Modulation of human lymphocyte proliferation by salivary gland extracts of ixodid ticks (Acari: Ixodidae): effect of feeding stage and sex

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1 FOLIA PARASITOLOGICA 5: , 3 Modultion of humn lymphocyte prolifertion y slivry glnd extrcts of ixodid ticks (Acri: Ixodide): effect of feeding stge nd sex Terézi Rolníková 1, Mári Kzimírová 1 nd Miln Buc 2 1 Institute of Zoology, Slovk Acdemy of Sciences, Dúrvská cest 9, SK-8456 Brtislv, Slovki; 2 Institute of Immunology, School of Medicine, Comenius University, Ssinkov 4, SK-8118 Brtislv, Slovki Key words: ticks, slivry glnds, humn lymphocytes, Rhipicephlus ppendicultus, Ixodes ricinus, Amlyomm vriegtum Astrct. Ixodid ticks remin ttched to their hosts for severl dys to weeks. During this extended feeding process new proteins involved in the modultion of host immune responses re expressed in tick slivry glnds. In our study stimultory or inhiitory effect of slivry glnd extrcts (SGE) of unfed nd prtilly fed femle Ixodes ricinus (Linneus, 1758), femle nd mle Amlyomm vriegtum (Fricius, 1794) nd Rhipicephlus ppendicultus Neumnn, 191 ticks on humn lymphocyte prolifertion induced y Concnvlin A (ConA) nd phytohemgglutinin (PHA), respectively, ws investigted. SGE of ll femle ticks exmined suppressed prolifertion of ConA-induced lymphocytes; highly significnt suppression ws oserved in the presence of unfed I. ricinus nd 9-dy fed A. vriegtum SGE. SGE of prtilly fed A. vriegtum nd I. ricinus femles suppressed PHA responses of lymphocytes. Lymphocytes showed reduced PHA nd ConA responses in the presence of SGE of unfed nd 2-dy fed R. ppendicultus femles, while SGE of 6-dy fed femles enhnced PHA responses, ut reduced their ConA responses; generlly SGE of 2-dy fed femles displyed the strongest inhiition. Amlyomm vriegtum mle SGE slightly enhnced PHA, ut significntly reduced ConA responses of lymphocytes nd their inhiitory effect incresed during feeding. SGE of unfed nd 2-dy fed R. ppendicultus mles enhnced PHA nd ConA responses nd those of 6-dy fed mles suppressed lymphocyte prolifertion. The results suggest tht (i) species- nd sex-specific differences exist in the effects of tick slivry glnd ntigens on humn lymphocyte prolifertion nd (ii) effect of SGE on humn lymphocyte responses to mitogens vries depending on the tick feeding sttus. Ticks re oligte lood-feeding ectoprsites, with mny species remining ttched to their hosts nd cquiring loodmel over period rnging from dys to weeks (Sonenshine 1991). The long contct etween tick nd its host provides host time to develop innte nd dptive host immune responses ginst the prsites. Not surprisingly, ticks hve developed numer of countermesures to evde the host immune response. Severl genes re induced in tick slivry glnds during the feeding process, leding to the expression of new proteins. These proteins re secreted into the tick sliv nd re potentilly involved in modultion of host immune nd hemosttic responses. Tick sliv contins lrge numer of phrmcologiclly ctive molecules tht possess ntihemosttic, vsoctive nd immunosuppressive properties (Rieiro 1995,, Wikel nd Bergmn 1997, Rieiro nd Frncischetti 3). Consequently, modultion of the host immune response fcilittes oth tick feeding nd trnsmission of vrious tick-orne pthogens (Wikel nd Alrcon-Chidez 1). The effect of slivry glnd extrcts (SGE) or sliv of different tick species hs een minly demonstrted in lortory models (e.g., mouse, rit, guine pig), however, tht on humn immune cells hs een limited to in vitro studies. The inhiitory effect of tick SGE on NK cells (Kueš et l. 1994, 2, Kopecký nd Kuthejlová 1998) nd on the production of nitric oxide y mcrophges (Kuthejlová et l. 1) were reported. An IL-2 