Diminished Monocyte Function in Microfilaremic Patients with Lymphatic Filariasis and Its Relationship to Altered Lymphoproliferative Respon
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感染与免疫杂志 2005年第6期
Centre for Biotechnology, Anna University, Chennai, India
Department of Public Health and Preventive Medicine, Government of Tamil Nadu, Chennai, India
Centre for Biotechnology, Jawaharlal Nehru University, New Delhi, India
Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892
ABSTRACT
Antigen-specific hyporesponsiveness to filarial antigens is a phenomenon observed in patent infection with lymph-dwelling filarial parasites of humans. This phenomenon has been attributed to a multitude of factors, one of which is altered monocyte function. To examine the role played by monocytes in filarial infection, we assessed the responses of monocytes obtained from normal and filarial parasite-infected individuals to both crude filarial antigen and purified recombinant filarial antigen WbSXP-1 and attempted to relate these to the altered lymphoproliferative responses seen in filarial infection. Monocytes from microfilaremic (MF) patients demonstrated an inability to respond to lipopolysaccharide compared to monocytes from endemic normal persons or from lymphedema patients. Indeed, interleukin 1 (IL-1) production was severely limited, a finding that did not extend to monocyte responses to filarial antigens. Serum from MF patients reduced adherence and spreading of normal monocytes, a finding not seen with serum from the other clinical groups. Interestingly, there was a significant correlation between the production of IL-1 and adherence. Moreover, the levels of spontaneous production of IL-1 correlated with high levels of spontaneous secretion of IL-10. The effects observed were not a result of diminished viability or alteration in the expression of the cell surface markers CD14 and HLA-DR. These data suggest that monocyte function is dampened in MF patients, a finding which could alter lymphoproliferative responses during patent infection.
INTRODUCTION
Understanding the induction of differentiated T-cell responses in helminth infections has been a major goal for parasite immunologists. The source of cytokines involved in the skewing of T-cell responses in helminth infections has been of particular interest, as the cytokine microenvironment is considered to be the most important factor influencing the differentiation of naive T cells and the generation of immunoregulatory cells (1, 30, 31, 36, 37). Among individuals with human lymphatic filariasis, those with patent infection (microfilaremic or antigenemic) exhibit parasite antigen-specific down-regulation of T-cell proliferation and gamma interferon production. These responses are different from those in patients with lymphedema/elephantiasis (mixed Th1/Th2 response) and infection-free endemic normal (EN) persons (predominant Th1 response to parasite antigen). The occurrence of filarial parasite-associated modulation of the immune response in microfilaremic patients is supported by extensive clinical (35, 38, 41) and animal (27) data. To date, however, no single mechanism can explain the modulatory effects of filarial infection in patients with patent infection.
The induction and maintenance of Th2-type immune responses are based largely on studies with murine models showing that antigen affinity for the T-cell receptor (6), the dose or route by which antigen is administered (11, 14), costimulatory molecule interactions (32, 49), the prevailing in vivo cytokine milieu (45), and the nature of the antigen-presenting cells (APCs) that initially encounter the antigen (2) can influence the development of a T-cell subset response. Helminth antigens have been shown to selectively induce naive human T-cell differentiation in vitro away from a Th1 phenotype (50). However, defining the factors responsible for the induction and maintenance of analogous human immune responses to infections in vivo has been difficult because of the many variables that affect host immunity and that cannot be readily controlled. These include (i) genetic heterogeneity among and within human populations (20), (ii) age-related changes in antigen-specific immunity (7, 24), (iii) immunomodulation that may occur during the course of chronic infections, (iv) neonatal immunotolerance or sensitization resulting from maternal infections during pregnancy (25, 26, 34), and (v) variability in exposure to or transmission of infective parasites (34, 43). Moreover, the concept of suppressor molecules, adherent cells, and/or T cells (42) in these infections has some experimental foundation. In addition, recent reports of results obtained with experimental murine models suggest that APCs that are activated in an alternative manner (12, 28) may play a role in suppressing T-cell responses. Further, regulatory T cells that express CD4+ CD25+ represent another cell population that may influence responses to parasite antigens (40).
To understand and unravel the complex immunological interactions between parasites and their hosts, it is important to examine both the specific responses of hosts to individual parasite antigens and the responses of various cell populations driven by those antigens. Thus, in the present study we examined the responses of purified monocytes and peripheral blood mononuclear cells (PBMCs) from filarial parasite-infected individuals to crude adult Brugia malayi antigen (BmA) and to recombinant filarial antigen WbSXP-1, a well-characterized 26-kDa protein. We compared these responses to those of uninfected EN individuals. Our data suggest that APCs from individuals with patent infection clearly influence the lymphoproliferative response in patent filarial infection and may do so because of their relative inability to respond to standard stimuli that reflect alterations in their intrinsic function.
MATERIALS AND METHODS
Materials. RPMI 1640 and fetal calf serum were obtained from HiMedia, Mumbai, India. HEPES was obtained from USB, Amersham Life Sciences, Cleveland, Ohio. NaHCO3, phenylmethylsulfonyl fluoride, and bovine serum albumin were obtained from Sigma (St. Louis, MO). Lymphoprep was obtained from Nycomed Pharma, AS (Oslo, Norway). Percoll was purchased from Fluka. L-Naphthyl acetate and pararosaniline were obtained from CDH Chemicals (New Delhi, India). Fluorescein isothiocyanate-conjugated antibodies to the cell surface molecules CD14 and HLA-DR were purchased from Sigma (St. Louis, MO). Gentamicin was obtained from Ranbaxy Pharmaceuticals (New Delhi, India).
Study population. Standardized histories were obtained from and physical examinations were done for all of the participing residents during epidemiological surveys in and around Chennai, India, an area endemic for Wuchereria bancrofti infections. Patients were recruited through the National Filaria Control Units under the Department of Public Health and Preventive Medicine (Chennai, India) after informed consent was obtained with protocols approved by the institutional review board of Anna University. Parasitological examination of all individuals was done by detection of microfilariae in blood smears taken from endemic individuals after 10 pm. The study population was divided into three categories—EN individuals (n = 10), individuals with chronic lymphedema and/or lymphatic obstruction (CP; n = 10), and individuals with microfilaremia (MF; n = 7)—based on clinical and parasitological evaluations. With the informed consent of all of the individuals or their parents, venous blood samples were taken. The MF patients had not received any treatment for their filarial infections prior to blood collection. Sera for adherence studies were from individuals in the same area of endemicity that were used previously (30, 32). MF individuals and members of their households were administered diethylcarbamazine after withdrawal of blood by officials of the Department of Public Health and Preventive Medicine (Chennai, India). The demographic and clinical details of the patient population are shown in Table 1.
