Histamine-binding proteins

Histamine is a key mediator of inflammation and also affects immune functions. It is secreted by mast cells, basophils and (in some species) blood platelets. In mast cells, histamine is stored within granules and released following activation of the cells by crosslinking of IgE receptors. Apart from its role in the brain, this biogenic amine is believed to function as a primitive means of controlling parasites, hence its abundance in the gastrointestinal tract and in skin (Stebbings, 1974). Following extracellular release, histamine binds to its receptors, triggering a cascade of inflammatory and immune responses that result in swelling (oedema), redness (erythema) and irritation. The importance of histamine as a mediator in successful anti-tick responses, particularly in previously exposed hosts that have developed an immune (anamnestic) response, has been demonstrated in several host species, including cattle, rabbits and guinea pigs (Willadsen, Wood & Riding, 1979; Brossard, 1982; Wikel, 1982).

Histamine plays a role in allergies and other inflammatory diseases. Most drugs that control the action of histamine are receptor antagonists, acting on one of the histamine receptors. Ticks have adopted a different strategy to control the effects of histamine: they produce saliva molecules that bind histamine directly (Paesen et al. 1999, 2000). Tick histamine-binding proteins are structurally related to a large family of barrel-shaped ligand-binding proteins known as lipocalins. They have been named histacalins. Their high affinity for histamine enables them to out-compete histamine receptors and thereby prevent a histamine-mediated inflammatory response. Histacalins have been isolated from several ixodid tick species including Rhipicephalus appendiculatus and Ixodes hexagonus. However, they have not been detected in the important vector species, I. ricinus (although anti-histamine activity has been detected) and O. moubata (G. C. Paesen, personal communication). Homologues of the R. appendiculatus histacalins have been identified in the salivary glands of I. scapularis (see the chapters by Brossard & Wikel and Valenzuela in this Supplement) but it remains to be determined whether they bind histamine. In addition, a histamine release factor homologue is secreted by at least one ixodid tick species, suggesting ticks have a multifaceted control mechanism for histamine (Mulenga et al. 2003).

The apparent absence of histacalins from I. ricinus and O. moubata, if confirmed, suggests that they are not SAT factor candidates for tick-borne pathogens transmitted by these two important vector species. However, this does not preclude a role for histacalins in SAT or a role for other anti-histamine molecules. A site of inflammation is a hostile environment for invading pathogens. Anti-histamines are likely to benefit tick-borne pathogens even if they are not the key factor in facilitating their transmission. Histamine upregulates certain cytokines such as tumour necrosis factor alpha (TNFα), a suppressor of B. burgdorferi infections, and activates natural killer (NK) cells, which have antiviral activity. Through the H₄ receptor, histamine is involved in leukocyte trafficking, including chemotaxis of eosinophils and mast cells, and recruitment of neutrophils (Takeshita et al. 2003). In a mouse model of allergic asthma, a histacalin of R. appendiculatus largely abrogated this inflammatory disease (Couillin et al. 2004). If ticks can effectively manipulate the many functions of histamine, all tick-borne pathogens would probably benefit to some degree.

Complement inhibitors

The complement system is the principal effector arm of the humoral immune system in vertebrates, involved in inflammation and innate immunity. The two activation pathways (classical and alternative) comprise parallel cascades that converge on the C3 protein resulting in complement activation via the formation of similar catalytic C3 and C5 convertases (Law & Reid, 1995). Complement activation results in the release of the anaphylatoxins, C3a and C5a, by the C3 and C5 convertases, respectively. These acute inflammatory peptides induce damage by recruiting and stimulating granulocytes to release proinflammatory mediators, tissue degradative enzymes, and oxygen free radicals (Ember, Jagels & Hugli, 1998). The anaphylatoxins also increase adhesion molecule and inflammatory cytokine expression (Kohl, 2001). The terminal membrane attack complex of the complement system can disrupt pathogens or infected host cells and has proinflammatory activity. Additional effects of complement activation include opsonisation and phagocytosis of pathogens, clearance of immune cell complexes by recruitment of macrophages, and increased efficiency of antigen presentation to B cell receptors. Clearly, complement is as much a threat to tick-borne pathogens as it is to ticks.

