Macrophages Behaving Badly: Infected Cells and Subversion of Immune Responses to Theileria annulata

Reviews

Macrophages Behaving Badly: Infected Cells and Subversion of Immune Responses to Theileria annulata J.D.M. Campbell and R.L. Spooner The protozoan parasite Theileria annulata is the causative agent of the tick-borne disease tropical theileriosis, responsible for morbidity and mortality of cattle in many developing countries. Here, John Campbell and Roger Spooner discuss how the parasite might evade immune destruction during an acute primary infection. Theileria annulata macroschizontinfected macrophages act as over-efficient antigen-presenting cells within the infected draining lymph node. Infected cells activate CD4+ and CD8+ T cells abnormally, giving rise to a cascade of cytokine production. This altered immune response does not reject the parasitized cells, and might actively participate in the growth of the developing parasite. Theileria annulata is a tick-borne, protozoan parasite that infects cattle, causing the disease tropical theileriosis. Transmitted by Hyalomma spp ticks, the disease extends from southern Europe and North Africa in the west, through the Middle East and the Indian subcontinent, to China in the east. In 1982, it was estimated that 250 million cattle were at risk from the disease1, with extremely high losses to the agricultural output of developing countries. Losses of US$800 million per annum due to tropical theileriosis are reported in India alone2. To place this in context with the other principal Theileria parasite of cattle, the total losses from East Coast fever, caused by Theileria parva, are US$168 million per annum3. The principal differences between T. annulata and T. parva are highlighted in Box 1. Mortality rates owing to tropical theileriosis in cattle introduced to endemic areas are high (40–90%, varying from country to country)1,4,5, and this is a major constraint on development. Mortality as a result of T. annulata infection in less productive local breeds is low5. Theileria annulata sporozoites invade host mononuclear cells, subsequently developing to the intracellular macroschizont stage in the lymph node draining the site of the tick bite (or experimental subcutaneous injection). Macroschizont-infected cells divide rapidly in the node. After the disruption of synchrony between division of the host cell and the parasite, the merozoite stage forms, a process often coincident with pyrexia6. Following host-cell rupture, the merozoites are released to invade red blood cells (RBCs) to form piroplasms. Animals die from acute infection 15–25 days after infection (depending on the sporozoite dose), showing anaemia, leukopaenia, inappetance, cachexia, mucous membrane discharge, cessation of rumination, dyspnoea and haemorrhagic diarrhoea6–8. It is thought that the majority of disease pathogenesis is driven by macroschizont-infected John Campbell is at the Department of Medicine, University of Glasgow, Glasgow Royal Infirmary, 10 Alexandra Parade, Glasgow, UK G31 2ER. Roger Spooner is at the Centre for Tropical Veterinary Medicine, University of Edinburgh, Easter Bush, Roslin, Midlothian, UK EH25 9RG. Tel: +44 141 211 5455, Fax: +44 141 552 2953, e-mail: [email protected] 10

cells. Certainly, disease symptoms are induced using macroschizont-infected cells that do not produce piroplasms9. Removal of piroplasm-infected RBCs in the liver has been suggested as a cause of anaemia10; however, anaemia can also be seen without any piroplasms9. Although macroschizont-infected cells are responsible for a major part of the disease pathology, these cells also appear to be the principal targets of the immune response in recovering animals. Such animals can destroy macroschizont-infected cells through lysis by major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes (CTLs)11. Strong protective CTL responses12,13 are also produced through vaccination with macroschizont-infected cells that have been attenuated by prolonged in vitro culture. The reasons why macroschizont-infected cells are not destroyed during a primary infection are discussed in this review. Evasion is clearly linked to the properties of macroschizont-infected cells, with the cells targeted by the parasite and their subsequent post-infection phenotypes crucial to immune subversion. Mononuclear cells infected by T. annulata sporozoites Theileria annulata sporozoites invade many different cell types, including bovine fibroblastoid cells, peripheral blood monocytes, bone marrow macrophages, lymphocytes, and ovine and caprine peripheral blood cells14–19. (Theileria annulata infection can also occur in sheep and goats, but the disease is usually subclinical.) The ability of sporozoites to enter cells and cause disease in several species strongly suggests that there might not be a highly restricted lineage or species-specific ligand for attachment and entry. The ligand must be highly conserved between many cell types from bovid species. The efficiency with which sporozoite infection is established in a cell varies widely. Therefore, when considering which cells are infected by T. annulata, it is imperative to distinguish between infection – the ability of a sporozoite to enter the cell – and ‘transformation’ – the ability of the parasite to pass through the trophozoite stage and undergo binary fission to form the schizont20, driving the host cell to continuous division. Although many cell types are invaded by sporozoites, not all transform efficiently into continuously growing cell lines; thus, it appears that development of macroschizont-infected cells depends on intracellular regulatory processes. Gene products characteristic of transformed cells such as Ki-67 (found in the nucleus of all cycling cells)21, the serine/threonine kinase casein kinase II (CKII)22 and the transcription factor AP-1 (Ref. 23) are synthesized in T. annulata-infected cells. The factors controlling the expression of these genes, whether host or parasite derived, remain unknown. A further factor in T. annulata-induced cell transformation might also be a requirement for growth factors to maintain or stabilize

