Transmission of Borrelia garinii OspA serotype 4 to BALB/c mice by Ixodes ricinus ticks collected in the field

In Europe, Borrelia garinii OspA serotype 4 has been isolated from the cerebrospinal fluid of patients but, up to now, has never been identified among culture isolates from Ixodes ricinus ticks. This information raises the question of whether OspA serotype 4 is transmitted by I. ricinus in nature. In the present study, I. ricinus nymphs collected in an area of endemicity in southern Germany were allowed to feed on mice. Cultivation of ear biopsy specimens showed that six of seven B. garinii-infected mice were infected by OspA serotype 4. In contrast, very few B. garinii OspA serotype 4 organisms were isolated directly from the ticks which infected the mice; most isolates were B. afzelii. The infected mice transmitted mainly OspA serotype 4 to xenodiagnostic ticks, preferentially in combination with B. afzelii.     

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.     

Infiltration of CD4+ CD8+ T cells, and expression of ICAM-1, Ia antigens, IL-1 alpha and TNF-alpha in the skin lesion of BALB/c mice undergoing repeated infestations with nymphal Ixodes ricinus ticks.

The skin cellular immune response of BALB/c mice was examined during three successive infestations with nymphal Ixodes ricinus ticks. An immunohistochemical analysis of skin cryostat sections 72 hr post-tick attachment revealed that CD4+ T cells outnumbered CD8+ T cells in all infestations. The CD4+:CD8+ T-cell ratio was 2.2:1 in the primary infestation, then increased to 3.2:1 and 4.7:1 in the secondary and tertiary infestations. No B lymphocytes (CD45R) were detected in the skin of control and infested mice. A positive staining of intercellular adhesion molecule-1 (ICAM-1) on vascular endothelial cells, dendritic cells and some other mononuclear cells was observed in the dermis. Also, a strong positive staining of Ia antigens on dendritic cells and infiltrated mononuclear cells was noted. The staining pattern was more intense and positive cells increased in number in the skin of re-infested mice compared to the primary infestation. In addition, cells such as epidermal keratinocytes, dermal dendritic cells and infiltrated mononuclear cells positive for the 'pro-inflammatory' cytokines interleukin-1 alpha (IL-1 alpha) and tumour necrosis factor-alpha (TNF-alpha) were localized in the skin of infested mice, as detected at the mRNA level by in situ hybridization and at protein level by immunostaining with antibodies. These results suggest that an antigen was presented to infiltrating T lymphocytes which then became activated. This event may explain the cutaneous delayed-type hypersensitivity previously described in tick-infected BALB/c mice. Importantly, this cutaneous reaction was not sufficient to protect the mouse against tick re-infestation. Furthermore, ICAM-1 could mediate, at least in part, the extravasation of inflammatory cells into the skin of infested mice.     

Early detection of Borrelia burgdorferi sensu lato infection in Balb/c mice by co-feeding Ixodes ricinus ticks

In Europe, Borrelia burgdorferi is transmitted by Ixodes ricinus to animals and human. When infected and uninfected ticks co-feed on a host, spirochetes are transmitted from ticks to animal and also to uninfected ticks. Here, we used uninfected ticks to co-feed with infected ticks on mice to evaluate this method to detect early infection in mice. A total of 128 mice were challenged by infected nymphs placed in capsules glued on the back of the mice. Three days later uninfected larvae were added in the capsule to co-feed with infected nymphs and were examined for Borrelia infection after natural detachment. Infection in mice was also determined by xenodiagnosis and by spirochete isolation from ear skin biopsy and back skin biopsy taken at the tick attachment site one month after infection. A total of 111 mice were found to be infected by at least one of these four methods. Borrelia infection was observed in 95% of mice by the co-feeding method, in 92% of mice by xenodiagnosis, in 69% and in 68% of mice by cultivation of ear and back skin biopsies, respectively. Our results demonstrate that the co-feeding method is a very sensitive method which can be used to detect very early infection in mice infected by tick bites.     

