Immunologic tolerance in lymphatic filariasis. Diminished parasite-specific T and B lymphocyte precursor frequency in the microfilaremic state.

To explore the mechanisms of antigen-specific immune unresponsiveness seen in microfilaremic patients with bancroftian filariasis, T and B cell precursor frequency analysis was performed using PBMC from individuals with either asymptomatic microfilaremia (MF, n = 7) or chronic lymphatic obstruction (CP, n = 20). Highly purified CD3+ cells were partially reconstituted with adherent cells and their proliferative response to parasite antigens determined in cultures of T cells by limiting dilution analysis. A filter immunoplaque assay also assessed the frequency of both total and parasite-specific Ig-producing B cells. While the lymphocyte proliferation to mitogens and to a nonparasite antigen (Streptolysin-O, [SLO]) were similar in all groups of patients, the frequency of parasite-specific CD3+ T cells was significantly lower (geometric mean [GM], 1/3,757) in MF patients when compared to that in CP patients (GM 1/1,513; P less than 0.001). Similarly, the proportion of lymphocytes producing parasite-specific IgE or IgG was significantly lower in MF patients (IgE mean, 0.2%; IgG mean, 0.33%) compared with CP patients (IgE mean, 3.2%; IgG mean, 1.76%; P less than 0.05 for both comparisons). These observations imply that low numbers of parasite-specific T and B lymphocytes may be partially responsible for the severely diminished capacity of lymphocytes from patients with MF to produce parasite-specific antibody and to proliferate to parasite antigen in vitro. Such differences in parasite-specific lymphocyte responses suggest that tolerance by clonal anergy may be a critical mechanism for maintaining the microfilaremic state.

Use of monoclonal antibodies to quantify subclasses of human IgG. II. Enzyme immunoassay to define antigen specific (anti-filarial) IgG subclass antibodies.

Because of their single epitope specificity, monoclonal antibodies (Mcabs) may perform with different levels of efficiency in immunoassays depending on the accessibility of the particular epitope recognized. In order to develop assays capable of detecting specific antibodies of each of the four human IgG subclasses, we have evaluated by ELISA the performance characteristics of a panel of Mcabs raised to the subclass proteins. At least one Mcab to each of the four subclasses was identified that was specific in its ability to capture its own relevant IgG subclass without any associated light chain, allotype or isoallotype activity and that was able to function effectively as a probe in an optimized, quantitative ELISA. When IgG subclass antibodies were measured in sera from patients with filariasis using specific filarial antigen, the sensitivities of each subclass antibody assay varied; for IgG1 and IgG4 antibodies the sensitivity of detection was 50 ng/ml and for IgG2 and IgG3, 10 ng/ml. The potency of the Mcab, determined by its titration for use as a probe, did not correlate with the sensitivity of the assay. These Mcabs were also capable of defining IgG subclass antibody responses qualitatively in immunoblot analyses with little or no non-specific binding. The availability of such highly characterized Mcabs for use in quantitative and qualitative definition of specific IgG subclass antibody responses should greatly improve our detection and subsequent understanding of the role of these IgG subclasses in various disease states.

Use of monoclonal antibodies to quantify subclasses of human IgG. I. Development of two-site immunoenzymometric assays for total IgG subclass determinations.

Dissection of the IgG antibody response into its subclass components has been difficult largely because of the lack of adequate supplies of specific reagents. The development of monoclonal antibodies (Mcab) promises to overcome this problem, but the use of such antibodies has certain inherent problems. It has been shown recently that Mcabs which were avid, potent and specific for well defined epitopes may partially or completely lose their activity depending on the assay system in which they were used. In order to identify Mcabs that would be specific and useful as capture antibodies in a simple two-site enzymometric assay, a panel of 18 Mcabs was screened and one Mcab to each of the four IgG subclasses was identified for quantitation of subclass levels in human serum.

Antigens from the surface and excretions/secretions of the filariform larva of Strongyloides stercoralis.

