To study the effect of repeated challenge of the innate immune system with pathogen-associated molecular patterns, cytokine responses to schistosomal lipids and bacterial lipopolysaccharide (LPS) were analyzed in schoolchildren living in an area in Gabon where schistosomiasis, a helminth infection that is chronic in nature, is endemic. A schistosomal phosphatidylserine (PS) fraction containing the Toll-like receptor (TLR)-2 ligand lyso-PS stimulated the production of interleukin (IL)-8, IL-10, IL-6, and tumor necrosis factor (TNF)-alpha in children without Schistosoma haematobium infection. However, in infected children, the responses to this stimulus were lower, in particular for production of IL-8 and TNF-alpha. Responses to the TLR4 ligand, LPS, followed a similar pattern. In contrast, schistosomal adult worm glycolipids that did not stimulate any of the TLRs tested induced IL-8 and IL-6 responses that were significantly higher in schistosome-infected children than in schistosome-uninfected children. These results indicate that relentless exposure to pathogens can lead to altered responses to TLR ligands.

Schistosome infections are characterized by prominent T cell hyporesponsiveness during the chronic stage of infection. We found that schistosome-specific phosphatidylserine (PS) activated TLR2 and affected dendritic cells such that mature dendritic cells gained the ability to induce the development of IL-10-producing regulatory T cells. Using mass spectrometry, schistosomal lysophosphatidylserine (lyso-PS) was identified as the TLR2-activating molecule. This activity appears to be a unique property of schistosomal lyso-PS, containing specific acyl chains, because neither a synthetic lyso-PS (16:0) nor PS isolated from the mammalian host activates TLR2. Taken together, these findings provide evidence for a novel host-parasite interaction that may be central to long term survival of the parasite and limited host pathology with implications beyond parasitology.

This study reports reversals in microfilarial density and the accompanying changes in cellular immune responses to filarial antigens of 39 individuals (11 microfilaria-positives, 22 microfilaria-negatives and six converters) living in an area endemic for brugian filariasis. Microfilarial counts decreased from April, the end of the rainy season to July, middle of the dry season (g.m. 88 mf/ml and 38 mf/ml, respectively; P = 0.001) and subsequently increased in November, the beginning of the rainy season (P = 0.088). Whereas the proliferative responses remained low throughout the study period in microfilaraemic individuals, in amicrofilaraemics these responses changed in the opposite direction to that of microfilarial densities. In three converters, proliferation changed in the opposite direction to the presence or absence of microfilariae. Cytokine analysis in the converters revealed that interferon-gamma was most affected by the shifts in microfilarial densities. In contrast, interleukin-4 responses showed little correlation with changes in parasite densities.

To understand the intricate balance and the coordinate expression of the Th1 and Th2 cytokines following a natural mode of T cell triggering, antigen-stimulated IL-4, IL-13 and IFN-gamma production was studied in primary peripheral blood mononuclear cell cultures at a single-cell level. Cells from filariasis patients who respond to parasite antigen by producing not only IFN-gamma but also IL-4 and IL-13 were stimulated with Brugia malayi adult worm antigen and analyzed for co-expression of cytokines by intracellular staining. IL-4 and IL-13 were frequently co-expressed (54% of IL-4+ cells stained for IL-13 and 29% of IL-13+ cells expressed IL-4 at all time points), whereas IFN-gamma expression was totally segregated from both IL-4 and IL-13. These data indicate that in human peripheral T cells the co-expression of the dominant Th1 and Th2 cytokines within a single cell is a rare event and that IL-13 is clearly more frequently associated with a Th2 than a Th1 type response in primary T cell cultures.

IgG4 and IgE isotypes contribute marginally to the pool of circulating antibodies in healthy individuals, but are elevated during atopic diseases and particularly upon helminth infections. To examine whether the high levels of these isotypes in circulation are reflected in a higher capacity of PBMC to produce IgG4 and IgE, we examined cells from patients infected with filarial nematodes that exhibit high levels of IgG4 and IgE. indeed, IgG4 production by PBMC correlated strongly with plasma levels of IgG4 (r = 0.534, P = 0.002), but such correlation was not found for IgE. The replacement of CD19(+) cells from PBMC by IgD(+) cells abrogated the high capacity of PBMC to make IgG4. This indicates that an altered B cell compartment accounts for the high IgG4-producing capacity of the PBMC. The high production of IgG4 in vitro was not dependent on IL-4 and IL-13, as neutralizing antibodies to these cytokines did not inhibit IgG4. However, IgE release by PBMC was dependent on IL-4 and IL-13. Antifilarial IgG4 was detected in culture supernatants from filarial patients and its production was independent of IL-4 and IL-13. These results demonstrate that in individuals with elevated IgG4, the B cell compartment in PBMC carries cells that are already committed to IgG4 production and are independent of IL-4 and IL-13.

