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An enduring association? Microfilariae and immunosupression in lymphatic filariasis
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Vol.19 No.12 December 2003
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An enduring association? Microfilariae and immunosupression in lymphatic filariasis Richard A. O’Connor1, Jessica S. Jenson2, Julie Osborne3 and Eileen Devaney 2 1
Trudeau Institute, 100 Algonquin Avenue, Saranac Lake, NY 12983, USA Veterinary Parasitology, Institute of Comparative Medicine, University of Glasgow, Bearsden Road, Glasgow, UK G61 1QH 3 Leukaemia Research Fund, Veterinary Pathology Institute of Comparative Medicine, University of Glasgow, Bearsden Road, Glasgow, UK G61 1QH 2
Human filarial infection is characterized by a defect in T-cell proliferative responses which is most pronounced among actively infected individuals. This article reviews the immunomodulatory potential of the first larval stage, the blood-borne microfilariae, which has long been associated with the most profound suppression of cellular responses. In particular, we focus on the induction of host cell apoptosis following murine infection with microfilariae. Promoting the apoptotic elimination of potentially reactive T cells could represent an important means of both facilitating parasite survival and limiting inflammatory pathology. Chronic helminth infections present a considerable challenge in terms of successful regulation of immune responses in the face of high antigenic loads: how does the immune system achieve a balance between protection and pathology? Filarial worms of the genus Brugia and Wuchereria are the causative agents of lymphatic filariasis (LF), a debilitating mosquito-borne infection of the tropics, which contributes significantly to the socio-economic burden faced by endemic countries. LF provides a good example of an infectious disease in which the equilibrium achieved by the parasite and the immune system results in the persistence of infection in the absence of overt pathology [1]. Recent estimates suggest that in the order of 120 million individuals are infected with lymphatic filarial worms, with , 20% of the global population living in endemic areas [2]. The adult parasites live in the afferent vessels of the lymphatic system and, following mating, the female worms release an abundance of first-stage larvae [microfilariae (Mf)], which circulate in the peripheral blood. The life cycle proceeds when Mf are ingested by a mosquito during blood feeding. Mf develop to infective third-stage larvae (L3) in the vector, and are transmitted to a new host when the infected mosquito next takes a bloodmeal. Both stages of the parasite in the mammalian host (the adult and the Mf) are long lived: the adult has an estimated reproductive life span of 5 – 8 years [3], and the Mf live for around one year. These parasites have Corresponding author: Eileen Devaney ([email protected]).
evolved myriad ways of evading host defenses and promoting their own survival and transmission. As each parasite stage in the mammalian host interacts with an immunologically distinct compartment (adults in the lymphatics, Mf in the peripheral blood, while L3 enter through the skin), the immune response elicited by each stage probably has its own distinctive features. This is supported by work on mouse models, which has provided much information on the immunomodulatory potential of individual life cycle stages (reviewed in Ref. [4]). At the same time, the Brugia malayi expressed-sequence tag (EST) project (http://nema.cap.ed.ac.uk/fgn/ests.html) has provided a wealth of molecular information on Brugia spp., allowing identification of important diagnostic and immunomodulatory proteins, and resulting in refinements in filarial immunobiology [5]. Not surprisingly, all indications are of a deep and complex association between host and parasite, the evolutionary fine-tuning of an intimate, longterm relationship. This review focuses on the immune responses elicited by the first larval stage, the Mf, and the mechanisms by which Mf could contribute to the proliferative defect that characterizes the immune response in human LF. Microfilariae and their role in immune regulation Mf can reach extraordinarily high numbers in the peripheral circulation. For example, in areas of high transmission of Wuchereria bancrofti, counts of several hundred Mf per ml of blood are not exceptional. Infection with the related parasite, Loa loa, can occasionally result in Mf counts that surpass 20 000 per ml. Mf represent the reservoir of infection for the mosquito population and, as such, have developed several evolutionary adaptations to ensure their transmission. The best characterized of these is the periodicity by which Mf peak in the peripheral blood when mosquito biting is maximal [6]. Recent evidence indicates that numbers of circulating Mf can also show a seasonal periodicity, with peaks and troughs corresponding to wet and dry seasons, reflecting the availability of vector mosquitoes [7]. Sustained transmission, however, requires more than simply being in the right place at the right time. It demands that huge numbers of organisms, representing a significant antigenic burden, be maintained
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in the circulatory system over prolonged periods. This is facilitated by a profound suppression of antifilarial immune responses in microfilaraemic individuals. In this case, ‘immunosuppression’ is most characteristically manifested as the inability of peripheral blood mononuclear cells (PBMC) from infected individuals to proliferate or to secrete interferon (IFN)-g in response to parasite antigen (Ag). Historically, the presence of circulating Mf was thought to be a crucial determinant of immune unresponsiveness. However, more recent studies with larger groups of B. malayi-infected individuals have shown impaired proliferative responses among amicrofilaraemic individuals and patients displaying chronic pathology (who are also typically amicrofilaraemic) [8]. Furthermore, cytokine profiling of infected individuals has shown that human infection cannot be accurately described in terms of the highly polarized T helper (Th)1 and Th2 responses seen in murine models. In B. malayi infection, production of Ag-specific IFN-g and interleukin (IL)-5 are both reduced among Mf þ individuals versus Mf- individuals, whereas IL-4 responses are equivalent [9]. Thus, the suppressive effects of infection are not restricted to the Th1 arm of the immune response. In the case of W. bancrofti, occult infections (i.e. adult worms, but no Mf) can be detected using tests for circulating antigen (CAg) and proliferative suppression in amicrofilaraemics is associated with the presence of CAg, indicative of active infection [10]. To add a further level of complexity, studies conducted in areas of markedly different transmission intensity suggest that the level of exposure to L3 can directly influence proliferative capacity. In areas of high transmission, proliferative responses were profoundly suppressed compared to those in low transmission areas, an effect unrelated to levels of CAg or circulating Mf [11]. However, while suppression is not solely associated with the presence of Mf, proliferative responses do tend to be most profoundly impaired and most difficult to restore in microfilaraemics [12]. In addition, in one study in which infected individuals were treated with ivermectin (a drug which