The surface and excretory/secretory (ES) antigens of the infective, filariform larva (L3) of Strongyloides stercoralis were identified. These studies provide a basis for the purification of these proteins as diagnostic allergens for human strongyloidiasis. The Mr values of the surface and ES molecules were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, fluorography, or silver staining following the recovery of these molecules after the radiolabelling of living parasites. At least 10 surface proteins were radioiodinated extrinsically using chloroglycoluril as the catalyst for iodination, and then extracted with detergents and/or beta-mercaptoethanol. Several surface molecules of the L3 were immunogenic in humans, as determined by immunoprecipitation with sera (IHS) from infected patients. About 30 proteins were present in the ES preparation. Many ES antigens were labelled biosynthetically during the culture of larvae in media supplemented with either [35S]methionine or [14C]glucose. Furthermore, several of the surface proteins of the L3 were found with the ES antigens recoverable by culturing larvae in vitro. About 10 of the ES proteins were immunogenic as determined by immunoaffinity chromatography using IHS; and two of these antigens with Mr 50,000 and 90,000 incorporated [35S]methionine during culture of larvae. Moreover, some ES proteins were allergenic when tested in an in vitro assay of histamine release from basophils from infected humans or monkeys. The isotype of the homocytophilic antibodies involved in this immediate hypersensitivity assay, which is the basis of a diagnostic skin test for human strongyloidiasis, appears to be IgE.

Modulation of the host response in human schistosomiasis. II. Humoral factors which inhibit lymphocyte proliferative responses to parasite antigens.

Humoral suppressive factors were identified in the plasma of 13 of 14 patients with schistosomiasis mansoni. These factors suppressed lymphocyte proliferative responses to parasite antigens but did not affect lymphocyte responses to either nonparasite antigens or mitogens. Furthermore, they had no inhibitory effect on proliferative responses of lymphocytes of normal, noninfected individuals. Three patients studied sequentially before and after therapy with niridazole developed positive lymphocyte responses to parasite antigens within the first month of treatment, but this enhanced responsiveness was not accompanied by clearance of these humoral supressive elements, which persisted in the patients' plasmas. Such suppressive factors which are specific for responses to parasite antigens probably form one of the mechanisms which determine the state of partial immunosuppression found in patients with chronic schistosome infection.

Detection, quantitation, and specificity of antiparasite IgE antibodies in human schistosomiasis mansoni.

Sera from 15 patients with acute or chronic Schistosoma mansoni infection were evaluated for IgE antibodies directed against soluble cercarial, adult worm, and egg antigens. Both the antigen-induced release of histamine from passively sensitized human basophils and specific radioimmunoassays were used to detect these IgE antibodies, and determination of serum IgE levels before and after specific immunosorption permitted their quantification. While chronically infected patients made IgE antibodies to all three stages of the parasite, only egg antigens induced an appreciable IgE antibody response in acutely infected individuals. Despite the fact that patients with chronic infection had significantly greater levels of total serum IgE than patients with acute disease, the percentage of this IgE that was parasite specific was similar for both groups, ranging between 4% and 28%. An ancillary observation was the fact that soluble egg antigen can trigger basophil histamine release through IgE-dependent reactions and through "nonimmunologic" mechanisms that require further characterization.

The clinical and immunologic responses of normal human volunteers to low dose hookworm (Necator americanus) infection.

Five normal human volunteers were exposed to approximately 50 infective larvae of Necator americanus and were observed for the development of clinical signs or symptoms and for changes in blood eosinophil levels, IgG antibody titers, total and parasite-specific IgE, and lymphocyte blastogenic responses for 6-10 weeks. Bronchoalveolar lavage was performed on four subjects prior to infection and at times when larval migration through the pulmonary tree was likely. Eggs were demonstrated in the stools of four volunteers who remained untreated for more than 6 weeks; one volunteer had to be treated at day 40 because of severe gastrointestinal symptoms. All others also complained of abdominal pain and flatulence between days 35-40. All volunteers developed marked blood eosinophilia which peaked between days 38-64 and ranged from 1,350-3,828 eosinophils/mm3. Small increases in total and parasite-specific IgE and IgG were noted in some volunteers. One volunteer showed a significant lymphocyte blastogenic response. With the exception of mucosal erythema, bronchoalveolar lavage results were unremarkable. Our data indicate that a single small inoculum of hookworm larvae is capable of producing significant transient gastrointestinal morbidity and marked blood eosinophilia but does not induce other prominent T cell- and B cell-dependent immune responses.