inding protein ws identified in the sliv of the Lyme disese vector, Ixodes scpulris (Gillespie et l. 1). The nti-chemokine ctivity of SGE from the ixodid ticks Amlyomm vriegtum, Dermcentor reticultus nd Rhipicephlus ppendicultus ws demonstrted y Hjnická et l. (1) nd Kocáková et l. (2). It ws lso reported tht ticks suppressed production of mcrophge proinflmmtory cytokines s well s secretion of T H 1 cytokines, however, the synthesis of T H 2 cytokines ws up-regulted, indicting T H 2 polristion of the immune response (Fuchserger et l. 1995, Ferreir nd Silv 1999, Kovář et l. 1, 2, Leoulle et l. 2). The tick-induced suppression of the host immune defences is lso chrcterized y reduced ility of murine nd humn lymphocytes to proliferte in vitro in the presence of T-lymphocyte mitogens (Kovář et l. 1, Leoulle et l. 2). There re only few reports on chnges of the immunomodultory effects of tick sliv during their Address for correspondence: M. Kzimírová, Institute of Zoology, Slovk Acdemy of Sciences, Dúrvská cest 9, SK-8456 Brtislv, Slovki. Phone: ; Fx: ; E-mil: mri.kzimirov@sv.sk 35

2 feeding (e.g., Kueš et l., 2, Bior et l. 2, Rolníková et l. 2). In the present study, the effects of SGE derived from mle nd femle Amlyomm vriegtum (Fricius, 1794), Rhipicephlus ppendicultus Neumnn, 191 nd femle Ixodes ricinus (Linneus, 1758) ticks on in vitro prolifertion of humn peripherl T-lymphocytes were compred nd chnges in lymphocyte responses to SGE of unfed nd feeding ticks were investigted. MATERIALS AND METHODS Ticks nd tick slivry glnd extrcts (SGE) Adult I. ricinus were collected y flgging the vegettion in selected loclities of south-western Slovki known to e free of tick-orne encephlitis virus. Their slivry glnd homogentes were screened for Borreli urgdorferi y PCR nd only negtive smples were used in susequent ssys. Rhipicephlus ppendicultus nd A. vriegtum were otined from lortory colonies mintined t the Institute of Zoology of the Slovk Acdemy of Sciences. Slivry glnds were dissected from unfed dult ticks or ticks tht were llowed to feed in groups within retining cells ttched to the cks of lortory nimls. Femles of I. ricinus were fed on lortory mice for 2 or 5 dys, femles nd mles of R. ppendicultus were fed on guine pigs for 2 or 6 dys. Adult A. vriegtum were fed on Cliforni rits for 2 or 9 dys; hosts were lwys exposed to mles for 4 dys efore femles were dded. Ticks were gently removed from lortory nimls t different stges of feeding. Both removed nd unfed ticks were wshed in wter, rinsed quickly in 7% ethnol nd susequently in distilled wter. Their slivry glnds were dissected under chilled.15 M NCl, wshed twice with.15 M NCl nd pooled in eppendorf tues (1 5 SG pirs/5 µl.15 M NCl, depending on tick species, sex nd feeding stge) nd immeditely frozen t 7 C until required. Prior to the ssys, tches of slivry glnds were quickly thwed, homogenized using pestle nd centrifuged t 12, g for 1 min. Superntnts were removed, pellets resuspended in.15 M NCl nd re-centrifuged. Superntnts otined (SGE) were pooled nd the protein concentrtion of the SGE ws determined using the Brdford ssy (Brdford 1976). SGE were diluted in 1% RPMI 16 medium t concentrtions.1 µg,.5 µg nd 1. µg solule protein/5 µl medium. Cell culture Blood ws otined from helthy humn donors. Lymphocytes were isolted from heprinized venous lood y seprtion on Ficoll-Verogrfin grdient (specific grvity 1.78, centrifugtion t 4 g for 3 min t room temperture). Lymphocytes from the interfce were removed, wshed three times in lnced sline solution nd resuspended (1 6 cells/ml) in RPMI 16 medium supplemented with 2 mm L- glutmine, 1 U/ml penicillin, 1 µg/ml streptomycin, 1 µg/ml gentmicin, nd 1% het-inctivted humn serum. Lymphocyte suspensions (1 µl/well) were distriuted to flt-ottom 96-well pltes (TC Microwell 96, NUNC). Lymphocyte prolifertion ssy (LPA) To ech well of the 96-well plte contining lymphocyte suspension either 5 µl of RPMI medium (non-stimulted cells) or 5 µl of Concnvlin A (ConA, Sigm; 1 µg/5 µl RPMI) or phytohemgglutinin (PHA, Sigm; 5 µg/5 µl RPMI) nd 5 µl SGE (contining.1 µg,.5 µg or 1. µg solule protein/5 µl RPMI, respectively) were dded. Mitogens were dded simultneously with SGE. Lymphocyte cultures were incuted for 66 h t 37 C in 5% CO 2. To mesure cell prolifertion, 37 Bq of tritited thymidine (Amershm) ([ 3 H]TdR)/ µl 1% RPMI 16) were dded to ech well 6 h efore cell hrvest. The cells were hrvested on glss fire filters (Whtmn) using n utomtic cell hrvester (Auto-Msh, Dynex). The mount of incorported [ 3 H]TdR into DNA of proliferting lymphocytes ws determined y liquid scintilltion counting on et counter (Spectrl, LKB). All smples were tested in triplicte nd ech ssy ws repeted 6 8 times, using lood from different donors. Men disintegrtions per minute (DPM) were determined for ech smple. Asolute vlues mesured for individul donors were trnsformed to reltive vlues nd re expressed s percentge of inhiition/stimultion of cell prolifertion s compred to control lymphocytes (without ddition of SGE). The percentge of stimultion/inhiition ws clculted s follows: Percentge of stimultion/inhiition of cells without ddition of mitogens (S/I): SGE Control DPM S (I) = 1 Control DPM Percentge of stimultion/inhiition of cells with ddition of mitogens (S M /I M ): SGE mitogens DPM S (I) = 1 mitogens DPM Sttisticl nlysis The effects of tick feeding stge nd SGE concentrtions on lymphocyte prolifertion were evluted using one-wy nlysis of vrince (ANOVA), followed y the Bonferroni test for pir-wise comprisons. Responses of lymphocytes to SGE of mle nd femle ticks of the sme species nd feeding stge were compred y two-smple nlysis (t-test). For sttisticl nlyses, percentges were trnsformed to rcsine p to meet the criteri of prmetric sttisticl tests. In ll sttisticl nlyses, P.5 ws considered significnt. RESULTS The in-vitro prolifertive responses of humn lymphocytes were significntly ffected y exposure to tick slivry glnd extrcts. Different ptterns of inhiition or enhncement of prolifertion were oserved for non-stimulted nd mitogen-stimulted lymphocytes treted with SGE of the three tick species. For ech tick species, lymphocyte responses lso vried when cultured with SGE of femles nd mles s well s with SGE of unfed nd prtilly engorged individuls. Responses of lymphocytes to tick SGE were generlly dose-dependent, however, for most of the tretments the differences in percentge of stimultion/ inhiition of prolifertion of cells exposed to.1,.5 nd 1. µg SGE solule proteins were not sttisticlly significnt (Tles 1 3). Consequently, responses of 36

3 Rolníková et l.: Modultion of lymphocyte prolifertion y tick SGE Tle 1. Stimultion/inhiition of humn lymphocyte prolifertion (% chnge compred with controls) in cultures treted with vrious concentrtions of femle Ixodes ricinus slivry glnd extrcts (SGE), without mitogens nd induced y phytohemgglutinin (PHA) nd Concnvlin A (ConA). Tick feeding stge Unfed Dy 2 Dy 5 Tretment No mitogens (% chnge) PHA (% chnge) ConA (% chnge) SGE proteins (µg/well) n Men (S.E.) n Men (S.E.) n Men (S.E.) (6.) (3.2) (4.2) (8.4) (3.6) (6.9) (8.3) (2.8) (3.) (22.4) (7.3) (4.5) (9.6) (7.9) (6.6) (13.8) (6.3) (7.3) (12.2) (4.2) (3.8) (6.6) (7.3) 8 9. (4.4) (12.6) (5.5) (3.9) n numers of replictes in tretments; mens indicted y the sme superscripts etween SGE concentrtions for ech tretment re not significntly different (one-wy ANOVA followed y Bonferroni test, P.5). Tle 2. Stimultion/inhiition of