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Reviews Box 1. Comparison of Theileria annulata (causes tropical theileriosis) and T. parva (causes East Coast fever) Differences Similarities • Theileria annulata is present from North Africa through • Theileria annulata and T. parva are both tick-transmitted southern former USSR, Middle East to India and China. protozoa that cause serious diseases of cattle. • Theileria parva is localized in East and Central Africa, south of • Infective sporozoite stage is transmitted by ticks. the Sahara. • Sporozoites infect mononuclear host cells to form schizont • Theileria annulata is transmitted by Hyalomma spp ticks.. stage, and stimulate division of host cells in the lymph node draining the site of sporozoite injection. • Theileria parva is transmitted by Rhipicephalus spp ticks.• • Theileria annulata principally infects and transforms monocyte/ • Schizont-infected cells leave the lymph node via efferent macrophage lineage cells, some B cells, but not T cells. lymph and metastasize around the host animal. • Theileria parva principally infects and transforms T cells and • Schizont develops into merozoite stage, which subsequently B cells, but not macrophages. invades red blood cells to form piroplasms. • Theileria annulata infection is often accompanied by anaemia. • Schizont-infected mononuclear cells can be effectively • Theileria parva is not accompanied by anaemia. transformed and grown indefinitely in vitro.

division, and the parasite’s ability to turn on such growth factor genes might determine the viability of the transformed cell (see discussion below). Theileria annulata-transformed cells are primarily of monocyte/macrophage origin In vitro infection. The preferred bovine cell for T. annulata-induced transformation has been a matter of debate for some time. The majority of the first isolations of T. annulata-infected cell lines were from lymph nodes, and the term lymphoblastoid was generally applied to infected cells, and their origin presumed to be lymphocytic. When the phenotypes of infected cells were examined24, it was found that the T. parva-infected cells studied expressed T-cell markers, whereas T. annulatainfected cells did not. In vitro infection studies showed that T. annulata infected macrophages, and to a lesser extent B cells, but never T cells15. Theileria parva infected T and B cells, but not macrophages15. Theileria annulata has since been shown to infect and transform blood, lymph, mammary and bone marrow macrophages16–18. Sergent et al. reported as early as 1945 that T. annulata infected reticuloendothelial cells of the lymph node and Kuppfer cells of the liver25, so the preference of the parasite for macrophage-lineage cells should not have come as a great surprise. Although macrophage-lineage cells from different tissues, encompassing small monocytes through to large macrophages, have widely varying phenotypes, they all express CD14 [the lipopolysaccharide (LPS) receptor]17,26. This appears to be a prerequisite for T. annulata infection: CD142 mononuclear cells do not transform after infection17. Although all CD14+ cells are readily infected, the transformation rates may vary slightly; for example, large macrophages might transform with a slightly higher efficiency than small monocytes15,17. The reasons for this are not fully understood, but might lie in the ability of the infected cells to produce cytokines involved in macrophage activation and development – this will be discussed later. The inability to infect CD142 cells from peripheral blood with sporozoites raises questions about infection and transformation of B cells, as the vast majority of these cells do not produce CD14. There is, however, a small number of CD14+ B cells in bovine peripheral blood (D.J. Brown, unpublished), and these may be the cells described as transformable by Spooner and colleagues15. Parasitology Today, vol. 15, no. 1, 1999

Phenotypes of macroschizont-infected cell lines. Theileria annulata macroschizont-infected cells acquire a basic uniform phenotype: they produce high levels of MHC class II15,17 but lose several macrophage lineage markers, including CD11b, CD14 and elastin receptors17,18 (Fig. 1). This is reversible: eliminating the parasite with the drug buparvaquone restores the expression of normal lineage markers18. Most infected cell lines retain the expression of the macrophage marker recognized by the monoclonal antibody (mAb) IL-A24, which can be expressed at varying intensities, commonly accompanied by CD2 (Ref. 27). Expression of CD2 is not necessarily an indicator of T-cell origin, as the antigen recognized by mAb IL-A24, and CD2, can be co-expressed on normal macrophages28. There are also reports of anti-CD3 mAbs and other lymphocyte markers recognizing occasional infected cell lines27. As T. annulata-infected cells are transformed, it is not surprising that they express unexpected molecules on the surface. Infection in vivo. Following experimental subcutaneous injection of sporozoites, infected cells are probably generated from infection of lymph node cells, rather than migration of infected cells from the site of sporozoite injection. Pseudoafferent and efferent cannulation has shown that sporozoites migrate to the lymph node within minutes of subcutaneous injection, and are trapped there, never leaving in the efferent lymph (A.K. Nichani and P. Goel, unpublished). Immunohistology of infected lymph nodes after in vivo sporozoite infection has shown that macroschizont-infected cells developing in lymph nodes are of monocyte/ macrophage lineage29. Specifically, they are MAC-1+ mononuclear cells29, express MHC class II molecules and do not express either CD3 or the generic B-cell marker VPM30 (J.D.M. Campbell, unpublished). Interactions between T cells and infected cells Theileria-infected cells activate autologous T cells from naïve donors. A key feature of T. annulata-infected cells is that they are professional antigen-presenting cells (APCs), expressing high levels of MHC class II: they can efficiently present antigens to T cells30. In addition, macroschizont-infected cells of both T. annulata and T. parva possess an ability to activate and induce proliferation of autologous T cells from animals that have not been exposed to the parasite30–33. This has been dubbed the Theileria mixed lymphocyte reaction (MLR) and will be referred to as ‘MLR activity’ of infected cells throughout. In T. parva infection, there is as yet no clear evidence for 11