Syndrome resembling tropical pulmonary eosinophilia but of non-filarial aetiology: serological findings with filarial antigens.

Although the tropical pulmonary eosinophilia (TPE) syndrome of filarial aetiology has very distinctive clinical and immunological features, its clinical profile is not unique; other helminths sometimes induce similar presentations. We carefully evaluated 7 individuals with non-filarial TPE-like syndromes and found that serological tests based on detection of 'antifilarial' immunoglobulin (Ig) G, IgG4, and IgE antibodies that are usually considered diagnostic for filarial TPE were equally elevated in patients with non-filarial, TPE-like syndromes and were therefore unhelpful diagnostically. The apparent reasons were immunological hyper-responsiveness of such individuals and the shared (i.e., cross-reactive) antigenicity found in the filarial antigen preparations used routinely for diagnosis. Because appropriate treatment for those different pulmonary eosinophilia conditions requires different drugs and management, and because delay in effective treatment results in significant morbidity in such patients, diagnostic capabilities must be improved by identifying and obtaining unique antigens that can serologically discriminate between filarial TPE and other similar, but non-filarial, pulmonary eosinophilia syndromes.

Control of allergic reactivity in human filariasis. Predominant localization of blocking antibody to the IgG4 subclass.

Patients with chronic helminth infections, despite having abundant basophils and mast cells specifically sensitized with antiparasite IgE and often exposed repeatedly to parasite Ag, rarely manifest allergic symptoms. This control of clinical allergic reactivity likely results from Ag-specific IgG "blocking antibodies" shown previously to be abundant in the sera of such patients. In the present study we used two approaches to determine in which of the four IgG subclasses this blocking activity was localized. First, specific antifilarial antibodies of each of the four IgG subclasses were quantified in the sera of 28 patients with Bancroftian filariasis and correlated with the levels of blocking activity in these sera (determined by histamine release assays). A significant correlation with blocking activity was seen only for antibodies of the IgG4 subclass, and, indeed, the correlation was especially strong in the group of totally asymptomatic patients (but with microfilariae circulating in the blood) in whom blocking antibody levels were highest. Interestingly, however, if the analysis excluded these asymptomatic microfilaremic patients and focused instead on those with lymphatic inflammatory pathology (who had relatively low levels of both serum blocking activity and specific IgG4 antibodies), then the small amount of blocking activity found in these sera correlated only with the levels of IgG1 subclass antibodies. The second approach utilized selective depletion of IgG4 (by anti-IgG4 affinity columns) from the sera of three microfilaremic patients with high levels of blocking activity and demonstrated clearly that removal of IgG4 abolished the majority of the blocking activity in these sera (53, 78, and 81%). These two sets of findings demonstrate a predominant role for specific IgG4 antibodies in blocking IgE-mediated allergic responses to the parasite Ag in vitro, but they also indicate that in some situations IgG1 antibodies can block such reactions. Furthermore, the correlation demonstrated between patients' clinical presentations and the levels of both their specific IgG4 antibodies and serum blocking activity suggests that these antibodies play a similar role in vivo as well.

'Long-distance' lymphocyte study. The effects of transporting blood in lymphocyte blastogenic responses.

A comparison of blastogenic responsiveness to antigens and mitogen by human lymphocytes was made between cells which had been processed for culture immediately following blood collection and cells obtained from blood collected 9-11 hr previously and transported via commercial airline from the patients' homes to our laboratory. There were no significant differences in the responses of transported and non-transported cells if the blood was maintained at ambient temperature during the period of shipment. Chilling the blood during transport, however, resulted both in decreased stimulation of the cells and increased 'background' activity in unstimulated cultures. These findings indicate the feasibility of carrying out both limited immunological evaluations and extended periods of follow-up for patients located at considerable distances from a research laboratory.

Egg laying is delayed but worm fecundity is normal in SCID mice infected with Schistosoma japonicum and S. mansoni with or without recombinant tumor necrosis factor alpha treatment.