is largely microfilaricidal), restoration of Ag-specific proliferative responses was observed in a significant proportion of individuals over the short term [13]. However, a long-term follow-up study of individuals who converted from Mf þ to Mf- revealed only marginal increases in cellular responses among the converters, with cytokine responses in subjects who had cleared their circulating Mf remaining lower than those of endemic normals [14]. This could indicate that infection imprints a degree of unresponsiveness on the host, which remains active even in the absence of circulating Mf. There is also evidence from the Brugia pahangi – jird model of infection to support an active role for Mf in downregulating proliferative responses. In this experimental system, development of proliferative suppression following infection was shown to correlate with the onset of Mf production [15]. The antigen-specific proliferative defect in infected humans The mechanistic basis of immune suppression in LF has been the focus of considerable study. A variety of mechanisms that could contribute to the inability of http://parasites.trends.com
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T cells to proliferate or to secrete IFN-g in response to re-stimulation with parasite Ag have been reported (reviewed in Ref. [1]). These include a cytokine imbalance in favour of Th2 responses, the expansion of T-regulatory cells, selective tolerance of Th1 cells or clonal deletion. Cytokine responses in microfilaraemic individuals are dominated by IL-4 and IL-10 production. Using PBMC from W. bancrofti microfilaraemic individuals, neutralization of IL-10 resulted in some restoration of proliferation [16], with neutralization of transforming growth factor b (TGF-b) having a similar, but less pronounced effect. Additional studies have demonstrated that the high level of IL-10 produced in vitro by cells from microfilaraemic individuals was primarily associated with re-stimulation by Mf-derived Ags [17]. More recently, characterization of cellular populations from Onchocerca volvulus-infected individuals demonstrated a subset of parasite-specific T cells expressing high levels of IL-10 and/or TGF-b, but low IL-2, a cytokine profile similar to that of T-regulatory cells [18]. While the elevated levels of regulatory cytokines reported in microfilaraemic individuals undoubtedly influences the proliferative capacity of T cells, additional underlying causes might contribute to impaired proliferation. For example, comparison of the precursor frequency of Ag-specific lymphocytes in microfilaraemic individuals or chronic pathology patients (who are usually amicrofilaraemic), demonstrated that levels of both Ag-specific T and B cells were significantly reduced in microfilaraemic individuals [19]. Furthermore, children born of W. bancrofti microfilaraemic mothers exhibit long-term proliferative unresponsiveness [20] and are more likely to become microfilaraemic [21] than children born of amicrofilaraemic mothers. By contrast, paternal infection status has no effect on the outcome of infection. Intriguingly, a similar phenomenon has been observed in the B. pahangi– jird model of infection [22]. Taken together, these findings imply that in utero exposure to filarial Ag might result in central tolerance due to thymic deletion of Ag-specific T cells. However, the recovery of proliferative and IFN-g responses post-chemotherapy [23] indicates that thymic deletion is unlikely to account fully for proliferative suppression and suggests that active infection could also induce a form of peripheral tolerance. Filarial Ag can also modulate T-cell responses indirectly by affecting the function of antigen-presenting cells (APC), as demonstrated by several studies in both animal models and human infection. When dendritic cells (DC), the most important initiators of immune responses, are exposed to ES-62, the major excretory – secretory (ES) product of the animal filarial worm Acanthocheilonmea viteae, they promote the subsequent differentiation of CD4þ cells to Th2 [24]. Likewise, exposure of human DC to Mf extracts during their differentiation in vitro results in impaired function, as assessed by the ability of Mf-exposed DC to support a mixed lymphocyte response [25]. ES-62 not only affects the initiation phase of an immune response but also modulates macrophage function [26] and signaling through the B- and T-cell receptors. Exposure to adult B. malayi or their ES products results in the generation of a population of alternatively activated macrophages that
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profoundly suppress proliferation of a range of cell types [27]. Together, such studies indicate the multitude of factors brought to bear on immunity over the course of a long-term infection. They illustrate how changes in clinical and parasitological status, levels of CAg, and fluctuations in exposure to L3 can all influence immune responsiveness. Thus, in human infection, it is probably most useful to think in terms of a dynamic response reflecting the cumulative input of all these factors, which is subject to both qualitative and quantitative changes over the course of chronic infection. Mf induce an antigen-specific proliferative defect The contribution of each life cycle stage to the overall development of the immune response is difficult to assess in human infection. In this respect, single-stage infections in mouse models have proved invaluable in allowing dissection of stage-specific responses. Recent studies in BALB/c mice infected with B. pahangi have demonstrated that infection with Mf alone is sufficient to induce proliferative suppression [28]. This proliferative defect is Ag-specific in nature, reflecting the situation most often observed in human filariasis. However, splenic lymphocytes from Mf-infected animals are not entirely nonresponsive to filarial Ag, and retain their capacity for Ag-specific cytokine production, which is intrinsically linked to proliferative suppression. A remarkable and consistent observation from murine infections is that distinct life cycle stages of Brugia elicit development of differentially polarized responses. Infection with adult worms or L3 leads to development of highly polarized Th2 responses [29]. Intriguingly, and most unexpectedly, infection with Mf, a life cycle stage associated with the most profound suppression of IFN-g production in human infection, uniquely elicits a Th1-like IFN-g dominated response in mice [28,29]. This differential polarization is stable in several strains of mice and following infection with either B. malayi or B. pahangi by a variety of routes [28,30]. Intravenous injection of live Mf elicits IFN-g production by splenocytes over a range of doses (2.5 £ 104 – 2.5 £ 105). Interestingly, given the high degree of crossreactivity between life cycle stages, Mf are also capable of inducing IFN-g production in the face of a pre-established Th2 response (following implantation of adult males) [30]. Immunization with Mf extract also elicits IFN-g production and following both immunization with extract or infection with live Mf, CD4þ T cells have been identified as the major source of IFN-g [31,32]. Our studies have shown that the proliferative suppression observed following Mf infection is dependent on the IFN-g-induced production of high levels of nitric oxide (NO) [28]. Infection of IFN-g receptor knockout (IFN-gR-/mice with Mf induces a strong proliferative response, as does the use of inducible nitric oxide synthase (iNOS) inhibitors in vitro [28,33]. iNOS inhibition also increased IFN-g production by splenocytes from Mf-infected mice, consistent with a negative regulatory effect of NO on IFN-g-producing cells. The ability to restore proliferative capacity clearly demonstrates that Ag-specific T cells are present and primed by infection. To assess the balance between cell division and cell death, we used http://parasites.trends.com
