Effects of Ixodes scapularis and Borrelia burgdorferi on modulation of the host immune response: induction of a TH2 cytokine response in Lyme disease-susceptible (C3H/HeJ) mice but not in disease-resistant (BALB/c) mice.

Previous studies have demonstrated that both Ixodes scapularis saliva and Borrelia burgdorferi antigens modulated lymphokines and monokines in vitro. The studies presented here were designed to delineate the role of I. scapularis and B. burgdorferi in modulation of the host immune response in vivo. Infestation of C3H/HeJ mice with infected I. scapularis resulted in an up regulation of IL-4 as early as 8 days after tick infestation, while the levels of T helper cell type 1 (TH1) cytokines, interleukin-2 (IL-2) and gamma interferon (IFN-gamma), were significantly decreased by days 10 to 12. In contrast, the cytokine profile of BALB/c mice exposed to infected nymphal ticks resulted in only transient alterations in IL-4, IL-2, and IFN-gamma production throughout a 12-day period postinfestation. Although the IL-10 level was elevated in both C3H/HeJ and BALB/c mice infested with infected nymphal ticks, no significant difference in the levels of IL-10 was noted between the mouse strains. Flow-cytometric analysis demonstrated increases in the numbers of splenic B-cell and CD4+ lymphocytes in C3H/HeJ but not BALB/c mice exposed to infected ticks. Cell depletion experiments with C3H/HeJ mice demonstrated that CD4+ cells were the sole producers of IFN-gamma and IL-10 while both CD4+ and CD8+ splenocytes contributed to the production of IL-2 and IL-4. These findings suggest that B and CD4+ splenocytes are activated, increase in number, and produce a polarized TH2 response in C3H/HeJ mice exposed to infected I. scapularis. Given that C3H/HeJ mice are susceptible to Lyme disease and the initial TH2 polarization is not evident in BALB/c mice, effective control of this response may have ramifications for spirochete transmission in vivo.     

Immunosuppression and cytokine production in mice infested with Ixodes ricinus ticks: a possible role of laminin and interleukin-10 on the in vitro responsiveness of lymphocytes to mitogens.

T cells from BALB/c mice infested 9 days before with Ixodes ricinus nymphs had a suppressed response to in vitro concanavalin A (Con A) stimulation compared to cells from uninfested mice. When laminin (the main component of the extracellular matrix) was used as a coating agent, the Con A response of naive mice was characterized by a decrease in cell proliferation, whereas there was no significant effect on the mitogen response of cells from infested mice. In contrast, an increased response to lipopolysaccharide (LPS) was observed when assaying lymph node cells of infested mice, probably reflecting an increase in B-lymphocyte number or activity. LPS cell stimulation was not modified by laminin. Supernatants of lymph node cells, taken 9 days after the first infestation of mice, stimulated with Con A in vitro, contained interleukin-10 (IL-10) but no significant levels of IL-5 as tested by enzyme-linked immunosorbent assay. At this stage of the infestation all T cells reactive with tick antigens generated in lymph nodes that drain the tick fixation site, were CD4+ cells, as determined by CD4+ depletion. With cells taken 9 days after the third infestation an increase of IL-5 and IL-10 was observed. The IL-10 levels were higher than the IL-5. According to these observations, we conclude that the reduction of T-cell proliferation in response to Con A observed in lymph node cells from infested mice, may be due to the combined effect of laminin interaction with T lymphocytes during migration and IL-10 production by these lymphocytes.     