humn lymphocyte prolifertion (% chnge compred with controls) in cultures treted with vrious concentrtions of femle nd mle Rhipicephlus ppendicultus slivry glnd extrcts (SGE), without mitogens nd induced y phytohemgglutinin (PHA) nd Concnvlin A (ConA). Tick feeding stge Tretment No mitogens (% chnge) PHA (% chnge) ConA (% chnge) SGE proteins (µg/well) n Men (S.E.) n Men (S.E.) n Men (S.E.) Femles (4.7) (3.5) (5.9) Unfed (7.5) (8.3) (5.9) (7.8) (7.3) (5.7) Femles (9.2) (7.7) (4.) Dy (9.2) (7.7) (4.) (6.6) (7.) (8.) Femles (4.7) (6.) (2.) Dy (6.4) (4.8) (4.3) (7.3) (1.3) (5.5) Mles (7.) (12.7) (3.1) Unfed (4.5) (16.1) (19.1) (9.4) (19.4) (11.3) Mles (7.1) (8.) (6.7) Dy (21.1) (4.8) (4.8) (31.8) 5.7 (6.1) (11.4) Mles (7.4) (6.) (6.1) Dy (6.4) (5.6) 8.9 (6.4) (7.) (4.2) (7.6) n numers of replictes in tretments; mens indicted y the sme superscripts etween SGE concentrtions for ech tretment re not significntly different (one-wy ANOVA followed y Bonferroni test, P.5). lymphocytes to SGE of the three tick species nd sexes nd to SGE from vrious feeding stges were further evluted only for the dose 1. µg. Responses of non-stimulted lymphocytes to SGE of unfed I. ricinus femles were suppressed, ut they were enhnced y SGE of prtilly engorged ticks (Fig. 1). In contrst, no significnt differences etween responses of non-stimulted cells were found when treted with SGE of unfed nd prtilly fed R. ppendicultus femles (Fig. 2) nd A. vriegtum mles (Fig. 3). SGE derived from unfed nd 6-dy fed R. ppendicultus mles suppressed cell prolifertion, however, SGE of 2-dy fed mles hd 94.5% stimultory effect. Consequently, differences etween the effect of SGE of 2-dy fed mle nd femle R. ppendicultus were highly significnt (Fig. 2). SGE of femle A. vriegtum suppressed cell prolifertion nd this effect significntly incresed with feeding, ut, except unfed ticks, the differences etween sexes were not significnt (Fig. 3). Responses of lymphocytes induced y mitogens were suppressed y ll feeding stges of I. ricinus, ut SGE of unfed ticks hd the strongest effect (Fig. 1). In mjority of tretments, responses of mitogen-stimulted lymphocytes to mle nd femle SGE of the sme 37

4 Tle 3. Stimultion/inhiition of humn lymphocyte prolifertion (% chnge compred with controls) in cultures treted with vrious concentrtions of femle nd mle Amlyomm vriegtum slivry glnd extrcts (SGE), without mitogens nd induced y phytohemgglutinin (PHA) nd Concnvlin A (ConA). Tick feeding stge Tretment No mitogens (% chnge) PHA (% chnge) ConA (% chnge) SGE proteins (µg/well) n Men (S.E.) n Men (S.E.) n Men (S.E.) Femles (6.9) (5.5) 8.1 (9.8) Unfed (1.3) (11.3) (1.5) (17.6) (7.9) 6.6 (6.7) Femles (1.7) (5.7) (4.4) Dy (4.3) (5.3) (5.8) (5.5) (4.8) (6.3) Femles (4.8) (6.9) (4.8) Dy (1.9) (7.3) (7.9) (8.2) (7.5) (8.4) Mles (5.4) (5.) (5.3) Unfed (5.1) (2.9) (6.1) (8.4) (5.2) (3.9) Mles (6.6) (5.6) (4.8) Dy (6.4) (6.2) 8.4 (4.1) (7.4) 6.3 (8.5) (3.9) Mles (2.1) (2.3) (4.1) Dy (3.1) (3.) (2.9) (7.8) (7.5) (3.5) n numers of replictes in tretments; mens indicted y the sme superscripts etween SGE concentrtions for ech tretment re not significntly different (one-wy ANOVA followed y Bonferroni test, P.5). Percent stimultion/inhiition 6 8 Ixodes ricinus femles Unfed Dy 2 Dy 5 species significntly differed within feeding sttus (Figs. 2, 3). Prolifertive responses of mitogen-induced lymphocytes to SGE of R. ppendicultus lso differed etween the two mitogens pplied (Fig. 2). While prolifertion of Con-A-induced lymphocytes ws suppressed y R. ppendicultus femle SGE, prolifertion of PHA-induced lymphocytes ws suppressed only y 2-dy fed femles. SGE of unfed nd 2-dy fed mles hd stimultory effect on mitogen-induced prolifertion, nd only 6-dy fed mles suppressed