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Am. J. Trop. Med. Hyg., 77(5), 2007, pp. 860-865
Copyright © 2007 by The American Society of Tropical Medicine and Hygiene

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Basophil Competence during Hookworm (Necator americanus) Infection

David I. Pritchard, Doreen S. W. Hooi, Alan Brown*, Moses J. Bockarie, Rebecca Caddick, AND Rupert J. Quinnell
The Boots Science Institute, School of Pharmacy, University of Nottingham, University Park, Nottingham, United Kingdom; Papua New Guinea Institute of Medical Research, Madang Province, Papua New Guinea; Institute of Integrative and Comparative Biology, University of Leeds, Leeds, United Kingdom


ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A popular hypothesis to explain parasite survival in the presence of a pronounced T helper 2 phenotype in helminth-parasitized populations has been Fc{epsilon}RI blockade by parasite-induced polyclonal IgE. To begin to test the hypothesis that Fc{epsilon}RI-bearing cells would be refractory to activation in parasitized populations, we investigated basophil function in 43 individuals from a hookworm endemic area. Study individuals had high levels of total IgE and eosinophilia and a mean hookworm burden of 2,257 epg. Basophils from all members of this parasitized population were shown to release histamine to a number of agonists, including anti IgE and a hookworm allergen, calreticulin. These data would indicate that Fc{epsilon}RI blockade at the level of the basophil did not occur in this parasitized population despite the presence of possible immunologic blocking agents. This would suggest that this effector arm of the T helper 2 phenotype remains operative in infected populations.


INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hookworms survive relatively well in an immunologically hostile environment, although the T helper 2 phenotype is associated with partial protection. The parasite may promote its survival by secreting a molecular screen of immune-suppressive agents and, possibly, by stimulating the appearance of regulatory T-cell populations.1–3 However, it is also possible that the parasite induces immunologic incompetence in one of the effector arms of the T helper 2 response, eliciting Fc{epsilon}R1 blockade in mast cells and basophils. This could be achieved through the upregulation of non–parasite-specific polyclonal IgE and autoantibodies acting in isolation and, possibly, in tandem.4,5 Therefore, it is reasonable at this stage to hypothesize that these and other immunologic components downregulate the capacity of Fc{epsilon}R1 to signal, and for cells of the basophil/mast cell series to respond biochemically to antigenic insult in infected individuals by releasing histamine. Such mast cell/basophil blockade could also partially explain the protective effect of hookworms against the development of respiratory wheeze against environmental allergens6 and the apparent asymptomatic nature of hookworm infection at mucosal surfaces,7 although investigating this is not the primary aim of the present study.

To test the hypothesis that Fc{epsilon}R1 blockade and basophil incompetence could indeed occur in hookworm-infected individuals, blood was collected from a parasitized population in Madang Province, Papua New Guinea, and cells exposed to a range of biologically relevant agonists including a known hookworm allergen and anti-human IgE, to assess the capability of basophils to release histamine. As a result, we have been able to demonstrate clearly the competence of basophils to release histamine in blood from a community infected with the hookworm Necator americanus. These data would indicate that blockade at this level is probably not responsible for parasite survival, nor for the protective effects of hookworm infection against the development of allergic disease.


MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Fieldwork parasitology and blood collection. The study sample comprised 43 inhabitants from the villages of Haven and Wasab in lowland Madang province, Papua New Guinea. This was an unselected sample taken during a parasitological survey of all individuals aged 4 years or over and formed part of a larger study assessing the immune biology of hookworm infection (Table 1Go).8–10 Necator americanus is the only hookworm species in this area. Informed consent was obtained from all subjects and their parents, and the study was approved by the Medical Research Advisory Committee of Papua New Guinea. Fecal samples were taken during Sep-tember 2001; helminth eggs were counted by a modified Mc-Master salt-flotation technique and expressed as eggs per gram feces (epg). Blood samples were taken during October 2001; 10–20 mL venous blood was taken into heparin (1000 U/ml). An aliquot of the blood collected was also diluted 1:20 into Kimura’s stain to carry out differential white cell counts using an improved Neubauer chamber. Most inhabitants of the study area, including 39 of 43 of the study sample, had been treated with anthelmintics in 1998. All sampled individuals were offered anthelmintic treatment at the conclusion of the study.