Activation of host complement occurs when ticks feed, contributing to resistance to tick infestation (Wikel & Allen, 1977; Allen, Khalil & Graham, 1979). Not surprisingly, ticks have evolved complement inhibitors. Saliva or SGE of several Ixodes spp. (I. scapularis, I. ricinus, I. hexagonus and I. uriae) inhibit the alternative complement pathway (Ribeiro & Spielman, 1986; Ribeiro, 1987; Astigarraga et al. 1997; Lawrie, Randolph & Nuttall, 1999) while Ornithodoros spp. target both the classical and alternative pathways (Astigarraga et al. 1997; Nunn et al. 2004). The complement inhibitor of I. scapularis is a 18·5 kD protein that appears to destabilise the active convertase, C3bBb (Valenzuela et al. 2000). Attempts to isolate an homologous protein from I. ricinus have been unsuccessful but activity was characterized as strongly inhibiting generation of the C3a anaphylatoxin (Lawrie, Sim & Nuttall, 2004). The complement inhibitor of O. moubata is a 17 kD lipocalin-like protein, which prevents production of the C5a but not the C3a anaphylatoxin (Nunn et al. 2004). Characterization of complement inhibitors from other tick species is needed to determine whether the different strategies (targeting the alternative pathway alone or inhibiting both alternative and classical pathways) is a feature of ixodid compared with argasid species, and its impact on pathogen transmission.

For I. ricinus, anti-complement activity is demonstrated by SGE from unfed as well as feeding ticks (Lawrie et al. 1999). This reflects the fact that activation of the alternative complement cascade is one of the first events following tick attachment, and a constant threat throughout tick feeding. However, the existence of significant anti-complement activity in unfed I. ricinus SGE indicates the complement inhibitor is not the SAT factor of TBEV, as SGE from unfed adult female I. ricinus does not demonstrate SAT activity (Labuda et al. 1993b). Nevertheless, complement inhibitors should be explored in relation to transmission of Lyme disease spirochaetes.

Complement sensitivity is a major influence in Lyme disease ecology and pathogenicity (Kurtenbach et al. 1998; van Dam, 2002; see also chapter by Piesman & Gern in this Supplement). When different members of the Borrelia burgdorferi sensu lato complex were incubated in vitro in serum from different vertebrate species, a striking correlation was observed with host susceptibility. Thus B. garinii strain ZQ 1 survives in avian serum but is rapidly lysed by rodent serum. By contrast, B. afzelii survives rodent serum but is killed by avian serum (Kurtenbach et al. 1998). Similar observations have been made for B. bissettii (Ullmann et al. 2003). The borreliacidal effect is due to the activity of the alternative complement pathway, and specific mechanisms appear to mediate resistance (Kurtenbach et al. 1998; Hellwage et al. 2001; Pausa et al. 2003). Several B. garinii strains are sensitive to human serum (van Dam et al. 1997). Despite this sensitivity, B. garinii is able to infect humans and cause neuroborreliosis. This may well be a property of particular B. garinii strains. But could it be that anti-complement factors in tick saliva provide sufficient protection of B. garinii from the borreliacidal effects of human complement, enabling the tick-transmitted borreliae to seek out an immunoprotected site within the tick-bitten human host?

If tick complement inhibitors promote transmission and aid survival of Lyme disease spirochaetes (and other tick-borne pathogens), another influential factor could be the host specificity of anti-complement activity (Lawrie et al. 1999). For example, the cosmopolitan I. ricinus demonstrates anti-complement activity against a range of mammalian (including human) and avian hosts. By contrast, the hedgehog tick, I. hexagonus, has high levels of activity against hedgehog serum, but no anti-complement activity against serum from avian species. However, complement inhibitors cannot be the only SAT factor for B. burgdorferi sl as I. ricinus (adult females) SGE can inhibit red deer (Cervus scoticus) serum complement and yet deer (including red deer) are not hosts for the Lyme disease spirochaete although they are important hosts for the principal Ixodes vector species (reviewed by Piesman & Gern in this Supplement).