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carboxyfluoresein diacetate succinimidyl ester (CFSE) labeling to follow cell division in Ag-stimulated culture and employed the TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labelling) method to investigate levels of T-cell apoptosis [32]. These studies demonstrated that, upon antigenic re-stimulation, CD4þ T cells from Mf-infected mice failed to proliferate and displayed high levels of NO-mediated apoptosis in vitro. This contrasts markedly with the situation seen following infection with L3, where robust proliferation is associated with low levels of T-cell apoptosis [32]. Such findings suggest that IFN-g production leads to macrophage activation and production of NO, which in turn suppresses proliferation by inducing the apoptosis of Ag-reactive T cells. In this way, Ag-specific IFN-g-producing T cells could indirectly limit their own expansion. There is growing evidence that such a negative feedback mechanism acts to eliminate activated T cells in both infectious [34] and autoimmune disease [35] in which pro-inflammatory responses could exacerbate pathology. The ability of Mf to stimulate high levels of IFN-g production and T-cell apoptosis upon re-stimulation in vitro raises several questions: do Mf induce apoptosis in vivo? How and why do Mf uniquely elicit IFN-g production? What is the significance of NO production in filarial infection? What relevance could this have to human infection? Could the proliferative defect observed in most in vitro studies with human PBMC be associated with T-cell apoptosis? Mf infection induces apoptosis in vivo In vivo studies have revealed evidence of a wave of apoptosis in the spleen following Mf infection. Using the TUNEL method in situ, a clustering of apoptotic cells in the follicles of the spleen was observed in Mf-infected mice (Figure 1a and b), whereas in L3-infected and uninfected control animals, only single apoptotic nuclei were seen scattered throughout the spleen (Figure 1c and d). Heightened levels of apoptosis in the spleen persist for up to one month following Mf infection. Intravenous or subcutaneous routes of infection produced identical patterns of apoptosis (J.S. Jenson et al., unpublished). When double-stained with peanut agglutinin, the TUNELpositive cells were found to be largely restricted to the T-cell areas of germinal centers in Mf-infected mice. By contrast, the scattered apoptotic nuclei identified in sections from L3-infected and noninfected mice did not display any organized distribution patterns. Molecules and pathways The observation that apoptosis is specific to infection with Mf might be indicative of the greater susceptibility of Th1 cells to apoptosis, as observed in several previous studies [36,37]. Because infection with L3 induces a Th2-dominated response, activated CD4 þ cells might be protected by IL-4 and IL-10, cytokines that are reported in other systems to have an anti-apoptotic effect via the upregulation of survival molecules such as cellular FLICEinhibitory protein (c-FLIP), B-cell lymphoma-x (bcl-x) [38] or B-cell leukaemia/lymphoma 2 (bcl-2) [39]. By contrast, IFN-g upregulates cell death-inducing caspases
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Figure 1. Infection of BALB/c mice with microfilariae results in apoptosis in vivo. Mice were infected with Mf or L3 of Brugia pahangi or received HBSS. At 12 days post-infection, spleens were fixed in formalin and processed for TUNEL staining, as described by Gavrieli et al. [55]. Following fixation and blocking of endogenous peroxidase activity, sections were incubated with TdT and dUTP-biotin in TdT buffer for 60 min at 37 8C. Negative controls were incubated in TdT buffer alone. The sections were washed and then exposed to HRP-strepavidin, followed by the chromagen 3-amino-9-ethyl-carbazole and counterstained with Mayer’s haematoxylin. TUNEL-positive nuclei are stained red. (a,b) Sections from Mf-infected mice, showing clusters of TUNEL-positive nuclei in the follicles of the spleen. (c) A section from a L3-infected mouse. (d) Control mouse. Scale bar: 100 mm (a,c,d); 50 mm (b). Abbreviations: dUTP, uridine triphosphate; HBSS, Hanks balanced salt solution; L3, third-stage larvae; Mf, microfilariae; TdT, terminal deoxynucleotidyl transferase; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labelling.
elicited apoptosis of human DC and resulted in a diminished capacity to promote CD4þ T-cell production of IFN-g and IL-5 [47]. DC exposed to live Mf upregulate the expression of a variety of pro-inflammatory cytokine messenger RNA (mRNA), although there was no evidence of a role for NO in inducing DC apoptosis. Helminth infections are classically acknowledged as potent inducers of Th2 responses, but the complexity of these infections suggests that strict application of the Th1 –Th2 paradigm is unlikely to adequately describe responses over the course of infection [48]. Indeed, NO production, which is most potently induced by the Th1 cytokine IFN-g, has been described in other helminth infections, most notably S. mansoni, coincident with egg deposition in the tissues [49]. However, while the Th1 to Th2 shift observed following egg laying in murine models of S. mansoni is thought to be regulated at least in part by T-cell apoptosis, there is no evidence that NO drives apoptosis in this system. Rather, NO appears to limit hepatic pathology at the onset of egg laying [49], whereas IL-10 is pro-apoptotic [50]. Most pertinently, it was recently demonstrated that PBMC from S. mansoniinfected individuals are capable of NO production following in vitro re-stimulation with parasite Ag and that iNOS inhibition exacerbated the granulomatous response [51]. Such results are compatible with a regulatory role for NO in human helminth infection and serve to demonstrate an additional way in which oxidative stress might act to limit the extent of inflammatory pathology.
in both lymphoid [40] and non-lymphoid cells [38]. The nature of the Mf Ags which elicit IFN-g production are of great interest, given that other life-cycle stages of filarial worms promote Th2 responses in infected mice. Recent studies have shown that a recombinant serpin, an abundant Mf-specific transcript, induces high levels of IFN-g upon injection into mice [41]. Filarial cystatins have also been independently identified as potent inducers of NO from IFN-g-activated macrophages [42]. The potential immunomodulatory influence of the endosymbiotic bacteria of the Wolbachia species family found within all lifecycle stages of Brugia is an important consideration in this respect, and deserving of further study [43]. Thus, filarial parasites might contain many Ags capable of inducing IFN-g and/or NO, but their effects could be masked by the dominance of Th2-inducing Ags in extracts of whole parasites. It is noteworthy that ES products or soluble Ags derived from a range of helminth parasites have now been shown to elicit apoptosis of various cell types. For example, an ES product of Schistosoma mansoni skinstage schistosomulae promoted T-cell apoptosis via the induction of caspase activity and the upregulation of Fas-L. By biochemical fractionation, the pro-apoptotic activity was shown to be associated with a 23-kDa protein [44]. Similarly, ES products from Nippostrongylus brasiliensis induced apoptosis in a rat epithelial cell line [45], whereas a protein extract from the cattle filarial nematode, Setaria digitata induced apoptosis in Hep-2 cells (a human epithelial cell line), an effect that could be reversed by the overexpression of bcl-2 in these cells [46]. A recent study reported that exposure to live Mf or to Mf ES in vitro