Reviews Up to 70% of autologous ab T cells from normal peripheral blood express the activation markers CD25 and MHC class II following interaction with macroschizont-infected cells in vitro, accompanied by a strong proliferative response31. This includes CD4+ and CD8+ T cells, although the direct contact-driven activation is only directed towards CD4+ cells36 (see below). Theileria annulata-infected cells can also activate gd T-cell receptor (TCR)-bearing T cells – a major circulating T-cell population in ruminants37. The ligand recognized by these T cells on infected cells remains unknown37. Proliferation is again cytokine dependent, with the gd T cells relying on exogenous IL-2 for their proliferation37. The MLR activity of T. annulata macroschizont-infected cells suggests the expression of a superantigen (SAg) similar to those of bacteria, viruses and other parasites. These antigens are commonly presented by MHC molecules but bind outside the normal antigen-binding groove of the MHC molecule. The SAg stimulates T cells by binding to conserved regions of the Vb domain of the TCR, bypassing the normal recognition site38. In this way, a single antigen can activate a large number of T cells in the peripheral T-cell pool, hence the prefix ‘super’. The ligand expressed by T. annulata-infected cells that induces T-cell proliferation is MHC class II associated, activating Fig. 1. Infection and transformation of mononuclear cells of various phenotypes induces only CD4+ T cells (Ref. 36; J.D.M. a uniform macroschizont-infected cell phenotype. There is no conclusive evidence to confirm or refute that T cells are invaded by sporozoites, and such cells certainly are Campbell, unpublished). Although not transformed. Macrophages form the main bulk of infected cells, although CD14+ B a full picture is yet to be established cells may also transform. NB The levels of IL-A24 antigen expression on macroschizontin outbred cattle, two or three Vb infected cells appear to vary with the cell cycle – cells in logarithmic growth phase tend families (expressed by ~10% of the to be brighter (J.D.M. Campbell, unpublished). ER, elastin receptors; FcR, Fc receptors; peripheral T-cell pool) appear to be IL-A24, macrophage-specific antigen recognized by monoclonal antibody (mAb) IL-A24; the targets for antigen-induced actiMHC, major histocompatibility complex; sIg, surface immunoglobulin; TCR, T-cell vation (J.D.M. Campbell and R.L. receptor; +/–, expressed on subsets of cells. Spooner, unpublished). This corresponds well with the number of Vb this T-cell activating mechanism in the pathogenesis of families targeted by bacterial SAg39, although further disease. However, this mechanism has been suggested work will be required to determine the antigenic elas a possible way for T. parva (which infects T cells) ement, and how ‘super’ it really is. However, as disto increase the production of growth factors, such as cussed above, many more than 10% of all T cells, ininterleukin 2 (IL-2), which can help division of paracluding CD8+ cells, which are not apparently targeted sitized cells34. In T. annulata infection, T-cell activation by the antigen, acquire activation markers in the by the MLR activity of macroschizont-infected cells Theileria MLR. Most of these will be activated as a remay be the root cause of many aspects of pathogenesis. sult of cytokine production from both the infected cells Theileria annulata-infected macrophages activate auand the antigen (Ag)-activated T cells. Therefore, it tologous T cells from naïve cattle through a combination might not be entirely valid to class the MLR activity of of cytokines and contact between infected cells and T T. annulata as SAg mediated, as a combination of an cells31,35. Macroschizont-infected cells express mRNA antigenic element on the surface of infected cells and for IL-1a, IL-1b, IL-6, IL-10, tumour necrosis factor a cytokines are required for the mass activation of T cells (TNF-a)35 and IL-12 (J.D.M. Campbell and D.J. Brown, observed. unpublished). The levels of IL-1a and IL-6 mRNA Normal pathways of T-cell development are disrupted by expressed by infected cells correlate directly with the macroschizont-infected cells in vivo. The first clue that the amount of proliferation induced in activated T cells35. MLR activity of T. annulata-infected cells was important 12