Immunosuppressive effects of<Emphasis Type="Italic"> Ixodes ricinus</Emphasis> tick saliva or salivary gland extracts on innate and acquired immune response of BALB/c mice

Saliva and salivary gland extract (SGE) of Ixodes ricinus ticks have suppressive effects on the innate immune response of BALB/c mice. Tick saliva prevents hemolysis of sheep red blood cells (SRBC) by the human alternative pathway of complement. The adaptive immune response is also modulated by tick antigens (saliva or SGE). When stimulated in vitro with increasing doses of tick antigens, the proliferation and IL-4 production of draining lymph node T cells of mice infested with nymphal ticks increase, peak and then decrease. These results indicate that immunostimulative and immunosuppressive molecules have competing effects in tick saliva or in SGE. I. ricinus saliva inhibits, in a dose-dependent manner, splenic T cell proliferation in response to concanavalin A (ConA). Tick SGE or saliva injected intraperitoneally to BALB/c mice simultaneously with SRBC systemically immunosuppress the anti-SRBC response as shown in vitro by the reduced responsiveness of sensitized splenic T cells to restimulation with SRBC. In brief some components of SGE or tick saliva reduce the responsiveness of draining lymph node T cells and of sensitized splenic T cells in vitro. The responsiveness of naive splenic T cells to ConA stimulation in vitro is also decreased by tick saliva. Modulation of host responses by tick antigens may facilitate tick feeding, transmission and the propagation of pathogens.     

Salivary gland extract from <Emphasis Type="Italic">Ixodes ricinus</Emphasis> tick modulates the host immune response towards the Th2 cytokine profile

In our previous work, the salivary gland extract (SGE) from Ixodes ricinus ticks impaired T-lymphocyte proliferation and clearly modulated the immune response towards the Th2 pattern in human peripheral blood mononuclear cell culture. In the present work, the results obtained on mouse splenocytes are compared with those on human leukocytes. ELISA (protein level) and RNAse protection assay (mRNA level) showed that SGE enhanced interleukin (IL)-1alpha, IL-1beta, IL-1Ra, IL-6, and IL-12p40 cytokines, whereas production of IL-2, IL-5, IL-10, and IL-13 was decreased. The minute levels of IL-9, IL-15 and IL-12p70 were not changed after the addition of tick saliva. IL-4 was upregulated, whereas the production of gamma interferon and migratory inhibition factor was downregulated after the addition of SGE. Tick saliva decreased concanavalin A-stimulated spleen cell proliferation and the percentage of activated T-cells. We conclude that the Th2 polarization did not involve all of the cytokines tested. However, the Th2 subset-augmenting effect of tick saliva was confirmed.     

Cytokine Production by Lymph Node Cells from Mice Infested with Ixodes ricinus Ticks and the Effect of Tick Salivary Gland Extracts on IL-2 Production

In BALB/c mice repeatedly infested with nymphal Ixodes ricinus ticks, lymphocytes from axillary and brachial lymph nodes which drain the tick attachment site produced significant levels of IL-2, TNF-α and GM-CSF when stimulated in vitro with Con A or anti-CD3 antibodies. Cytokine production by cells from lymph nodes of the opposite flank was equivalent to that of cells from uninfested mice. Nine days after the first infestation and IL-2, GM-CSF were produced primarily by the CD4⁺ T cells, while some other cell types contributed also to the TNF-α production. In mice repeatedly infested, a gradual increase of lymph node cell production of IL-2 was observed. The IL-2 levels regularly increased from the first to the third infestation compared to TNF-α levels which gradually decreased. The in vitro production of GM-CSF was not affected by successive infestations. Spleen lymphocytes from naive mice produced higher levels of IL-2 than lymphocytes from axillary and brachial lymph nodes. Both tick salivary gland extracts and D-mannose inhibited IL-2 production by these lymphocytes.     

In vitro production of interleukin-4 and interferon-gamma by lymph node cells from BALB/c mice infested with nymphal Ixodes ricinus ticks.

In this study we compared the ability of lymphocytes taken from axillary and brachial lymph nodes of BALB/c mice that had been infested once three times with 15 nymphal Ixodes ricinus ticks, to produce interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) after in vitro stimulation with concanavalin A (Con A). They released high levels of IL-4 and low levels of IFN-gamma. An increase of IFN-gamma between the first and the third tick infestation was observed. Salivary gland extracts from female I. ricinus ticks induced specific in vitro proliferation of lymphocytes from infested mice. IL-4 production was correlated with the salivary gland extracts' ability to stimulate tick-specific lymphocyte proliferation. Its levels remained high from the first to the third infestation. IFN-gamma production was not necessarily associated with tick salivary gland antigen stimulation. In BALB/c mice, anti-tick immune response induction is regional and the contribution of other similar secondary lymphoid organs is negligible. Only cells from the lymph nodes which drained the tick-fixation site proliferated in vitro in the presence of tick antigens, and when stimulated with Con A produced IL-4 and IFN-gamma.     