What is the significance of apoptosis in terms of the infection? It is difficult to distinguish whether elevated lymphocyte apoptosis during Mf infection represents an attempt by the host to maintain homeostasis and avoid immunopathology, or whether apoptosis is actively induced by the parasite to subvert the host immune response. It is conceivable that both ends are equally well served by the same means. Following Mf infection in the murine model, re-exposure to Ag in vitro results in the apoptotic death of Ag-responsive CD4þ T cells. Given that Mf compose a large antigenic burden in infected humans, it is conceivable that repeated re-stimulation in vivo might also result in T-cell death. Such a scenario could contribute to the lower precursor frequencies of Ag-specific T cells recovered from microfilaraemic individuals [19]. Interestingly, previous studies in filarial-infected humans suggested that the chronicity of infection leads to the downregulation of Mf-induced Th1 responses and could facilitate the emergence of a Th2 response. In the mouse model, following multiple immunizations with Mf extract and subsequent Mf infection, the Mf-specific IFN-g response was downregulated by IL-10 [31]. At later time points post-Mf infection, the magnitude of the IFN-g response has also been shown to correlate inversely with the initial infective dose of parasites [30]. Intriguingly, our studies have shown that challenge infection with Mf induces a secondary, more extensive wave of apoptosis in the spleen, as observed by in situ TUNEL staining (J.S. Jenson et al., unpublished). In parallel, secondary infection with Mf also significantly decreases the magnitude of the IFN-g response in vitro in
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the absence of a coincident increase in Th2 cytokine production (R.A. O’Connor, et al., unpublished). These data are compatible with the hypothesis that apoptosis deletes a population of pro-inflammatory Ag-specific T cells. They do not, however, directly support a role for Th2 cytokines in driving down IFN-g responses following exposure to live Mf. Could apoptosis of effector T cells underlie the defective immune responses reported in many chronic parasitic infections of humans? One study reported high levels of apoptosis within PBMC of individuals infected with a range of gastrointestinal helminths and a reduction in levels of apoptosis following treatment with anthelmintics [52]. In filarial-infected humans, the Mf-induced apoptosis of T cells could help explain many previous observations, all from in vitro studies, which link the most profound defect in proliferation and IFN-g production to active infection with Mf. Furthermore, the uptake of apoptotic cells by APC (macrophages or dendritic cells) is now recognized to have a profound downregulatory influence on the effector phenotype of the APC by induction of immunosuppressive cytokines such as IL-10 or TGF-b [53]. Ag-specific T cells undergoing apoptosis are also reported to produce significant quantities of IL-10, which, via its activity on APC, can polarize T helper responses to Type 2, resulting in the downregulation of inflammatory responses and peripheral tolerance [54]. Both IL-10 and TGF-b have been implicated in the suppression of T-cell responses and IFN-g production in microfilaraemic individuals [16]. Filarial worms have evolved many different strategies for downregulating potentially pathogenic proinflammatory responses and prolonging the patent phase of infection. Whether the selective apoptosis of Ag-reactive CD4þ T cells secreting IFN-g can be added to the list of mechanisms that contribute to the tolerance and/or immune deviation induced by filarial infection in the human host awaits further study. Acknowledgements This work was supported by grants from the Wellcome Trust and the MRC. R.O’C. was in receipt of a University of Glasgow studentship. Thanks go to Iain MacMillan and the staff of the histopathology laboratory, Department of Veterinary Pathology, for their help in establishing the TUNEL stain and to Tom Nutman (NIH) for access to unpublished data.
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apoptosis of CD4þ T lymphocytes: a mechanism of immune unresponsiveness in filariasis? Eur. J. Immunol. 32, 858– 867 Allen, J.E. et al. (1996) APC from mice harbouring the filarial parasite, Brugia malayi, prevent cellular proliferation but not cytokine production. Int. Immunol. 8, 143– 151 Dalton, D.K. et al. (2000) Interferon-g eliminates responding CD4 T cells during mycobacterial infection by inducing apoptosis of activated CD4 T cells. J. Exp. Med. 192, 117 – 122 Chu, C-Q. et al. (2000) Failure to suppress the expansion of the activated CD4 T cell population in interferon-g-deficient mice leads to exacerbation of experimental autoimmune encephalomyelitis. J. Exp. Med. 192, 123 – 128 Zhang, X. et al. (1997) Unequal death in T helper cell (Th) 1 and Th2 effectors: Th1, but not Th2, effectors undergo rapid Fas/FasL-mediated apoptosis. J. Exp. Med. 185, 1837 – 1849 Zhang, L. et al. (1996) Characterisation of apoptosis resistant antigen specific T cells in vivo. J. Exp. Med. 183, 2065– 2073 Stassi, G. et al. (2000) Control of target cell survival in thyroid autoimmunity by T helper cytokines via regulation of apoptotic proteins. Nat. Immunol. 1, 483 – 488 Cohen, S.B. et al. (1997) Interleukin-10 rescues T cells from apoptotic cell death: association with an upregulation of Bcl-2. Immunology 92, 1–5 Refaeli, Y. et al. (2002) Interferon-g is required for activation – induced death of T lymphocytes. J. Exp. Med. 196, 999 – 1005 Zang, X. et al. (2000) The serpin secreted by Brugia malayi microfilariae, Bm-SPN-2, elicits strong, but short-lived, immune responses in mice and humans. J. Immunol. 165, 5161– 5169 Hartmann, S. et al. (2002) Cystatins of filarial nematodes up-regulate the nitric oxide production of interferon-gamma-activated murine macrophages. Parasite Immunol. 24, 253– 262 Taylor, M.J. et al. (2000) Inflammatory responses induced by the filarial nematode Brugia malayi are mediated by lipopolysaccharidelike activity from endosymbiotic Wolbachia bacteria. J. Exp. Med. 191, 1429 – 1435
