Antihemostatic, antiinflammatory, and immunosuppressive properties of the saliva of a tick, Ixodes dammini

Hematophagous organisms must overcome host hemostasis in order to locate blood and maintain its flow during ingestion (1, 2). Platelet aggregation provides the main hemostatic obstacle because duration of bleeding of lacerated small vessels depends mainly upon platelet function (3, 4). Important stimuli inducing platelet aggregation include ADP (released by injured cells), collagen fibrils (exposed in subendotheliai tissues), thrombin (produced after activation of the coagulation cascade) and PAF I (platelet-aggregating factor, released by leuko-cytes). Activated platelets may release thromboxane A2 (a potent vasoconstrictor and platelet-aggregating stimulus), ADP, and serotonin (which further enhance platelet aggregation and vessel constriction) (5). In addition, platelet-derived factors will also contribute to thrombin formation and clotting, thus providing rigidity to the platelet plug (4). Thus, one can anticipate that any antihemostatic properties of blood-feeding arthropods will focus mainly on platelet aggregation and, secondarily, on vascular contraction or coagulation. Hard ticks feed solely on blood, each engorgement generally extending over at least several days, thereby providing ample time for inflammation to promote hemostasis at the feeding site, while increasing the tick's need to antagonize this process. Leukocyte-derived products, particularly PAF, would enhance hemosta-sis at the point of tick attachment. Immune mechanisms may further reduce feeding success by enhancing inflammatory reactions (6, 7). Interestingly, saliva of some ticks includes an anticoagulant, an antihistamine, and prostaglan-dins(PG); these compounds may facilitate feeding (8). Other enzymes, including esterases and glycosidases have been identified, but their functions remain unknown (8). Antiplatelet activity, however, has not been described. Their prolonged period of contact with a narrow range of hosts suggests that hard ticks may possess effective antihemostatic mechanisms peculiarly adapted to interfere with the inflammatory mechanisms of particular hosts. Accordingly, we sought to describe the salivary armamentarium of such a tick, and to ascribe

function to its various components. In particular, we attempted to identify platelet antiaggregating activity, as well as antiinflammatory components in saliva harvested from Ixodes dammini.

Materials and Methods
Materials. PG were kindly provided by Dr. J. E. Pike (Upjohn Co. Kalamazoo, MI).
Glucose and inorganic salts used were American Chemical Society-standard. Other reagents were obtained from Sigma Chemical Co., St. Louis, MO. Anti-Thy-1 monoclonal antibody (mAb) was kindly provided by Dr. Ethan Shevach (Laboratory of Immunology, National Institutes of Health (9).
Source of Ticks. Adult I. dammini ticks were collected by flagging on Great Island, in West Yarmouth on Cape Cod in southern Massachusetts, during the spring of 1984.
Harvest of Saliva. Piiocarpine was used to induce ticks to salivate (10,11). Ticks were permitted to engorge for 4-5 d on the ear of a rabbit, after which they were removed by traction. Ticks weighed 25-175 mg. To collect saliva, 1 #1 of a 5% (wt/vol) pilocarpine hydrochloride solution in 0.7 M NaCI was injected through glass micropipettes near the corner of the scutum while the ticks were restrained on glass slides by adhesive tape. Saliva was collected in glass tubes throughout 2 h postinjection at 35°C in a humid chamber. 1-25 mg of saliva were collected per tick, and samples were stored at -27°C. Collections <3 mg were pooled until ___ 3 mg was obtained. <5% of samples were small enough to require such pooling. Platelet Aggregation. Platelet aggregation was monitored in an aggregometer equipped with a 0.1 ml cuvette (12,13) using human citrated platelet-rich plasma (0.38% final citrate). Five or more saliva samples were pooled for each experiment.