Immunoglobulin-binding proteins

When ticks feed, a small proportion of host plasma proteins escape digestion and pass through the gut wall, into the haemocoel. These host proteins include immunoglobulin G (IgG) molecules, some of which may be pathogen-specific antibodies (Fujisaki, Kamio & Kitaoka, 1984; Jasinskas, Jaworski & Barbour, 2000). The fate of host immunoglobulins that enter the tick haemocoel was unknown until the discovery that adult female R. appendiculatus excrete intact IgG in their saliva (Wang & Nuttall, 1994). Further investigations revealed a family of immunoglobulin G-binding proteins (IGBPs) in the haemolymph and salivary glands of adult R. appendiculatus, and proteins with similar activity in other ixodid species, including I. ricinus and Amblyomma variegatum, and a sequence homologue in I. scapularis (Wang & Nuttall, 1999; Packila & Guilfoile, 2002). Although IGBPs have not been reported for argasid tick species, circumstantial evidence suggests they may occur (Minoura, Chinzei & Kitamura, 1985).

The prevalence and abundance of IGBPs indicate that they play an important role in blood feeding. Possibly they provide a Tick Immunoglobulin Excretion System (TIES) that enables ticks to ferry potentially damaging antibodies safely through the haemocoel to their salivary glands from where they are excreted (Wang & Nuttall, 1999). If such a TIES exists, it may benefit tick-borne pathogens by protecting them (and possibly the infected tick) from pathogen-specific antibodies taken up in the bloodmeal. An indication of such protection was obtained in studies of Thogoto virus infection of R. appendiculatus ticks in the presence of antibody to Thogoto virus (Jones & Nuttall, 1989).

The difficulty with the TIES hypothesis is that it does not explain why ticks have such an elaborate system for transporting biologically active immunoglobulins through their body; a simpler solution would be to ensure that all immunoglobulins in the blood-meal are degraded. The answer to this apparent conundrum is that the tick appears to use the excreted immunoglobulins against its host. Thus when guinea pigs were immunised with IGBPMC, a secreted IGBP from adult male R. appendiculatus, the feeding performance of its female mate was impaired. In this particular case, the male tick seems to use the host's immunoglobulins to induce local immunosuppression for the benefit of the female, a novel form of ‘mate guarding’ (Wang et al. 1998). Whether such an effect aids in the transmission or acquisition of tick-borne pathogens by feeding ticks remains to be explored.

Cytokine inhibitors

Cytokines are the chemical mediators of inflammation and immunity. The type 1 interferons are a cytokine superfamily comprising four subfamilies of which IFNα and IFNβ are induced by viral infections. Most cell types produce them. A common inducer is dsRNA, a product of viral replication. Infected cells synthesise and release IFN, which then binds to IFN receptors of neighbouring cells, triggering more that 50 genes via the JAK/STAT signalling pathway (Stark et al. 1998). Several factors are activated (including dsRNA-dependent protein kinase R, the 2′–5′ oligoadenylate synthestase, and the Mx proteins) that create an antiviral state in which inhibition occurs at different stages of viral replication and assembly (Goodbourn, Didcock & Randall, 2000).