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44 Chen, L. et al. (2002) Skin-stage schistosomula of Schistosoma mansoni produce an apoptosis-inducing factor that can cause apoptosis of T cells. J. Biol. Chem. 277, 34329 – 34335 45 Hyoh, Y. et al. (2002) Activation of caspases in intestinal villus epithelial cells of normal and nematode infected rats. Gut 50, 71– 77 46 Krishnamoorthy, B. et al. (1998) Apoptosis induced by filarial parasitic sheath protein in Hep2 cell lines blocked by ectopic expression of bcl-2. Cell Biol Int. 22, 483– 492 47 Semnani, R.T. et al. (2003) Brugia malayi microfilariae induce cell death in human dendritic cells, inhibit their ability to make IL-12 and IL-10, and reduce their capacity to activate CD4(þ) T cells. J. Immunol. 171, 1950– 1960 48 Allen, J.E. and Maizels, R.M. (1997) Th1-Th2: reliable paradigm or dangerous dogma? Immunol. Today 18, 387 – 392 49 Brunet, L.R. et al. (1999) Nitric oxide and the Th2 response combine to prevent severe hepatic damage during Schistosoma mansoni infection. J. Immunol. 163, 4976– 4984 50 Estaquier, J. et al. (1997) Interleukin-10-mediated T cell apoptosis during the T helper type 2 cytokine response in murine Schistosoma mansoni parasite infection. Eur. Cytokine Netw. 8, 153– 160 51 Oliveira, D.M. et al. (1998) Role of nitric oxide on human schistosomiasis mansoni: upregulation of in vitro granuloma formation by N omega-nitro-L-arginine methyl ester. Nitric Oxide 2, 57 – 65 52 Kalinkovich, A. et al. (1998) Decreased CD4 and increased CD8 counts with T cell activation is associated with chronic helminth infection. Clin. Exp. Immunol. 114, 414 – 421 53 McDonald, P.P. et al. (1999) Transcriptional and translational regulation of inflammatory mediator production by endogenous TGF-b in macrophages that have ingested apoptotic cells. J. Immunol. 163, 6164– 6172 54 Gao, Y. et al. (1998) Anti-inflammatory effects of CD95 ligand (FasL)induced apoptosis. J. Exp. Med. 188, 887 – 896 55 Gavrieli, Y. et al. (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell Biol. 119, 493– 501
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An enduring association? Microfilariae and immunosupression in lymphatic filariasis Richard A. O’Connor1, Jessica S. Jenson2, Julie Osborne3 and Eileen Devaney 2 1
Trudeau Institute, 100 Algonquin Avenue, Saranac Lake, NY 12983, USA Veterinary Parasitology, Institute of Comparative Medicine, University of Glasgow, Bearsden Road, Glasgow, UK G61 1QH 3 Leukaemia Research Fund, Veterinary Pathology Institute of Comparative Medicine, University of Glasgow, Bearsden Road, Glasgow, UK G61 1QH 2
Human filarial infection is characterized by a defect in T-cell proliferative responses which is most pronounced among actively infected individuals. This article reviews the immunomodulatory potential of the first larval stage, the blood-borne microfilariae, which has long been associated with the most profound suppression of cellular responses. In particular, we focus on the induction of host cell apoptosis following murine infection with microfilariae. Promoting the apoptotic elimination of potentially reactive T cells could represent an important means of both facilitating parasite survival and limiting inflammatory pathology. Chronic helminth infections present a considerable challenge in terms of successful regulation of immune responses in the face of high antigenic loads: how does the immune system achieve a balance between protection and pathology? Filarial worms of the genus Brugia and Wuchereria are the causative agents of lymphatic filariasis (LF), a debilitating mosquito-borne infection of the tropics, which contributes significantly to the socio-economic burden faced by endemic countries. LF provides a good example of an infectious disease in which the equilibrium achieved by the parasite and the immune system results in the persistence of infection in the absence of overt pathology [1]. Recent estimates suggest that in the order of 120 million individuals are infected with lymphatic filarial worms, with , 20% of the global population living in endemic areas [2]. The adult parasites live in the afferent vessels of the lymphatic system and, following mating, the female worms release an abundance of first-stage larvae [microfilariae (Mf)], which circulate in the peripheral blood. The life cycle proceeds when Mf are ingested by a mosquito during blood feeding. Mf develop to infective third-stage larvae (L3) in the vector, and are transmitted to a new host when the infected mosquito next takes a bloodmeal. Both stages of the parasite in the mammalian host (the adult and the Mf) are long lived: the adult has an estimated reproductive life span of 5 – 8 years [3], and the Mf live for around one year. These parasites have Corresponding author: Eileen Devaney ([email protected]).
evolved myriad ways of evading host defenses and promoting their own survival and transmission. As each parasite stage in the mammalian host interacts with an immunologically distinct compartment (adults in the lymphatics, Mf in the peripheral blood, while L3 enter through the skin), the immune response elicited by each stage probably has its own distinctive features. This is supported by work on mouse models, which has provided much information on the immunomodulatory potential of individual life cycle stages (reviewed in Ref. [4]). At the same time, the Brugia malayi expressed-sequence tag (EST) project (http://nema.cap.ed.ac.uk/fgn/ests.html) has provided a wealth of molecular information on Brugia spp., allowing identification of important diagnostic and immunomodulatory proteins, and resulting in refinements in filarial immunobiology [5]. Not surprisingly, all indications are of a deep and complex association between host and parasite, the evolutionary fine-tuning of an intimate, longterm relationship. This review focuses on the immune responses elicited by the first larval stage, the Mf, and the mechanisms by which Mf could contribute to the proliferative defect that characterizes the immune response in human LF. Microfilariae and their role in immune regulation Mf can reach extraordinarily high numbers in the peripheral circulation. For example, in areas of high transmission of Wuchereria bancrofti, counts of several hundred Mf per ml of blood are not exceptional. Infection with the related parasite, Loa loa, can occasionally result in Mf counts that surpass 20 000 per ml. Mf represent the reservoir of infection for the mosquito population and, as such, have developed several evolutionary adaptations to ensure their transmission. The best characterized of these is the periodicity by which Mf peak in the peripheral blood when mosquito biting is maximal [6]. Recent evidence indicates that numbers of circulating Mf can also show a seasonal periodicity, with peaks and troughs corresponding to wet and dry seasons, reflecting the availability of vector mosquitoes [7]. Sustained transmission, however, requires more than simply being in the right place at the right time. It demands that huge numbers of organisms, representing a significant antigenic burden, be maintained
