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What causes lymphocyte hyporesponsiveness during filarial nematode infection?
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TRENDS in Parasitology
Vol.22 No.3 March 2006
Immunoparasitology series
What causes lymphocyte hyporesponsiveness during filarial nematode infection? William Harnett1 and Margaret M. Harnett2 1 2
Department of Immunology, University of Strathclyde, Glasgow, UK, G4 0NR Division of Immunology, Infection and Inflammation, University of Glasgow, Glasgow, UK, G11 6NT
Filarial nematodes persist in the parasitized host by modulating immune responsiveness. A feature of this that has been observed in a multitude of studies dating back several decades is an inability of lymphocytes to respond appropriately to filarial nematode antigens and, in some cases, to other stimuli. The consistency of this observation, allied to the ease of measurement of lymphocyte hyporesponsiveness, has resulted in many attempts to understand its cause. Lymphocyte hyporesponsiveness Adult filarial nematodes are noted for their longevity, with estimates of the lifespan of Wuchereria bancrofti varying between eight and 15 years [1]. It is generally accepted that the nematodes facilitate their long survival by modulating the human immune system. Consistent with this, numerous defects in immune responsiveness have been observed in studies from several decades ago. Although there are some disparities regarding the nature of these defects, an observation that has been consistently reported is a defective lymphocyte proliferative response – often called lymphocyte hyporesponsiveness. This has been observed with respect to both B cells and T cells, and although in many studies the defect seems to be antigen specific, in others it extends to heterologous antigens. Lymphocyte hyporesponsiveness was first noticed in the 1970s, when it was observed that lymphocytes isolated from infected people failed to respond to filarial nematode antigens [2]. At around the same time, similar observations were made when investigating animal models of filariasis [3]. In this article we review the literature on filarial nematode-induced lymphocyte hyporesponsiveness in an attempt to elucidate its cause. The field is somewhat confusing because it encompasses studies of several different parasites of humans, in addition to various parasites in model systems. Nevertheless, it is apparent that some patterns have emerged that give a degree of insight into how filarial nematodes promote an immunological defect that one assumes is paramount to their survival. Corresponding author: Harnett, W. ([email protected]).
Is lymphocyte hyporesponsiveness due to microfilariae? There has long been a belief that lymphocyte hyporesponsiveness in filarial nematode infection is associated with patency. The term refers to the stage of infection in which the larval microfilaria forms are detected and before which there is usually a period of immunological responsiveness. As early as 1976, Dalesandro [4] observed that in hamsters infected with Acanthocheilonema viteae, the ability to produce antibody- secreting lymphocytes in response to sheep red blood cells was diminished in animals infected for ten weeks (patent) but not in those infected for five weeks (nonpatent). Subsequently, it was shown that A. viteae microfilaraemia was associated with impaired lymphocyte proliferative responses to filarial antigens and to B- and T-cell mitogens [3,5]. The same picture emerged when studying Brugia pahangi in jirds as the model system [6,7]. The initial human study referred to earlier [2] also showed that inhibition of antigen-specific lymphocyte hyporesponsiveness was associated with microfilaraemia. Since these early studies, there have been numerous additional investigations reporting similar findings, leading to the conclusion that microfilariae are the cause of lymphocyte hyporesponsiveness [8]. Infected individuals can harbour millions of these larval forms within their bodies, although this might be predicted to lead more to apoptosis than to hyporesponsiveness. Indeed, lymphocyte apoptosis was observed when B. pahangi microfilariae were injected into mice [8]. However, it has also been shown that the presence of microfilariae can lead to the suppression of splenic antigen-specific lymphocyte responses ex vivo [9], and microfilaria-derived molecules that can inhibit lymphocyte proliferative responses to concanavalin A have been reported [10]. Nevertheless, careful analysis of the data in some studies [3,11–13] suggests that suppression of lymphocyte responses might slightly precede the onset of patency. Furthermore, although chemotherapeutic treatments that lead to elimination of microfilariae can be associated with recovery of T-cell proliferative responses [14], this does not always happen. For example, in human infection with W. bancrofti, it has been observed that a state of hyporesponsiveness still exists two to three years after treatment to remove microfilariae [15]. Thus,
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additional explanations for hyporesponsiveness must be sought. Can other parasite stages induce lymphocyte hyporesponsiveness? The answer to this question is ‘yes’. For example, spleen cells derived from mice exposed to B. pahangi demonstrate considerably reduced proliferative responses to concanavalin A, 12 days postinfection [16], and B. malayi infective larvae can inhibit anti-CD3-dependent T-cell proliferation in the presence of Langerhans cells [17]. In addition, studies in humans in Papua New Guinea indicate that lymphocyte hyporesponsiveness is strongly correlated with the degree of exposure to infective larvae when parasitized individuals are matched for infection level [18]. Interestingly, a high degree of exposure might predispose to hyporesponsiveness to heterologous antigens. Is lymphocyte hyporesponsiveness due to excretory–secretory products? Filarial nematode-derived molecules can be found in the culture fluid of worms maintained in vitro or in the bloodstream of infected humans and animals [19]. In humans, excretory–secretory (ES) products are most readily detected in people in whom microfilariae are present [20], and, indeed, positive correlations between the two can be observed in animal models, even when the ES product being studied is not produced by the microfilariae [21]. However, recent studies in humans indicate that lymphocyte hyporesponsiveness correlates more closely with levels of circulating antigen than with microfilariae [18,22]. Several studies during the late 1970s and early 1980s demonstrated that serum obtained from infected humans or animals could interfere with lymphocyte proliferative responses in vitro [3,23], and Dasgupta et al. [24] later correlated this with the presence of filarial nematode antigen. Furthermore, chemotherapeutic treatments that reduce microfilaria numbers can also reduce circulating antigen levels [25], indicating that this should be taken into account when considering reasons for recovery of proliferative responses in drug-treated individuals. Studies in the 1980s and 1990s showed that in vitro culture products could inhibit lymphocyte proliferation [10,26,27], and in one case at least, this seemed to be the result of direct interaction with the lymphocytes [28]. What is the mechanism of lymphocyte hyporesponsiveness? King et al. [29] noted that the microfilaraemic state in human filarial nematode infection was associated with a diminished parasite-specific lymphocyte precursor frequency. This was observed with respect to both B cells and T cells but did not extend to non-parasite antigens or mitogens. Moreover, Steel et al. [30] observed that children born to microfilaria-positive mothers remained hyporesponsive to microfilaria antigens throughout their lives, raising the possibility that clonal deletion had occurred. These data provide explanations for the lack of antigen-mediated proliferative lymphocyte responses in www.sciencedirect.com
