the Middle East, and North Africa. However, little is known about the immunological response or course of infection in these small rodents. In this study, 40 commercially obtained inbred Meriones unguiculatus were divided into equal groups and injected in the right hind footpad with various doses of L. major promastigotes or with medium only. At regular intervals, blood was collected from the animals for subsequent evaluation of the kinetics of anti-L. major serum antibody production. Footpad lesions were measured periodically for 13 wk, beginning just before infection. The humoral response to infection and the course and severity of disease were dose related. However, metastasis lymph nodes, liver, spleen, and secondary cutaneous sites occurred at each of the doses tested.

Differential recognition of two cloned Brugia malayi antigens by antibody class.

The humoral and cellular immune response to filarial parasites is complex. Numerous studies have shown that antibodies to a large number of protein and non-protein antigens may be produced over the course of infection and that immune recognition of any given antigen may vary by disease manifestation and by immunoglobulin class. We have used the techniques of molecular cloning to attempt to dissect this complex interaction, and describe here two clones, isolated from an expression library constructed from Brugia malayi genomic DNA, whose products are recognized by distinct immunoglobulin classes. A lambda gt11 fusion protein containing part of the B. malayi myosin tail region is recognized by antibodies of the IgG class from a high percentage of bancroftian filariasis patients. A fusion protein containing a collagen-like sequence is less frequently and weakly recognized under the same experimental conditions, but is almost universally recognized when the developing reagent is specific for IgE. We thus identify specific filarial proteins against which the infected human host responds preferentially with antibodies of a specific immunoglobulin class.

Monokine production by hypersensitivity (Schistosoma mansoni egg) and foreign body (Sephadex bead)-type granuloma macrophages. Evidence for sequential production of IL-1 and tumor necrosis factor.

The present study examined the potential role of IL-1 and TNF in granuloma formation. Mice were given i.v. injections of Schistosoma mansoni eggs or Sephadex beads to induce synchronous immune T cell-mediated (hypersensitivity type) or nonimmune (foreign-body type) granulomas, respectively. Granuloma macrophages isolated at 2, 4, 8, 16, and 32 days of granuloma formation were evaluated for their capacity to produce IL-1 and TNF in response to 1 microgram/ml LPS. This was related to circulating levels of the acute phase protein, serum amyloid P (SAP) and expression of Ia Ag by monocytes and macrophages. Macrophages from nonimmune bead lesions were generally weak producers of IL-1 and TNF. In contrast, those from T cell-mediated egg lesions produced significant levels of both monokines. Moreover, there was a clear pattern of sequential monokine production such that IL-1 was produced in greatest amounts early (2 to 4 days), whereas TNF was produced later (8 to 16 days). Levels of SAP showed an initial sharp rise following particle embolization, then decreased rapidly in bead injected animals. However, mice with immune granulomas showed a prolonged elevation in SAP levels that corresponded to the period of maximal IL-1 production (2 to 4 days). Macrophage/monocyte Ia Ag expression was greatest at 8 to 16 days, corresponding to the period of TNF production. Bead injected animals showed low levels of Ia expression over the study period. These findings suggest that IL-1 may be important in the early recruitment stages of granuloma formation while TNF may take part in later maintenance or effector functions. The extent of production of both is likely influenced by the local or systemic milieu of lymphokines.

Flow-cytometric evaluation of lymphocyte subpopulations in synchronously developing Schistosoma mansoni egg and Sephadex bead pulmonary granulomas.