Mice with severe combined immunodeficiency (SCID mice) lack functional B and T cells. Egg laying by Schistosoma mansoni and S. japonicum was delayed in SCID mice, but in a matter of weeks worm fecundity was equivalent to that in intact mice. SCID mice formed smaller hepatic granulomas and showed less fibrosis than did intact mice. The reduction in egg-associated pathology in SCID mice correlated with marked reductions in interleukin-4 (IL-4), IL-5, IL-13, and gamma interferon mRNA expression in the liver. S. mansoni infections were frequently lethal for SCID mice infected for more than 9 weeks, while S. japonicum-infected SCID mice died at the same rate as infected intact mice. We were unable to affect hepatic granuloma formation or egg laying by worms in SCID mice by administration of recombinant murine tumor necrosis factor alpha (TNF-alpha). In fact, SCID and BALB/c mice appeared to express nearly equivalent levels of TNF-alpha mRNA in their granulomatous tissues, suggesting that there is little or no deficit in TNF-alpha expression in infected SCID mice. The data indicate that TNF-alpha may be in large part derived from a non-T-cell source. Together, these findings provide little evidence that TNF-alpha alone can reconstitute early fecundity, granuloma formation, or hepatic fibrosis in schistosome-infected SCID mice.

Immunologic correlates of the hyperresponsive syndrome of loiasis.

The usual clinical picture of loiasis in long-term visitors to endemic areas differs from that in residents of these areas, with allergic symptoms, hypergammaglobulinemia, profound hypereosinophilia, and increased serum levels of IgE being more prominent. In further analyzing the immunologic correlates of this apparent hyperreactivity in 20 patients, we have found the following: (1) parasite-specific IgG (in all) and IgE (in some) were extremely elevated in the patients; (2) qualitative analysis by immunoblotting indicated multiple antigens recognized by both IgG and IgE antibodies in these patients; (3) filaria antigen-specific, lymphocyte proliferative immune responses were vigorous in all patients and, in each individual, exceed the response to other soluble antigens; (4) spontaneous and antigen-driven, parasite-specific antibody production in vitro was elevated in all six patients studied; (5) there was a significant increase in the ratio of CD4/CD8+ T cells. These observations suggest that both specific dysregulation of the immune response to the parasite antigen, as well as nonspecific immune activation, accounts for the clinically apparent hyperresponsive state seen in expatriates acquiring loiasis.

Prominence of IgG4 in the IgG antibody response to human filariasis.

The four subclasses of IgG are distinct in structure, function, and degree of participation in the response to complex antigens. Because these differences could have important pathogenetic significance, we analyzed total and filaria antigen-specific IgG of each subclass in 31 patients with different clinical manifestations of Bancroftian filariasis. Subclass-specific, affinity-purified polyclonal antibodies were prepared from antisera raised in sheep immunized with purified myeloma IgG subclass proteins. These were radiolabeled (125I) and used to detect IgG1, IgG2, IgG3, and IgG4 in solid phase radioimmunoassays (SPRIA). The antigen-specific SPRIA was used with Brugia malayi adult antigen (BmA) bound to Sepharose 4B, whereas measurement of total IgG subclass levels in each serum was with goat anti-human IgG bound to the solid matrix. Quantification of total subclass levels was by reference to the WHO 67/97 standard, and of specific subclass antibody by development of standards from high titered sera. Although there were modest increases of total IgG1 and IgG2 in patients with filariasis compared with normals, the most striking finding was the extreme elevation of both total, and particularly, filaria antigen-specific IgG4. These elevations were seen for essentially all patients, but the relative proportion of the total IgG antibody response accounted for by IgG4 antibody was particularly marked (up to 95%) in patients with either microfilaremia or the tropical pulmonary eosinophilia syndrome. The meaning of this special prominence of the IgG4 antibody response to filarial infection is not yet clear, but the question of whether these antibodies play a role in immediate hypersensitivity reactions as either reagins or blocking antibodies is being investigated for its potential pathogenetic significance.

An IL-12-based vaccination method for preventing fibrosis induced by schistosome infection.