Antigens and mitogens. BmA was prepared as a saline extract as described previously (17). Phytohemagglutinin (PHA; 10 μg/ml) and lipopolysaccharide (LPS; 100 ng/ml) (Sigma Chemical Co., St. Louis, MO) were used for PBMC stimulation and monocyte stimulation, respectively. The Wuchereria bancrofti orthologue of Brugia malayi SXP, WbSXP-1, was expressed and purified as described previously (44). Assessment of endotoxin contamination was done by using a Limulus amoebocyte lysate assay, which showed <1 pg of LPS/12.5 μg of protein.
Antigen-driven and polyclonally stimulated proliferation of human PBMCs. The peripheral mononuclear cell fractions of normal healthy volunteers and infected patients were isolated from heparinized venous blood by density gradient sedimentation with Ficoll-Hypaque (Lymphoprep; Nycomed Pharma, AS, Oslo, Norway). Cells were washed in RPMI 1640 (Sigma) for 10 min at 1,200 rpm. Cells were resuspended in medium supplemented with 10% fetal calf serum (HiMedia) or heat-inactivated human AB serum, HEPES (25 mM), and 80 mg gentamicin per liter of RPMI 1640. PBMCs (0.2 x 106/well) were cultured in 96-well round-bottom tissue culture plates (Sigma) at 37°C in a humidified 5% CO2 incubator and stimulated with 10 μg/ml of PHA (Sigma) or 10 μg/ml of soluble crude extract of BmA. A dose-response study was carried out with PBMCs from normal healthy volunteers to determine the optimum proliferative dose of WbSXP-1, which was found to be 12.5 μg/ml. Antigen-stimulated PBMCs were cultured for 96 h and PBMCs stimulated with PHA were cultured for 48 h in the presence or absence of polymyxin B sulfate. Antigen-specific proliferation was quantified as [3H]thymidine (Amersham-Life Science, United Kingdom) incorporation during the last 20 h of incubation. All experiments were performed in triplicate. The viability of the cells in the presence of recombinant protein WbSXP-1 was monitored by trypan blue exclusion.
Monocyte/macrophage cultures. Monocytes were isolated from PBMCs by density gradient centrifugation. Briefly, the PBMC fraction was overlaid on 46% Percoll prepared in 2x phosphate-buffered saline and centrifuged at 550 x g for 30 min. The interphase containing the monocytes was removed and washed in RPMI 1640, and the yield was estimated by using a Neubaeur hemocytometer. The viability of the cells was assessed by trypan blue exclusion. The purity of the isolated monocytes was verified by immunofluorescence analysis with fluorescein isothiocyanate-conjugated monoclonal antibodies to CD14 and HLA-DR. The monocytes (in general) were found to be >90% pure, and the expression of CD14 and HLA-DR was seen in monocytes from all clinical categories.
Characterization of monocytes. Purified monocytes were assessed for their basic characteristics by adherence, phagocytosis, spreading, and nonspecific esterase staining analyses. Although blinded studies were not done, a single reader did all of the comparative adherence and spreading analyses.
Adherence studies. The monocytes obtained were plated at 106 cells/well in a 24-well plate (Corning) with RPMI 1640. After 3 h of adherence, the cells were washed in RPMI 1640 to remove nonadherent or weakly adhering populations of cells. The adherent population was incubated overnight in RPMI 1640 containing 10% pooled sera from patients in each of the clinical categories (MF, EN, and CP). The wells were washed gently to remove nonadherent cells, and the percentage of adherent cells was calculated and compared to the percentage of cells adhering in complete RPMI 1640 (with 10% AB serum).
Phagocytic index. The monocytes obtained were plated at 106 cells on glass coverslips in a 24-well plate and incubated with 100 x 106 blue latex beads for 4 h. The cells were washed with phosphate-buffered saline, and cells containing latex beads were enumerated.
Spreading. Spreading of the monocytes in cultures was monitored every 12 h for a 5-day culture period. Spreading of the monocytes was observed as a flattened appearance and subsequent protrusion of pseudopodium-like arms (filopodia).
Nonspecific esterase staining. The cells were fixed with 1.5% glutaraldehyde for 15 min and incubated with 0.01% (wt/vol) naphthyl acetate in 0.06 M phosphate buffer (containing 1.5% of pararosaniline hydrochloride that is subjected to 6-azo group modification) for 40 min at 37°C. The cells were rinsed with water, counterstained with 2% methyl green, and examined microscopically.
In vitro activation of monocytes/macrophages. The monocytes were plated at 0.1 x 106 cells/well in a 48-well plate (Corning) and stimulated in quadruplicate alone, with recombinant WbSXP-1, or with soluble BmA. LPS (100 ng/ml) was used as a positive control.
Cytokine analysis. Cytokine levels in culture supernatants of PBMCs and of purified monocytes/macrophages were measured by enzyme-linked immunosorbent assays and expressed in pg/ml by interpolation from standard curves based on recombinant lymphokines in accordance with manufacturer protocols. Culture supernatants were used at 24, 48, and 72 h. The supernatants were screened for the release of IL-10, IFN-, and IL-1 by using commercially available enzyme-linked immunosorbent assays (Pharmingen, San Diego, CA, and R&D Systems, Minneapolis, MN). In separate cultures, PBMCs were stimulated with various suboptimal concentrations of LPS in the presence or absence of polymyxin B sulfate, WbSXP-1 protein, and WbSXP-1 protein spiked with suboptimal concentrations of LPS in the presence or absence of polymyxin B sulfate (10 μg/ml; Sigma). The levels of IL-10, IL-1, IL-6, and IL-12 p70 were measured by using a multiplex cytokine protein array (Seachlight; Pierce-Endogen, Boston, MA).
Statistical methods. The Wilcoxon signed-rank test and the Mann-Whitney U test were used throughout. Correlations were determined by the Spearman rank correlation test. All statistical analyses were performed with STATVIEW 5 (SAS Institute, Cary, NC).