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filariasis but not for the existence of nonspecific defects. Furthermore, some studies indicate that chemotherapy can reverse the defect in antigen-specific responses [31]. Thus, filarial nematode infection promotes a defective antigen-specific lymphocyte response by mechanisms additional to the loss of a responsive pool. Antigen-specific lymphocytes exist but are unresponsive, and, in relation to this, it is of interest that increased surface expression of a molecule called cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) on T lymphocytes has been reported [32,33]. CTLA-4 is a high-affinity receptor for the costimulatory molecule B7 on antigen-presenting cells (APCs). Interaction of B7 with CTLA-4 results in the delivery of inhibitory signals and inactivation of the T cell (Figure 1). It is also possible to provide stimuli in the test tube that can reverse lymphocyte hyporesponsiveness associated with filarial nematode infection. Thus, exposing hyporesponsive lymphocytes to a variety of reagents – cytokines, anti-cytokine antibodies, phorbol esters and inhibitors of inducible nitric oxide synthase – can partially restore the proliferative lymphocyte response [9,34–36]. However, this is not always observed, and contradictory results are particularly prevalent with respect to a suppressive role for the inhibitory cytokine interleukin (IL) 10 [37,38]. Furthermore, the variability in the nature of the stimulus that was effective with individual samples in their study led Sartono et al. [35] to conclude that hyporesponsiveness might reflect more than one downregulatory mechanism. Thus, it is perhaps prudent to observe that a variety of immunologically active molecules could have an adverse impact on lymphocyte proliferation. The source of such molecules could be the lymphocytes themselves [36] but other cells might also contribute. Thus, lymphocyte proliferation can be inhibited by induction of T-regulatory (Treg) cells by filarial nematodes [33] (Figure 1) or by effects that the parasites have on APCs [39,40] (Figure 1). Interestingly, in one study, the inhibition was shown to be dependent on cell–cell contact [41] and, although initially shown with living adult worms, could be replicated with ES products [42]. However, as mentioned, it seems that secreted filarial nematode products can directly inhibit lymphocyte proliferation. The mechanism underlying this has been investigated using ES-62, a phosphorylcholine (PC)containing glycoprotein that was discovered in A. viteae [43] and that, along with other secreted PC-containing molecules, is found in human filarial nematodes [44,45]. ES-62 inhibits the polyclonal activation of murine B cells by mitogenic antibodies directed against surface immunoglobulin (sIg) [28]. Furthermore, the molecule is active in vivo at concentrations found during natural infection [46]. This effect seems to be the result of the PC moiety because it can be mimicked by PC conjugated to proteins such as albumin [28,47], consistent with earlier studies by Lal et al. [48] that had shown that PC-containing extracts of Brugia could inhibit T-cell proliferation. The mechanism of action of ES-62 is shown in Figure 2. ES-62 failed to inhibit sIg-mediated generation of the second messenger inositol trisphosphate (IP3) but it reduced the level of an important downstream enzyme in the activation process,
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Figure 1. Potential mechanisms of filaria-induced hyporesponsiveness. Infection with filarial nematodes can induce T-cell hyporesponsiveness by directly or indirectly (through APCs) suppressing T-cell activation. For example, infection can result in the induction of CTLA-4-expressing Treg cells that are refractory to antigen and can suppress heterologous T-cell activation. Alternatively, the worms can secrete immunomodulatory ES molecules that can directly inhibit key proliferative signalling pathways such as PKC and MAPK in target T cells. Finally, ES products can act on B1 cells to result in the production of IL-10, which inhibits the upregulation of co-stimulatory molecule expression on APCs, resulting in reduced T-cell activation. CD80/86 denotes CD80 and CD86. Abbreviation: TCR, T-cell receptor.
protein kinase C (PKC) within the cells. More-detailed analysis indicated that only certain isoforms of PKC – in particular, those associated with the transduction of proliferative signals, such as PKCa and PKCb – were targeted [49]. Inhibitor studies suggest that the effects on PKC levels are due mainly to stimulation of proteolytic degradation. Interestingly, IL-4 can protect B cells from the PKC degradation induced by ES-62 [49]. ES-62 has also been shown to activate certain molecules associated with the activation process in B cells – specifically, protein tyrosine kinase and mitogen-activated protein kinase (MAPK) signal-transduction elements [50]. This is insufficient to induce proliferation but serves to desensitize the cells to subsequent activation of the phosphoinositide-3kinase and Ras–MAPK pathways and, in collaboration with the effects on PKC isoforms, to proliferation through the antigen receptor. Essentially identical data were obtained when examining the effect of ES-62 on the human Jurkat T-cell line [51]. ES-62 mediates uncoupling of sIg from B-cell signal-transduction pathways by priming for the induction of the tyrosine phosphatase SH2-domain-containing tyrosine phosphatase (SHP)-1 [52]. Specifically, this molecule negatively regulates activation through sIg by dephosphorylating tyrosine residues on accessory molecules that are necessary for the recruitment of Ras–MAPK pathway components. Interestingly, ES-62 does not cause hyporesponsiveness in B1 cells, a population of cells of limited antigen receptor diversity based mainly in the peritoneal cavity. Instead, it www.sciencedirect.com