Synchronous models of T-cell-mediated and foreign body granulomas were induced in mice by intravenous embolization of Schistosoma mansoni eggs and Sephadex beads, respectively. The authors then performed flow-cytometric analysis of lymphocytes from dispersed granulomas, spleens, and peripheral blood at 4, 8, 16, and 32 days corresponding to the induction, growth, and maintenance, and resolution of these lesions. Lymphocytes were identified on the basis of light scatter characteristics, and the nature of the cells was confirmed by cell sorting and electron-microscopic examination. Lymphocyte subpopulations were characterized with antibodies to lymphocyte surface markers, specifically Ig, Thy 1.2, Lyt 1, Lyt 2, and L3T4. Natural killer cells were identified with anti-asialo GM1. Egg-induced granulomas had more lymphocytes of all phenotypes at all time points. Surprisingly, there was a significant number of cells staining positive for asialo GM1. On Day 16 after embolization there was a greater percentage of helper T cells, as defined by positive staining with L3T4, in the egg model, compared with the bead model. There was no obvious shift of lymphocytes from either the blood or spleen into the granuloma. These data confirm the importance of T cells in the direct participation of granulomatous inflammation, and the large numbers of asialo GM1-positive cells suggest a role for natural killer cells.

Suppression of natural killer cytolytic activity in mice undergoing pulmonary granulomatous inflammation.

CBA/J mice undergoing pulmonary granulomatous inflammation exhibited depressed NK cytolytic activity. Granulomas induced by i.v. embolization of Schistosoma mansoni eggs (hypersensitivity type) or Sephadex beads (foreign body type) both caused reduced NK activity, although hypersensitivity granulomas induced a significantly higher level of NK suppression. Kinetic analysis of hypersensitivity lesions at 4, 8, 16, and 32 days post-embolization indicated that NK activity was significantly suppressed by day 8, maximally suppressed by day 16 (at the peak of the inflammatory response) then returned to near control values by day 32 (as the granulomas resolved). Suppression of NK activity ranged from three- to 15-fold in different experiments. NK cells obtained from both spleen and peripheral blood demonstrated reduced NK activity with kinetic patterns similar to the granuloma NK cells. Suppression was not due to reduced splenic NK cells as the frequency of YAC-1 binding cells, as well as asialo GM1+ or laminin+ cells remained constant over the entire study period. Suppression of NK activity did not appear to be due to serum components or suppressor cells present in the spleen preparations. However, the suppression of NK activity could be reversed by overnight incubation of spleen cells at 25 or 37 degrees C or daily treatment of the mice with indomethacin. Suppression also appeared relatively specific for NK cells as the generation and expression of cytotoxic T lymphocyte activity was not affected.

Cutaneous leishmaniasis in Jordan: biochemical identification of human and Psammomys obesus isolates as Leishmania major.

As part of a series of epidemiological and ecological studies of leishmaniasis in Jordan, we have made functional studies of four isolates from human lesions and from ear lesions of three field-collected Psammomys obesus. Primary isolates were subcultured, frozen stabilates prepared and BALB/c mouse infectivity experiments initiated. Each mouse was inoculated with 4-8 x 10(4) promastigotes into a hind footpad. Quantitative evaluation of the footpads showed enlargement three to four weeks postinoculation. Amastigotes were readily identified in smears from footpad lesions and promastigotes in culture. At 47 days, liver and spleen samples grew out promastigotes. Biochemical characterization of these seven isolates was made by isozyme analysis using cellulose acetate membrane electrophoresis of fructokinase, phosphoglucose isomerase, phosphoglucomutase, aspartate aminotransferase, malate dehydrogenase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Reference isolates used for comparison were Leishmania major, L. tropica minor, L. donovani, L. aethiopica and L. m. mexicana. All seven Jordan isolates showed enzyme electromorphs identical to L. major, confirming our ecological/epidemiological studies that P. obesus is a major reservoir for human cutaneous leishmaniasis in Jordan.

Modulation of murine schistosomiasis by exogenously administered prostaglandins.

The effect of parenteral administration of prostaglandins, 15-(s)-15-methyl PGE1 (M-PGE) and PGF2 alpha (PGF) on the pathophysiologic manifestations of active murine Schistosoma mansoni infection was examined. Both M-PGE and PGF resulted in a nearly 50% suppression of granuloma size following a 7-day course of treatment. M-PGE and PGF appeared to act by different mechanisms. The former caused a broad-spectrum immunosuppression manifested as reduced splenomegaly, B-cell proliferation, and antigen-specific interleukin-2 (IL-2) production as well as decreased granuloma macrophage Ia antigen expression, superoxide anion (O2-) production, and interleukin-1 (IL-1) production. In contrast, PGF did not ameliorate splenomegaly, but caused increases in splenic asialo-GM1 (natural killer cells) and L3T4 (helper) positive T cells. Prostaglandin F also reduced IL-2 production, but to a lesser extent that M-PGE. Although PGF caused reduced granuloma macrophage Ia expression and O2- production, it did not suppress IL-1 production. Overall, these data show that PGs can significantly modulate immunopathologic events in chronic granulomatous disease states.