Susceptibility of BALB/c mice to nymphs and larvae of Ixodes ricinus after modulation of IgE production with anti-interleukin-4 or anti-interferon-γ monoclonal antibodies

BALB/c mice infested three times with nymphs or larvae of Ixdoes ricinus ticks do not acquire resistance as assessed by evaluation of both tick attachment and the weight of engorged nymphs or larvae. Tick challenge causes a gradual increase in total IgE antibody production from the first to the third infestation. Anti-tick IgG antibodies are never detected. When the mice are treated with anti-interleukin-4 (anti-IL-4) or anti-interferon-gamma (anti-IFN-γ) monoclonal antibodies (mAbs) 1 day before each infestation, they produce fewer or more IgE antibodies, respectively. No effect is observed on IgG antibodies. In IL-4-deficient mice, no IgE or IgG antibody is produced. However, these treatments and the use of IL-4-deficient mice have no negative effect on either tick attachment or the weight of engorged nymphs or larvae. Treatment with anti-IL-4 mAb and the use of IL-4-deficient mice inhibits and abolishes the switching of IgE, respectively, but these are apparently not sufficient to shift the response toward Th1 cells. Received: 18 July 1997 / Accepted: 12 November 1997     

Enhanced interferon-gamma (IFN) production by lymph node cells from autoimmune (MRL/1, MRL/n) mice.

Interferons (IFNs) have been implicated in the aetiopathogenesis of the autoimmune disease systemic lupus erythematosus (SLE). The ability of unstimulated and Sepharose-bound concanavalin A (ConA) stimulated spleen or lymph node (LN) cells from normal CBA/T6 mice and histocompatible autoimmune strains (MRL/1, MRL/n) of various ages to produce IFN-gamma was measured. Levels of IFN-gamma produced by ConA-stimulated spleen cells from CBA/T6, MRL/1 and MRL/n mice at 6 weeks of age were not statistically different (mean +/- SEM: 786 +/- 182, 1147 +/- 282, 1024 +/- 146 IU/ml, respectively). At this age only stimulated LN cells from MRL/l mice produced detectable IFN-gamma (538 +/- 44 IU/ml) and these levels remained constant up to 6 months. IFN production by stimulated LN cells from young MRL/n mice (66 +/- 21 IU/ml at 3 months, 44 +/- 41 IU/ml at 6 months) increased at 1 year (463 +/- 97 IU/ml) corresponding to the age of disease onset. The failure of stimulated CBA/T6 LN cells to produce IFN-gamma was not due to cell-cell suppression, defective IL-2 production or the generation of soluble inhibitors. Stimulated LN cells from other normal inbred (C57Bl/6, Balb/c, A/J) outbred and FI hybrid mouse strains (Swiss, [Swiss x Balb/c] F1, (CBA/T65 x C57Bl/6]FI) produced undetectable or low levels of IFN compared to MRL/1 and MRL/n mice. These results show that autoimmune mouse LNs generate more IFN compared to normal controls and that the increase in IFN levels (at least in MRL/n) corresponds to the age of disease onset.     

In vivo and in vitro responses to sheep erythrocytes by lymph node cells from mice with trichinellosis

Mice infected with Trichinella spiralis for 20 days have decreased numbers of plaque forming cells (PFC) in the draining lymph nodes following subcutaneous immunization with sheep erythrocytes. However, when immunized in vitro, lymph node cells from infected mice generate more PFC than normal controls. Splenectomy has no effect on suppression observed in vivo. There is a large increase in the proportion and numbers of B cells in the lymph nodes of infected mice, and these cells may account for the enhanced PFC responses in vitro.     

In vitro studies on antibody production by lymph node cells using cell electrophoresis

Investigations were undertaken on rat lymph node cell suspensions using cell electrophoretic techniques: (a) to examine the specificity of antigen—antibody binding, (b) to determine any relationship between the electrophoretic mobility pattern and antibody production, and (c) to estimate the potentiality of a single cell to produce antibody to more than one antigen at the same time. The results, in brief, indicate that a drastic reduction in the mean mobility is caused when the immunized cells are treated with the same antigen used for immunization. These interactions have antigenic specificity. In addition, it has been shown that not all cells in a lymph node produce antibodies and the percentage of cells that produce antibodies changes from day to day. The maximum response occurs between the 4th and 5th day of the second antigenic administration. Studies with simultaneous administration of two bacterial antigens support the concept that by and large a lymph node cell produces specific antibodies to only one antigen at a time.     

In vitro tolerance induction to a thymus-dependent antigen with BALB/c and C57BL/6 lymph node cells: effect of tolerogen concentration and duration of exposure.