causes their activation, resulting in the secretion of IL-10 [53], which might contribute to T-cell unresponsiveness (Figure 1). Concluding remarks A summary of the mechanisms of hyporesponsiveness referred to in this article is shown in Table 1. Although there is evidence that filarial nematode infection can result in a reduction in the specific lymphocyte repertoire, there is no doubt that a hyporesponsive lymphocyte population is induced during active infection. Such hyporesponsiveness of lymphocytes might be inducible by products of microfilariae. However, it is likely that all stages in the vertebrate host have the potential to induce this state. ES-62, for example, is not restricted to one stage but is found in both L4 and adult (both male and female) parasites [43]. There are probably three main mechanisms by which a hyporesponsive lymphocyte population could be generated (Figure 1). First, hyporesponsiveness might reflect the cytokine environment; for example, parasite-induced production of IL-10 might inhibit expression of costimulatory molecules on macrophages and dendritic cells, thereby resulting in incomplete T-cell activation. Second, ES products have been shown to have a direct effect on lymphocyte signal-transduction pathways, thereby rendering them hyporesponsive. Finally, filarial nematode infection might result in the induction of a regulatory T-cell population that fails to proliferate in
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Figure 2. Modulation of B-cell antigen receptor signalling by ES-62. Following ligation of the B-cell antigen receptor (BCR), the protein tyrosine kinase Lyn phosphorylates the immunoreceptor tyrosine-activation motifs (ITAMs) on the accessory transducing molecules Ig-a and Ig-b, resulting in the recruitment and activation of the phospholipase C (PLC)g signalling pathway. PLCg activation induces IP3 and diacylglycerol (DAG) generation, resulting in the activation of classical PKC isoforms such as PKCa and PKCb. The binding of the adaptor proteins SH2-containing transforming protein C1 (Shc) and B-cell linker protein (BLNK) to the phosphorylated ITAMs leads to the recruitment of the growth-factor-receptor-bound protein 2 (Grb2)–Sos (son of sevenless) complexes (Grb2 is an adaptor protein that binds to the guanine-nucleotide-exchange factor Sos), which are required for activation of the GTPase Ras. Active Ras initiates the extracellular-signal-regulated kinase (ERK)–MAPK cascade by binding to and activating the serine/threonine kinase Raf. This leads to stimulation of the threonine/tyrosine kinase MAPK kinase (MEK) and the consequent activation and nuclear translocation of the serine/threonine kinase ERK. ES-62–PC signalling disrupts BCR coupling to proliferative signalling by selectively downregulating the expression of key PKC isoforms such as PKCa, in addition to targeting major negative regulatory sites in the control of the ERK–MAPK cascade. Thus, ES-62 signalling promotes the BCR activation of SHP-1 tyrosine phosphatase to prevent initiation of BCR signalling by maintaining the ITAMs in a resting dephosphorylated state; hence, it prevents recruitment of the Shc–Grb2–Sos complexes, which are required to activate the Ras–MAPK and Rac–MAPK cascades. ES-62 signalling also promotes the BCR-mediated recruitment of GTPase-activating protein (GAP) to terminate ongoing Ras signals. In addition, ES-62 is likely to target MAPK activation by downregulating PKC expression. Finally, ES-62-signalling promotes the BCR-driven association of the nuclear MAPK dual (threonine/tyrosine) phosphatase Pac-1 with ERK to terminate any ongoing ERK signals. This multipronged mechanism results in a rapid and profound desensitization of BCR coupling to the MAPK cascades. Co-stimulation with IL-4 can rescue B cells from hyporesponsiveness, at least in part, by preventing the downregulation of PKCa expression. Abbreviation: IL-4R, IL-4 receptor.
response to antigen and also prevents the proliferation of other T cells. It can be concluded, therefore, that the hyporesponsiveness of lymphocytes during filarial nematode infection might be a consequence of more than one parasite stage, several parasite-derived molecules and several different induction mechanisms. What remains to be established is the relative importance of each factor and the degree of redundancy in operation with respect to the latter. A final point here is that, in addition to furthering the understanding of lymphocyte hyporesponsiveness from the viewpoint of parasite survival, such elucidation might be of relevance to filaria-induced pathogenesis. It has always been assumed that the induction of lymphocyte
hyporesponsiveness is an adaptation by the parasite to facilitate its survival. However, there has been recent interest in the idea that by reducing the potential for potentially damaging (to the host) inflammation, such hyporesponsiveness might contribute to the maintenance of host health. Indeed, it has been suggested that understanding the mechanism by which filarial nematodes promote an anti-inflammatory immunological phenotype, as manifested by alterations such as lymphocyte hyporesponsiveness, might ultimately lead to the development of novel strategies for treating human inflammatory diseases. Proof of principle for this exciting idea is shown by the recent observation that ES-62 can ameliorate arthritis in a murine model [54].
Table 1. Mechanisms to explain lymphocyte hyporesponsiveness in filarial nematode infection Mechanism Decreased parasite-specific lymphocytes Apoptosis induced by microfilariae Effects on APCs Induction of anti-inflammatory cytokines Upregulation of CTLA-4 Induction of Treg cells Modulation of signal-transduction pathways www.sciencedirect.com
Study Wuchereria bancrofti (human) Brugia pahangi (mouse) Brugia malayi (mouse) W. bancrofti (human) B. pahangi (mouse) W. bancrofti (human) Onchocerca volvulus (human) Litomosoides sigmodontis (mouse) ES-62 (mouse)
Refs [29] [8] [40] [37] [39] [32] [36] [33] [49–52]
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References 1 Subramanian, S. et al. (2004) The dynamics of Wuchereria bancrofti infection: a model-based analysis of longitudinal data from Pondicherry, India. Parasitology 128, 467–482 2 Ottesen, E.A. et al. (1977) Specific cellular immune unresponsiveness in human filariasis. Immunology 33, 413–421 3 Weiss, N. (1978) Dipetalonema viteae: in vitro blastogenesis of hamster spleen and lymph node cells to phytohemagglutinin and filarial antigens. Exp. Parasitol. 46, 283–299 4 Dalesandro, D.A. (1977) Evidence for immunodepression of Syrian hamsters and Mongolian jirds by Dipetalonema viteae infections. Trans. R. Soc. Trop. Med. Hyg. 70, 534–535 5 Weller, P.F. (1978) Cell-mediated immunity in experimental filariasis: lymphocyte reactivity to filarial stage-specific antigens and to B- and T-cell mitogens during acute and chronic infection. Cell. Immunol. 37, 369–382 6 Lammie, P.J. and Katz, S.P. (1983) Immunoregulation in experimental filariasis. I. In vitro suppression of mitogen-induced blastogenesis by adherent cells from Jirds chronically infected with Brugia pahangi. J. Immunol. 