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Reviews for the generation of immune responses came when attempts were made to isolate T-cell lines from immunized donors. In the case of T. parva, MLR T-cell activating ability does not hinder the generation of parasitespecific CD4+ or CD8+ T-cell lines from immune animals in vitro40. However, the ability of T. annulata macroschizont-infected cells to induce nonspecific T-cell activation is sufficiently powerful to block the isolation of parasite-specific, T-cell lines of any type from immune animals (J.D.M. Campbell and K. Odling, unpublished). If this interference mechanism, which has been demonstrated in vitro, of the nonspecific MLR blocking ‘genuine’ T-cell responses actually takes place during the development of primary immune responses, it could seriously affect the ability of the immune response to deal with the growing pathogen. Indeed, studies of the activation pathways of CD4+ T cells in draining lymph nodes during the course of a primary infection found that the development of this essential arm of the immune response was altered. Normal T-cell responses. During the initiation of normal T-cell responses, CD4+ T cells enter the paracortex of the lymph node from the blood via high endothelial venules (HEVs). Here, they interact with antigen-bearing APCs, which traffic from the afferent lymph to the paracortex of the lymph node. Taking place in the first week of a response to an antigen41,42, this provides a highly efficient immune surveillance mechanism. Antigen is exposed to the many millions of T cells passing through the lymph node. The small numbers of T cells passing through the node that can react to the antigen presented are then activated and proliferate, generating an expanded pool of T cells. These cells go on to form an appropriate immune response within the lymph node during the second and third weeks after introduction of the antigen41,42. The T. annulata-modified response. In T. annulata infection, an extra element is added to the system – large numbers of infected APCs with augmented MLR capacity are developing in the lymph node medulla within 48 h of infection. Instead of the usual interactions between APCs and T cells in the lymph node paracortex, large numbers of CD4+ T cells are activated by the MLR capacity of infected APCs in the medulla of the node within four days31. This strongly suggests that the MLR activity of macroschizont-infected cells is active early in development – when the host cell is induced to divide for the first time. Thus, the usual selection of small numbers of T cells for further expansion is bypassed, and a rapid ‘firing off’ of the immune response, which has no resemblance to normal development pathways, is generated. Large numbers of activated CD4+ T cells are detected within the node within four days of infection. Furthermore, instead of remaining in the node as normal, the activated T cells leave the node – large numbers of IL-2-responsive, CD25+ CD4+ T cells are found in the efferent lymph from Day 6 onwards13. Infected lymph nodes and efferent lymph are essentially denuded of activated T cells by Day 10 post-infection13,31. Even if the activated T cells were capable of antiparasite responses (and it appears that this is not the case)13, they are therefore no longer present at the site where an antiparasite response is required. Alternatively, if there are any activated T cells in the lymph node, they appear to be ‘turned off’, again resulting in a non-functional response. Parasitology Today, vol. 15, no. 1, 1999

Therefore, the development of T. annulata macroschizont-infected cells in the lymph node draining the site of infection affects T-cell activation in three distinct steps: (1) a large number of APCs with augmented MLR activity develop in the medulla, outside the normal Tcell/APC interaction sites; (2) much larger numbers of T cells than in a normal response are activated by the infected APC MLR activity in the medulla; (3) activated T cells do not remain in the lymph node to expand and mediate immunity. T cells activated by macroschizont-infected cells produce a non-protective cytokine response. In addition to blocking the development of normal immunity, non-specific MLR activation of T cells by infected cells also drastically alters the cytokines produced by these T cells. When T cells are stimulated by T. annulata macroschizontinfected cells in vitro, the principal cytokine mRNA species produced are IL-2 and interferon g (IFN-g), while IL-4 mRNA disappears43. Thus T. annulata induces a skewing to a T helper-1 (Th1)-type response in MLRactivated T cells. Th1 T-cell responses are characterized by the production of cytokines such as IFN-g and IL-2, which stimulate and maintain cellular rather than humoral immune responses. It seems likely that the Th1 phenotype is induced by IL-12, produced by infected cells. This must also raise the question of whether the IL-10 detected at the mRNA level in infected cells35 is active or relevant, as there is no evidence to date of any IL-10-like biological effect. T-cell responses in vivo. Efferent lymph IFN-g protein production is greatly elevated43 from Days 4–5 of primary infection, corresponding to the time when many T cells are activated by infected cells. Conversely, only two small bursts of IFN-g production43 are seen in the efferent lymph of immune animals undergoing rechallenge. Net IFN-g production 4–10 days after experimental infection is 20 times greater in non-protected naïve animals than in protected immune animals43. Thus, very high levels of IFN-g alone do not control acute infections. A role for IFN-g in infected cell development? The failure of IFN-g production to control T. annulata infection flies in the face of current thinking about mechanisms for rejection of several protozoan parasites that are macrophage resident, such as Toxoplasma and Leishmania44. IFN-g activation of infected macrophages induces the production of nitric oxide (NO), which damages intracellular parasites and leads to their rejection44. This mechanism can be protective in the early stages of T. annulata infection – IFN-g-induced NO inhibits trophozoite establishment after sporozoite infection of bovine cells45. NO (but not IFN-g generated) also slows the growth of fully formed, macroschizont-infected cells in vitro and might cause infected cells to apoptose46. Why do the very high levels of IFN-g produced during a primary infection not lead to a resolution of infection? When the kinetics of IFN-g production induced by acute infection are examined, a possible role for the cytokine in infected-cell development becomes apparent (Fig. 2). Even after acute experimental infection, IFN-g is not seen in efferent lymph for at least 3–4 days post infection43. Experiments in our laboratory suggest that this is the length of time required for the parasite to invade host macrophages, stimulate division and first activate surrounding T cells (J.D.M. Campbell and R. Nelson, unpublished). This is followed by 4–5 days of very high 13