RESULTS
Antigen specific proliferation to WbSXP-1. To examine if purified recombinant filarial protein behaves similarly to BmA in the induction of a parasite specific responses, PBMCs obtained from EN, CP and MF individuals were stimulated with 12.5 μg/ml of WbSXP-1 or 10 μg/ml of BmA. As shown previously (3, 21), BmA induced proliferation of PBMCs was diminished in the MF group when compared to either the EN (P = 0.0009) or the CP (P = 0.0015) group (Fig. 1). In contrast. WbSXP-1 induced a proliferative responses that exceeded the BmA induced responses in both MF (P = 0.04) using paired comparisons and EN (P = 0.02) groups, but not in the CP group (P = 0.79). Proliferation studies with endemic normal lymphocytes in the presence or absence of polymixin B sulfate suggested that the proliferation induced was WbSXP-1 specific and not due to low levels of contaminating LPS (Fig. 2). The proliferative responses to PHA were found to have no significant differences among the groups (21). These data suggest that the proliferative suppression commonly seen to parasite antigen in MF individuals may be the aggregate responses to both potentiating and inhibitory antigens since a single purified filarial antigen induced proliferation of PBMC from MF patients.
Production of IL-1 by monocytes. High amounts of spontaneous IL-1 was observed in unstimulated monocytes from all patient groups, but was most apparent in those with CP compared to those from EN (P = 0.009) or MF (P = 0.009) (Fig. 3A). Stimulation with WbSXP-1 or BmA does not increase IL-1 above baseline for any of the groups (Fig. 3B). However, LPS induced monocyte IL-1 production in both the CP (P = 0.04) and EN (P = 0.04) groups, but not the MF group to respond to LPS. These data suggest that at least some functions associated with monocyte activation may be compromised in MF individuals.
Antigen-stimulated T cells may activate autologous macrophages to produce proinflammatory cytokines such as IL-1 by direct cell-cell contact. To examine this aspect of monocyte function, PBMCs were examined for IL-1 production. In contrast to what was seen in purified monocytes from the same individuals, there was generally more IL-1 produced spontaneously from CP and MF but not EN patients (Fig. 4A), while the percent change in IL-1 was heightened for the EN group in response to WbSXP-1 (P = 0.04) and BmA (P = 0.04) (Fig. 4B). Interestingly, IL-1 production for all groups was apparently not inhibited by IL-10, as there was a significant positive correlation between IL-10 and IL-1 produced spontaneously (r = 0.627, P = 0.0016) or following stimulation with BmA (r = 0.794, p = <0.0001) or WbSXP-1 (r = 0.588, P = 0.0027) (data not shown).
MF serum affects monocyte adherence and spreading. To assess the effect of serum factors on the morphological and functional properties of monocytes, endemic normal monocytes were incubated with heat-inactivated sera from filarial parasite-infected and non-filaria-infected patients. The capacity of monocytes to adhere to plastic was significantly inhibited when incubated with sera from MF patients, in contrast to the adherence of the same monocytes in the presence of control AB serum or of serum from ENs, CP or nonendemic sera (NEN) (P < 0.008) (Table 2). There was a significant positive correlation observed with spontaneous IL-1 levels in the monocyte supernatants and the percent adherence (r = 0.706, P = 0.0083). There was no significant difference in the phagocytic capability of the monocytes, nonspecific esterase staining or viability among the groups, but a loss in the relative spreading of the monocytes assessed over a period of 5 days in culture was more pronounced in presence of sera from MF patients compared to all others (Table 2). These data indicates that the serum of MF patients contain factors that can affect some monocyte physiology that might be reflected in altered accessory function.
Preferential secretion of IL-10 by filarial antigens. Comparative analysis of IFN- response of PBMC from filarial parasite-infected patients to BmA or WbSXP-1 indicated no significant difference in the percentage change of IFN- secretion on stimulation with filarial antigens BmA and WbSXP-1 in MF and CP (Fig. 5A and B). A significant (P = 0.03) increase in the IFN- levels was observed in the EN over MF, which was not observed with CP. IFN- secretion was augmented (5 out of 10) and inhibited (5 out of 10) equally in the CP in comparison with unstimulated controls. Though, on an individual basis, IFN- responses to WbSXP-1 were enhanced over BmA in MF and CP (Fig. 5C), the overall responses was diminished which corroborates with the preferential inhibition of IFN- secretion by IL-10 as observed in earlier studies (32). There were high level of IL-10 being secreted spontaneously in both the MF and CP individuals when compared to those of the EN (P = 0.002) (Fig. 6A). Moreover, cells from the MF individuals produced more IL-10 that did cells from the other clinical groups. Stimulation with crude BmA resulted in a significant reduction of IL-10 secretion in all the groups when compared with the IL-10 produced in absence of stimulation (Fig. 6B). There was not much of a difference seen in the MF and CP on stimulation with BmA while significantly increased levels of IL-10 were observed in the EN in comparison with MF (P = 0.01) and CP (P = 0.04). On stimulation with WbSXP-1, increased levels of IL-10 were released by all the three groups EN (P = 0.005), MF (P = 0.01) and CP (P = 0.005) in comparison with the corresponding BmA stimulated levels. However, only the EN (P = 0.006) exhibited enhanced IL-10 levels compared to the MF group, while no significant change was observed in the others. This increase in IL-10 levels was more pronounced with a threefold increase in the EN and twofold in the CP.
To ensure that low-levels of contaminating LPS were not stimulating the cytokine secretion seen, PBMC from EN were stimulated with various concentrations of WbSXP-1 protein or LPS (at levels greater than, equal to and less than that found in the LAL assay, i.e., 10 ng, 1 ng, 100 pg, 10 pg, and 1 pg of LPS) in the presence or absence of polymixin B sulfate. The results indicate that the IL-10 release on stimulation with WbSXP-1 was antigen-specific, in that the addition of polymixin did not have any effect on the secretion of IL-10 (data not shown).
DISCUSSION
In the present study, we compared the response of PBMC from EN, CP and MF patients to a purified recombinant filarial protein WbSXP-1 and crude filarial extract (BmA) and attempted to elucidate the role played by monocytes in influencing the PBMC responses to these antigens. WbSXP-1 was used because it is a well characterized immunodominant filarial protein (23, 44) expressed in all the stages of the parasite life cycle that stimulates elevated levels of antigen specific IgG4 antibodies in individuals with patent infection. We were able to stimulate PBMC from MF individuals with WbSXP-1 but not crude extract (BmA). In addition, filarial antigens derived from intact MF impair APC function that results in PBMC hyporesponsiveness (46, 47); similar data was generated using a BmSXP-1 orthologue. These data suggest that responses to WbSXP-1 might be due to the absence of posttranslational modifications or the presence of additional products in the crude extract that reduce cell activation processes. Although contaminating LPS can confound cell proliferation studies using recombinant proteins, it is unlikely that this was a factor in our studies given the results of the LAL assay done on WbSXP-1 and BmSXP-1 preparations since the concentration of LPS detected was <1 pg/12.5 μg protein used in the stimulation assays in the presence or absence of polymixin B sulfate.