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in the circulatory system over prolonged periods. This is facilitated by a profound suppression of antifilarial immune responses in microfilaraemic individuals. In this case, ‘immunosuppression’ is most characteristically manifested as the inability of peripheral blood mononuclear cells (PBMC) from infected individuals to proliferate or to secrete interferon (IFN)-g in response to parasite antigen (Ag). Historically, the presence of circulating Mf was thought to be a crucial determinant of immune unresponsiveness. However, more recent studies with larger groups of B. malayi-infected individuals have shown impaired proliferative responses among amicrofilaraemic individuals and patients displaying chronic pathology (who are also typically amicrofilaraemic) [8]. Furthermore, cytokine profiling of infected individuals has shown that human infection cannot be accurately described in terms of the highly polarized T helper (Th)1 and Th2 responses seen in murine models. In B. malayi infection, production of Ag-specific IFN-g and interleukin (IL)-5 are both reduced among Mf þ individuals versus Mf- individuals, whereas IL-4 responses are equivalent [9]. Thus, the suppressive effects of infection are not restricted to the Th1 arm of the immune response. In the case of W. bancrofti, occult infections (i.e. adult worms, but no Mf) can be detected using tests for circulating antigen (CAg) and proliferative suppression in amicrofilaraemics is associated with the presence of CAg, indicative of active infection [10]. To add a further level of complexity, studies conducted in areas of markedly different transmission intensity suggest that the level of exposure to L3 can directly influence proliferative capacity. In areas of high transmission, proliferative responses were profoundly suppressed compared to those in low transmission areas, an effect unrelated to levels of CAg or circulating Mf [11]. However, while suppression is not solely associated with the presence of Mf, proliferative responses do tend to be most profoundly impaired and most difficult to restore in microfilaraemics [12]. In addition, in one study in which infected individuals were treated with ivermectin (a drug which is largely microfilaricidal), restoration of Ag-specific proliferative responses was observed in a significant proportion of individuals over the short term [13]. However, a long-term follow-up study of individuals who converted from Mf þ to Mf- revealed only marginal increases in cellular responses among the converters, with cytokine responses in subjects who had cleared their circulating Mf remaining lower than those of endemic normals [14]. This could indicate that infection imprints a degree of unresponsiveness on the host, which remains active even in the absence of circulating Mf. There is also evidence from the Brugia pahangi – jird model of infection to support an active role for Mf in downregulating proliferative responses. In this experimental system, development of proliferative suppression following infection was shown to correlate with the onset of Mf production [15]. The antigen-specific proliferative defect in infected humans The mechanistic basis of immune suppression in LF has been the focus of considerable study. A variety of mechanisms that could contribute to the inability of http://parasites.trends.com
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T cells to proliferate or to secrete IFN-g in response to re-stimulation with parasite Ag have been reported (reviewed in Ref. [1]). These include a cytokine imbalance in favour of Th2 responses, the expansion of T-regulatory cells, selective tolerance of Th1 cells or clonal deletion. Cytokine responses in microfilaraemic individuals are dominated by IL-4 and IL-10 production. Using PBMC from W. bancrofti microfilaraemic individuals, neutralization of IL-10 resulted in some restoration of proliferation [16], with neutralization of transforming growth factor b (TGF-b) having a similar, but less pronounced effect. Additional studies have demonstrated that the high level of IL-10 produced in vitro by cells from microfilaraemic individuals was primarily associated with re-stimulation by Mf-derived Ags [17]. More recently, characterization of cellular populations from Onchocerca volvulus-infected individuals demonstrated a subset of parasite-specific T cells expressing high levels of IL-10 and/or TGF-b, but low IL-2, a cytokine profile similar to that of T-regulatory cells [18]. While the elevated levels of regulatory cytokines reported in microfilaraemic individuals undoubtedly influences the proliferative capacity of T cells, additional underlying causes might contribute to impaired proliferation. For example, comparison of the precursor frequency of Ag-specific lymphocytes in microfilaraemic individuals or chronic pathology patients (who are usually amicrofilaraemic), demonstrated that levels of both Ag-specific T and B cells were significantly reduced in microfilaraemic individuals [19]. Furthermore, children born of W. bancrofti microfilaraemic mothers exhibit long-term proliferative unresponsiveness [20] and are more likely to become microfilaraemic [21] than children born of amicrofilaraemic mothers. By contrast, paternal infection status has no effect on the outcome of infection. Intriguingly, a similar phenomenon has been observed in the B. pahangi– jird model of infection [22]. Taken together, these findings imply that in utero exposure to filarial Ag might result in central tolerance due to thymic deletion of Ag-specific T cells. However, the recovery of proliferative and IFN-g responses post-chemotherapy [23] indicates that thymic deletion is unlikely to account fully for proliferative suppression and suggests that active infection could also induce a form of peripheral tolerance. Filarial Ag can also modulate T-cell responses indirectly by affecting the function of antigen-presenting cells (APC), as demonstrated by several studies in both animal models and human infection. When dendritic cells (DC), the most important initiators of immune responses, are exposed to ES-62, the major excretory – secretory (ES) product of the animal filarial worm Acanthocheilonmea viteae, they promote the subsequent differentiation of CD4þ cells to Th2 [24]. Likewise, exposure of human DC to Mf extracts during their differentiation in vitro results in impaired function, as assessed by the ability of Mf-exposed DC to support a mixed lymphocyte response [25]. ES-62 not only affects the initiation phase of an immune response but also modulates macrophage function [26] and signaling through the B- and T-cell receptors. Exposure to adult B. malayi or their ES products results in the generation of a population of alternatively activated macrophages that
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profoundly suppress proliferation of a range of cell types [27]. Together, such studies indicate the multitude of factors brought to bear on immunity over the course of a long-term infection. They illustrate how changes in clinical and parasitological status, levels of CAg, and fluctuations in exposure to L3 can all influence immune responsiveness. Thus, in human infection, it is probably most useful to think in terms of a dynamic response reflecting the cumulative input of all these factors, which is subject to both qualitative and quantitative changes over the course of chronic infection. Mf induce an antigen-specific proliferative defect The contribution of each life cycle stage to the overall development of the immune response is difficult to assess in human infection. In this respect, single-stage infections in mouse models have proved invaluable in allowing dissection of stage-specific responses. Recent studies in BALB/c mice infected with B. pahangi have demonstrated that infection with Mf alone is sufficient to induce proliferative suppression [28]. This proliferative defect is Ag-specific in nature, reflecting the situation most often observed in human filariasis. However, splenic lymphocytes from Mf-infected animals are not entirely nonresponsive to filarial Ag, and retain their capacity for Ag-specific cytokine production, which is intrinsically linked to proliferative suppression. A remarkable and consistent observation from murine infections is that distinct life cycle stages of Brugia elicit development of differentially polarized responses. Infection with adult worms or L3 leads to development of highly polarized Th2 responses [29]. Intriguingly, and most unexpectedly, infection with Mf, a life cycle stage associated with the most