Reviews

Fig. 2. A putative role for interferon g (IFN-g) in the development of infected cells. Once the sporozoite has entered the target cell, it undergoes binary fission to form the first macroschizonts, and induces host-cell division. The macroschizont-infected cell then induces activation of neighbouring T cells and, probably through the action of interleukin 12 (IL-12), causes T cells to secrete IFN-g. In turn, IFN-g induces IL-1 and tumour necrosis factor a (TNF-a) production by the developing infected cell, which act as autocrine growth/apoptosis-rescue factors, stabilizing the developing macroschizont-infected cell.

levels of IFN-g production, which drop again quickly, once the activated T cells are no longer present in the lymph node13,31,43. At this stage, the majority of the cells in the lymph node are easily detected as fully formed, macroschizont-infected cells; that is, the majority of IFN-g production induced by the parasite takes place between the IFN-g-sensitive sporozoite invasion stage and the fully formed, potentially NO-sensitive macroschizont stage. This points to a role for IFN-g in an intermediate stage of schizont development, between inducing the first division of the parasitized cell and the stable division of the fully mature, infected cell (Fig. 2). Macrophage lineage cells do not normally undergo many cell divisions after leaving the bone marrow, and inducing division commonly results in apoptosis of the cell47. High levels of apoptotic cells are seen in the foci of rapidly dividing cells in lymph nodes following sporozoite infection31. Located in the macrophage-rich medulla, it is likely that many of these cells are parasite infected. Macrophages can be rescued from apoptosis to a large extent through the action of IFN-g, which induces the production of IL-1 and TNF-a by the macrophage48. These cytokines 14

act as autocrine growth factors that stabilize the dividing cell48. TNF-a and IL-1 are detected in fully formed (ie. undergoing stable proliferation in vitro) macroschizontinfected cells35. It is tantalizing to suggest that induction of IFN-g from T cells by unstable, developing, macroschizont-infected cells results in an upregulation in their own IL-1 and TNF-a production, thereby effecting rescue from apoptosis. Once this rescue is no longer needed, as infected cells are producing their own growth factors, the potentially negative effects of IFN-g are avoided, as the majority of the activated T cells have left the node in the efferent lymph by Day 10 post-acute infection13. CTL responses are turned off by the parasite. CTLs are a major mechanism for parasite clearance, both after drug treatment and vaccination, but no CTL activity is found in acutely infected animals undergoing primary infection11,13. However, activated (CD25+, MHC class II+) CD8+ T cells are present in the lymph nodes and efferent lymph of acutely infected animals13. It is now known that these CD8+ cells have altered surface expression of CD2 – a molecule that is essential in adherence to target cells, activation of CTL activity and target lysis49. CD2 epitope expression is significantly altered on CD4+ T cells or on any T-cell subsets of immune animals undergoing immune challenge. The loss of CD2 epitopes from CD8+ T cells is associated directly with the presence of growing macroschizont-infected cells – normal CD22 expression is restored immediately when animals are treated with drugs to kill the parasite. Whether the CD22 CD8+ cells are non-functional CTLs, or a separate population of cells induced to swamp genuine CTL responses, requires further investigation. However, restoration of normal CD2 expression on CD8+ cells results in an immediate strong CTL response against macroschizont-infected cells13. Future prospects What immune responses will induce protection against T. annulata challenge? We have here highlighted the mechanisms used by T. annulata to evade destruction by the immune system in a susceptible host. The rapid manifestation of T-cell activating capabilities by the macroschizont-infected cell after sporozoite infection allows the parasite to interfere with activation of T cells extremely early during acute infection. The cytokine and CTL responses generated do not reject the parasite, and might actively aid its growth. Any strategy based on the induction of macrophage activation to reject the parasite through the production of NO seems unlikely to work outside the early stages of infection. The IFN-g/TNF-a/IL-1 associated with these strategies appear to be beneficial to the parasite within four or five days of infection. In addition, IFN-g (and presumably the induction of NO) does not appear to form a major part of the response to the invasive stages of the parasite in immunized, re-challenged animals. The small increases in IFN-g production in efferent lymph seen in these animals take place approximately six days after sporozoite infection. The ‘Holy Grail’ for any subunit vaccine against T. annulata remains the isolation of schizont-specific antigens responsible for the extremely efficient CTL response and parasite clearance in animals that have undergone infection and treatment. In any successful generation of an immune response, the race must be won between Parasitology Today, vol. 15, no. 1, 1999