Schistosomiasis in an American medical investigator.

A 42-year-old American male researcher contracted schistosomiasis from environmental sources in the course of his observations on human behavior in Upper Egypt. After a long asymptomatic period, he developed various symptoms and Schistosoma haematobium was found in a urine examination. After treatment with Metrifonate, urine examination became negative. However, abdominal pain persisted and most diagnostic tests were negative. Colonoscopic examination and biopsy of the mucosa revealed schistosomiasis. Treatment with Praziquantel was thoroughly effective in clearing the persistent Schistosoma haematobium infection. It is necessary to maintain a high index of suspicion in cases of potential schistosomiasis. The availability of nontoxic treatment is discussed.

Ultrastructural observations on the fate of Brugia malayi in jirds previously vaccinated with irradiated infective stage larvae.

Vaccination of inbred jirds (Meriones unguiculatus) with 60cobalt radiation-attenuated Brugia malayi infective stage larvae (L3) protected against homologous challenge given either subcutaneously (sc) or by the intraperitoneal route (ip). At necropsy numerous nodules were recovered from the peritoneal cavities of jirds which had been vaccinated sc and challenged ip. Histopathologic analysis showed these to be granulomas containing dead and dying larvae and transmission electron microscopy showed that eosinophils were present in high numbers around and within the larvae. Structural damage to the L3 cuticle was apparent in discrete areas and eosinophils actively entering the breached cuticle at the time of fixation were observed. Coalescence of eosinophil secretion granules and the formation of degranulation vacuoles were seen in eosinophils throughout the granulomas. Degranulation resulted in the deposition of electron-dense material on the surface of the larval cuticle. The jird vaccine model for B. malayi thus appears to be a potentially useful tool for investigation of immune mechanisms in filariasis.

Species-dependent regulation of monocyte/macrophage Ia antigen expression and antigen presentation by prostaglandin E.

The expression of Ia antigen by various murine and human macrophage populations and the ability of prostaglandins of the E series to regulate Ia antigen expression were explored. Monocytes and macrophages from human and murine populations demonstrated a dichotomy in the expression of Ia antigen. Both human monocytes and macrophages expressed elevated levels of Ia antigen compared to their murine counterpart. Murine macrophages appear to express elevated levels of Ia antigen only when actively interacting with T lymphocytes in vivo or with lymphokines in vitro. Prostaglandins of the E series can suppress murine macrophage Ia antigen expression, but have little effect on the expression of Ia antigen by human monocytes and macrophages. Also, prostaglandins of the E series do not modulate the ability of human monocytes to present antigen to autologous lymphocytes when studied over a broad concentration range. These data suggest that prostaglandin E compounds do not profoundly affect human monocyte/macrophage Ia antigen expression or human monocyte antigen presenting activity.

Dynamics of arachidonic acid metabolism in macrophages from delayed-type hypersensitivity (Schistosoma mansoni egg) and foreign-body-type granulomas.

The present study examines the kinetics of arachidonic acid (AA) metabolism by murine macrophages isolated from sites of experimentally induced pulmonary granulomatous inflammation. Macrophages of T-cell-mediated hypersensitivity lesions induced by Schistosoma mansoni eggs (SE-GM) and non-T-cell-mediated foreign-body-type lesions (FB-GM) induced by Sephadex beads were examined. Overall, macrophages from both types of lesions produced mainly lipoxygenase pathway metabolites, leukotrienes, and monohydroxyeicosatetraenoic acids (mono-HETEs). Early after induction (4 days [4D]), SE-GM showed an augmented zymosan-stimulated AA release and metabolism compared to resident peritoneal macrophages. Macrophages from mature lesions (8-32D) showed constitutive synthesis of metabolites and were refractory to zymosan stimulation. Both SE-GM and FB-GM showed augmented AA uptake incorporating a large proportion into neutral lipids. A direct comparison of SE-GM and FB-GM revealed that the T-cell-mediated lesion produced lesser amounts of prostaglandins and leukotrienes and showed reduced incorporation of AA into phosphatidylcholine. These data suggest that AA metabolism by granuloma macrophages is sequentially modified during recruitment and activation at sites of chronic inflammation.