Mechanisms of eosinophilia in mice infested with larval Haemaphysalis longicornis ticks.

Infestation of larval Haemaphysalis longicornis ticks induced a threefold increase of eosinophils in the peripheral blood of normal WBB6F1- +/+ mice 2 days after tick infestation. In genetically mast cell-deficient WBB6F1- W/Wv mice, a threefold increase of blood eosinophils was observed 6 days after the tick infestation. However, marked infiltration of eosinophils was detected in the tick infestation sites of the WBB6F1- +/+ mice but not the WBB6F1- W/Wv mice. When the mast cell deficiency of WBB6F1- W/Wv mice had been rescued locally by intradermal injections of WBB6F1- +/+ mouse-derived cultured mast cells, a rapid increase of blood eosinophils and tissue infiltration of eosinophils were revealed following tick infestation. The intravenous (i.v.) injection of immune spleen or lymph node cells obtained from WBB6F1- +/+ mice 10 days after tick infestation led to significant eosinophilia in naive recipient mice. Treatment with anti-Thy-1.2 or anti-CD4 monoclonal antibody (mAb) and complement (C) completely abolished the eosinophilia; the early response (2 days after tick challenge) is dependent on mast cells at the feeding site, and the late response (6 days after tick challenge) is dependent on T lymphocytes. Since amplified interleukin-5 (IL-5) cDNA was detectable in the spleen cells 4 days after tick infestation, the late response might be mediated by IL-5. The infiltration of eosinophils at the feeding site of skin appeared to be dependent on mast cells.     

Saponin adjuvant primes for a dominant interleukin-10 production to ovalbumin and to Trypanosoma cruzi antigen.

The adjuvant activity of saponin for T-cell responses was evaluated and compared with that of complete Freund's adjuvant (CFA) in two antigen systems: a lysate of the protozoa Trypanosoma cruzi and ovalbumin (OA). Strong delayed-type hypersensitivity and T-cell proliferate responses, comparable with those stimulated by CFA, were observed for both antigens following immunization with saponin as adjuvant. Upon in vitro secondary antigen stimulation, high interleukin-10 (IL-10) and low interferon-gamma (IFN-gamma) levels were observed in lymph node (LN) cell cultures from saponin-immunized mice in contrast with the high IFN-gamma and decreased IL-10 production by LN cells from CFA-immunized mice. Production of IL-10 and IFN-gamma in these conditions was CD4-activation dependent. Concanavalin A (Con A)-stimulated interleukin-4 (IL-4) production was higher in saponin-immunized mice than in CFA-immunized mice. IL-10 produced by LN cells from saponin-immunized mice suppressed IFN-gamma production and Con A-induced proliferation. Taken together, these data are consistent with in vivo stimulation of both T-helper (Th)1 and Th2-type cells by immunization with saponin; in vitro a Th2-type cytokine response with high IL-10 production predominates, indicating preferential priming towards a Th2-type response. Immunization with CFA induced a Th1-type cytokine response. To our knowledge this is the first report in which an adjuvant is shown to prime for a dominant IL-10 production.     

Local Th1/Th2 cytokine production during experimental vaginal candidiasis: potential importance of transforming growth factor-beta.