Arboviruses are not generally recognised as strong inducers of IFNα/β and there have been few (if any) studies of IFN induction following vector-borne virus transmission. A notable exception as a strong IFN inducer is vesicular stomatitis virus, an insect-borne rhabdovirus. This virus was used to study the effect of tick SGE on viral replication in vitro, although it is not tick-borne. Cultures of mouse L cells were treated with SGE from partly fed adult R. appendiculatus or D. reticulatus and then infected with low doses of vesicular stomatitis virus (Hajnicka et al. 1998). Viral yield was increased by 100- to 1000-fold 16–23 h post-infection compared with untreated cultures, and corresponded with the earlier appearance of vesicular stomatitis virus nucleocapsid protein. This was the first published evidence that tick SGE can promote virus replication in vitro. A follow-up study showed that the effect was likely to result from inhibition of the antiviral effect of IFN by salivary gland products (Hajnicka et al. 2000). The suppressive effect was most apparent when cells were treated with SGE prior to virus infection, suggesting that the effect may be directed against the IFNα/β receptor rather than a direct interaction with IFN.

IFNα/β-induced viral resistance is mediated by antiviral factors such as Mx gene products that are active against viruses of several different families including the Orthomyxoviridae, Bunyaviridae and Togaviridae. The interferon-induced mouse Mx1 protein has intrinsic antiviral activity against influenza A and B viruses, and the tick-borne orthomyxovirus, Thogoto virus (Haller et al. 1995). Mice carrying the Mx1 gene are resistant to Thogoto virus infection by needle injection. However, they are susceptible to tick-borne virus challenge (non-viraemic transmission from infected to uninfected co-feeding ticks) and, to a lesser degree, injection of virus mixed with tick SGE (Dessens & Nuttall, 1998). These data are consistent with the ability of tick SGE to interfere with the antiviral action of IFNα/β. Cells exposed to tick saliva in the skin of Mx1⁺ mice may not respond to released interferon (through the putative effect of the saliva on the IFNα/β receptor) thus preventing induction of the Mx1 protein (a large nuclear GTPase) and the resulting antiviral state. The biological significance of these observations is unknown, particularly as Thogoto virus produces a potent IFNα/β antagonist of its own (Hagmaier et al. 2003). However, it is curious that Mx1-based resistance is found in approximately 75% of wild mice and yet no murine influenza viruses have been recorded, begging the question why mice should have a potent defence mechanism against influenza A and B viruses (Haller, Acklin & Staehli, 1987; Haller et al. 1995). Inhibition of IFNα/β appears to be a good candidate for the SAT factor of Thogoto virus. However, studies to date have been confined to D. reticulatus and need to be extended to the vector of Thogoto virus, R. appendiculatus.

In addition to the action on IFN (described above), several studies have reported effects of tick feeding or tick salivary gland products on cytokine expression or activities (see chapter by Brossard & Wikel in this Supplement). Many of the effects on cytokine activities appear to be due to a wealth of cytokine binding molecules produced by ixodid tick species and secreted in their saliva (Gillespie et al. 2001; Hajnická et al. 2001 and unpublished data). Cytokine binders react with IL-2, IL-4, IL-8, MCP-1, MIP-1α, RANTES and eotaxin, and probably more. The IL-2 binding protein of I. scapularis provides a mechanism for suppressing T cell proliferation and other IL-2-stimulated immune responses (Gillespie et al. 2001). The IL-8 binder of D. reticulatus out-competes IL-8 receptors on neutrophils, inhibiting IL-8-induced chemotaxis of neutrophils (Hajnická et al. 2001). Biological activities of other putative cytokine binders have not yet been investigated. Nevertheless, the apparent strategy of manipulating the cytokine network most likely overcomes redundancy in this innate immune system, and should greatly facilitate blood feeding.

By binding different cytokines, ixodid ticks provide a potential gateway for tick-borne pathogens. To what extent this gateway is exploited remains to be determined. The only indication to date is that inoculation of C3H/HeJ mice with a mixture of TNF-α, IFN-γ and IL-2 at the time of tick feeding suppressed B. burgdorferi transmission by I. scapularis, suggesting that cytokine manipulation by ticks might aid borrelia transmission (Zeidner et al. 1996). Clearly, the extent to which ticks manipulate the cytokine network, and the consequences for tick-borne pathogen transmission, need to be explored.