130, 1381–1385 7 Lammie, P.J. and Katz, S.P. (1983) Immunoregulation in experimental filariasis. II. Responses to parasite and nonparasite antigens in jirds with Brugia pahangi. J. Immunol. 130, 1386–1389 8 O’Connor, R.A. et al. (2003) An enduring association? Microfilariae and immunosuppression in lymphatic filariasis. Trends Parasitol. 19, 565–570 9 O’Connor, R.A. et al. (2000) NO contributes to proliferative suppression in a murine model of filariasis. Infect. Immun. 68, 6101–6107 10 Wadee, A.A. et al. (1987) Characterization of immunosuppressive proteins of Brugia malayi microfilariae. Acta Trop. 44, 343–352 11 Soboslay, P.T. et al. (1991) Experimental onchocerciasis in chimpanzees. Cell-mediated immune responses, and production and effects of IL-1 and IL-2 with Onchocerca volvulus infection. J. Immunol. 147, 346–353 12 Ghosh, R.P. et al. (1999) Longitudinal cellular immune responses in asymptomatic and symptomatic Brugia malayi-infected Indian leaf monkey Presbytis entellus. J. Parasitol. 85, 861–866 13 Graham, S.P. et al. (2001) Down-regulated lymphoproliferation coincides with parasite maturation and with the collapse of both gamma interferon and interleukin-4 responses in a bovine model of onchocerciasis. Infect. Immun. 69, 4313–4319 14 Mahanty, S. and Nutman, T.B. (1995) Immunoregulation in human lymphatic filariasis: the role of interleukin 10. Parasite Immunol. 17, 385–392 15 Gopinath, R. et al. (1999) Long-term persistence of cellular hyporesponsiveness to filarial antigens after clearance of microfilaremia. Am. J. Trop. Med. Hyg. 60, 848–853 16 Osborne, J. et al. (1996) Anti-interleukin-4 modulation of the Th2 polarized response to the parasitic nematode Brugia pahangi. Infect. Immun. 64, 3461–3466 17 Semnani, R.T. et al. (2004) Filaria-induced immune evasion: suppression by the infective stage of Brugia malayi at the earliest host-parasite interface. J. Immunol. 172, 6229–6238 18 King, C.L. (2001) Transmission intensity and human immune responses to lymphatic filariasis. Parasite Immunol. 23, 363–371 19 Harnett, W. and Parkhouse, R.M.E. (1995) Structure and function of nematode surface and excretory-secretory products. In Perspectives in Nematode Physiology and Biochemistry (Sood, S.M., ed.), pp. 207–242, M/S Narendra Publication House 20 Weil, G.J. (1990) Parasite antigenemia in lymphatic filariasis. Exp. Parasitol. 71, 353–356 21 Harnett, W. et al. (1990) Association between circulating antigen and parasite load in a model filarial system, Acanthocheilonema viteae in jirds. Parasitology 101, 435–444 22 Dimock, K.A. et al. (1996) Th1-Like antifilarial immune-responses predominate in antigen- negative persons. Infect. Immun. 64, 2962–2967 23 Piessens, W.F. et al. (1980) Antigen-specific suppressor cells and suppressor factors in human filariasis with Brugia malayi. N. Engl. J. Med. 302, 833–837 24 Dasgupta, A. et al. (1987) Cellular unresponsiveness in patients with soluble circulating antigens in bancroftian filariasis. Indian J. Med. Res. 85, 136–139 www.sciencedirect.com
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25 Nicolas, L. et al. (1997) Reduction of Wuchereria bancrofti adult worm circulating antigen after annual treatments of diethylcarbamazine combined with ivermectin in French Polynesia. J. Infect. Dis. 175, 489–492 26 Elkhalifa, M.Y. et al. (1991) Suppression of human lymphocyte responses to specific and non-specific stimuli in human onchocerciasis. Clin. Exp. Immunol. 86, 433–439 27 Hartmann, S. et al. (1997) A filarial cysteine protease inhibitor downregulates T cell proliferation and enhances interleukin-10 production. Eur. J. Immunol. 27, 2253–2260 28 Harnett, W. and Harnett, M.M. (1993) Inhibition of murine B cell proliferation and down-regulation of protein kinase C levels by a phosphorylcholine-containing filarial excretory-secretory product. J. Immunol. 151, 4829–4837 29 King, C.L. et al. (1992) Immunologic tolerance in lymphatic filariasis. Diminished parasite-specific T and B lymphocyte precursor frequency in the microfilaremic state. J. Clin. Invest. 89, 1403–1410 30 Steel, C. et al. (1994) Long-term effect of prenatal exposure to maternal microfilaraemia on immune responsiveness to filarial parasite antigens. Lancet 343, 890–893 31 Piessens, W.F. et al. (1981) Effect of treatment with diethylcarbamazine on immune responses to filarial antigens in patients infected with Brugia malayi. Acta Trop. 38, 227–234 32 Steel, C. and Nutman, T.B. (2003) CTLA-4 in filarial infections: implications for a role in diminished T cell reactivity. J. Immunol. 170, 1930–1938 33 Taylor, M.D. et al. (2005) Removal of regulatory T cell activity reverses hyporesponsiveness and leads to filarial parasite clearance in vivo. J. Immunol. 174, 4924–4933 34 King, C.L. et al. (1993) Cytokine control of parasite-specific anergy in human lymphatic filariasis. Preferential induction of a regulatory T helper type 2 lymphocyte subset. J. Clin. Invest. 92, 1667–1673 35 Sartono, E. et al. (1995) Specific T cell unresponsiveness in human filariasis: diversity in underlying mechanisms. Parasite Immunol. 17, 587–594 36 Doetze, A. et al. (2000) Antigen-specific cellular hyporesponsiveness in a chronic human helminth infection is mediated by T(h)3/T(r)1-type cytokines IL-10 and transforming growth factor-beta but not by a T(h)1 to T(h)2 shift. Int. Immunol. 12, 623–630 37 Mahanty, S. and Nutman, T.B. (1995) Immunoregulation in human lymphatic filariasis – the role of interleukin-10. Parasite Immunol. 17, 385–392 38 Soboslay, P.T. et al. (1999) Regulatory effects of Th1-type (IFNgamma, IL-12) and Th2-type cytokines (IL-10, IL-13) on parasite-specific cellular responsiveness in Onchocerca volvulusinfected humans and exposed endemic controls. Immunology 97, 219–225 39 Osborne, J. and Devaney, E. (1999) Interleukin-10 and antigenpresenting cells actively suppress Th1 cells in BALB/c mice infected with the filarial parasite Brugia pahangi. Infect. Immun. 67, 1599–1605 40 Allen, J.E. et al. (1996) APC from mice harbouring the filarial nematode, Brugia malayi, prevent cellular proliferation but not cytokine production. Int. Immunol. 8, 143–151 41 Loke, P. et al. (2000) Alternatively activated macrophages induced by nematode infection inhibit proliferation via cell-to-cell contact. Eur. J. Immunol. 30, 2669–2678 42 Allen, J.E. and MacDonald, A.S. (1998) Profound suppression of cellular proliferation mediated by the secretions of nematodes. Parasite Immunol. 20, 241–247 43 Harnett, W. et al. (1989) Origin, kinetics of circulation and fate in vivo of the major excretory-secretory product of Acanthocheilonema viteae. Parasitology 99, 229–239 44 Weil, G.J. and Liftis, F. (1987) Identification and partial characterization of a parasite antigen in sera from humans infected with Wuchereria bancrofti. J. Immunol. 138, 3035–3041 45 Stepek, G. et al. (2004) Stage-specific and species-specific differences in the production of the mRNA and protein for the filarial nematode secreted product, ES-62. Parasitology 128, 91–98