Host defense mechanisms against vaginal Candida albicans infections are poorly understood. Despite the protective role of T helper (Th)1-type cell-mediated immunity (CMI) against mucosal C. albicans infections, studies using an estrogen-dependent murine model of vaginal candidiasis have shown a lack of effect of systemic Th1-type CMI against a vaginal C. albicans infection, and a lack of changes in local T cells during infection. In the present study, the local Thl- (interleukin [IL]-2, interferon [IFN]-gamma and IL-12) and Th2- (IL-4, IL-10 and transforming growth factor [TGF]-beta1) type cytokines were evaluated in vaginal tissue during an experimental C. albicans infection. Results showed constitutive expression of TGF-beta1 in vaginal tissue of naive mice that was two-fold higher than the levels of the other cytokines examined. These high levels of TGF-beta1 were further increased as a result of pseudoestrus and/or infection, and were corroborated at the messenger RNA level. Furthermore, the levels of TGF-beta in naive or infected mice were significantly higher in the vagina compared to other areas of the genital tract. Finally, TGF-beta1 predominated as well in the draining, but not non-draining, lymph nodes during infection. These results suggest that TGF-beta1, a potent immunoregulatory cytokine, may be important in the lack of demonstrable CMI at the vaginal mucosa against C. albicans.     

Cellular immune responses to the murine nematode parasite Trichuris muris. II. Differential induction of TH-cell subsets in resistant versus susceptible mice.

We have analysed the production of a wide variety of cytokines by in vitro concanavalin A (Con A) stimulated mesenteric lymph node cells (MLNC) from strains of mice experiencing chronic (B10.BR, AKR) versus acute (BALB/K) infection with the nematode parasite Trichuris muris. MLNC from infected BALB/K mice produced elevated levels of the Th2-specific cytokines interleukin-5 (IL-5) and IL-9. IL-3 and IL-4 remained at or just above normal. Interferon-gamma (IFN-gamma) (a Th1-type cytokine) was secreted only in small amounts. MLNC from infected susceptible B10.BR and AKR mice produced large amounts of IFN-gamma in the relative absence of IL-4 and IL-9. IL-5 levels failed to rise significantly above normal in B10.BR mice whilst in AKR mice high levels of IL-5 were detected early post-infection (p.i.) but levels decreased dramatically as the infection proceeded to reach normal levels by Day 34. IL-3 levels were depressed below normal. Our results are consistent with the polarization of the Th-cell response during T. muris infection to give predominantly IFN-gamma-secreting Th1 cells in strains of mice unable to expel the parasite and mainly IL-4, IL-5 and IL-9 producing Th2-type cells in resistant strains.     

Dendritic cells have a crucial role in the production of cytokines in mesenteric lymph nodes of B10.BR mice infected with Trichuris muris

Dendritic cells bridge innate and adaptive immunity and establish protective immunity to pathogens. Protection against the murine nematode parasite Trichuris muris depends on the T helper 2 (Th2) response and requires the Th2 cytokines interleukin 4 (IL-4), IL-10, or IL-13. To examine if the Th2 response to T. muris infection is regulated by CD11c⁺B220⁻ dendritic cells in mesenteric lymph nodes, dendritic cell-enriched and dendritic cell-depleted fractions were obtained from mesenteric lymph node cells of T. muris-infected mice, and production of cytokines in cultures of these fractions was measured. At day 14 postinfection, no worm expulsion was observed, and high levels of interferon γ production occurred in dendritic cell-enriched fractions. Expulsion of worms occurred on days 20 and 25 postinfection, and IL-10 production was induced in dendritic cell-enriched fractions on these 2 days. No cytokine production was observed in mesenteric lymph node cells and dendritic cell-depleted fractions during T. muris infection. The occurrence of worm expulsion was consistent with IL-10 production in dendritic cell-enriched fractions. IL-10 inhibits Th1 cells and promotes the Th2 response, and results from this study suggest that CD11c⁺B220⁻ dendritic cells in the mesenteric lymph nodes are required for IL-10 production and the IL-10-dependent protective Th2 response.     