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TABLE 1
Parasitological and immunological parameters of the study population
 
Parasite maintenance and basophil agonist preparation. Necator americanus was maintained in the laboratory in syn-geneic DSN hamsters.11 Two- to 4-day-old neonate hamsters were infected percutaneously with 100 infective larvae and adult worms harvested from the small intestine 35 days post-infection. All animal work was licensed under the UK Animals (Scientific Procedures) Act 1986. Adult ES products were prepared as described previously.12 The protein content of ES products was determined using the BioRAD protein assay. ES products were finally dialyzed against distilled water, freeze dried, and stored at -20°C prior to shipping to Papua New Guinea. Some ES products were boiled for 30 min as described by Brown and others13 to inactivate the protein-ase activities present, given that hookworm proteinases themselves can cause basophil activation.14

Proteolytic activity of ES products. The proteolytic activity of normal and heat-inactivated ES products was determined using fluorescein isothiocyanate labeled casein (FITC-casein) as described by Brown and others13; 12 µg of ES (20 µL) products were mixed with 10 µL of FITC-casein (stock 0.5 mg/ml) and 170 µL of 50 mM phosphate buffer, pH 6.5 containing 5 mM cysteine and incubated at 37°C for 2 h. The reaction was stopped with 120 µL of 5% w/v trichloroacetic acid and the tubes allowed to stand at room temperature for 1 h. Precipitated protein was removed by centrifugation at 13,000 g for 10 minutes. Triplicate, 20 µL aliquots of the supernatant were added to 80 µL of 0.5 M Tris pH 8.5 and the fluorescence measured (excitation 490 nm, emission detection 525 nm) using a Dynex MFX microplate fluorimeter. In this assay, untreated ES products gave a specific activity of 3387 ± 91.6 fluorescence units/min/mg. No activity was detected in heat-treated ES products.

Recombinant N. americanus calreticulin. Recombinant N. americanus calreticulin was expressed in culture as described previously15 and purified using a combination of ‘Bugbuster’ protein extraction reagent (Novagen) and a His Bind Purification kit (Novagen) following the manufacturer’s instructions. Fractions containing calreticulin as determined by SDS-PAGE16 were pooled, dialyzed against PBS, and stored at -20°C until required. The authenticity of the purified calreticulin was confirmed by MALDI-TOF mass spectrometry peptide sequencing.

Assessment of LPS in agonists. To eliminate any possible stimulatory effects of LPS in agonist preparations, N. ameri-canus ES products and recombinant calreticulin were assessed for LPS content using the Sigma E-TOXATE assay kit in accordance with the manufacturer’s instructions. No LPS was detected in the ES and recombinant calreticulin samples used in this study.

Western blotting. Fifty µg of adult somatic extract, 120 µg of ES products (untreated and heat inactivated), and 10 µg of recombinant calreticulin were resolved by 12% SDS-PAGE under reducing conditions and transferred onto nitrocellu-lose.17 Western blots were blocked with 5% non-fat dried milk powder in Tris buffered saline (TBS) for 1 h and incubated overnight at 4°C with either rabbit anti N. americanus calreticulin (1:600 in blocking agent) or pooled post-infection human sera (1:20 in blocking agent) obtained during this study and a previous study in 1996.18 After washing with TBS/0.05% Tween 20, the blots were incubated with either alkaline phosphatase conjugated goat anti-rabbit IgG (Sigma, 1: 10000 in blocking agent) or alkaline phosphatase conjugated goat anti-human IgE (Sigma, 1:500 in blocking agent). After further washing with TBS/0.05% Tween 20, antibody binding was visualized by incubation with 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium in 0.75 M Tris pH 9.6.

Human whole blood challenge for histamine release. To assess histamine release from challenged basophils 100 µL of whole blood (collected into heparinized tubes as described previously) was challenged with untreated ES products (10–0.001 µg/ml), heat-inactivated ES products (10–0.001 µg/ml), recombinant calreticulin (40–0.001 µg/ml), goat anti-human IgE (50–0.001 µg/ml), goat anti-human IgG (isotype control, 50 µg/ml), and ovalbumin (10–0.01 µg/ml) in a total volume of 500 µL made up with Pipes buffer pH 7.4 (0.01 M Piperazine-N'N-bis[2-ethaneculfonic acid], 0.14 M sodium acetate, 5 mM potassium acetate, 0.1% glucose, 1 mM CaCl2, and 0.03% human serum albumin). All challenges and spontaneous histamine release (100 µL of whole blood in Pipes buffer) were assessed after incubation for 1 h at 37°C. Total histamine release was assessed by mixing 50 µL of whole blood in 450 µL dH2O and freeze-thawing three times. Standard histamine calibrators of 0, 10, 25, 50, 100, and 250 ng/ml (Hycor Biomedical Ltd., Penicuik, UK) were included with the whole blood challenges.