Reviews attacking the macroschizont and its initiation of the evasion mechanisms. This will only be achieved if a pool of parasite-specific, Theileria MLR non-reactive T cells have been generated through vaccination. There might be a complementary role for antisporozoite antibody responses such as those induced by immunization with the sporozoite antigen SPAG-1 (Ref. 50). Such antibody responses do not provide satisfactory protection alone, but can slow the onset of disease50. As immune dysfunction is related to the development of large numbers of macroschizont-infected cells, the mopping up of a proportion of the sporozoites by antibody probably results in slowing of the induction of non-protective T-cell responses, and ultimately delays but does not stop the onset of disease. A parallel to this might be the infection of naïve animals with low sporozoite doses – commonly a low sporozoite dose has delayed symptoms (presumably as a result of the slower build-up of macroschizont-infected cells), but can still lead to a fatal infection8. If an animal has already been primed with an antischizont vaccine, a preformed antibody response may be of some benefit, slowing the development of macroschizont-infected cells and allowing the previously generated antischizont recall responses to become operative. Perhaps of more immediate practical benefit, the association between activation of T cells and pathology can be exploited in the selection of T. annulata vaccine cell lines. Currently, the mechanism of attenuation is poorly understood and involves the culture of cells for several years. In addition, long-term passage lines might not give lasting protection in the field without re-challenge51. In an ongoing study, cloned, infected cell lines selected for low production of T-cell activating cytokines, but not long-term attenuated in the conventional manner, have immunized successfully in a small trial with no side effects (D.J. Brown et al., unpublished). This selection method is potentially attractive as it combines short selection time with safe immunization and is now undergoing further testing. Acknowledgements Much of the work in our laboratory is supported by the European Union under the INCO-DC programme. We would like to thank David Taylor, Louise Taylor, Sarah Howie and Liz Glass for their constructive criticism, and David Brown for access to unpublished data. References 1 Robinson, P.M. (1982) Theileriosis annulata and its transmission – a review. Trop. Anim. Health Prod. 14, 3–12 2 Devendra, C. (1995) in Global Agenda for Livestock Research, pp 41–48, EDS ILRI, Nairobi 3 Mukhebi, A.W. (1992) in The Epidemiology of Theileriosis in Africa (Norval, R., Perry, B. and Young, A., eds), pp 379–403, Academic Press 4 Hashemi-Fesharki, R. (1988) Control of Theileria annulata in Iran. Parasitol. Today 4, 36–40 5 Oudich, H. et al. (1993) in Resistance or Tolerance of Animals to Disease and Veterinary Epidemiology and Diagnostic Methods (Uilenberg, G. and Hamers, R., eds), pp 78–81, CIRAD-EMVT 6 Barnett, S.F. (1977) in Parasitic Protozoa (Vol. IV) (Kreier, J.P., ed.), pp 77–113, Academic Press 7 Neitz, W.O. (1957) Theileriosis, gonderioses and cytauxzoonoses: a review. Onderstepoort J. Vet. Res. 27, 275–431 8 Preston, P.M. et al. (1992) Tropical theileriosis in Bos taurus and Bos taurus cross Bos indicus calves: response to infection with graded doses of sporozoites of Theileria annulata. Res. Vet. Sci. 53, 230–243 9 Hooshmand-Rad, P. (1976) The pathogenesis of anaemia in Theileria annulata infection. Res. Vet. Sci. 20, 324–329