prolifertion. Responses of mitogen-induced lymphocytes to A. vriegtum SGE lso vried with feeding stge nd sex (Fig. 3). Prolifertion of lymphocytes ws enhnced when treted with SGE of unfed nd 2-dy fed femles, ut it ws suppressed y SGE of 9-dy fed femles. The suppressive effect of mle A. vriegtum SGE on Con- A-stimulted prolifertion significntly incresed with durtion of feeding, however, this effect ws not oserved in PHA-stimulted lymphocytes. Fig. 1. Prolifertion of humn lymphocytes (% chnge compred with control lymphocytes) fter tretment with 1 µg of SGE proteins of unfed nd prtilly fed Ixodes ricinus femles. Lymphocytes were non-stimulted with mitogens (Lymph) or stimulted with PHA nd ConA. Vlues represent mens ± SEM. Mens within tretment indicted y the sme letters re not significntly different (one-wy ANOVA followed y Bonferroni test, P.5). Results of ANOVA: non-stimulted lymphocytes (F = 12.79, P <.1); lymphocytes stimulted with PHA (F = 3.5, P <.1); lymphocytes stimulted with ConA (F =.62, P <.1). DISCUSSION Ticks hve evolved vrious strtegies to evde the immune response of hosts they feed on. Biologiclly ctive compounds in their slivry glnds ply the min role in immunosuppression nd immunomodultion. Tick-induced immunosuppression of the host is chrcterized y decresed primry ntiody responses to T-cell-dependent ntigens, decresed production of T H 1 cytokines (IL-2, IFN-γ) nd enhnced production of T H 2 cytokines (IL-4, IL-5, IL-6, IL-1, IL-13) (Willdsen nd Jongejn 1999, Gillespie et l., Schoeler nd Wikel 1, Wikel nd Alrcon-Chidez 1, Rieiro nd Frncischetti 3). 38

5 Rolníková et l.: Modultion of lymphocyte prolifertion y tick SGE Rhipicephlus ppendicultus femles Amlyomm vriegtum femles Percent stimultion/inhiition 6 8 n.s. ** * ** ** ** ** ** n.s. Percent stimultion/inhiition ** n.s. n.s. ** ** * ** ** ** c c Unfed Dy 2 Dy 6 Unfed Dy 2 Dy 9 Rhipicephlus ppendicultus mles Amlyomm vriegtum mles Percent stimultion/inhiition Percent stimultion/inhiition 8 c Unfed Dy 2 Dy 6 Unfed Dy 2 Dy 9 Fig. 2. Prolifertion of humn lymphocytes (% chnge compred with control lymphocytes) fter tretment with 1 µg of SGE proteins of unfed nd prtilly fed R. ppendicultus femles nd mles. Lymphocytes were non-stimulted with mitogens (Lymph) or stimulted with PHA nd ConA. Vlues represent mens ± SEM. Mens within tretment indicted y the sme letters re not significntly different (one-wy ANOVA followed y Bonferroni test, P.5). Results of ANOVA for femle ticks: non-stimulted lymphocytes (F = 1.31, n.s.); lymphocytes stimulted with PHA (F = 26., P <.1); lymphocytes stimulted with ConA (F = 5.67, P <.5). Results of ANOVA for mle ticks: non-stimulted lymphocytes (F = 12.5, P <.1); lymphocytes stimulted with PHA (F = 19.13, P <.1); lymphocytes stimulted with ConA (F = 21.65, P <.1). Differences etween responses to mle nd femle SGE re indicted ove or elow the vlues for femles s n.s. (not significnt), * P <.5 or ** P <.1 (t-test). Fig. 3. Prolifertion of humn lymphocytes (% chnge compred with control lymphocytes) fter tretment with 1 µg of SGE proteins of unfed nd prtilly fed A. vriegtum femles nd mles. Lymphocytes were non-stimulted with mitogens (Lymph) or stimulted with PHA nd ConA. Vlues represent mens ± SEM. Mens within tretment indicted y the sme letters re not significntly different (one-wy ANOVA followed y Bonferroni test, P.5). Results of ANOVA for femle ticks: non-stimulted lymphocytes (F = 78.4, P <.1); lymphocytes stimulted with PHA (F = , P <.1); lymphocytes stimulted with ConA (F = 39.26, P <.1). Results of ANOVA for mle ticks: nonstimulted lymphocytes (F = 3.8, n.s.); lymphocytes stimulted with PHA (F = 4.4, P <.5); lymphocytes stimulted with ConA (F = 33.48, P <.1). Differences etween