Brugia malayi: vaccination of jirds with 60cobalt-attenuated infective stage larvae protects against homologous challenge.

Vaccination of inbred jirds (Meriones unguiculatus) with 60cobalt radiation-attenuated Brugia malayi infective stage larvae (L3) protected against homologous challenge given either subcutaneously (sc) or by the intraperitoneal (ip) route. Groups of jirds vaccinated once sc with 75, 15 Krad L3 showed from 69% to 91% reduction in recovered worms after ip challenge infection compared to infection in non-vaccinated control jirds, while 75% reduction in mean worm burden was seen in jirds receiving sc challenge infection. A single sc vaccination with 75, 10 or 20 Krad L3 produced no protection (10 Krad) and 64% reduction in recovered worms (20 Krad). Therefore the 15 Krad dose appeared to be best. A marked increase in anti-B. malayi antibody in vaccinated jirds was seen (by ELISA) immediately after challenge infection and an immunofluorescence assay showed that L3 incubated in serum from vaccinated jirds were completely and uniformly covered with specific antibody. Eosinophil-rich granulomas containing dead and moribund L3 were recovered from vaccinated jirds. This model of protective immunity in a Brugia-susceptible small rodent may provide a useful system for identification of molecularly defined filarial-protective immunogens.

Activation of the alternative complement pathway in normal human serum by Loa loa and Brugia malayi infective stage larvae.

Infective stage larvae (L3) of Loa loa and Brugia malayi upon in vitro incubation with normal human serum activated the alternative complement pathway. C3 conversion products were detected on larval cuticles by eosinophil adherence and by immunofluorescence with C3c antiserum. No evidence for cuticle binding of IgG, IgA, IgM, Clq, or C4 was found by immunofluorescence. L3-induced C3 activation was inhibited by 10 mM EDTA but unaffected by 10 mM Mg++-EGTA. Human sera deficient in C2, C4, or C6 incubated with L3 resulted in C3 activation. However, sera treated with zymosan or heated for 1 h, 56 degrees C were unreactive with L3. Immunoelectrophoresis of fresh serum exposed to L3 for 1 h at 37 degrees C showed C3 cleavage products. The results indicate that these nematode L3 activate the alternative complement cascade via cuticular surface components. Larval viability was unaffected by complement activation or by adherence of eosinophils.

Schistosoma haematobium in Upper Egypt: analysis of dispersion patterns.

Statistical methods analyzing changes in dispersion patterns of parasites among hosts were applied to Schistosoma haematobium egg excretion data from a five-year study of a cohort of 1,400 boys in 3 villages in Upper Egypt. Despite significant changes in mean density of the parasites in the first 4 years, the degree of aggregation of S. haematobium among the cohort did not change markedly in analysis of egg count data obtained semiannually. Two other related measures of dispersion, mean crowding and patchiness, were also obtained and compared to prevalence and mean intensity, the components determining mean density of schistosomes among hosts. Anti-schistosomal chemotherapy with metrifonate in the cohort did not succeed in appreciably reducing the mean density of S. haematobium over a one-year follow-up. The dispersion pattern of S. haematobium among the study group was not markedly altered. Prevalence and mean intensity were useful indicators of changes in the degree of aggregation of S. haematobium among hosts. The inability of chemotherapy to provide a persistent decline in the mean density parameter is reflected in the rise of mean intensity and prevalence of S. haematobium 1 year post-therapy. It is suggested that analysis of quantifiable changes in dispersion patterns of S. haematobium among their hosts can be a useful aid in planning and assessment of control strategies.