Results
Platelet Antiaggregating Activity. To detect platelet antiaggregating activity, 4 ~1 of tick saliva were added to 100 #1 of citrated human plateletorich plasma in an aggregometer cuvette. Platelet aggregation was triggered by adding ADP, collagen suspension, or PAF, all used in concentrations that triggered maximum platelet response. A minor and transient episode of platelet aggregatioo followed addition of saliva to the platelet-rich plasma (Figs. 1-3), but subsequent aggregation was aborted or delayed. We conclude that the saliva of this tick contains antiplatelet activity that is effective against the main stimuli of platelet aggregation anticipated at the tick's feeding site.
Anticoagulant Activity. Because coagulation not only consolidates the already formed platelet plug, but contributes to its formation by promoting platelet aggregation (4-5), we sought evidence of an anticoagulant in the tick saliva. First, we determined whether saliva may delay recalcification time of citrated human plasma. Samples that normally clotted in 88 + 1 s, clotted in 131 _+ 10 s when 2.5 t~i of saliva were added (k _+ SE; n = 6; P < 0.01, paired t test). Addition of saliva did not affect prothrombin time. Because this assay depends on factors VII, X, and thrombin as well as fibrinogen, we conclude that saliva delays coagulation by acting on the intrinsic pathway of the coagulation cascade before factor X activation. This activity may help prevent coagulation and thrombin formation at the feeding site.
Apyrase Activity. Apyrase enzymes have recently been described in the saliva of blood-sucking bugs (26), tse-tse flies (28), and mosquitoes (29), where they account, at least in part, for the anti-platelet-aggregating properties of the saliva of these unrelated blood-feeding insects. Accordingly, we investigated whether L dammini may have evolved a similar antiplatelet system. Indeed, tick saliva hydrolyzed both ATP and ADP (Table I), but not AMP (not shown), and this characterizes apyrase activity. When estimating this activity, provision was made for the presence of 2.5 + 0.3 mM orthophosphate (n = 10) in the saliva.  Preliminary characterization of this enzyme activity in pooled saliva indicated a requirement for divalent cations, and that Mg ++ was a better activator than Ca ++. Optimum pH was 8.5-9.0 both for ATP and ADP hydrolysis, and both activities were stimulated by 20 mM 2-mercaptoethanol. The ratio of ATP to ADP hydrolysis at pH 7.5 was 1.10 __. 0.04 to 1 (n --10), demonstrating close correlation between the two hydrolytic activities, despite a broad range of activity when individual samples were compared (Table I). We conclude that tick saliva contains an enzyme with apyrase activity.
PGE2 Characterization. Anti-platelet-aggregating activity seemed too great to be explained solely by apyrase, particularly the powerful inhibition of PAFinduced platelet aggregation (Fig. 3) (Fig. 5) and the slope of the regression line (0.88) did not differ from unity (as shown by analysis of variance). This suggests that most, if not all, rat fundus-contracting activity can be attributed to PGE2.
Immunosuppressive Activity. Because PGE~ is immunosuppressive (23), we asked whether tick saliva inhibits T lymphocyte activation. In this system, a cloned T cell hybridoma, ES.A 1 was activated by the anti-Thy-1 mAb to secrete IL-2. The presence of IL-2 in conditioned media from E8.A1 cells was deter-      (Table II) show that addition of <1 #1 of saliva from four individual ticks caused marked suppression of IL-2 secretion by the T cell hybridomas. Although accurate quantitative comparisons cannot be made on the basis of this data, the degree of suppression of IL-2 secretion is in the general range expected from the measured quantities of PGE~ in these saliva specimens (38). We tentatively conclude that the suppression of IL-2 production by tick saliva can be accounted for by their content of PGE2.

Effect of Tick Saliva on the Production of lL-2 by ES.A1 T Hybridoma Cells Stimulated by Anti-Thy-1 mAb as Detected by [SH]Thymidine Uptake by IL-2dependent Cells
Kininase Activity. PGE2 is hyperalgesic, increasing the sensitivity of a lesion to the pain-producing effects of bradykinin (24), which would stimulate hosts to remove ticks. PGE2 also potentiates the edema promoted by histamine, serotonin, and bradykinin (25), a reaction that is present in lesions of hosts resistant to ticks (32). But ticks feed successfully and without causing pain, possibly because tick saliva antagonizes bradykinin. To test this hypothesis, we used a guinea pig ileum assay to determine whether saliva prevented bradykinin activity that was elicited by contact activation of human plasma (21). In effect, 1 #1 of saliva prevented contractions brought on by the addition of 0.1 ml of citrated plasma plus kaolin to the preparation (Fig. 6). This effect is consistent with that of a kininase, because, in another demonstration, bradykinin incubated in Tyrode's solution became progressively inactivated in the presence of saliva (Fig. 7). Such activity was destroyed by heat (100 °C for 1 min), and abolished in the presence of EDTA (3 mM), cysteine (3 mM), Zn ++ (0.25 mM), or Co ++ (3 mM). The presence of this kininase in tick saliva resolves the paradox presented by the otherwise hyperalgesia-and edema-promoting activities of PGE2.