responses to mle nd femle SGE re indicted ove or elow the vlues for femles s n.s. (not significnt), * P <.5 or ** P <.1 (t-test). Inhiition of T-cell responsiveness to ConA could result from the direct effect of slivry glnd proteins on lymphocytes or from their production of IL-1 (Wikel 1999, Schoeler nd Wikel 1, Wikel nd Alrcon- Chidez 1). The suppression of T-lymphocyte prolifertion cn lso e due to prostglndin E 2 which inhiits IL-2 nd IFN-γ production nd T-lymphocyte prolifertion nd is present in high concentrtions in tick sliv (Bowmn et l. 1996). PGE 2 inhiits in vitro cytokine production y T H 1 cells; however, it hs no effect on T H 2 cells. Up-regultion of IL-4 nd IL-1 proly leds to the development of T H 2 response, 39

6 resulting in reduction of the T H 1 response (Rmchndr nd Wikel 1992, Wikel 1999, Kovář et l. 1, Schoeler nd Wikel 1, Wikel nd Alrcon- Chidez 1). Tick-medited suppression of the T H 1-lymphocyte rectivity my inhiit expnsion of ntigen specific T- lymphocyte clones (IL-2), differentition of B-lymphocytes (IL-4, IL-6, IL-13), ctivtion of mcrophges (IFN-γ), nd nturl killer cell ctivity (IL-12, IL-15, IFN-γ). The tick-induced T H 2 cytokine profile seems to e dvntgeous for the survivl of the tick ecuse of the nti-inflmmtory effect of T H 2 cytokines. These nti-inflmmtory mechnisms my lso enhnce the trnsmission of tick orne pthogens (Schoeler nd Wikel 1, Wikel nd Alrcon-Chidez 1). In our experiments, Rhipicephlus ppendicultus, Amlyomm vriegtum nd Ixodes ricinus SGE modulted prolifertion of humn peripherl lood T-lymphocytes in vitro in vrious wys, depending on feeding stge nd sex. In mjority of tretments, responses of lymphocytes to tick SGE when stimulted with mitogens simultneously or 2 h fter exposure to SGE showed no significnt differences (Rolníková et l., unpulished results). The suppressive effect of the R. ppendicultus SGE on the production of numer of cytokines (IFN-α, IFN-γ, IL-1, IL-5, IL-6, IL-7 nd IL-8) in humn leucocytes hs lredy een demonstrted (Fuchserger et l. 1995). Kueš et l. () reported tht slivry glnd extrcts derived from A. vriegtum femles decresed in vitro nturl killer (NK) ctivity of helthy persons. Hjnická et l. (1) showed tht the A. vriegtum SGE reduced the level of IL-8, resulting in inhiition of chemotxis of neutrophils. As the SGE-induced recruitment of leucocytes provides defence ginst prsitic ttck, the nti-il-8 ctivity my fcilitte tick lood-feeding. Additionlly, Kocáková et l. (2) reported tht R. ppendicultus nd A. vriegtum SGE reduced levels of oth, IL-8 nd MIP-1α, chemokine responsile for chemotxis of humn monocytes. In generl, in-vitro prolifertive responses of mouse, humn, or cttle lymphocytes to SGE of prtilly engorged femle ticks were found to e suppressed (Bergmn et l. 1995, Schoeler et l., Kovář et l. 1, Turni et l. 2), however, no dt hve een ville on the effects of SGE derived from unfed femle or mle ixodid ticks. Sexul ehviour of some tick species evokes specultion out possile coopertion etween sexes in the interction with the host (Bior et l. 2). The specific function of mle R. ppendicultus ticks in femle tick feeding hs een reported (Wng et l. 1999). Rhipicephlus ppendicultus mle ticks mte on the host with the feeding femles, nd then help their mtes to complete successful engorgement y secreting immunosuppressive sliv components into the co-feeding site. Differences in mle nd femle SGE protein profiles presented y Gšperík et l. () demonstrted the existence of sexul dimorphisms in R. ppendicultus, Dermcentor reticultus nd A. vriegtum. The support of femle tick feeding y mle SGE indictes different functions of sliv from oth sexes nd the effective coopertion etween mles nd femles during their feeding on