Performance of female Rhipicephalus sanguineus (Acari: Ixodidae) fed on dogs exposed to multiple infestations or immunization with tick salivary gland or midgut tissues

This investigation compared the effects of repeated infestations to immunization of dogs with tick salivary gland or midgut extracts on the feeding and fecundity performances of female Rhipicephalus sanguineus (Latrielle). In each immunized group, three tick-naive dogs were immunized three times with tick salivary gland or midgut extracts, and twice challenged at 21-d intervals by allowing 80 female and 40 male adult ticks to feed on each host. The repeated infestation group of three naive dogs was infested five times at 21-d intervals by the same numbers of ticks. The repeated infestation group showed a trend of reduced tick performance after the third infestation, but some of the tick performance parameters had recovered by the fifth infestation. Tick attachment was reduced by immunization with either tick salivary gland or midgut extract. Immunization with tick salivary gland extract had the greatest impact on the feeding period and engorgement weight of the female ticks. Immunization with tick midgut extract resulted in the greatest reduction of tick fecundity parameters, which included preoviposition, oviposition, and egg-incubation periods in addition to reduced egg production and egg viability. These results confirm that dogs can become resistant to R. sanguineus, and demonstrate that immunization with tick salivary gland or midgut extract has different effects on tick feeding and fecundity.     

Th1-type immune responses by Toll-like receptor 4 signaling are required for the development of myocarditis in mice with BCG-induced myocarditis

The immunological aspects of autoimmune myocarditis are difficult to understand because of the existence of many infectious agents and animal models suggesting different mechanisms in autoimmune myocarditis. To overcome these difficulties, two strains of mice, C3H/HeN and C3H/HeJ, showing different immune responses to mycobacteria, were immunized with myosin mixed with BCG. The C3H/HeN mice with a wild-type Toll-like receptor 4 (TLR4) showed severe myocarditis, whereas the C3H/HeJ mice with nonfunctional mutated TLR4 did not. CD4(+) cells from both strains of mice exhibited appreciable proliferative responses following myosin stimulation; however, the cytokines from these cells differed between these two strains. The C3H/HeN mice showed T helper (Th)1-type cytokine responses, whereas the expressions of mRNA in C3H/HeJ mice were Th2-type cytokine. When both of these strains of immunized mice were inoculated with a plasmid encoding cDNA of interleukin (IL)-4 or agonistic IL-4, the development of myocarditis was inhibited in C3H/HeN mice. Moreover, C3H/HeJ mice, in which development of myocarditis was not induced by immunization of myosin mixed with BCG, showed myocarditis after injection of IL-4 antagonistic mutant DNA for the induction of Th1-type immune responses. The results suggested that the induction of autoimmune myocarditis by myosin is affected by Th1-type immune responses.     

Cytokines (IL-4 and IFN-γ) and antibodies (IgE and IgG2a) produced in mice infected with Borrelia burgdorferi sensu stricto via nymphs of Ixodes ricinus ticks or syringe inoculations

Mice were tolerant to tick bites during three infestations with nymphs of Ixodes ricinus infected with Borrelia burgdorferi sensu stricto. To determine whether tick bites influence the immune response against B. burgdorferi, we examined the production of cytokines IL-4 and IFN-γ by lymph node cells of BALB/c mice and IL-4 deficient BALB/c mice after tick inoculation versus syringe inoculation of B. burgdorferi. We also measured IgG2a anti-borrelial antibodies and total IgE in these mice. Results showed that BALB/c mice developed a Th2 immune response against B. burgdorferi after tick inoculation and a mixed Th1/Th2 response after syringe inoculation of B. burgdorferi. IL-4 deficient mice produced a Th1 immune response in both cases. IL-4 produced following tick bites greatly decreased the production of anti-borrelial IgG2a antibodies by comparison with the production of anti-borrelial IgG2a antibodies produced following syringe injection of B. burgdorferi. Received: 23 November 1999 / Accepted: 9 December 1999     

Immune responses of interferon gamma (IFN-gamma) knock out mice to repeated Haemaphysalis longicornis (Acari: Ixodidae) nymph infestations