Leukocyte modulators

Numerous effects of tick saliva or SGE on T cell and macrophage function have been described in vitro (see chapter by Wikel & Brossard in this Supplement). Many of them are mediated through the effects on cytokines. The potential benefits to tick-borne pathogens are obvious but the real benefits of leukocyte modulators have not been elucidated. For example, neutrophils phagocytize B. burgdorferi hence the ability of their tick vector's saliva to inhibit neutrophil function could be highly significant (Ribeiro, Weis & Telford, 1990; Suhonen, Hartiala & Viljanen, 1998). Langerhans cells are thought to play a key role in non-viraemic transmission of TBEV (see ‘The “red herring” hypothesis’ in Nuttall & Labuda, 2003). Obviously the effect of SGE on Langerhans cells needs to be examined in the search for the SAT factor of TBEV. The function of the homologue of Macrophage Migration Inhibitory Factor (MIF, a mammalian pro-inflammatory cytokine) is unknown. It has been detected in both salivary glands and midgut tissues of unfed and feeding A. americanum adult females and, in vitro, inhibited macrophage migration (Jaworski et al. 2001). Potentially it may reduce macrophage microbicidal activity.

Tick infestation is characterized by polarization towards a Th2 response. This appears to benefit tick-borne pathogens such as B. burgdorferi and Babesia bovis (Zeidner et al. 1996, 1997; Christe, Rutti & Brossard, 2000; Goff et al. 2003). Indirect evidence of the potential benefit for TBEV was demonstrated by the reduction of co-feeding transmission in laboratory mice in which a Th1-like response was induced by immunization with a recombinant tick salivary gland protein (Labuda and Lickova, unpublished data). Mice repeatedly infested with I. scapularis nymphs, and showing a Th2-polarized response, became resistant to B. burgdorferi transmission indicating that the benefits of such polarization may be overcome by the host (Wikel et al. 1997).

Direct immunosuppression of B cells by I. ricinus SGE (Hannier et al. 2003) may explain the reduced ability of tick-infested hosts to produce antibodies (Wikel, 1985; Christe et al. 2000). Such immunosuppression is also likely to affect B cell production of immune regulatory cytokines such as IFNγ. B cells play a crucial role in antimicrobial immunity (Ochsenbein et al. 1999; Baumgarth, 2000). Tick-borne pathogens such as B. burgdorferi express surface B cell mitogens (Ma & Weis, 1993). Obviously, secretion of a B cell inhibitor in tick saliva, through inhibiting T cell-dependent and T cell-independent B cell activation, could provide a safe haven in the skin feeding site for tick-borne pathogens. As yet, the B cell inhibitor has not been characterized.

The role of natural killer (NK) cells includes control of viral infections through the killing of cells that express viral antigens on their surface. They may even kill extracellular bacteria. NK cells secrete cytokines, providing an important means by which innate immunity communicates with the acquired immune system (Lanier, 2000). Direct evidence of a role for NK cells during tick feeding is lacking, nevertheless tick salivary glands contain a protein that suppresses NK cell activity (Kubeš et al. 1994). Such activity was demonstrated with SGE from partially fed Dermacentor reticulatus, Amblyomma variegatum and Haemaphysalis inermis, but not from Ixodes ricinus or Rhipicephalus appendiculatus (Kubeš et al. 2002). The apparent absence of activity for the latter two important vectors suggests that control of NK cells does not play an important role in promoting tick-borne pathogen transmission, at least for these two tick species. However, a suppressive effect on NK cell activity was observed in a mouse model with SGE from partially fed female I. ricinus (Kopecky & Kuthejlova, 1998). Interactions between dendritic cells and NK cells, during the early stages of B. burgdorferi infection, influence development of a protective humoral response in mice (Mbow et al. 2001). Suppression of NK cell activity might thus affect B. burgdorferi, although possibly at a later stage than SAT.