the host. The different role of the sexes in host immunomodultion is lso supported y our study, showing, in some cses, controversil responses of humn lymphocytes to mle nd femle SGE isolted from R. ppendicultus nd A. vriegtum, respectively. The strong stimultion of lymphocytes y 2-dy fed R. ppendicultus mles is lso very interesting. The results of different, sometimes quite opposite effects of mle nd femle SGE on lymphocyte prolifertion points to the importnce of coopertion of sexes during their feeding on the host. The mechnism how femle nd mle ixodid ticks modulte the immune response of their hosts needs to e studied in more detil. Our experiments with SGE of I. ricinus femles, derived from vrious stges of engorgement, showed their inhiitory effect on PHA- nd ConA-induced prolifertion of humn peripherl lood T-lymphocytes. Very interesting is our finding on the strongest inhiitory effect of SGE of unfed I. ricinus femles. The presence of immunosuppressive sustnces in unfed ticks is not surprising, s they hve to counterct host defences s soon s they ttch to their hosts. Also the expression of novel proteins during engorgement of hrd ticks is well known, leding to production of wide rry of iologiclly ctive compounds in their slivry glnds (Sonenshine 1991, Suer et l. 1995). We treted humn lymphocytes with certin concentrtions of solule SGE proteins (i.e. cocktil of immunosuppressive s well s ntigenic compounds). Bsed on our results we suppose tht the rtio of immunosuppresive compounds in I. ricinus SGE chnges during feeding, eing proly lower in prtilly engorged ticks. Recently, Leoulle et l. (2) hve detected novel protein immunosuppressor (Iris) in the SGE of 5-dy fed femle I. ricinus. They found tht expression of Iris ws induced in the slivry glnds of I. ricinus during the feeding process nd tht Iris ws secreted into the tick sliv. It suppressed T-lymphocyte prolifertion nd induced T H 2 type immune response nd inhiited the production of pro-inflmmtory cytokines (IL-6 nd TNF). Kovář et l. (1) investigted the tick-medited (I. ricinus SGE) modultion of humn T-cell prolifertion nd cytokine synthesis using humn peripherl lood mononucler cells (PBMCs) stimulted with ConA or LPS. The SGE significntly inhiited the in vitro production of IL-2 nd IFN-γ. In contrst, the production of IL-4, IL-6, nd IL-1 ws significntly incresed. In susequent study the uthors showed tht the SGE of prtilly engorged I. ricinus inhiited T-lymphocyte prolifertion (Kovář et l. 2). 31

7 Rolníková et l.: Modultion of lymphocyte prolifertion y tick SGE In generl, our results confirmed the suppressive effect of the SGE from prtilly engorged femle ixodid ticks tested on mitogen-stimulted humn peripherl lood T-lymphocyte prolifertion. In contrst to I. ricinus, stimultory effect ws oserved in unfed nd 2- dy fed A. vriegtum femles, while 9-dy fed femles strongly suppressed lymphocyte prolifertion. We suggest tht this is not the consequence of sence of immunosuppressive compounds in the sliv of unfed ticks, ut more proly results from the undnce of compounds hving no immunosuppressive properties. In R. ppendicultus femles nd A. vriegtum mles, differences in responses of PHA- nd ConAstimulted lymphocytes hve een found. These differences could result, in prt, from different effects of SGE on vrious susets of T-lymphocytes, s PHA stimultes preferentilly CD4 +, while ConA stimultes more CD8 + T-lymphocytes (e.g. Buc 1). However, these questions should e ddressed in susequent studies deling with effects of tick SGE on prolifertion of vrious susets of T-lymphocytes. Acknowledgements. The reserch ws finncilly supported y Evolutec Ltd. nd prtly y Grnt VEGA, No. 2/1129/21, from the Slovk Acdemy of Sciences nd Project APVT The uthors wish to thnk V. Trgeľová for screening tick smples y PCR. REFERENCES BERGMAN D.K., RAMACHANDRA R.N., WIKEL S.K. 1995: Dermcentor ndersoni: slivry glnd proteins suppressing T-lymphocyte responses to Concnvlin A in vitro. Exp. Prsitol. 