Earlier studies on the regulation of antigen specific responses have implicated cytokines as important mediators in the suppression of immune responses in the MF patients (9, 30). IL-10 has been directly implicated in this cytokine-mediated suppression to BmA. Distinct fractions of crude antigens elicit differential cytokine responsiveness, that affect cell proliferation (9). The pleiotropic effects of IL-10 may partially explain the observed effects in responses to a given antigen. IL-10 suppresses the proliferative responses in vitro, by inhibiting IL-2 secretion (15, 52) and by modulating the surface expression of important costimulatory molecules. IL-10 has been shown to regulate BmA driven IL-12 production by maintaining endogenously secreted IL-12 at low levels; exogenous supplementation of IL-12 overcomes the inhibitory effect of IL-10 stimulated by BmA (32) in one study. The present study also indicates that cells from MF individuals secrete high levels of IL-10 spontaneously when compared to the CP and EN corroborating observations made in the past (31). The contribution of B-cells (making up a small fraction of the total PBMC pool) in IL-10 secretion is likely to be small. However, the possibility of traces of LPS activating B cells in the PBMCs to release IL-10 was evaluated by using the recombinant protein in the presence of polymixin B sulfate and/or exogenously added LPS. The results as assayed by highly sensitive cytokine release of IL-1, IL-6, IL-10, and IL-12 p70, suggest that contaminating LPS as a nonspecific cytokine inducer in the present study is remote. Furthermore IL-10 has the ability to suppress IFN- production, downregulate MHC class II expression and reduces the expression of costimulatory molecules (8, 10). It has also been demonstrated that individuals with active B. malayi infection have lower levels of filarial antigen driven IFN- secretion when compared to EN and CP patients (33). This shift in the Th1/Th2 balance has been attributed more to the inhibition of IFN- than to the induction of IL-4-producing T cells (28). In the present study, IFN- responses to the recombinant WbSXP-1 indicated that although individually the MF respond to WbSXP-1, the aggregate responses to a set of crude antigens suppresses levels of IFN- in MF patients (data not shown). Previous studies in our lab have shown that the IFN- responses are restored upon IL-10 inhibition (32). Moreover, the increased IL-10 production seen in the MF patients provides a rationale for the high levels of antigen specific IgG4 seen in the MF state as IL-10 has been shown to preferentially induce switching to IgG4 (18).
Th1-type cytokines have been shown to have a preferential effect on macrophages to produce IL-1, while Th2 cytokines such as IL-4, IL-10 downregulate its production by inducing IL-1R (19, 22, 48). IL-1 responses of the macrophages have been directly correlated with IFN- production and appears to be independent of IL-4 (5). Furthermore, several pairs of receptors and ligands mediate the contact-dependent activation of IL-1. CD40-CD40L, CD11b-CD18, CD11c-CD18, CD23 interactions each has been shown to regulate IL-1 production (54). Alternatively, the presence of both IL-1 and IL-10 spontaneously in the unstimulated controls supports the studies in which IL-1 has been shown to provide costimulatory signals along with the T-cell receptor in inducing Th2 proliferation that is independent of IL-4 (16). Although we found IL-1 in high levels presumably released by the monocytes in the PBMCs, we still find a profound suppressed proliferation of the PBMCs in individuals with asymptomatic patent infections when compared to normal and symptomatic patients (ENs and CPs). Whether this may be secondary to the low frequency and reduced number of parasite antigen-responsive T cells in the MF (21, 35) remains to be clarified.
The positive nonspecific esterase activity staining (specific for monocyte) in conjunction with CD14 and HLA-DR rules out the possibility of contaminating cells in the observed responses. Adherence of monocytes stimulates the production of IL-1, which in-turn enhances the expression of adherence molecules ICAM (CD54) and 2-integrins (CD11a/CD18 and CD11b/CD18). This effect is inhibited by IL-10 (8). 2-integrins have been shown to regulate the spreading of monocytes on adherence (29). The loss of adherence in this instance could reflect on the spreading of the monocytes using sera from MF. Early studies (51) on the adherence of the peripheral blood lymphocytes (PBL) to microfilariae in vitro in the presence of various sera show that sera from all clinical groups promote adhesion of PBL to the microfilariae except the MF sera. Recently, it has been shown that the absence of adherence of monocytes prevented the inhibitory effects of IL-10 on IL-1 production following LPS stimulation (39). Whether this holds true for filarial immune responses remains to be investigated. The most intriguing finding is while the EN and CP secrete IL-1 in great excess compared to the unstimulated controls, the monocytes from MF responded poorly not only to filarial antigens alone, but also to the very potent stimulator LPS. LPS signaling at low doses is CD14 dependent. Though the kinetics of CD14 expression were not done, monocytes obtained from PBMCs in all the groups were positive for CD14 as assessed by immunofluorescence using monoclonal antibodies to CD14. This suggests that the monocytes from the MF are modulated to the point where intrinsic responses to LPS is inhibited or lost. Alternatively, LPS tolerance may be at play as an adaptive host immune response. This is characterized by decreased production of TNF-, IL-1, IL-6 and IL-10 and concurrently increased production of IL-1R antagonist upon ex vivo restimulation of PBMCs with LPS (13, 53). One source of LPS in the filaria-infected patients may be from the endosymbiotic bacterium Wolbachia (4).
Serum suppressive factors apart from adherent suppressive cells also have been implicated as a partial mediator of the antigen specific hyporesponsiveness seen in patent filarial infections. The significant inhibition of the adherence capacity of the normal monocytes by serum of MF individuals strengthens the original finding (42) and suggests that the serum does contribute to the functional loss of monocyte accessory functions.
In summary, our data suggest that monocytes from MF are in some way down modulated. The diminished responsiveness and function of monocytes in patent filarial infection provide additional evidence that APCs are one component in the cascade of events that suppresses the Th1-type T-cell response to filarial antigens.
ACKNOWLEDGMENTS
This study was supported by grants from the INDO-US PL480 program, from the University Grants Commission (UGC), New Delhi, India, and from the All India Council of Technical Education (AICTE), New Delhi, India. B. Sasisekhar is the recipient of a senior research fellowship awarded by the Council of Scientific and Industrial Research (CSIR), New Delhi, India.