Histamine released in each whole blood challenge was determined using a Histamine Assay Kit (Hycor Biomedical Ltd.); 50 µL of challenged whole blood was added to histamine-coated 96 well plates followed by 50 µL of mouse antihistamine monoclonal antibody conjugated to alkaline phos-phatase. The plates were incubated for 1 h at room temperature followed by three washes with a provided EIA wash solution. Antibody binding was visualized by the addition of 100 µL of 1 mg/ml p-nitrophenyl phosphate (pNPP) substrate. The plates were developed for 1 h at 37°C. Absorbance was measured at 405 nm using a Dynex MRX absorbance microplate reader. A standard curve was constructed from each histamine calibrator and the levels of histamine released in each challenge determined from this curve. Histamine levels were analyzed after subtraction of background histamine production release. The data presented are combined data obtained from all 43 individuals; not all individuals were examined at every agonist concentration indicated.

Total IgE assay. Total IgE concentrations in plasma samples were measured in an enzyme-linked immunosorbent assay (ELISA). Ninety-six well polystyrene plates were coated with 50 µL of a monoclonal mouse anti-human IgE (clone GE-1, ascites fluid, Sigma, Poole, UK, 5 µg/ml diluted in 0.05 M carbonate/bicarbonate buffer, pH 9.6) overnight at 4°C. The plates were washed with phosphate buffered saline/0.05% Tween 20, pH 7.2 (PBS/Tween) and blocked with 100 µL of 1% human serum albumin in PBS (HSA/PBS) for 1 h at room temperature. After blocking, the plates were washed again and 50 µL of human serum (diluted in the range 1:50 to 1:5000 in HSA/PBS) added to each well and incubated overnight at 4°C. In addition, 50 µL of human IgE standards (doubling dilutions from 31.25 to 1.953 IU/ml, National Institute of Biologic Standards and Control, UK) were included on each plate. All assays were carried out in duplicate. After overnight incubation the plates were washed again and 50 µL of alkaline phosphatase conjugated goat anti-human IgE ({epsilon}-chain specific, diluted 1:1000 HSA/PBS) was added to each well and the plate incubated at room temperature for 2 h. Antibody binding was visualized by the addition of 100 µL of 1 mg/ml pNPP substrate. Absorbance was measured at 405 nm using a Dynex MRX microplate reader.

Assay of IgG anti-IgE autoantibodies. Human IgG anti-IgE autoantibodies were measured in an ELISA with an immobilized (2 µg/ml) myeloma IgE (kind gift from Binding Site, Birmingham) bound to a 96-well plate. After blocking with 1% human serum albumin in PBS (HSA/PBS), plasma samples were diluted 1:5 in HSA/PBS with 0.05% Tween 20 and 50 µL added to the IgE-coated wells. (All samples were assayed in duplicate.) After overnight incubation at 4°C, the plates were washed and bound anti-IgE autoantibodies were detected with a peroxidase conjugated sheep anti-human IgG (Binding Site, Birmingham) diluted 1:000, 50 µL per well and the plate incubated at room temperature for 2 h. After further washing the plate was developed with 100 µL per well of 1 mg/ml tetramethylbenzidine (Sigma). Assay development was halted with the addition of 50 µL per well of 2.5 M H2SO4 and the absorbance read at 450 nm using a Dynex MRX microplate plate reader. The mean of duplicate absorbance readings was taken after background correction (serum wells with IgE coating omitted) and values above 0.05 were taken as positive.


RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Immunoepidemiological parameters. The study sample consisted of 43 individuals—27 males and 16 females (Table 1Go). All sampled individuals in the study were infected with hookworms, with a mean egg count of 2257 epg. Study individuals did not differ significantly by age, sex, or egg count from the total sampled population of the study villages (data not shown). Study individuals had elevated IgE levels, ranging from 677 to 26467 IU/ml compared with baseline values obtained in the UK, which ranged from 12 to 308 IU/ml.