81: BIOR A.D., ESSENBERG R.C., SAUER J.R. 2: Comprison of differentilly expressed genes in the slivry glnds of mle ticks, Amlyomm mericnum nd Dermcentor ndersoni. Insect Biochem. Mol. Biol. 32: BOWMAN A.S., DILLWITH J.W., SAUER J.R. 1996: Tick slivry prostglndins: presence, origin nd significnce. Prsitol. Tody 12: BRADFORD M.M. 1976: A rpid nd sensitive method for the quntittion of microgrm quntities of protein utilizing the principle of protein-dye inding. Anl. Biochem. 72: BROSSARD M., WIKEL S.K. 1997: Immunology of interctions etween ticks nd hosts. Med. Vet. Entomol. 11: BUC M. 1: Imunológi. Ved, Brtislv, 454 pp. FERREIRA B.R., SILVA J.S. 1999: Successive tick infesttions selectively promote T-helper 2 cytokine profile in mice. Immunology 96: FUCHSBERGER N., KITA M., HAJNICKÁ V., IMANISHI J., LABUDA M., NUTTALL P.A. 1995: Ixodid tick slivry glnd extrcts inhiit production of lipopolyscchride-induced mrna of severl different humn cytokines. Exp. Appl. Acrol. 19: GAŠPERÍK J., KOCÁKOVÁ P., SLOVÁK M., HAJNICKÁ V., FUCHSBERGER N., NUTTALL P.A. : Differences in slivry glnd extrcts derived from mle nd/or femle ticks. In: M. Kzimírová, M. Lud nd P.A. Nuttll (Eds.), Proceedings of the 3 rd Interntionl Conference Ticks nd Tick-orne Pthogens: Into the 21 st Century. Slovk Acdemy of Sciences, Brtislv, pp GILLESPIE R.D., DOLAN M.C., PIESMAN J., TITUS R.G. 1: Identifiction of n IL-2 inding protein in the sliv of the Lyme disese vector tick, Ixodes scpulris. J. Immunol. 166: GILLESPIE R.D., MBOW M.L., TITUS R.G. : The immunomodultory fctors of loodfeeding rthropod sliv. Prsite Immunol. 22: HAJNICKÁ V., KOCÁKOVÁ P., SLÁVIKOVÁ M., SLOVÁK M., GAŠPERÍK J., FUCHSBERGER N., NUTTALL P.A. 1: Anti-interleukin-8 ctivity of tick slivry glnd extrcts. Prsite Immunol. 23: KOCÁKOVÁ P., HAJNICKÁ V., SLÁVIKOVÁ M., SLO- VÁK M., GAŠPERÍK J., VANČOVÁ I., FUCHS- BERGER N., NUTTALL P.A. 2: Anti-chemokine ctivity of tick slivry glnd extrcts. In: 4 th Interntionl Conference on Ticks nd Tick-Borne Pthogens, July 2, The Bnff Centre, Bnff, Alert, Cnd, Progrm nd Astrcts, p. 94. KOPECKÝ J., KUTHEJLOVÁ M. 1998: Suppressive effects of Ixodes ricinus slivry glnd extrct on mechnisms of nturl immunity in vitro. Prsite Immunol. : KOVÁŘ L., KOPECKÝ J., ŘÍHOVÁ B. 1: Slivry glnd extrct from Ixodes ricinus tick polrizes the cytokine profile towrd Th2 nd suppresses prolifertion of T lymphocytes in humn PBMC culture. J. Prsitol. 87: KOVÁŘ L., KOPECKÝ J., ŘÍHOVÁ B. 2: Slivry glnd extrct from Ixodes ricinus tick modultes the host immune response towrds the Th2 cytokine profile. Prsitol. Res. 88: KUBEŠ M., FUCHSBERGER N., LABUDA M., ŽUFFOVÁ E., NUTTALL P.A. 1994: Slivry glnd extrcts of prtilly fed Dermcentor reticultus ticks decrese nturl killer cell ctivity in vitro. Immunology 82: KUBEŠ M., KOCÁKOVÁ P., SLOVÁK M., FUCHS- BERGER N., NUTTALL P.A. : Slivry glnd extrcts of different prtilly fed ticks ffects humn nturl killer cell ctivity in different mnner. In: M. Kzimírová, M. Lud nd P.A. Nuttll (Eds.), Proceedings of the 3 rd Interntionl Conference Ticks nd Tick-orne Pthogens: Into the 21 st Century. Slovk Acdemy of Sciences, Brtislv, pp KUBEŠ M., KOCÁKOVÁ P., SLOVÁK M., SLÁVIKOVÁ M., FUCHSBERGER N., NUTTALL P.A. 2: Heterogeneity in the effect of different ixodid tick species on humn nturl killer cell ctivity. Prsite Immunol. 24: KUTHEJLOVÁ M., KOPECKÝ J., ŠTĚPÁNOVÁ G., MACELA A. 1: Tick slivry glnd extrct inhiits 311

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