The harmful fibrosis which often occurs in the context of infectious disease involves the excessive deposition of connective tissue matrix, particularly collagen, and is mostly resistant to pharmacological and immunological intervention. In schistosomiasis, fibrosis is associated with the granulomatous response to parasite eggs trapped in the liver. We have previously shown that interleukin (IL)-12 administered peritoneally with eggs prevents subsequent pulmonary granuloma formation on intravenous challenge with eggs. Here we show that sensitization with eggs plus IL-12 partly inhibits granuloma formation and dramatically reduces the tissue fibrosis induced by natural infection with Schistosoma mansoni worms. These results are an example of a vaccine against parasites which acts by preventing pathology rather than infection. IL-12 is known to favour the priming of TH1 rather than Th2 cells, and the effects on fibrosis are accompanied by replacement of the Th2-dominated pattern of cytokine expression characteristic of S. mansoni infection with one dominated by Th1 cytokines. Elevated Th2 cytokine expression and fibrosis are common manifestations of a wide variety of infectious diseases and atopic disorders which might be ameliorated by vaccination with antigen and IL-12.

Frequency analysis of IgE-secreting B lymphocytes in persons with normal or elevated serum IgE levels.

The immunoregulatory mechanisms that determine the high serum IgE antibody levels in disorders such as helminth parasite infections and the hyper-IgE recurrent infection syndrome (HIE) remain poorly understood. To assess whether elevated serum IgE levels result from an increased number of B lymphocytes committed to IgE production, the proportion of IgE-producing B lymphocytes was determined by a filter immunoplaque assay using PBMC from persons with a broad range of serum IgE levels that included normal persons (n = 9) and patients with loiasis (n = 12), tropical pulmonary eosinophilia (TPE) (n = 6), lymphatic filariasis (n = 28), and HIE (n = 8). PBMC from these persons were assessed for production of in vitro IgE. The geometric mean number of IgE-secreting cells in 10(5) B lymphocytes in PBMC was 0.42 (range 0-2.2) in normal persons, 5.6 (range 0.1-35.5) among patients with loiasis, 9.4 (range 0-53.2) among patients with lymphatic filariasis, 52 (range 31.5-115) among patients with TPE, and 218 (range 56-1404) among patients with HIE. When all study subjects were grouped, there were significant correlations with serum IgE levels (r2 = 0.78; p less than 0.0001) and net spontaneous in vitro IgE production (r2 = 0.8; p less than 0.0001). Estimates of the amount of IgE production per B lymphocyte were similar among normal persons, patients with filarial infections, and patients with TPE (geometric means of 134, 96, and 141 pg/ml/cell, respectively); in contrast, for HIE patients, IgE production by individual B cells was significantly lower (geometric mean 28 pg/ml/cell; p less than 0.001). These observations demonstrate that clonal expansion of IgE-producing B lymphocytes is a major mechanism underlying the elevated serum IgE levels seen in persons with hyper-IgE states.

Suppressive effect of interleukin-4 neutralization differs for granulomas around Schistosoma mansoni eggs injected into mice compared with those around eggs laid in infected mice.

The principal pathological manifestation of murine Schistosoma mansoni infection is the egg-induced granuloma. Synchronous pulmonary granulomas forming around intravenously injected schistosome eggs are widely used to study the immunopathology of schistosomiasis. A number of anticytokine antibody treatments have a remarkable effect in modulating granulomas in this model but little effect on the size of hepatic granulomas around laid eggs during experimental infection. To examine this discrepancy, we examined the effects of anticytokine antibodies on liver and lung granulomas around injected eggs and around eggs laid during infection in both locations. Anti-interleukin-4 (IL-4) treatment greatly reduced the volume of granulomas around eggs injected into the liver via the portal vein and around eggs injected into the lung via the tail vein. On the contrary, granulomas around eggs laid by worms in either the liver or the lung during the course of infection were not significantly decreased in size by anti-IL-4 treatment. Thus, site is not important for the disparate effects of anti-IL-4 in granuloma formation around injected versus laid eggs. This effect is seen in naive and sensitized animals and is most probably due to differences in the quality of injected eggs versus those laid in situ by the worms.