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46 Wilson, E.H. et al. (2003) Hyporesponsiveness of murine B lymphocytes exposed to the filarial nematode secreted product ES-62 in vivo. Immunology 109, 238–245 47 Harnett, W. et al. (1999) Immunomodulatory properties of a phosphorylcholine-containing secreted filarial glycoprotein. Parasite Immunol. 21, 601–608 48 Lal, R.B. et al. (1990) Phosphorylcholine-containing antigens of Brugia malayi non-specifically suppress lymphocyte function. Am. J. Trop. Med. Hyg. 42, 56–64 49 Deehan, M. et al. (1997) A filarial nematode secreted product differentially modulates expression and activation of protein kinase C isoforms in B lymphocytes. J. Immunol. 159, 6105–6111 50 Deehan, M.R. et al. (1998) A phosphorylcholine-containing filarial nematode-secreted product disrupts B lymphocyte activation by targeting key proliferative signaling pathways. J. Immunol. 160, 2692–2699
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51 Harnett, M.M. et al. (1998) Induction of signalling anergy via the T-cell receptor in cultured Jurkat T cells by pre-exposure to a filarial nematode secreted product. Parasite Immunol. 20, 551–563 52 Deehan, M.R. et al. (2001) A filarial nematode-secreted phosphorylcholine-containing glycoprotein uncouples the B cell antigen receptor from extracellular signal-regulated kinase-mitogen-activated protein kinase by promoting the surface Ig-mediated recruitment of Src homology 2 domain-containing tyrosine phosphatase-1 and Pac-1 mitogen-activated kinase-phosphatase. J. Immunol. 166, 7462–7468 53 Wilson, E.H. et al. (2003) In vivo activation of murine peritoneal B1 cells by the filarial nematode phosphorylcholine-containing glycoprotein ES-62. Parasite Immunol. 25, 463–466 54 McInnes, I.B. et al. (2003) A novel therapeutic approach targeting articular inflammation using the filarial nematode-derived phosphorylcholine-containing glycoprotein ES-62. J. Immunol. 171, 2127–2133
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Immunoparasitology series
What causes lymphocyte hyporesponsiveness during filarial nematode infection? William Harnett1 and Margaret M. Harnett2 1 2
Department of Immunology, University of Strathclyde, Glasgow, UK, G4 0NR Division of Immunology, Infection and Inflammation, University of Glasgow, Glasgow, UK, G11 6NT
Filarial nematodes persist in the parasitized host by modulating immune responsiveness. A feature of this that has been observed in a multitude of studies dating back several decades is an inability of lymphocytes to respond appropriately to filarial nematode antigens and, in some cases, to other stimuli. The consistency of this observation, allied to the ease of measurement of lymphocyte hyporesponsiveness, has resulted in many attempts to understand its cause. Lymphocyte hyporesponsiveness Adult filarial nematodes are noted for their longevity, with estimates of the lifespan of Wuchereria bancrofti varying between eight and 15 years [1]. It is generally accepted that the nematodes facilitate their long survival by modulating the human immune system. Consistent with this, numerous defects in immune responsiveness have been observed in studies from several decades ago. Although there are some disparities regarding the nature of these defects, an observation that has been consistently reported is a defective lymphocyte proliferative response – often called lymphocyte hyporesponsiveness. This has been observed with respect to both B cells and T cells, and although in many studies the defect seems to be antigen specific, in others it extends to heterologous antigens. Lymphocyte hyporesponsiveness was first noticed in the 1970s, when it was observed that lymphocytes isolated from infected people failed to respond to filarial nematode antigens [2]. At around the same time, similar observations were made when investigating animal models of filariasis [3]. In this article we review the literature on filarial nematode-induced lymphocyte hyporesponsiveness in an attempt to elucidate its cause. The field is somewhat confusing because it encompasses studies of several different parasites of humans, in addition to various parasites in model systems. Nevertheless, it is apparent that some patterns have emerged that give a degree of insight into how filarial nematodes promote an immunological defect that one assumes is paramount to their survival. Corresponding author: Harnett, W. ([email protected]).
Is lymphocyte hyporesponsiveness due to microfilariae? There has long been a belief that lymphocyte hyporesponsiveness in filarial nematode infection is associated with patency. The term refers to the stage of infection in which the larval microfilaria forms are detected and before which there is usually a period of immunological responsiveness. As early as 1976, Dalesandro [4] observed that in hamsters infected with Acanthocheilonema viteae, the ability to produce antibody- secreting lymphocytes in response to sheep red blood cells was diminished in animals infected for ten weeks (patent) but not in those infected for five weeks (nonpatent). Subsequently, it was shown that A. viteae microfilaraemia was associated with impaired lymphocyte proliferative responses to filarial antigens and to B- and T-cell mitogens [3,5]. The same picture emerged when studying Brugia pahangi in jirds as the model system [6,7]. The initial human study referred to earlier [2] also showed that inhibition of antigen-specific lymphocyte hyporesponsiveness was associated with microfilaraemia. Since these early studies, there have been numerous additional investigations reporting similar findings, leading to the conclusion that microfilariae are the cause of lymphocyte hyporesponsiveness [8]. Infected individuals can harbour millions of these larval forms within their bodies, although this might be predicted to lead more to apoptosis than to hyporesponsiveness. Indeed, lymphocyte apoptosis was observed when B. pahangi microfilariae were injected into mice [8]. However, it has also been shown that the presence of microfilariae can lead to the suppression of splenic antigen-specific lymphocyte responses ex vivo [9], and microfilaria-derived molecules that can inhibit lymphocyte proliferative responses to concanavalin A have been reported [10]. Nevertheless, careful analysis of the data in some studies [3,11–13] suggests that suppression of lymphocyte responses might slightly precede the onset of patency. Furthermore, although chemotherapeutic treatments that lead to elimination of microfilariae can be associated with recovery of T-cell proliferative responses [14], this does not always happen. For example, in human infection with W. bancrofti, it has been observed that a state of hyporesponsiveness still exists two to three years after treatment to remove microfilariae [15]. Thus,