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10 Uilenberg, G. (1981) in Diseases of Cattle in the Tropics (Ristic, M. and McIntyre, I., eds), pp 411–427, Martinus Nijhoff 11 Preston, P.M., Brown, C.G.D. and Spooner, R.L. (1983) Cellmediated cytotoxicity in Theileria annulata infection of cattle with evidence for BoLA restriction. Clin. Exp. Immunol. 53, 88–100 12 Innes, E.A. et al. (1989) The development and specificity of cytotoxic cells in cattle immunized with autologous or allogeneic Theileria annulata infected lymphoblastoid cell lines. Parasite Immunol. 11, 57–68 13 Nichani, A.K. et al. (1994) in Proceedings of the Third EU Coordination Meeting on Tropical Theileriosis (Spooner, R. and Campbell, J., eds), pp 117–122, Roslin Institute 14 Brown, C.G.D. and Gray, M.A. (1981) Infection of a bovine fibroblastic cell line with Theileria parva and T. annulata. Trans. R. Soc. Trop. Med. Hyg. 75, 323–324 15 Spooner, R.L. et al. (1989) Theileria annulata and T. parva infect and transform different bovine mononuclear cells. Immunology 66, 284–288 16 Glass, E.J. et al. (1989) Infection of bovine monocyte/macrophage populations with Theileria annulata and Theileria parva. Vet. Immunol. Immunopathol. 22, 355–368 17 Campbell, J.D.M. et al. (1994) Theileria annulata sporozoite targets. Parasite Immunol. 16, 501–505 18 Sager, H. et al. (1997) Macrophage–parasite relationship in theileriosis. Reversible phenotypic and functional dedifferentiation of macrophages infected with Theileria annulata. J. Leukocyte Biol. 61, 459–468 19 Steuber, S. et al. (1986) In vitro susceptibility of different mammalian lymphocytes to sporozoites of Theileria annulata. Z. Parasitkd. 72, 831–834 20 Jura, W.G.Z.O., Brown, C.G.D. and Kelly, B. (1983) Fine structure and invasive behaviour of the early developmental stages of Theileria annulata in vitro. Vet. Parasitol. 12, 31–44 21 Shayan, P. et al. (1994) in Proceedings of the Third EU Coordination Meeting on Tropical Theileriosis (Spooner, R. and Campbell, J., eds), pp 98–100, Roslin Institute 22 Shayan, P. and Ahmed, J.S. (1997) Theileria-mediated constitutive expression of the casein kinase II-alpha subunit in bovine lymphoblastoid cells. Parasitol. Res. 83, 526–532 23 Baylis, H.A., Megson, A. and Hall, R. (1995) Infection with Theileria annulata induces expression of matrix metalloproteinase-9 and transcription factor AP-1 in bovine leukocytes. Mol. Biochem. Parasitol. 69, 211–222 24 Spooner, R.L. et al. (1988) Bovine mononuclear cell lines transformed by Theileria parva or Theileria annulata express different subpopulation markers. Parasite Immunol. 10, 619–629 25 Sergent, E. et al. (1945) Etudes sur les piroplasmoses bovines. Annales l’Institut Pasteur d’Algerie 1945, 489–506 26 Sopp, P., Kwong, L.S. and Howard, C.J. (1996) Identification of bovine CD14. Vet. Immunol. Immunopathol. 52, 323–328 27 Howard, C.J. et al. (1993) Phenotypic analysis of bovine leukocyte cell lines infected with Theileria annulata. Vet. Immunol. Immunopathol. 39, 275–282 28 Haig, D.M., Thomson, J. and Dawson, A. (1991) Reactivity of the workshop monoclonal-antibodies with ovine bone-marrow cells and bone-marrow derived monocyte macrophage and mast-cell lines. Vet. Immunol. Immunopathol. 27, 135–145 29 Forsyth, L.M.G. et al. (1996) Bovine cells infected in vivo with Theileria annulata express CD11b, the C3bi complement receptor. Vet. Res. Commun. 21, 249–263 30 Glass, E.J. and Spooner, R.L. (1990) Parasite-accessory cell interactions in theileriosis. Antigen presentation by Theileria annulatainfected macrophages and production of continuously growing antigen-presenting cell lines. Eur. J. Immunol. 20, 2491–2497 31 Campbell, J.D.M. et al. (1995) Theileria annulata induces aberrant T cell activation in vitro and in vivo. Clin. Exp. Immunol. 99, 203–210 32 Pearson, T.W. et al. (1979) Cell-mediated immunity to Theileriatransformed cells. Nature 281, 678–680 33 Goddeeris, B.M. and Morrison, W.I. (1987) The bovine autologous Theileria mixed leucocyte reaction: influence of monocytes and phenotype of the parasitised stimulator cell on proliferation and parasite specificity. Immunology 60, 63–69 34 Morrison, W.I., Taracha, E.L.N. and McKeever, D.J. (1995) Contribution of T cell responses to immunity and pathogenesis in infections with Theileria parva. Parasitol. Today 11, 14–18 35 Brown, D.J. et al. (1995) T cell activation by Theileria annulata infected macrophages correlates with cytokine production. Clin. Exp. Immunol. 102, 507–514 36 Campbell, J.D.M. et al. (1997) Theileria annulata ‘superantigen’ activity – TCR usage by responding bovine CD4+ T cells from uninfected donors. Biochem. Soc. Trans. 25, S277

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Reviews 37 Collins, R.A. et al. (1996) Bovine gamma/delta TCR+ T-lymphocytes are stimulated to proliferate by autologous Theileria annulatainfected cells in the presence of interleukin-2. Scand. J. Immunol. 44, 444–452 38 Torres, B.A., Griggs, N.D. and Johnson, H.M. (1993) Bacterial and retroviral superantigens share a common binding region on class II MHC antigens. Nature 364, 142–154 39 Marrack, P., Kushnir, E. and Kappler, J. (1991) A maternally inherited superantigen encoded by a mammary tumour virus. Nature 349, 524–526 40 Goddeeris, B.M. and Morrison, W.I. (1988) Techniques for the generation, cloning, and characterization of bovine cytotoxic T cells specific for the protozoan parasite Theileria parva. J. Tissue Cult. Methods 11, 101–110 41 Bogen, S.A., Weinberg, D.S. and Abbas, A.K. (1991) Histologic analysis of T lymphocyte activation in reactive lymph nodes. J. Immunol. 147, 1537–1541 42 Bogen, S.A. et al. (1993) Analysis of IL-2, IL-4, and IFN-gproducing cells in situ during immune responses to protein antigens. J. Immunol. 150, 4197–4205 43 Campbell, J.D.M. et al. (1997) A non-protective T helper 1 response against the intra-macrophage protozoan Theileria annulata. Clin. Exp. Immunol. 108, 463–470