Discussion
Saliva ejected by adult I. dammini contains antihemostatic, antiinflammatory, and immunosuppressive components, properties that appear to facilitate blood~ feeding success of this tick during its prolonged period of host attachment. This combination of pharmacologically active attributes of saliva in ticks are described for the first time.
Piatelet antiaggregating activity has previously been described for bloodsucking bugs (27), tsetse flies (28), and mosquitoes (2,29) and is here reported for the first time in the saliva of a tick. Indeed, L dammini saliva inhibits platelet aggregation induced by ADP, collagen, or PAF (Figs. 1-3), thus counteracting the main expected stimuli of platelet aggregation at the tick's feeding site. Thrombin formation, a similarly anticipated factor, is prevented by a salivary anticoagulant. L dammini, thus, is well equipped to prevent host hemostasis.
The crucial role of ADP in aggregating vertebrate platelets (5,30,31), and a common requirement for blocking host hemostasis, must have promoted the apparently convergent development of salivary apyrase enzymes in such distantly related arthropods as the mosquito Aedes aegypti (29), the blood-sucking bug Rhodnius prolixus (26), the tse-tse fly Glossina tachinoides (28), and the tick L dammini. The observed apyrase activity may account, at least in part, for the inhibitory effect of saliva on platelet aggregation. Because platelet aggregation is redundantly stimulated (4,5), effective prevention of hemostasis requires a redundant pharmacological cocktail. PGE2 (22), and possibly other as yet unidentified components, may contribute to the inhibitory effect of saliva on platelet aggregation.
Salivary apyrase may promote other effects important to a tick's successful feeding. In addition to inhibiting hemostasis by degrading ADP, salivary apyrase may prevent those inflammatory processes stimulated by ATP (34), including mast cell degranulation (35), and aggregation of neutrophils (36). These processes are associated with release of prohemostatic compounds such as thromboxane, PAF, and vasoactive amines (33). Apyrase converts ATP to AMP, which is pharmacologically inactive or even inhibitory to purinergic P2 receptors (37). Thus, apyrase contributes to antiinflammatory as well as antihemostatic activity.
The cardinal signs of inflammation, erythema, edema, and pain, each may affect the outcome of a tick's attempt to draw blood from its host. The erythematous reaction would help by increasing the flow of blood to the feeding site (8,32). Edema, on the other hand, reduces blood flow or induces bleb formation, as in the skin of cattle resistant to Boophilus microplus, but not in succeptible animals (32). Pain, which focuses attention of the host on the parasite's feeding site, would increase grooming behavior. Because inflammation both helps and hinders feeding, ticks may modulate inflammation selectively.
One salivary component, PGE~, presents a particular problem in this regard by producing a spectrum of effects that may hinder as well as help feeding. The helpful category includes erythema (increasing the flow of blood to the feeding tick), inhibition of mast cell degranulation (which helps minimize release of platelet-aggregating, edema-promoting, and vasoconstrictive factors) (33), and immunosupression (23) (potentially preventing the production of antibodies against salivary antigens). On the other hand, PGE2 potentiates pain produced by bradykinin (24), as well as edema caused by substrates that increase vascular permeability (25). The tick's salivary kininase may counteract these "undesirable" PGE~ side effects by destroying bradykinin. In this manner, ticks antagonize their host's hemostatic and inflammatory responses by the actions of several distinct salivary components.
We found that I. dammini saliva is immunosuppressive, as measured by inhibition of T hybridoma activation. This inhibitory activity can be explained by the PGE2 content of saliva, although detailed quantitative studies have not yet been performed. This evidence may provide an explanation for the failure of salivary vaccines to induce host resistance to ticks (39), the poor mitogen responsiveness of T cells from tick-infected hosts (40), and the frequently described immunosuppressed state of tick-infected animals (7). Indeed, T cell activation occurs at the very beginning of the cascade of cellular events leading to antibody production, acting at the site where the antigen is deposited (41). This action of tick saliva may delay, reduce, or abolish the host's response to the tick's salivary antigens, thus reducing immune-mediated inflammatory responses at the tick's feeding site.
Tick-resistant hosts reject feeding ticks by means of immune-mediated, immediate inflammatory skin reactions (6,7). In nature, however, ticks are not rejected. But the mechanism that permits such stable, chronic associations of ticks and their hosts have not been described. It seems likely that the pharmacological armamentarium in saliva may specifically prevent antisalivary antibody production and antagonize chemical mediators of the host's inflammatory response. Different hosts have evolved characteristic methods for mediating immune and inflammatory responses. For example, rat and mouse mast cells contain important amounts of serotonin, whereas this amine may not be detectable in other mammalian mast cells (42). Some animals release more histamine than serotonin after platelet aggregation (43). Asthma in guinea pigs is mediated mostly by histamine, whereas leukotrienes predominate in the human disease (44). Stable host associations require that the tick match its host's defenses with an appropriate array of its own, suggesting that these defenses may fit as a "lock and key." These considerations support the idea that ticks, like other parasites, evade host reactions that would cause rejection, and that this adaptation is a component of host specificity.
Finally, the salivary components injected by ticks may promote invasion of the host by tick-borne pathogens. For example, by preventing macrophage activation and neutrophil activity (45), PGE~ would protect the pathogen during its initial phase of adaptation in the skin of a new host. L dammini is the vector of human babesiosis and Lyme disease (46); coinjected saliva may facilitate transmission of these newly discovered agents of human disease.

Summary
Pilocarpine-induced saliva of the tick, Ixodes dammini, inhibited platelet aggregation triggered by ADP and collagen, as well as platelet-aggregation factor. In addition, we found apyrase activity (which degrades ATP and ADP to AMP and orthophosphate) and an anticoagulant. We showed the presence of prostaglandin E2 (PGE~) by bioassay and radioimmunoassay. This saliva inhibited interleukin 2 production by T cell hybridomas, an activity consistent with that of PGE2. A kininase was demonstrated, and this may counteract the algesia-and edemapromoting properties of PGE~. Together, these salivary components produce antihemostatic, antiinflammatory, and immunosuppressive effects that may facilitate feeding, as well as transmission of tick-borne pathogens.