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additional explanations for hyporesponsiveness must be sought. Can other parasite stages induce lymphocyte hyporesponsiveness? The answer to this question is ‘yes’. For example, spleen cells derived from mice exposed to B. pahangi demonstrate considerably reduced proliferative responses to concanavalin A, 12 days postinfection [16], and B. malayi infective larvae can inhibit anti-CD3-dependent T-cell proliferation in the presence of Langerhans cells [17]. In addition, studies in humans in Papua New Guinea indicate that lymphocyte hyporesponsiveness is strongly correlated with the degree of exposure to infective larvae when parasitized individuals are matched for infection level [18]. Interestingly, a high degree of exposure might predispose to hyporesponsiveness to heterologous antigens. Is lymphocyte hyporesponsiveness due to excretory–secretory products? Filarial nematode-derived molecules can be found in the culture fluid of worms maintained in vitro or in the bloodstream of infected humans and animals [19]. In humans, excretory–secretory (ES) products are most readily detected in people in whom microfilariae are present [20], and, indeed, positive correlations between the two can be observed in animal models, even when the ES product being studied is not produced by the microfilariae [21]. However, recent studies in humans indicate that lymphocyte hyporesponsiveness correlates more closely with levels of circulating antigen than with microfilariae [18,22]. Several studies during the late 1970s and early 1980s demonstrated that serum obtained from infected humans or animals could interfere with lymphocyte proliferative responses in vitro [3,23], and Dasgupta et al. [24] later correlated this with the presence of filarial nematode antigen. Furthermore, chemotherapeutic treatments that reduce microfilaria numbers can also reduce circulating antigen levels [25], indicating that this should be taken into account when considering reasons for recovery of proliferative responses in drug-treated individuals. Studies in the 1980s and 1990s showed that in vitro culture products could inhibit lymphocyte proliferation [10,26,27], and in one case at least, this seemed to be the result of direct interaction with the lymphocytes [28]. What is the mechanism of lymphocyte hyporesponsiveness? King et al. [29] noted that the microfilaraemic state in human filarial nematode infection was associated with a diminished parasite-specific lymphocyte precursor frequency. This was observed with respect to both B cells and T cells but did not extend to non-parasite antigens or mitogens. Moreover, Steel et al. [30] observed that children born to microfilaria-positive mothers remained hyporesponsive to microfilaria antigens throughout their lives, raising the possibility that clonal deletion had occurred. These data provide explanations for the lack of antigen-mediated proliferative lymphocyte responses in www.sciencedirect.com
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filariasis but not for the existence of nonspecific defects. Furthermore, some studies indicate that chemotherapy can reverse the defect in antigen-specific responses [31]. Thus, filarial nematode infection promotes a defective antigen-specific lymphocyte response by mechanisms additional to the loss of a responsive pool. Antigen-specific lymphocytes exist but are unresponsive, and, in relation to this, it is of interest that increased surface expression of a molecule called cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) on T lymphocytes has been reported [32,33]. CTLA-4 is a high-affinity receptor for the costimulatory molecule B7 on antigen-presenting cells (APCs). Interaction of B7 with CTLA-4 results in the delivery of inhibitory signals and inactivation of the T cell (Figure 1). It is also possible to provide stimuli in the test tube that can reverse lymphocyte hyporesponsiveness associated with filarial nematode infection. Thus, exposing hyporesponsive lymphocytes to a variety of reagents – cytokines, anti-cytokine antibodies, phorbol esters and inhibitors of inducible nitric oxide synthase – can partially restore the proliferative lymphocyte response [9,34–36]. However, this is not always observed, and contradictory results are particularly prevalent with respect to a suppressive role for the inhibitory cytokine interleukin (IL) 10 [37,38]. Furthermore, the variability in the nature of the stimulus that was effective with individual samples in their study led Sartono et al. [35] to conclude that hyporesponsiveness might reflect more than one downregulatory mechanism. Thus, it is perhaps prudent to observe that a variety of immunologically active molecules could have an adverse impact on lymphocyte proliferation. The source of such molecules could be the lymphocytes themselves [36] but other cells might also contribute. Thus, lymphocyte proliferation can be inhibited by induction of T-regulatory (Treg) cells by filarial nematodes [33] (Figure 1) or by effects that the parasites have on APCs [39,40] (Figure 1). Interestingly, in one study, the inhibition was shown to be dependent on cell–cell contact [41] and, although initially shown with living adult worms, could be replicated with ES products [42]. However, as mentioned, it seems that secreted filarial nematode products can directly inhibit lymphocyte proliferation. The mechanism underlying this has been investigated using ES-62, a phosphorylcholine (PC)containing glycoprotein that was discovered in A. viteae [43] and that, along with other secreted PC-containing molecules, is found in human filarial nematodes [44,45]. ES-62 inhibits the polyclonal activation of murine B cells by mitogenic antibodies directed against surface immunoglobulin (sIg) [28]. Furthermore, the molecule is active in vivo at concentrations found during natural infection [46]. This effect seems to be the result of the PC moiety because it can be mimicked by PC conjugated to proteins such as albumin [28,47], consistent with earlier studies by Lal et al. [48] that had shown that PC-containing extracts of Brugia could inhibit T-cell proliferation. The mechanism of action of ES-62 is shown in Figure 2. ES-62 failed to inhibit sIg-mediated generation of the second messenger inositol trisphosphate (IP3) but it reduced the level of an important downstream enzyme in the activation process,
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Figure 1. Potential mechanisms of filaria-induced hyporesponsiveness. Infection with filarial nematodes can induce T-cell hyporesponsiveness by directly or indirectly (through APCs) suppressing T-cell activation. For example, infection can result in the induction of CTLA-4-expressing Treg cells that are refractory to antigen and can suppress heterologous T-cell activation. Alternatively, the worms can secrete immunomodulatory ES molecules that can directly inhibit key proliferative signalling pathways such as PKC and MAPK in target T cells. Finally, ES products can act on B1 cells to result in the production of IL-10, which inhibits the upregulation of co-stimulatory molecule expression on APCs, resulting in reduced T-cell activation. CD80/86 denotes CD80 and CD86. Abbreviation: TCR, T-cell receptor.