In an area in which brugian filariasis is endemic, when cytokine levels were analyzed as a function of clinical status comparing those who were asymptomatic and amicrofilaremic with those who were microfilaremic, it was found that both interferon (IFN)-gamma and interleukin (IL)-5 were suppressed in microfilariae carriers (P < .01 and P < .001, respectively), but IL-4 was unabated. Age had a significant effect on cytokine production in both groups. In asymptomatic amicrofilaremic subjects, IL-4 production was high in young persons and decreased with age, whereas in microfilaremic subjects, IL-4 increased significantly with age. Conversely, IFN-gamma showed a tendency to increase with age in asymptomatic amicrofilaremic subjects but not in microfilaremic subjects. IL-5 decreased significantly with increasing age in both asymptomatic amicrofilaremic and microfilaremic groups. These results indicate that the length of exposure to and infection with filarial parasites can each exert a substantial effect on the cytokine response profiles of host T cell populations.

The effect of interleukin-12 (IL-12) on human immunoglobulin G4 (IgG4) and IgE production was examined with cells derived from filarial patients and European controls. IL-12 inhibited IgE release but enhanced IgG4 production in cultures of peripheral blood mononuclear cells stimulated with anti-CD2 plus IL-2. When purified T- and B-cell cocultures were examined, IL-12 again markedly enhanced IgG4, whereas IgE production was no longer inhibited.

Parasitic worms of the genus Schistosoma excrete relatively large amounts of immunogenic glycoproteins (circulating cathodic antigen [CCA]) that contain polysaccharide side chains with the trisaccharide Lewis-x (L(ex)) as a repeating unit. These carbohydrates evoke high titers of specific IgM antibodies that cross-react with the repeating L(ex) units on the surface of granulocytes. Consequently this might lead, in the presence of complement, to lysis of the granulocytes. In the present study, this hypothesis was investigated using anti-CCA mouse monoclonal antibodies (MoAbs) and polyclonal antibodies purified from sera of infected humans. By flow cytometry, it was demonstrated that the mouse MoAbs directed against CCA strongly recognized the granulocytes. It could also be shown that these MoAbs, as well as anti-CCA IgM antibodies purified from infected human sera, caused lysis of granulocytes in a complement-dependent cytotoxicity assay. Sera from healthy controls or from patients with other helminth infections resulted in negligible granulocytotoxicity. These in vitro observed phenomena may explain the mild to moderate neutropenia that occurs in schistosomiasis patients.

Natural infection with filarial nematode parasites results in immune responses skewed towards T helper (Th)2, while infection with mycobacteria shows many characteristics of a Th1-dominated response. Cytokines typifying Th1, interferon (IFN)-gamma, and Th2, interleukin (IL)-4, were measured following stimulation of peripheral blood mononuclear cells from filarial patients with Brugia malayi adult worm antigen (BmA) and purified protein derivative of Mycobacterium tuberculosis (PPD). In response to PPD, only 1 out of 81 patients produced IL-4, and this at an amount (4.4 pg/ml) just above the detection limit, whereas 59% of patients responded to BmA by releasing IL-4. Conversely, substantial quantities of IFN-gamma were released in response to PPD (geometric mean 37.43 U/ml) compared to low BmA-stimulated IFN-gamma production in the same patients (geometric mean 5.02 U/ml). These results demonstrate that the strong skewing of the cytokine environment towards Th2 in filarial patients in vivo does not influence the predominance of a Th1 type immune response to PPD.

Cellular immune responses to filarial antigens were examined in persons before and 1 year after beginning treatment with diethylcarbamazine (DEC). The subjects (17 microfilaremics, 13 asymptomatic amcirofilaremics, and 13 with elephantiasis) had not responded to Brugia malayi adult worm antigen (BmA) before chemotherapy. T cell proliferative responses to BmA improved significantly after therapy in the 3 clinical groups (P < .05) but was highest in the elephantiasis patients and asymptomatic amicrofilaremics. Cytokine release profiles after stimulation with parasite antigen were analyzed, Production of interferon (IFN)-gamma by BmA-stimulated mono-nuclear cells increased significantly after DEC treatment (geometric mean, 39.6-55.7 U/mL; P < .05), largely due to improved responses in elephantiasis patients and asymptomatic amicrofilaremics, In contrast, BmA-induced interleukin (IL)-4 release did not change significantly in these same patients after treatment. Thus, both microfilaremic and amicrofilaremic infections with B. malayi are associated with similar down-regulation of proliferative T cell function and IFN-gamma release.