We thank the Department of Public Health and Preventive Medicine, Government of Tamil Nadu, Chennai, India, for help in the communities in which filariasis is endemic.
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Department of Public Health and Preventive Medicine, Government of Tamil Nadu, Chennai, India
Centre for Biotechnology, Jawaharlal Nehru University, New Delhi, India
Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892
ABSTRACT
Antigen-specific hyporesponsiveness to filarial antigens is a phenomenon observed in patent infection with lymph-dwelling filarial parasites of humans. This phenomenon has been attributed to a multitude of factors, one of which is altered monocyte function. To examine the role played by monocytes in filarial infection, we assessed the responses of monocytes obtained from normal and filarial parasite-infected individuals to both crude filarial antigen and purified recombinant filarial antigen WbSXP-1 and attempted to relate these to the altered lymphoproliferative responses seen in filarial infection. Monocytes from microfilaremic (MF) patients demonstrated an inability to respond to lipopolysaccharide compared to monocytes from endemic normal persons or from lymphedema patients. Indeed, interleukin 1 (IL-1) production was severely limited, a finding that did not extend to monocyte responses to filarial antigens. Serum from MF patients reduced adherence and spreading of normal monocytes, a finding not seen with serum from the other clinical groups. Interestingly, there was a significant correlation between the production of IL-1 and adherence. Moreover, the levels of spontaneous production of IL-1 correlated with high levels of spontaneous secretion of IL-10. The effects observed were not a result of diminished viability or alteration in the expression of the cell surface markers CD14 and HLA-DR. These data suggest that monocyte function is dampened in MF patients, a finding which could alter lymphoproliferative responses during patent infection.
INTRODUCTION
Understanding the induction of differentiated T-cell responses in helminth infections has been a major goal for parasite immunologists. The source of cytokines involved in the skewing of T-cell responses in helminth infections has been of particular interest, as the cytokine microenvironment is considered to be the most important factor influencing the differentiation of naive T cells and the generation of immunoregulatory cells (1, 30, 31, 36, 37). Among individuals with human lymphatic filariasis, those with patent infection (microfilaremic or antigenemic) exhibit parasite antigen-specific down-regulation of T-cell proliferation and gamma interferon production. These responses are different from those in patients with lymphedema/elephantiasis (mixed Th1/Th2 response) and infection-free endemic normal (EN) persons (predominant Th1 response to parasite antigen). The occurrence of filarial parasite-associated modulation of the immune response in microfilaremic patients is supported by extensive clinical (35, 38, 41) and animal (27) data. To date, however, no single mechanism can explain the modulatory effects of filarial infection in patients with patent infection.
The induction and maintenance of Th2-type immune responses are based largely on studies with murine models showing that antigen affinity for the T-cell receptor (6), the dose or route by which antigen is administered (11, 14), costimulatory molecule interactions (32, 49), the prevailing in vivo cytokine milieu (45), and the nature of the antigen-presenting cells (APCs) that initially encounter the antigen (2) can influence the development of a T-cell subset response. Helminth antigens have been shown to selectively induce naive human T-cell differentiation in vitro away from a Th1 phenotype (50). However, defining the factors responsible for the induction and maintenance of analogous human immune responses to infections in vivo has been difficult because of the many variables that affect host immunity and that cannot be readily controlled. These include (i) genetic heterogeneity among and within human populations (20), (ii) age-related changes in antigen-specific immunity (7, 24), (iii) immunomodulation that may occur during the course of chronic infections, (iv) neonatal immunotolerance or sensitization resulting from maternal infections during pregnancy (25, 26, 34), and (v) variability in exposure to or transmission of infective parasites (34, 43). Moreover, the concept of suppressor molecules, adherent cells, and/or T cells (42) in these infections has some experimental foundation. In addition, recent reports of results obtained with experimental murine models suggest that APCs that are activated in an alternative manner (12, 28) may play a role in suppressing T-cell responses. Further, regulatory T cells that express CD4+ CD25+ represent another cell population that may influence responses to parasite antigens (40).
To understand and unravel the complex immunological interactions between parasites and their hosts, it is important to examine both the specific responses of hosts to individual parasite antigens and the responses of various cell populations driven by those antigens. Thus, in the present study we examined the responses of purified monocytes and peripheral blood mononuclear cells (PBMCs) from filarial parasite-infected individuals to crude adult Brugia malayi antigen (BmA) and to recombinant filarial antigen WbSXP-1, a well-characterized 26-kDa protein. We compared these responses to those of uninfected EN individuals. Our data suggest that APCs from individuals with patent infection clearly influence the lymphoproliferative response in patent filarial infection and may do so because of their relative inability to respond to standard stimuli that reflect alterations in their intrinsic function.
MATERIALS AND METHODS
Materials. RPMI 1640 and fetal calf serum were obtained from HiMedia, Mumbai, India. HEPES was obtained from USB, Amersham Life Sciences, Cleveland, Ohio. NaHCO3, phenylmethylsulfonyl fluoride, and bovine serum albumin were obtained from Sigma (St. Louis, MO). Lymphoprep was obtained from Nycomed Pharma, AS (Oslo, Norway). Percoll was purchased from Fluka. L-Naphthyl acetate and pararosaniline were obtained from CDH Chemicals (New Delhi, India). Fluorescein isothiocyanate-conjugated antibodies to the cell surface molecules CD14 and HLA-DR were purchased from Sigma (St. Louis, MO). Gentamicin was obtained from Ranbaxy Pharmaceuticals (New Delhi, India).
Study population. Standardized histories were obtained from and physical examinations were done for all of the participing residents during epidemiological surveys in and around Chennai, India, an area endemic for Wuchereria bancrofti infections. Patients were recruited through the National Filaria Control Units under the Department of Public Health and Preventive Medicine (Chennai, India) after informed consent was obtained with protocols approved by the institutional review board of Anna University. Parasitological examination of all individuals was done by detection of microfilariae in blood smears taken from endemic individuals after 10 pm. The study population was divided into three categories—EN individuals (n = 10), individuals with chronic lymphedema and/or lymphatic obstruction (CP; n = 10), and individuals with microfilaremia (MF; n = 7)—based on clinical and parasitological evaluations. With the informed consent of all of the individuals or their parents, venous blood samples were taken. The MF patients had not received any treatment for their filarial infections prior to blood collection. Sera for adherence studies were from individuals in the same area of endemicity that were used previously (30, 32). MF individuals and members of their households were administered diethylcarbamazine after withdrawal of blood by officials of the Department of Public Health and Preventive Medicine (Chennai, India). The demographic and clinical details of the patient population are shown in Table 1.