profound suppression of IFN-g production in human infection, uniquely elicits a Th1-like IFN-g dominated response in mice [28,29]. This differential polarization is stable in several strains of mice and following infection with either B. malayi or B. pahangi by a variety of routes [28,30]. Intravenous injection of live Mf elicits IFN-g production by splenocytes over a range of doses (2.5 £ 104 – 2.5 £ 105). Interestingly, given the high degree of crossreactivity between life cycle stages, Mf are also capable of inducing IFN-g production in the face of a pre-established Th2 response (following implantation of adult males) [30]. Immunization with Mf extract also elicits IFN-g production and following both immunization with extract or infection with live Mf, CD4þ T cells have been identified as the major source of IFN-g [31,32]. Our studies have shown that the proliferative suppression observed following Mf infection is dependent on the IFN-g-induced production of high levels of nitric oxide (NO) [28]. Infection of IFN-g receptor knockout (IFN-gR-/mice with Mf induces a strong proliferative response, as does the use of inducible nitric oxide synthase (iNOS) inhibitors in vitro [28,33]. iNOS inhibition also increased IFN-g production by splenocytes from Mf-infected mice, consistent with a negative regulatory effect of NO on IFN-g-producing cells. The ability to restore proliferative capacity clearly demonstrates that Ag-specific T cells are present and primed by infection. To assess the balance between cell division and cell death, we used http://parasites.trends.com
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carboxyfluoresein diacetate succinimidyl ester (CFSE) labeling to follow cell division in Ag-stimulated culture and employed the TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labelling) method to investigate levels of T-cell apoptosis [32]. These studies demonstrated that, upon antigenic re-stimulation, CD4þ T cells from Mf-infected mice failed to proliferate and displayed high levels of NO-mediated apoptosis in vitro. This contrasts markedly with the situation seen following infection with L3, where robust proliferation is associated with low levels of T-cell apoptosis [32]. Such findings suggest that IFN-g production leads to macrophage activation and production of NO, which in turn suppresses proliferation by inducing the apoptosis of Ag-reactive T cells. In this way, Ag-specific IFN-g-producing T cells could indirectly limit their own expansion. There is growing evidence that such a negative feedback mechanism acts to eliminate activated T cells in both infectious [34] and autoimmune disease [35] in which pro-inflammatory responses could exacerbate pathology. The ability of Mf to stimulate high levels of IFN-g production and T-cell apoptosis upon re-stimulation in vitro raises several questions: do Mf induce apoptosis in vivo? How and why do Mf uniquely elicit IFN-g production? What is the significance of NO production in filarial infection? What relevance could this have to human infection? Could the proliferative defect observed in most in vitro studies with human PBMC be associated with T-cell apoptosis? Mf infection induces apoptosis in vivo In vivo studies have revealed evidence of a wave of apoptosis in the spleen following Mf infection. Using the TUNEL method in situ, a clustering of apoptotic cells in the follicles of the spleen was observed in Mf-infected mice (Figure 1a and b), whereas in L3-infected and uninfected control animals, only single apoptotic nuclei were seen scattered throughout the spleen (Figure 1c and d). Heightened levels of apoptosis in the spleen persist for up to one month following Mf infection. Intravenous or subcutaneous routes of infection produced identical patterns of apoptosis (J.S. Jenson et al., unpublished). When double-stained with peanut agglutinin, the TUNELpositive cells were found to be largely restricted to the T-cell areas of germinal centers in Mf-infected mice. By contrast, the scattered apoptotic nuclei identified in sections from L3-infected and noninfected mice did not display any organized distribution patterns. Molecules and pathways The observation that apoptosis is specific to infection with Mf might be indicative of the greater susceptibility of Th1 cells to apoptosis, as observed in several previous studies [36,37]. Because infection with L3 induces a Th2-dominated response, activated CD4 þ cells might be protected by IL-4 and IL-10, cytokines that are reported in other systems to have an anti-apoptotic effect via the upregulation of survival molecules such as cellular FLICEinhibitory protein (c-FLIP), B-cell lymphoma-x (bcl-x) [38] or B-cell leukaemia/lymphoma 2 (bcl-2) [39]. By contrast, IFN-g upregulates cell death-inducing caspases
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Figure 1. Infection of BALB/c mice with microfilariae results in apoptosis in vivo. Mice were infected with Mf or L3 of Brugia pahangi or received HBSS. At 12 days post-infection, spleens were fixed in formalin and processed for TUNEL staining, as described by Gavrieli et al. [55]. Following fixation and blocking of endogenous peroxidase activity, sections were incubated with TdT and dUTP-biotin in TdT buffer for 60 min at 37 8C. Negative controls were incubated in TdT buffer alone. The sections were washed and then exposed to HRP-strepavidin, followed by the chromagen 3-amino-9-ethyl-carbazole and counterstained with Mayer’s haematoxylin. TUNEL-positive nuclei are stained red. (a,b) Sections from Mf-infected mice, showing clusters of TUNEL-positive nuclei in the follicles of the spleen. (c) A section from a L3-infected mouse. (d) Control mouse. Scale bar: 100 mm (a,c,d); 50 mm (b). Abbreviations: dUTP, uridine triphosphate; HBSS, Hanks balanced salt solution; L3, third-stage larvae; Mf, microfilariae; TdT, terminal deoxynucleotidyl transferase; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labelling.
elicited apoptosis of human DC and resulted in a diminished capacity to promote CD4þ T-cell production of IFN-g and IL-5 [47]. DC exposed to live Mf upregulate the expression of a variety of pro-inflammatory cytokine messenger RNA (mRNA), although there was no evidence of a role for NO in inducing DC apoptosis. Helminth infections are classically acknowledged as potent inducers of Th2 responses, but the complexity of these infections suggests that strict application of the Th1 –Th2 paradigm is unlikely to adequately describe responses over the course of infection [48]. Indeed, NO production, which is most potently induced by the Th1 cytokine IFN-g, has been described in other helminth infections, most notably S. mansoni, coincident with egg deposition in the tissues [49]. However, while the Th1 to Th2 shift observed following egg laying in murine models of S. mansoni is thought to be regulated at least in part by T-cell apoptosis, there is no evidence that NO drives apoptosis in this system. Rather, NO appears to limit hepatic pathology at the onset of egg laying [49], whereas IL-10 is pro-apoptotic [50]. Most pertinently, it was recently demonstrated that PBMC from S. mansoniinfected individuals are capable of NO production following in vitro re-stimulation with parasite Ag and that iNOS inhibition exacerbated the granulomatous response [51]. Such results are compatible with a regulatory role for NO in human helminth infection and serve to demonstrate an additional way in which oxidative stress might act to limit the extent of inflammatory pathology.