protein kinase C (PKC) within the cells. More-detailed analysis indicated that only certain isoforms of PKC – in particular, those associated with the transduction of proliferative signals, such as PKCa and PKCb – were targeted [49]. Inhibitor studies suggest that the effects on PKC levels are due mainly to stimulation of proteolytic degradation. Interestingly, IL-4 can protect B cells from the PKC degradation induced by ES-62 [49]. ES-62 has also been shown to activate certain molecules associated with the activation process in B cells – specifically, protein tyrosine kinase and mitogen-activated protein kinase (MAPK) signal-transduction elements [50]. This is insufficient to induce proliferation but serves to desensitize the cells to subsequent activation of the phosphoinositide-3kinase and Ras–MAPK pathways and, in collaboration with the effects on PKC isoforms, to proliferation through the antigen receptor. Essentially identical data were obtained when examining the effect of ES-62 on the human Jurkat T-cell line [51]. ES-62 mediates uncoupling of sIg from B-cell signal-transduction pathways by priming for the induction of the tyrosine phosphatase SH2-domain-containing tyrosine phosphatase (SHP)-1 [52]. Specifically, this molecule negatively regulates activation through sIg by dephosphorylating tyrosine residues on accessory molecules that are necessary for the recruitment of Ras–MAPK pathway components. Interestingly, ES-62 does not cause hyporesponsiveness in B1 cells, a population of cells of limited antigen receptor diversity based mainly in the peritoneal cavity. Instead, it www.sciencedirect.com
causes their activation, resulting in the secretion of IL-10 [53], which might contribute to T-cell unresponsiveness (Figure 1). Concluding remarks A summary of the mechanisms of hyporesponsiveness referred to in this article is shown in Table 1. Although there is evidence that filarial nematode infection can result in a reduction in the specific lymphocyte repertoire, there is no doubt that a hyporesponsive lymphocyte population is induced during active infection. Such hyporesponsiveness of lymphocytes might be inducible by products of microfilariae. However, it is likely that all stages in the vertebrate host have the potential to induce this state. ES-62, for example, is not restricted to one stage but is found in both L4 and adult (both male and female) parasites [43]. There are probably three main mechanisms by which a hyporesponsive lymphocyte population could be generated (Figure 1). First, hyporesponsiveness might reflect the cytokine environment; for example, parasite-induced production of IL-10 might inhibit expression of costimulatory molecules on macrophages and dendritic cells, thereby resulting in incomplete T-cell activation. Second, ES products have been shown to have a direct effect on lymphocyte signal-transduction pathways, thereby rendering them hyporesponsive. Finally, filarial nematode infection might result in the induction of a regulatory T-cell population that fails to proliferate in
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Figure 2. Modulation of B-cell antigen receptor signalling by ES-62. Following ligation of the B-cell antigen receptor (BCR), the protein tyrosine kinase Lyn phosphorylates the immunoreceptor tyrosine-activation motifs (ITAMs) on the accessory transducing molecules Ig-a and Ig-b, resulting in the recruitment and activation of the phospholipase C (PLC)g signalling pathway. PLCg activation induces IP3 and diacylglycerol (DAG) generation, resulting in the activation of classical PKC isoforms such as PKCa and PKCb. The binding of the adaptor proteins SH2-containing transforming protein C1 (Shc) and B-cell linker protein (BLNK) to the phosphorylated ITAMs leads to the recruitment of the growth-factor-receptor-bound protein 2 (Grb2)–Sos (son of sevenless) complexes (Grb2 is an adaptor protein that binds to the guanine-nucleotide-exchange factor Sos), which are required for activation of the GTPase Ras. Active Ras initiates the extracellular-signal-regulated kinase (ERK)–MAPK cascade by binding to and activating the serine/threonine kinase Raf. This leads to stimulation of the threonine/tyrosine kinase MAPK kinase (MEK) and the consequent activation and nuclear translocation of the serine/threonine kinase ERK. ES-62–PC signalling disrupts BCR coupling to proliferative signalling by selectively downregulating the expression of key PKC isoforms such as PKCa, in addition to targeting major negative regulatory sites in the control of the ERK–MAPK cascade. Thus, ES-62 signalling promotes the BCR activation of SHP-1 tyrosine phosphatase to prevent initiation of BCR signalling by maintaining the ITAMs in a resting dephosphorylated state; hence, it prevents recruitment of the Shc–Grb2–Sos complexes, which are required to activate the Ras–MAPK and Rac–MAPK cascades. ES-62 signalling also promotes the BCR-mediated recruitment of GTPase-activating protein (GAP) to terminate ongoing Ras signals. In addition, ES-62 is likely to target MAPK activation by downregulating PKC expression. Finally, ES-62-signalling promotes the BCR-driven association of the nuclear MAPK dual (threonine/tyrosine) phosphatase Pac-1 with ERK to terminate any ongoing ERK signals. This multipronged mechanism results in a rapid and profound desensitization of BCR coupling to the MAPK cascades. Co-stimulation with IL-4 can rescue B cells from hyporesponsiveness, at least in part, by preventing the downregulation of PKCa expression. Abbreviation: IL-4R, IL-4 receptor.
response to antigen and also prevents the proliferation of other T cells. It can be concluded, therefore, that the hyporesponsiveness of lymphocytes during filarial nematode infection might be a consequence of more than one parasite stage, several parasite-derived molecules and several different induction mechanisms. What remains to be established is the relative importance of each factor and the degree of redundancy in operation with respect to the latter. A final point here is that, in addition to furthering the understanding of lymphocyte hyporesponsiveness from the viewpoint of parasite survival, such elucidation might be of relevance to filaria-induced pathogenesis. It has always been assumed that the induction of lymphocyte
hyporesponsiveness is an adaptation by the parasite to facilitate its survival. However, there has been recent interest in the idea that by reducing the potential for potentially damaging (to the host) inflammation, such hyporesponsiveness might contribute to the maintenance of host health. Indeed, it has been suggested that understanding the mechanism by which filarial nematodes promote an anti-inflammatory immunological phenotype, as manifested by alterations such as lymphocyte hyporesponsiveness, might ultimately lead to the development of novel strategies for treating human inflammatory diseases. Proof of principle for this exciting idea is shown by the recent observation that ES-62 can ameliorate arthritis in a murine model [54].