Antigens and mitogens. BmA was prepared as a saline extract as described previously (17). Phytohemagglutinin (PHA; 10 μg/ml) and lipopolysaccharide (LPS; 100 ng/ml) (Sigma Chemical Co., St. Louis, MO) were used for PBMC stimulation and monocyte stimulation, respectively. The Wuchereria bancrofti orthologue of Brugia malayi SXP, WbSXP-1, was expressed and purified as described previously (44). Assessment of endotoxin contamination was done by using a Limulus amoebocyte lysate assay, which showed <1 pg of LPS/12.5 μg of protein.
Antigen-driven and polyclonally stimulated proliferation of human PBMCs. The peripheral mononuclear cell fractions of normal healthy volunteers and infected patients were isolated from heparinized venous blood by density gradient sedimentation with Ficoll-Hypaque (Lymphoprep; Nycomed Pharma, AS, Oslo, Norway). Cells were washed in RPMI 1640 (Sigma) for 10 min at 1,200 rpm. Cells were resuspended in medium supplemented with 10% fetal calf serum (HiMedia) or heat-inactivated human AB serum, HEPES (25 mM), and 80 mg gentamicin per liter of RPMI 1640. PBMCs (0.2 x 106/well) were cultured in 96-well round-bottom tissue culture plates (Sigma) at 37°C in a humidified 5% CO2 incubator and stimulated with 10 μg/ml of PHA (Sigma) or 10 μg/ml of soluble crude extract of BmA. A dose-response study was carried out with PBMCs from normal healthy volunteers to determine the optimum proliferative dose of WbSXP-1, which was found to be 12.5 μg/ml. Antigen-stimulated PBMCs were cultured for 96 h and PBMCs stimulated with PHA were cultured for 48 h in the presence or absence of polymyxin B sulfate. Antigen-specific proliferation was quantified as [3H]thymidine (Amersham-Life Science, United Kingdom) incorporation during the last 20 h of incubation. All experiments were performed in triplicate. The viability of the cells in the presence of recombinant protein WbSXP-1 was monitored by trypan blue exclusion.
Monocyte/macrophage cultures. Monocytes were isolated from PBMCs by density gradient centrifugation. Briefly, the PBMC fraction was overlaid on 46% Percoll prepared in 2x phosphate-buffered saline and centrifuged at 550 x g for 30 min. The interphase containing the monocytes was removed and washed in RPMI 1640, and the yield was estimated by using a Neubaeur hemocytometer. The viability of the cells was assessed by trypan blue exclusion. The purity of the isolated monocytes was verified by immunofluorescence analysis with fluorescein isothiocyanate-conjugated monoclonal antibodies to CD14 and HLA-DR. The monocytes (in general) were found to be >90% pure, and the expression of CD14 and HLA-DR was seen in monocytes from all clinical categories.
Characterization of monocytes. Purified monocytes were assessed for their basic characteristics by adherence, phagocytosis, spreading, and nonspecific esterase staining analyses. Although blinded studies were not done, a single reader did all of the comparative adherence and spreading analyses.
Adherence studies. The monocytes obtained were plated at 106 cells/well in a 24-well plate (Corning) with RPMI 1640. After 3 h of adherence, the cells were washed in RPMI 1640 to remove nonadherent or weakly adhering populations of cells. The adherent population was incubated overnight in RPMI 1640 containing 10% pooled sera from patients in each of the clinical categories (MF, EN, and CP). The wells were washed gently to remove nonadherent cells, and the percentage of adherent cells was calculated and compared to the percentage of cells adhering in complete RPMI 1640 (with 10% AB serum).
Phagocytic index. The monocytes obtained were plated at 106 cells on glass coverslips in a 24-well plate and incubated with 100 x 106 blue latex beads for 4 h. The cells were washed with phosphate-buffered saline, and cells containing latex beads were enumerated.
Spreading. Spreading of the monocytes in cultures was monitored every 12 h for a 5-day culture period. Spreading of the monocytes was observed as a flattened appearance and subsequent protrusion of pseudopodium-like arms (filopodia).
Nonspecific esterase staining. The cells were fixed with 1.5% glutaraldehyde for 15 min and incubated with 0.01% (wt/vol) naphthyl acetate in 0.06 M phosphate buffer (containing 1.5% of pararosaniline hydrochloride that is subjected to 6-azo group modification) for 40 min at 37°C. The cells were rinsed with water, counterstained with 2% methyl green, and examined microscopically.
In vitro activation of monocytes/macrophages. The monocytes were plated at 0.1 x 106 cells/well in a 48-well plate (Corning) and stimulated in quadruplicate alone, with recombinant WbSXP-1, or with soluble BmA. LPS (100 ng/ml) was used as a positive control.
Cytokine analysis. Cytokine levels in culture supernatants of PBMCs and of purified monocytes/macrophages were measured by enzyme-linked immunosorbent assays and expressed in pg/ml by interpolation from standard curves based on recombinant lymphokines in accordance with manufacturer protocols. Culture supernatants were used at 24, 48, and 72 h. The supernatants were screened for the release of IL-10, IFN-, and IL-1 by using commercially available enzyme-linked immunosorbent assays (Pharmingen, San Diego, CA, and R&D Systems, Minneapolis, MN). In separate cultures, PBMCs were stimulated with various suboptimal concentrations of LPS in the presence or absence of polymyxin B sulfate, WbSXP-1 protein, and WbSXP-1 protein spiked with suboptimal concentrations of LPS in the presence or absence of polymyxin B sulfate (10 μg/ml; Sigma). The levels of IL-10, IL-1, IL-6, and IL-12 p70 were measured by using a multiplex cytokine protein array (Seachlight; Pierce-Endogen, Boston, MA).
Statistical methods. The Wilcoxon signed-rank test and the Mann-Whitney U test were used throughout. Correlations were determined by the Spearman rank correlation test. All statistical analyses were performed with STATVIEW 5 (SAS Institute, Cary, NC).