in both lymphoid [40] and non-lymphoid cells [38]. The nature of the Mf Ags which elicit IFN-g production are of great interest, given that other life-cycle stages of filarial worms promote Th2 responses in infected mice. Recent studies have shown that a recombinant serpin, an abundant Mf-specific transcript, induces high levels of IFN-g upon injection into mice [41]. Filarial cystatins have also been independently identified as potent inducers of NO from IFN-g-activated macrophages [42]. The potential immunomodulatory influence of the endosymbiotic bacteria of the Wolbachia species family found within all lifecycle stages of Brugia is an important consideration in this respect, and deserving of further study [43]. Thus, filarial parasites might contain many Ags capable of inducing IFN-g and/or NO, but their effects could be masked by the dominance of Th2-inducing Ags in extracts of whole parasites. It is noteworthy that ES products or soluble Ags derived from a range of helminth parasites have now been shown to elicit apoptosis of various cell types. For example, an ES product of Schistosoma mansoni skinstage schistosomulae promoted T-cell apoptosis via the induction of caspase activity and the upregulation of Fas-L. By biochemical fractionation, the pro-apoptotic activity was shown to be associated with a 23-kDa protein [44]. Similarly, ES products from Nippostrongylus brasiliensis induced apoptosis in a rat epithelial cell line [45], whereas a protein extract from the cattle filarial nematode, Setaria digitata induced apoptosis in Hep-2 cells (a human epithelial cell line), an effect that could be reversed by the overexpression of bcl-2 in these cells [46]. A recent study reported that exposure to live Mf or to Mf ES in vitro
What is the significance of apoptosis in terms of the infection? It is difficult to distinguish whether elevated lymphocyte apoptosis during Mf infection represents an attempt by the host to maintain homeostasis and avoid immunopathology, or whether apoptosis is actively induced by the parasite to subvert the host immune response. It is conceivable that both ends are equally well served by the same means. Following Mf infection in the murine model, re-exposure to Ag in vitro results in the apoptotic death of Ag-responsive CD4þ T cells. Given that Mf compose a large antigenic burden in infected humans, it is conceivable that repeated re-stimulation in vivo might also result in T-cell death. Such a scenario could contribute to the lower precursor frequencies of Ag-specific T cells recovered from microfilaraemic individuals [19]. Interestingly, previous studies in filarial-infected humans suggested that the chronicity of infection leads to the downregulation of Mf-induced Th1 responses and could facilitate the emergence of a Th2 response. In the mouse model, following multiple immunizations with Mf extract and subsequent Mf infection, the Mf-specific IFN-g response was downregulated by IL-10 [31]. At later time points post-Mf infection, the magnitude of the IFN-g response has also been shown to correlate inversely with the initial infective dose of parasites [30]. Intriguingly, our studies have shown that challenge infection with Mf induces a secondary, more extensive wave of apoptosis in the spleen, as observed by in situ TUNEL staining (J.S. Jenson et al., unpublished). In parallel, secondary infection with Mf also significantly decreases the magnitude of the IFN-g response in vitro in
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the absence of a coincident increase in Th2 cytokine production (R.A. O’Connor, et al., unpublished). These data are compatible with the hypothesis that apoptosis deletes a population of pro-inflammatory Ag-specific T cells. They do not, however, directly support a role for Th2 cytokines in driving down IFN-g responses following exposure to live Mf. Could apoptosis of effector T cells underlie the defective immune responses reported in many chronic parasitic infections of humans? One study reported high levels of apoptosis within PBMC of individuals infected with a range of gastrointestinal helminths and a reduction in levels of apoptosis following treatment with anthelmintics [52]. In filarial-infected humans, the Mf-induced apoptosis of T cells could help explain many previous observations, all from in vitro studies, which link the most profound defect in proliferation and IFN-g production to active infection with Mf. Furthermore, the uptake of apoptotic cells by APC (macrophages or dendritic cells) is now recognized to have a profound downregulatory influence on the effector phenotype of the APC by induction of immunosuppressive cytokines such as IL-10 or TGF-b [53]. Ag-specific T cells undergoing apoptosis are also reported to produce significant quantities of IL-10, which, via its activity on APC, can polarize T helper responses to Type 2, resulting in the downregulation of inflammatory responses and peripheral tolerance [54]. Both IL-10 and TGF-b have been implicated in the suppression of T-cell responses and IFN-g production in microfilaraemic individuals [16]. Filarial worms have evolved many different strategies for downregulating potentially pathogenic proinflammatory responses and prolonging the patent phase of infection. Whether the selective apoptosis of Ag-reactive CD4þ T cells secreting IFN-g can be added to the list of mechanisms that contribute to the tolerance and/or immune deviation induced by filarial infection in the human host awaits further study. Acknowledgements This work was supported by grants from the Wellcome Trust and the MRC. R.O’C. was in receipt of a University of Glasgow studentship. Thanks go to Iain MacMillan and the staff of the histopathology laboratory, Department of Veterinary Pathology, for their help in establishing the TUNEL stain and to Tom Nutman (NIH) for access to unpublished data.
References 1 Maizels, R.M. and Lawrence, R.A. (1991) Immunological tolerance: the key feature in human filariasis? Parasitol. Today 7, 271 – 276 2 Michael, E. and Bundy, D.A. (1997) Global mapping of lymphatic filariasis. Parasitol. Today 13, 472 – 477 3 Vanamail, P. et al. (1996) Estimation of the fecund life span of Wuchereria bancrofti in an endemic area. Trans. R. Soc. Trop. Med. Hyg. 90, 119 – 121 4 Lawrence, R.A. and Devaney, E. (2001) Lymphatic filariasis: parallels between the immunology of infection in humans and mice. Parasite Immunol. 23, 353 – 362 5 Maizels, R.M. et al. (2001) Immunological genomics of Brugia malayi: filarial genes implicated in immune evasion and protective immunity. Parasite Immunol. 23, 327 – 344 6 Hawking, F. et al. (1966) The periodicity of microfilariae. XI The effect of body temperature and other stimuli upon the cycles of Wuchereria bancrofti, Brugia malayi, B. ceylonensis and Dirofilaria repens. Trans. R. Soc. Trop. Med. Hyg. 60, 497 – 513 7 Sartono, E. et al. (1999) Reversal in microfilarial density and T cell http://parasites.trends.com
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apoptosis of CD4þ T lymphocytes: a mechanism of immune unresponsiveness in filariasis? Eur. J. Immunol. 32, 858– 867 Allen, J.E. et al. (1996) APC from mice harbouring the filarial parasite, Brugia malayi, prevent cellular proliferation but not cytokine production. Int. Immunol. 8, 143– 151 Dalton, D.K. et al. (2000) Interferon-g eliminates responding CD4 T cells during mycobacterial infection by inducing apoptosis of activated CD4 T cells. J. Exp. Med. 192, 117 – 122 Chu, C-Q. et al. (2000) Failure to suppress the expansion of the activated CD4 T cell population in interferon-g-deficient mice leads to exacerbation of experimental autoimmune encephalomyelitis. J. Exp. Med. 192, 123 – 128 Zhang, X. et al. (1997) Unequal death in T helper cell (Th) 1 and Th2 effectors: Th1, but not Th2, effectors undergo rapid Fas/FasL-mediated apoptosis. J. Exp. Med. 185, 1837 – 1849 Zhang, L. et al. (1996) Characterisation of apoptosis resistant antigen specific T cells in vivo. J. Exp. Med. 183, 2065– 2073 Stassi, G. et al. (2000) Control of target cell survival in thyroid autoimmunity by T helper cytokines via regulation of apoptotic proteins. Nat. Immunol. 1, 483 – 488 Cohen, S.B. et al. (1997) Interleukin-10 rescues T cells from apoptotic cell death: association with an upregulation of Bcl-2. Immunology 92, 1–5 Refaeli, Y. et al. (2002) Interferon-g is required for activation – induced death of T lymphocytes. J. Exp. Med. 196, 999 – 1005 Zang, X. et al. (2000) The serpin secreted by Brugia malayi microfilariae, Bm-SPN-2, elicits strong, but short-lived, immune responses in mice and humans. J. Immunol. 165, 5161– 5169 Hartmann, S. et al. (2002) Cystatins of filarial nematodes up-regulate the nitric oxide production of interferon-gamma-activated murine macrophages. Parasite Immunol. 24, 253– 262 Taylor, M.J. et al. (2000) Inflammatory responses induced by the filarial nematode Brugia malayi are mediated by lipopolysaccharidelike activity from endosymbiotic Wolbachia bacteria. J. Exp. Med. 191, 1429 – 1435
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