Table 1. Mechanisms to explain lymphocyte hyporesponsiveness in filarial nematode infection Mechanism Decreased parasite-specific lymphocytes Apoptosis induced by microfilariae Effects on APCs Induction of anti-inflammatory cytokines Upregulation of CTLA-4 Induction of Treg cells Modulation of signal-transduction pathways www.sciencedirect.com
Study Wuchereria bancrofti (human) Brugia pahangi (mouse) Brugia malayi (mouse) W. bancrofti (human) B. pahangi (mouse) W. bancrofti (human) Onchocerca volvulus (human) Litomosoides sigmodontis (mouse) ES-62 (mouse)
Refs [29] [8] [40] [37] [39] [32] [36] [33] [49–52]
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25 Nicolas, L. et al. (1997) Reduction of Wuchereria bancrofti adult worm circulating antigen after annual treatments of diethylcarbamazine combined with ivermectin in French Polynesia. J. Infect. Dis. 175, 489–492 26 Elkhalifa, M.Y. et al. (1991) Suppression of human lymphocyte responses to specific and non-specific stimuli in human onchocerciasis. Clin. Exp. Immunol. 86, 433–439 27 Hartmann, S. et al. (1997) A filarial cysteine protease inhibitor downregulates T cell proliferation and enhances interleukin-10 production. Eur. J. Immunol. 27, 2253–2260 28 Harnett, W. and Harnett, M.M. (1993) Inhibition of murine B cell proliferation and down-regulation of protein kinase C levels by a phosphorylcholine-containing filarial excretory-secretory product. J. Immunol. 151, 4829–4837 29 King, C.L. et al. (1992) Immunologic tolerance in lymphatic filariasis. Diminished parasite-specific T and B lymphocyte precursor frequency in the microfilaremic state. J. Clin. Invest. 89, 1403–1410 30 Steel, C. et al. (1994) Long-term effect of prenatal exposure to maternal microfilaraemia on immune responsiveness to filarial parasite antigens. Lancet 343, 890–893 31 Piessens, W.F. et al. (1981) Effect of treatment with diethylcarbamazine on immune responses to filarial antigens in patients infected with Brugia malayi. Acta Trop. 38, 227–234 32 Steel, C. and Nutman, T.B. (2003) CTLA-4 in filarial infections: implications for a role in diminished T cell reactivity. J. Immunol. 170, 1930–1938 33 Taylor, M.D. et al. (2005) Removal of regulatory T cell activity reverses hyporesponsiveness and leads to filarial parasite clearance in vivo. J. Immunol. 174, 4924–4933 34 King, C.L. et al. (1993) Cytokine control of parasite-specific anergy in human lymphatic filariasis. Preferential induction of a regulatory T helper type 2 lymphocyte subset. J. Clin. Invest. 92, 1667–1673 35 Sartono, E. et al. (1995) Specific T cell unresponsiveness in human filariasis: diversity in underlying mechanisms. Parasite Immunol. 17, 587–594 36 Doetze, A. et al. (2000) Antigen-specific cellular hyporesponsiveness in a chronic human helminth infection is mediated by T(h)3/T(r)1-type cytokines IL-10 and transforming growth factor-beta but not by a T(h)1 to T(h)2 shift. Int. Immunol. 12, 623–630 37 Mahanty, S. and Nutman, T.B. (1995) Immunoregulation in human lymphatic filariasis – the role of interleukin-10. Parasite Immunol. 17, 385–392 38 Soboslay, P.T. et al. (1999) Regulatory effects of Th1-type (IFNgamma, IL-12) and Th2-type cytokines (IL-10, IL-13) on parasite-specific cellular responsiveness in Onchocerca volvulusinfected humans and exposed endemic controls. Immunology 97, 219–225 39 Osborne, J. and Devaney, E. (1999) Interleukin-10 and antigenpresenting cells actively suppress Th1 cells in BALB/c mice infected with the filarial parasite Brugia pahangi. Infect. Immun. 67, 1599–1605 40 Allen, J.E. et al. (1996) APC from mice harbouring the filarial nematode, Brugia malayi, prevent cellular proliferation but not cytokine production. Int. Immunol. 8, 143–151 41 Loke, P. et al. (2000) Alternatively activated macrophages induced by nematode infection inhibit proliferation via cell-to-cell contact. Eur. J. Immunol. 30, 2669–2678 42 Allen, J.E. and MacDonald, A.S. (1998) Profound suppression of cellular proliferation mediated by the secretions of nematodes. Parasite Immunol. 20, 241–247 43 Harnett, W. et al. (1989) Origin, kinetics of circulation and fate in vivo of the major excretory-secretory product of Acanthocheilonema viteae. Parasitology 99, 229–239 44 Weil, G.J. and Liftis, F. (1987) Identification and partial characterization of a parasite antigen in sera from humans infected with Wuchereria bancrofti. J. Immunol. 138, 3035–3041 45 Stepek, G. et al. (2004) Stage-specific and species-specific differences in the production of the mRNA and protein for the filarial nematode secreted product, ES-62. Parasitology 128, 91–98
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46 Wilson, E.H. et al. (2003) Hyporesponsiveness of murine B lymphocytes exposed to the filarial nematode secreted product ES-62 in vivo. Immunology 109, 238–245 47 Harnett, W. et al. (1999) Immunomodulatory properties of a phosphorylcholine-containing secreted filarial glycoprotein. Parasite Immunol. 21, 601–608 48 Lal, R.B. et al. (1990) Phosphorylcholine-containing antigens of Brugia malayi non-specifically suppress lymphocyte function. Am. J. Trop. Med. Hyg. 42, 56–64 49 Deehan, M. et al. (1997) A filarial nematode secreted product differentially modulates expression and activation of protein kinase C isoforms in B lymphocytes. J. Immunol. 159, 6105–6111 50 Deehan, M.R. et al. (1998) A phosphorylcholine-containing filarial nematode-secreted product disrupts B lymphocyte activation by targeting key proliferative signaling pathways. J. Immunol. 160, 2692–2699
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51 Harnett, M.M. et al. (1998) Induction of signalling anergy via the T-cell receptor in cultured Jurkat T cells by pre-exposure to a filarial nematode secreted product. Parasite Immunol. 20, 551–563 52 Deehan, M.R. et al. (2001) A filarial nematode-secreted phosphorylcholine-containing glycoprotein uncouples the B cell antigen receptor from extracellular signal-regulated kinase-mitogen-activated protein kinase by promoting the surface Ig-mediated recruitment of Src homology 2 domain-containing tyrosine phosphatase-1 and Pac-1 mitogen-activated kinase-phosphatase. J. Immunol. 166, 7462–7468 53 Wilson, E.H. et al. (2003) In vivo activation of murine peritoneal B1 cells by the filarial nematode phosphorylcholine-containing glycoprotein ES-62. Parasite Immunol. 25, 463–466 54 McInnes, I.B. et al. (2003) A novel therapeutic approach targeting articular inflammation using the filarial nematode-derived phosphorylcholine-containing glycoprotein ES-62. J. Immunol. 171, 2127–2133
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