44 Mauel, J. (1996) Intracellular survival of protozoan parasites with special reference to Leishmania spp., Toxoplasma gondii and Trypanosoma cruzi. Adv. Parasitol. 38, 1–51 45 Visser, A.E. et al. (1995) Nitric oxide inhibits establishment of macroschizont-infected cells lines and is produced by macrophages of calves undergoing bovine tropical theileriosis or East Coast fever. Parasite Immunol. 17, 91–102 46 Richardson, J.O. et al. (1998) Nitric oxide causes the macroschizonts of Theileria annulata to disappear and the host cells to become apoptotic. Vet. Res. Commun. 22, 31–45 47 Cotter, T.G. et al. (1994) Cell death in the myeloid lineage. Immunol. Rev. 142, 93–112 48 Mangan, D.F. and Wahl, D.F. (1991) Differential regulation of human monocyte programmed cell death (apoptosis) by chemotactic factors and pro-inflammatory cytokines. J. Immunol. 147, 3408–3412 49 Bierer, B.E. et al. (1989) The biologic roles of CD2, CD4, and CD8 in T cell activation. Annu. Rev. Immunol. 7, 579–599 50 Boulter, N.R. et al. (1995) Theileria annulata sporozoite antigen fused to hepatitis-B core antigen used in a vaccination trial. Vaccine 13, 1152–1160 51 Dargouth, M.A. et al. (1996) A preliminary-study on the attenuation of Tunisian schizont-infected cell-lines of Theileria annulata. Parasitol. Res. 82, 647–655

Parasites as Accumulation Indicators of Heavy Metal Pollution B. Sures, R. Siddall and H. Taraschewski Parasites are attracting increasing interest from parasite ecologists as potential indicators of environmental quality because of the variety of ways in which they respond to anthropogenic pollution. However, until recently, little was known about the accumulation of toxins within parasites. Certain parasites, particularly intestinal acanthocephalans and cestodes of fish, can accumulate heavy metals at concentrations that are orders of magnitude higher than those in the host tissues or the environment. In this review, Bernd Sures, Roy Siddall and Horst Taraschewski discuss the recently described phenomenon of conspicuous metal accumulation by parasites and how this might be applied to environmental monitoring. They also suggest how environmental science and parasitology might profit from each other in the near future. Approximately 130–150 papers have been published since 1980 that are directly concerned with the relationship between pollution and parasitism, mainly in the aquatic environment. Attempts at using parasites as biological indicators in environmental impact studies have been the subject of several recent reviews (cited in Refs 1 and 2) and a European Symposium entitled ‘Fish parasites as indicators of environmental quality’*. The majority of investigations have examined the effects of various forms of pollution on the abundance and distribution of parasites, and the comBernd Sures and Horst Taraschewski are at the Zoologisches Institut I – Ökologie/Parasitologie, Geb. 30.43, Universität Karlsruhe, Kaiserstr. 12, 76128 Karlsruhe, Germany. Roy Siddall is at the School of Environmental and Applied Sciences, University of Derby, Kedleston Road, Derby, UK DE22 1GB. Tel: +49 721 6082701, Fax: +49 721 6087655, e-mail: [email protected] 16

bined effects of pollution and parasitism on the health of the hosts. However, interactions between the environment and host–parasite systems are complex and not easily interpreted as they are dependent on a wide variety of factors3,4. Recently, Lafferty2 drew attention to the conflicting evidence on, and inconsistent associations between, environmental impacts and parasites, concluding that few parasite–pollution combinations show predictable changes, despite the considerable effort that has been put into linking levels of parasitic infection with pollution. Nevertheless, parasites have alternative applications in environmental biomonitoring by using them as accumulation indicators of heavy metal contamination. In environmental impact studies, certain organisms provide valuable information about the chemical state of their environment, not through their presence or absence, but through their ability to concentrate environmental toxins within their tissues5. Free-living invertebrates, notably bivalve molluscs, are commonly employed in this role as ‘sentinel organisms’ to monitor the concentrations of bioavailable metals in aquatic ecosystems. Good sentinel species tend to be ubiquitous, sedentary and long-lived, coupled with a high pollution tolerance and the ability to accumulate high quantities of a toxin6. They are widely used to indicate contamination of the environment with toxicants at levels that vary during the day or year and whose bioavailability cannot therefore be determined. Many pollutants might be damaging in the long term, and sentinel species can indicate the risk of toxicant bioaccumulation in their predators. One * European Multicolloquium of Parasitology (EMOP) VII, 2–6 September 1996; articles published in Parassitologia 39(3), Paperna, I., ed., 1998

0169-4758/99/$ – see front matter © 1999 Elsevier Science. All rights reserved. PII: S0169-4758(98)01358-1

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