RESULTS
Antigen specific proliferation to WbSXP-1. To examine if purified recombinant filarial protein behaves similarly to BmA in the induction of a parasite specific responses, PBMCs obtained from EN, CP and MF individuals were stimulated with 12.5 μg/ml of WbSXP-1 or 10 μg/ml of BmA. As shown previously (3, 21), BmA induced proliferation of PBMCs was diminished in the MF group when compared to either the EN (P = 0.0009) or the CP (P = 0.0015) group (Fig. 1). In contrast. WbSXP-1 induced a proliferative responses that exceeded the BmA induced responses in both MF (P = 0.04) using paired comparisons and EN (P = 0.02) groups, but not in the CP group (P = 0.79). Proliferation studies with endemic normal lymphocytes in the presence or absence of polymixin B sulfate suggested that the proliferation induced was WbSXP-1 specific and not due to low levels of contaminating LPS (Fig. 2). The proliferative responses to PHA were found to have no significant differences among the groups (21). These data suggest that the proliferative suppression commonly seen to parasite antigen in MF individuals may be the aggregate responses to both potentiating and inhibitory antigens since a single purified filarial antigen induced proliferation of PBMC from MF patients.
Production of IL-1 by monocytes. High amounts of spontaneous IL-1 was observed in unstimulated monocytes from all patient groups, but was most apparent in those with CP compared to those from EN (P = 0.009) or MF (P = 0.009) (Fig. 3A). Stimulation with WbSXP-1 or BmA does not increase IL-1 above baseline for any of the groups (Fig. 3B). However, LPS induced monocyte IL-1 production in both the CP (P = 0.04) and EN (P = 0.04) groups, but not the MF group to respond to LPS. These data suggest that at least some functions associated with monocyte activation may be compromised in MF individuals.
Antigen-stimulated T cells may activate autologous macrophages to produce proinflammatory cytokines such as IL-1 by direct cell-cell contact. To examine this aspect of monocyte function, PBMCs were examined for IL-1 production. In contrast to what was seen in purified monocytes from the same individuals, there was generally more IL-1 produced spontaneously from CP and MF but not EN patients (Fig. 4A), while the percent change in IL-1 was heightened for the EN group in response to WbSXP-1 (P = 0.04) and BmA (P = 0.04) (Fig. 4B). Interestingly, IL-1 production for all groups was apparently not inhibited by IL-10, as there was a significant positive correlation between IL-10 and IL-1 produced spontaneously (r = 0.627, P = 0.0016) or following stimulation with BmA (r = 0.794, p = <0.0001) or WbSXP-1 (r = 0.588, P = 0.0027) (data not shown).
MF serum affects monocyte adherence and spreading. To assess the effect of serum factors on the morphological and functional properties of monocytes, endemic normal monocytes were incubated with heat-inactivated sera from filarial parasite-infected and non-filaria-infected patients. The capacity of monocytes to adhere to plastic was significantly inhibited when incubated with sera from MF patients, in contrast to the adherence of the same monocytes in the presence of control AB serum or of serum from ENs, CP or nonendemic sera (NEN) (P < 0.008) (Table 2). There was a significant positive correlation observed with spontaneous IL-1 levels in the monocyte supernatants and the percent adherence (r = 0.706, P = 0.0083). There was no significant difference in the phagocytic capability of the monocytes, nonspecific esterase staining or viability among the groups, but a loss in the relative spreading of the monocytes assessed over a period of 5 days in culture was more pronounced in presence of sera from MF patients compared to all others (Table 2). These data indicates that the serum of MF patients contain factors that can affect some monocyte physiology that might be reflected in altered accessory function.
Preferential secretion of IL-10 by filarial antigens. Comparative analysis of IFN- response of PBMC from filarial parasite-infected patients to BmA or WbSXP-1 indicated no significant difference in the percentage change of IFN- secretion on stimulation with filarial antigens BmA and WbSXP-1 in MF and CP (Fig. 5A and B). A significant (P = 0.03) increase in the IFN- levels was observed in the EN over MF, which was not observed with CP. IFN- secretion was augmented (5 out of 10) and inhibited (5 out of 10) equally in the CP in comparison with unstimulated controls. Though, on an individual basis, IFN- responses to WbSXP-1 were enhanced over BmA in MF and CP (Fig. 5C), the overall responses was diminished which corroborates with the preferential inhibition of IFN- secretion by IL-10 as observed in earlier studies (32). There were high level of IL-10 being secreted spontaneously in both the MF and CP individuals when compared to those of the EN (P = 0.002) (Fig. 6A). Moreover, cells from the MF individuals produced more IL-10 that did cells from the other clinical groups. Stimulation with crude BmA resulted in a significant reduction of IL-10 secretion in all the groups when compared with the IL-10 produced in absence of stimulation (Fig. 6B). There was not much of a difference seen in the MF and CP on stimulation with BmA while significantly increased levels of IL-10 were observed in the EN in comparison with MF (P = 0.01) and CP (P = 0.04). On stimulation with WbSXP-1, increased levels of IL-10 were released by all the three groups EN (P = 0.005), MF (P = 0.01) and CP (P = 0.005) in comparison with the corresponding BmA stimulated levels. However, only the EN (P = 0.006) exhibited enhanced IL-10 levels compared to the MF group, while no significant change was observed in the others. This increase in IL-10 levels was more pronounced with a threefold increase in the EN and twofold in the CP.
To ensure that low-levels of contaminating LPS were not stimulating the cytokine secretion seen, PBMC from EN were stimulated with various concentrations of WbSXP-1 protein or LPS (at levels greater than, equal to and less than that found in the LAL assay, i.e., 10 ng, 1 ng, 100 pg, 10 pg, and 1 pg of LPS) in the presence or absence of polymixin B sulfate. The results indicate that the IL-10 release on stimulation with WbSXP-1 was antigen-specific, in that the addition of polymixin did not have any effect on the secretion of IL-10 (data not shown).
DISCUSSION
In the present study, we compared the response of PBMC from EN, CP and MF patients to a purified recombinant filarial protein WbSXP-1 and crude filarial extract (BmA) and attempted to elucidate the role played by monocytes in influencing the PBMC responses to these antigens. WbSXP-1 was used because it is a well characterized immunodominant filarial protein (23, 44) expressed in all the stages of the parasite life cycle that stimulates elevated levels of antigen specific IgG4 antibodies in individuals with patent infection. We were able to stimulate PBMC from MF individuals with WbSXP-1 but not crude extract (BmA). In addition, filarial antigens derived from intact MF impair APC function that results in PBMC hyporesponsiveness (46, 47); similar data was generated using a BmSXP-1 orthologue. These data suggest that responses to WbSXP-1 might be due to the absence of posttranslational modifications or the presence of additional products in the crude extract that reduce cell activation processes. Although contaminating LPS can confound cell proliferation studies using recombinant proteins, it is unlikely that this was a factor in our studies given the results of the LAL assay done on WbSXP-1 and BmSXP-1 preparations since the concentration of LPS detected was <1 pg/12.5 μg protein used in the stimulation assays in the presence or absence of polymixin B sulfate.