Lymphatic filariasis presents a spectrum of manifestations with infection-free asymptomatics at one end and elephantiasis at the other. In order to determine if any HLA antigens are associated with the development of elephantiasis, we compared the HLA frequencies in 55 elephantiasis patients with those in 40 controls consisting of individuals older than 45 years of age without any signs of elephantiasis. The only significant difference in class I antigen frequencies was observed for B27, which was present in 11% of the patients and absent in the controls. More differences were observed in HLA class II antigen frequencies. Both DR3 and the 2B3 epitope (on DQ6, DQ8, and DQ9 molecules) were significantly decreased in patients with elephantiasis whereas the DQ5 frequency was significantly higher in patients than in controls. Analysis of specific antibody isotype profiles revealed that DQ5-positive individuals had increased levels of antifilarial IgG3, an isotype known to be involved in tissue damage. These data suggest that HLA class II genes may control the course of Brugian filariasis by influencing the T-cell-dependent antibody repertoire.

In an attempt to overcome T cell unresponsiveness to filarial antigens, 65 individuals belonging to the three clinical groups of elephantiasis patients, microfilaraemics, and asymptomatic amicrofilaraemics who exhibited unresponsiveness to Brugia malayi adult worm antigen (BmA) were studied. Peripheral blood mononuclear cells were cocultured with antigen and one of the following reagents that have been reported to be effective in reconstituting T cell proliferation: interleukin-2 (IL-2), interleukin-7 (IL-7), anti-interleukin-4, anti-interleukin-10, anti-CD2, anti-CD27, anti-CD28, indomethacin, phorbol myristate acetate (PMA), or calcium ionophore (A23187). We were able to overcome antigen-specific unresponsiveness in only a minority of the individuals studied. Co-culture with IL-2, IL-7, indomethacin and PMA were the only conditions which resulted in enhanced proliferation to BmA in these individuals. In general, unresponsiveness in elephantiasis patients was easier to reverse than in other clinical groups: in 50% of elephantiasis patients, in 12.5% of microfilaraemics and in 20% of asymptomatic amicrofilaraemics. The results indicate that more than one distinct immunological mechanism may account for the antigen-specific unresponsiveness in individuals exposed to and infected with brugian filariasis.

To establish the relationships among T and B cell responses, active infection, and clinical manifestations in lymphatic filariasis, filarial-specific lymphocyte proliferation, IgG antibody isotypes, and IgE levels were determined in an exposed population: 31 asymptomatic amicrofilaremics, 43 microfilaremics, 12 symptomatic amicrofilaremics, and 52 elephantiasis patients. Lymphocyte proliferation was higher in elephantiasis patients and asymptomatic amicrofilaremics than in microfilaremics (P < .004). A proportion of asymptomatic amicrofilaremics (32%), elephantiasis patients (37%), and symptomatic amicrofilaremics (58%) showed antigen-specific lymphocyte unresponsiveness, and lymphocyte proliferation to filarial antigens correlated negatively with specific IgG4 levels (rho = -0.315, P < .001). As elevated specific IgG4 is an indicator of active infection, it is argued that active infection may result in lymphocyte hyporesponsiveness irrespective of clinical category. Of those with elevated specific IgE levels and high T cell proliferative responses, 70% had elephantiasis, suggesting these factors have a role in pathology. However, the existence of a proportion of elephantiasis patients with low anti-filarial IgE and T cell unresponsiveness to filarial antigens suggests that elephantiasis can be caused by distinct processes.

To assess the immunological changes occurring during filarial infection with or without elephantiasis, 145 patients in different clinical groups from an endemic area in Indonesia were compared with respect to plasma levels of both soluble CD25 (sCD25) and sCD27; interleukin-4 (IL-4) and interferon-gamma release by peripheral blood mononuclear cells was also measured in a smaller subset of individuals. Levels of sCD27 were significantly elevated in elephantiasis and microfilaremic patients compared with endemic normals (p < 0.002), whereas sCD25 levels remained low in microfilaremics and was only slightly elevated in elephantiasis patients compared with endemic normals (p < 0.02). As activated T cell populations release both sCD27 and sCD25, these findings imply that there is filarial-driven activation of a T cell subset that releases sCD27 rather than sCD25. The expansion of a particular T cell population by filarial parasites is further suggested by the enhancement in both IL-4-producing and CD4+CD27- T cells in PBMC from elephantiasis and microfilaremic patients compared with endemic normals. More detailed characterization and comparison of CD27- lymphocytes from these individuals may identify mechanisms involved in the pathogenesis of lymphatic filariasis.