To investigate the immunological mechanisms of acquired resistance to tick infestation, interferon gamma (IFN-gamma) deficient mice (IFN-gamma mice) were used to assess interleukin-4 (IL-4) and antibody production levels against tick salivary gland antigen on three successive infestations with Haemoaphysalis longicornis Neumann nymphs. The engorged body weight of the ticks decreased during the second and third infestations. Similar observations were noted in IFN-gamma+/+ mice. However, the engorged body weight of the ticks from IFN-gamma +/+ mice were considerably lower than those from IFN-gamma-/- mice. A marked increase in antibody production during the second and third infestations was observed indicating that IFN-gamma-/- mice could acquire immunological resistance against H. longicornis nymphs. Moreover, IL-4 levels were higher during the first and third infestations but decreased during the second infestation. IL-4 levels were significantly higher in IFN-gamma-/- mice than in IFN-gamma+/+ mice. We have shown here that the statistically significant high IL-4 levels observed in IFN-gamma-/- mice may be a result of type 2 helper cell (Th2) polarization. However, the apparently higher IL-4 levels during the first and third infestations and the notable decline during the second infestation suggest that other cytokines or factors in the host immune system may play a part in regulating IL-4 levels.     

Low-grade B cell lymphomas of mucosa-associated lymphoid tissue (MALT-type) require CD40-mediated signaling and Th2-type cytokines for in vitro growth and differentiation.

To investigate the mechanisms of T cell dependence underlying the development of extranodal mucosa-associated lymphoid tissue (MALT)-type B cell lymphomas, the activation, proliferation, and differentiation of lymphoma B cells were studied using ligand binding to the CD40 membrane receptor. The activation and proliferative response of all investigated low-grade MALT-type lymphomas (n = 6) was strongly dependent on anti-CD40-mediated signals and was complemented by cytokines produced by T helper cells of the Th2 type (interleukin-4 (IL-4) and/or IL-10). Th1 cytokines (IL-2 and/or interferon-gamma) bad little effect. Low-grade, but less so high-grade, MALT-type lymphoma B cells were induced to secrete large amounts of tumor immunoglobulin in response to IL-10. In contrast, high-grade MALT-type lymphomas (n = 5) proliferated in response to both Th2- and Th1-type cytokines and CD40 stimulation, whereas Burkitt lymphomas (n = 3) could not be rescued from apoptosis by CD40 stimulation with or without cytokines. These results suggest that CD40 signaling in combination with Th2 cytokines are essential for the development and progression of low-grade MALT-type B cell lymphoma. We conclude that T cells, which activate B cells in a CD40-dependent fashion, may contribute to lymphoma pathogenesis.     

Regulatory Th2-type T cell Lines Against Insulin and GAD Peptides Derived from Orally- and Nasally-Treated NOD Mice Suppress Diabetes

Non-obese diabetic (NOD) mice spontaneously develop diabetes. Ourselves and others have previously shown that oral and nasal administration of insulin or glutamic acid decarboxylase (GAD) suppresses development of diabetes in the NOD mouse and that this suppression appears secondary to the generation of regulatory T cells that act by secreting anti-inflammatory cytokines such as IL-4 and TGF-beta. In the present study, we analysed cytokine patterns associated with mucosal administration of insulin B-chain, B-chain peptide 10-24 and GAD peptide 524-543 and derived lines and clones from mucosally-treated animals. Mice were fed five times (400-600 microg/feed) or nasally-treated three times (60 microg/application), and 2 days after the last treatment were immunized in the footpad with the mucosally administered antigen in CFA. Primary immune responses in the popliteal lymph node were measured 10 days after immunization and lines and clones were then established from the primary cultures. There was significantly less IFN-gamma production in mucosally-treated mice associated with increased production of IL-10 and TGF-beta. The nature of the antigen appeared to determine cytokine production as the B-chain given either orally or nasally primed for TGF-beta responses, whereas mucosally administered B-chain peptide 10-24 primed for IL-10. T cell clones, established from draining lymph nodes of fed or nasally-treated animals, secreted IL-4, IL-10 and TGF-beta whereas those from non-fed mice secreted IL-2 and IFN-gamma. Transfer of Th1 lines with splenocytes from diabetic NOD mice into NOD or NOD/SCID animals accelerated diabetes, whereas transfer of Th2 lines suppressed the development of diabetes. Our results further support a role for Th2-type cells in the regulation of diabetes in NOD mice.