Ten of 43 individuals (23.2%) produced detectable IgG anti-IgE. The number of eosinophils was also elevated, varying from 0.1–1.6 x 109 cells/l (normal range 0.04–0.4 x 109 cells/l). However, basophil numbers did not appear to be elevated in the study individuals (range 0–0.1 x 109 cells/l) when compared with uninfected individuals (normal range 0.01–0.1 x 109 cells/l).

Authentication of basophil agonists. To demonstrate the reactivity of N. americanus ES products and recombinant cal-reticulin for IgE prior to the study commencing in Papua New Guinea, Western blots of adult somatic extract (SE), ES products (ES), and recombinant calreticulin (rCal) were probed with either rabbit anti N. americanus calreticulin or pooled post-infection IgE obtained during a previous study in Madang Province in 1996 (Figure 1Go).18 Panel A illustrates the purity of the recombinant calreticulin used for the study and panel B shows myeloma IgE probed with the secondary antibody used for these studies. On return from Papua New Guinea to Nottingham the agonists were also tested against post-infection sera obtained during the current study. Recombinant calreticulin (rCal) reacted with both rabbit anti-calreticulin and human post-infection IgE from both studies (Figure 1C–1EGo). Human post-infection IgE from both studies also recognized a band of approximately 33 kDa in N. ameri-canus ES products (Figure 1D and 1EGo). The reactivity and specificity of the anti-human IgE (Sigma) used in this study has been authenticated in previous studies.18 Therefore, all agonists were biologically intact and capable of recognizing or being recognized by IgE, an important consideration if blockade is a distinct possibility.


Figure 1
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  		    FIGURE 1. Authentication of basophil agonists. Western blots of adult somatic extract (SE), ES products (ES), and recombinant calreticulin (rCal) were probed with rabbit anti N. americanus calreticulin (C), pooled post-infection sera obtained during a previous study in 1996 (1) (D), and pooled post-infection sera collected during the present study (E).

 
Basophil histamine release. Anti-human IgE induced histamine release from human basophils in a dose-dependent manner between 1 and 50 µg/ml (Figure 2AGo). Basophils challenged with anti-human IgG at a concentration of 50 µg/ml failed to release histamine (data not shown). Recombinant calreticulin, a hookworm allergen, induced basophil degranu-lation in a similar dose-dependent manner to anti-human IgE (Figure 2AGo). N. americanus ES products induced histamine release in a dose-dependent manner between 0.01 and 10 µg/ml (Figure 2BGo). Heat-inactivated ES products also induced similar levels of histamine release from five individuals tested, within the same dose range, indicating that the factor responsible for inducing basophil degranulation is not pro-teolytic in nature. Ovalbumin, a ‘bystander’ antigen control, failed to induce significant levels of histamine release over the range 0.01–10 µg/ml (N = 5).


Figure 2
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  		    FIGURE 2. The release of histamine by human basophils challenged with anti-human IgE, recombinant calreticulin, ES products, heat-inactivated ES products, and ovalbumin. In a whole blood assay human basophils were challenged with anti-human IgE (N = 43), recombinant calreticulin (N = 43) (A), ES products (N = 43), heat-inactivated (HI) ES products (N = 5), and ovalbumin (N = 5) (B). The resulting histamine released was measured using a Hycor Biomedical histamine assay kit. The results represent the mean concentration of histamine released in ng/ml + SE. Basophils challenged with anti-human IgG failed to release histamine. At the highest concentration of agonist tested, histamine release from the infected population was compared with that from a naive population (C). Data was analyzed using one way ANOVA followed by Holm Sidaks confirmatory test (*P ≤ 0.01).

 
These results are compared statistically to those obtained from UK naive individuals under the same conditions (Figure 2CGo). Basophils from non-parasitized donors released significantly less histamine after exposure to anti IgE, and to recombinant calreticulin, confirming the competence of baso-phils from a parasitized population to release histamine on challenge.

The higher levels of histamine release seen from basophils from parasitized individuals probably reflects the higher degree of Fc{epsilon}RI loading by IgE, given the differences seen in levels of total and specific IgE between the parasitized (mean 7594 I.U./ml) and non-parasitized (mean 136 I.U./ml). The higher background level seen with calreticulin possibly reflects the fact that it is both an allergen per se, and lectin.15,18

Ironically the eventuality that we are witnessing a combination of IgE-specific and non-specific events, where agonists can possibly engage IgE via Fab and Fc regions does not detract from our main argument that substances released from hookworms retain the capability to release histamine from basophils under conditions deemed by others to be prohibitive of the event.


DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study has demonstrated clear indications of basophil and Fc{epsilon}RI competence in an immunologically well-studied hookworm-infected population from Papua New Guinea.19–21 Histamine release was recorded to anti-IgE (but not to anti-IgG) and to the hookworm allergen calreticulin18 (Figure 2AGo), indicating an intact IgE - Fc{epsilon}RI axis in basophils from this population. No release was seen to the ‘bystander’ antigen ovalbumin (Figure 2BGo). This is a potentially significant finding, and indicates that polyclonal IgE of the levels recorded in this population were insufficient to block or saturate Fc{epsilon}RI, at least on basophils.

The histamine release data in Figure 2CGo indicate significance over background for all agonists, proving that basophils from parasitized individuals retain competence whether triggering is through polyclonal IgE, allergen-specific IgE, or a combination of both. The lower release values for anti-IgE probably reflect the fact that, in a whole blood assay, the presence of high levels of polyclonal IgE act to sequester agonist. Whatever the scenario, mast cell blockade is unlikely to be a realistic event in the parasitized, given that some parasite secretions may be able to act through IgE per se.

It has been reported previously that only 6% to 17% of the polyclonal IgE produced is parasite specific,22 allowing ample opportunity for non–parasite-specific IgE to interfere with histamine release. This was clearly not seen in the present study. In addition, the presence of autoantibodies to IgE had no deleterious effect on basophil function despite the ability of anti IgE antibodies to interfere with signalling23,24 and to potentially downregulate Fc{epsilon}RI expression.25

From a parasitological standpoint, these data show that potentially protective Fc{epsilon}RI-dependent immune responses are not blocked in human necatoriasis. This is consistent with observations that the Th2 phenotype is associated with both reduced worm size and fecundity and reduced re-infection with hookworms,10,26 and with post-vaccination immunity in murine models.27–30

There are a number of other potential mechanisms for helminth-mediated regulation of immunologic responses if Fc{epsilon}RI blockade is not operating to the extent previously thought. For example, the production of regulatory cytokines, such as IL-10, has been suggested to be responsible for the downregulation of allergic responses in schistosomiasis.31,32 Downregulated cytokine responses to bystander antigens10 and upregulated IL-10 responses33,34 have both also been reported in hookworm infection, though the functional relationship between them has not been investigated.

It is also possible that Fc{epsilon}RI blockade can indeed occur differentially at the mast cell level, although we consider this to be unlikely given the molecular conservation of this receptor across these cell types.

Whatever the mechanisms involved, we can categorically report at this stage that the IgE- Fc{epsilon}RI basophil histamine release axis is fully operational in a tropical population infected with hookworm exhibiting high levels of polyclonal IgE, and in the presence of autoantibodies to IgE. This is perhaps not too surprising given that the membrane frequency of Fc{epsilon}RI is in fact upregulated in the presence of high levels of IgE, which undoubtedly exist in hookworm infection. This effect presumably outweighs any downregulatory effect of anti IgE, leaving the original system intact. Therefore, we now suggest that hookworm-mediated downregulation of immune responses is instead mediated by secreted parasite immune suppressants and the induction of regulatory leukocyte populations3 rather than at the level of Fc{epsilon}RI blockade.

Received December 6, 2006. Accepted for publication July 3, 2007.

Acknowledgments: Fieldwork was supported by the Wellcome Trust and RJQ was supported by a MRC Career Development Award. We thank Moses Lagog (Papua New Guinea Institute of Medical Research) for technical support in the field, and the inhabitants of Haven and Wasab for their kind participation.

* Address correspondence to Alan Brown, University Park, Notting-ham, NG7 2RD. E-mail: alan.brown{at}nottingham.ac.uk Back

Authors’ addresses: David I. Pritchard, Doreen S. W. Hooi, Alan Brown, and Rebecca Caddick, The Boots Institute, School of Pharmacy, University of Nottingham, NG7 2RD. Telephone: +44 115 9516165. Fax: + 44 115 9515122. Moses J. Bockarie, Papua New Guinea Institute of Medical Research, Madang Province, Papua New Guinea. Rupert J. Quinnell, Institute of Integrative and Comparative Biology, University of Leeds, Leeds, LS2 9JT.

Reprint requests: Alan Brown, University Park, Nottingham, NG7 2RD. E-mail: alan.brown{at}nottingham.ac.uk


REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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