Earlier studies on the regulation of antigen specific responses have implicated cytokines as important mediators in the suppression of immune responses in the MF patients (9, 30). IL-10 has been directly implicated in this cytokine-mediated suppression to BmA. Distinct fractions of crude antigens elicit differential cytokine responsiveness, that affect cell proliferation (9). The pleiotropic effects of IL-10 may partially explain the observed effects in responses to a given antigen. IL-10 suppresses the proliferative responses in vitro, by inhibiting IL-2 secretion (15, 52) and by modulating the surface expression of important costimulatory molecules. IL-10 has been shown to regulate BmA driven IL-12 production by maintaining endogenously secreted IL-12 at low levels; exogenous supplementation of IL-12 overcomes the inhibitory effect of IL-10 stimulated by BmA (32) in one study. The present study also indicates that cells from MF individuals secrete high levels of IL-10 spontaneously when compared to the CP and EN corroborating observations made in the past (31). The contribution of B-cells (making up a small fraction of the total PBMC pool) in IL-10 secretion is likely to be small. However, the possibility of traces of LPS activating B cells in the PBMCs to release IL-10 was evaluated by using the recombinant protein in the presence of polymixin B sulfate and/or exogenously added LPS. The results as assayed by highly sensitive cytokine release of IL-1, IL-6, IL-10, and IL-12 p70, suggest that contaminating LPS as a nonspecific cytokine inducer in the present study is remote. Furthermore IL-10 has the ability to suppress IFN- production, downregulate MHC class II expression and reduces the expression of costimulatory molecules (8, 10). It has also been demonstrated that individuals with active B. malayi infection have lower levels of filarial antigen driven IFN- secretion when compared to EN and CP patients (33). This shift in the Th1/Th2 balance has been attributed more to the inhibition of IFN- than to the induction of IL-4-producing T cells (28). In the present study, IFN- responses to the recombinant WbSXP-1 indicated that although individually the MF respond to WbSXP-1, the aggregate responses to a set of crude antigens suppresses levels of IFN- in MF patients (data not shown). Previous studies in our lab have shown that the IFN- responses are restored upon IL-10 inhibition (32). Moreover, the increased IL-10 production seen in the MF patients provides a rationale for the high levels of antigen specific IgG4 seen in the MF state as IL-10 has been shown to preferentially induce switching to IgG4 (18).
Th1-type cytokines have been shown to have a preferential effect on macrophages to produce IL-1, while Th2 cytokines such as IL-4, IL-10 downregulate its production by inducing IL-1R (19, 22, 48). IL-1 responses of the macrophages have been directly correlated with IFN- production and appears to be independent of IL-4 (5). Furthermore, several pairs of receptors and ligands mediate the contact-dependent activation of IL-1. CD40-CD40L, CD11b-CD18, CD11c-CD18, CD23 interactions each has been shown to regulate IL-1 production (54). Alternatively, the presence of both IL-1 and IL-10 spontaneously in the unstimulated controls supports the studies in which IL-1 has been shown to provide costimulatory signals along with the T-cell receptor in inducing Th2 proliferation that is independent of IL-4 (16). Although we found IL-1 in high levels presumably released by the monocytes in the PBMCs, we still find a profound suppressed proliferation of the PBMCs in individuals with asymptomatic patent infections when compared to normal and symptomatic patients (ENs and CPs). Whether this may be secondary to the low frequency and reduced number of parasite antigen-responsive T cells in the MF (21, 35) remains to be clarified.
The positive nonspecific esterase activity staining (specific for monocyte) in conjunction with CD14 and HLA-DR rules out the possibility of contaminating cells in the observed responses. Adherence of monocytes stimulates the production of IL-1, which in-turn enhances the expression of adherence molecules ICAM (CD54) and 2-integrins (CD11a/CD18 and CD11b/CD18). This effect is inhibited by IL-10 (8). 2-integrins have been shown to regulate the spreading of monocytes on adherence (29). The loss of adherence in this instance could reflect on the spreading of the monocytes using sera from MF. Early studies (51) on the adherence of the peripheral blood lymphocytes (PBL) to microfilariae in vitro in the presence of various sera show that sera from all clinical groups promote adhesion of PBL to the microfilariae except the MF sera. Recently, it has been shown that the absence of adherence of monocytes prevented the inhibitory effects of IL-10 on IL-1 production following LPS stimulation (39). Whether this holds true for filarial immune responses remains to be investigated. The most intriguing finding is while the EN and CP secrete IL-1 in great excess compared to the unstimulated controls, the monocytes from MF responded poorly not only to filarial antigens alone, but also to the very potent stimulator LPS. LPS signaling at low doses is CD14 dependent. Though the kinetics of CD14 expression were not done, monocytes obtained from PBMCs in all the groups were positive for CD14 as assessed by immunofluorescence using monoclonal antibodies to CD14. This suggests that the monocytes from the MF are modulated to the point where intrinsic responses to LPS is inhibited or lost. Alternatively, LPS tolerance may be at play as an adaptive host immune response. This is characterized by decreased production of TNF-, IL-1, IL-6 and IL-10 and concurrently increased production of IL-1R antagonist upon ex vivo restimulation of PBMCs with LPS (13, 53). One source of LPS in the filaria-infected patients may be from the endosymbiotic bacterium Wolbachia (4).
Serum suppressive factors apart from adherent suppressive cells also have been implicated as a partial mediator of the antigen specific hyporesponsiveness seen in patent filarial infections. The significant inhibition of the adherence capacity of the normal monocytes by serum of MF individuals strengthens the original finding (42) and suggests that the serum does contribute to the functional loss of monocyte accessory functions.
In summary, our data suggest that monocytes from MF are in some way down modulated. The diminished responsiveness and function of monocytes in patent filarial infection provide additional evidence that APCs are one component in the cascade of events that suppresses the Th1-type T-cell response to filarial antigens.
ACKNOWLEDGMENTS
This study was supported by grants from the INDO-US PL480 program, from the University Grants Commission (UGC), New Delhi, India, and from the All India Council of Technical Education (AICTE), New Delhi, India. B. Sasisekhar is the recipient of a senior research fellowship awarded by the Council of Scientific and Industrial Research (CSIR), New Delhi, India.
We thank the Department of Public Health and Preventive Medicine, Government of Tamil Nadu, Chennai, India, for help in the communities in which filariasis is endemic.
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