Further studies on tolerance induction in vitro to bovine gamma globulin (BGG) in nonadherent BALB/c lymph node cells showed that it was dependent upon both the dose of tolerogen and the time of exposure. Nearly complete tolerance was achieved at a dose of 1.0 mg/ml and an incubation time of 12-18 hours. When C57BL/6 strain was used for comparison with the BALB/c strain because of its relative ease to become tolerant after in vivo injection of tolerogen, the rate of tolerance induction of its nonadherent lymph node cells was not different from that of BALB/c cells. Thus, in the absence of other host factors, the acquisition of tolerance by lymphocytes is gradual and may reflect the time required for a cell to reach a susceptible phase of the cell cycle and/or the activation of suppressor cells.     

Experimental production of actinomycetoma in BALB/c mice.

Chronic actinomycetoma associated with grain production was induced in BALB/c mice by subcutaneous inoculation of live Nocardia brasiliensis in Freund incomplete adjuvant into the hind footpads. Similar inoculation of N. asteroides and N. caviae resulted in local tumor formation which healed spontaneously after 5 months, the disease disseminating into the peritoneum, where masses or organisms could be detected. Grains were recovered from superficial skin lesions of N. caviae, but not from the N. asteroides-infected mice. Mycetoma lesions, appearing as early as 1 month after inoculation of 1.2 X 10(7) colony-forming units of N. brasiliensis per ml or as late as 3 months with inoculation of 1.0 X 10(5) colony-forming units per ml, became persistent and were readily detectable even 6 months after inoculation. No spontaneous healing occurred, and grains were recovered at different stages of the disease. Saline suspensions of N. brasiliensis also produced typical mycetoma lesions, although the incubation period was ca. 6 months. Adjuvant addition appeared to accelerate the onset of the disease. Experimental production of actinomycetoma in laboratory animals allows the study of many unanswered aspects of the disease and also provides a suitable model for therapeutic trials in the search for new and more effective chemotherapeutic agents.     

Simultaneous transmission of Borrelia burgdorferi and Babesia microti by individual nymphal Ixodes dammini ticks.

Nymphal Ixodes dammini ticks, selected from a group of ticks in which 22 of 31 (71%) contained dual Borrelia burgdorferi and Babesia microti infections, simultaneously transmitted B. burgdorferi and B. microti to 4 of 7 (57%) hamsters exposed to individual ticks.     

Enhanced sensitivity of DEAE-dextran treated spleen cells from normal BALB/c mice,B-lymphocyte depleted BALB/c mice and nude mice toin vitro antibody-complement mediated immune cytolysis

The sensitivity of thein vitro antibody-complement mediated dye exclusion cytotoxicity teat when carried out with rabbit antisera and mouse target cells was investigated with the polycation DEAE-dextran present in the cell suspension. One antiserum was raised against BALB/c mouse thymus cells (ATS) another against nude (nu/nu) mouse spleen cells (ANS). Target cells were from the spleens of normal BALB/c mice, cyclophosphatamide treated (B-cell depleted) BALB/c mice, and nude (T-cell depleted) mice. DEAE-dextran enhanced the sensitivity in all antiserum-target cell combinations, the enhancing effect being most pronounced when ANS was used against nude spleen cells.     

In vitro proliferative response of BALB/c mouse spleen cells stimulated with trinitrophenylated syngeneic spleen cells.

Spleen cells from normal BALB/c mice showed in vitro proliferative response against hapten-conjugated syngeneic spleen cells. Trinitrophenylated (TNP) spleen cells were prepared by treating normal spleen cells with sodium 2,4,6-trinitrobenzenesulphonate (TNBS). Four-day cultures of TNP-labelled spleen cells incorporated 2.5-7.4 times more [3H]thymidine than similar cultures of untreated spleen cells. An obviously positive mixed lymphocyte reaction (MLR) by normal spleen cells against mitomycin C (MC) treated TNP-labeled syngeneic spleen cells was observed after 4 days of culture. The MLR to TNP-labelled syngeneic cells was inhibited in the presence of epsilon-TNP-L-lysine by 23-37%. The spleen cells from the mice injected intraperitoneally with TNP-labelled syngeneic spleen cells showed a higher MLR against TNP-labelled spleen cells than normal spleen cells. The sensitized spleen cells also showed an increased response to MC-treated spleen cells. These results suggest that normal spleen cells include cells which can recognize the hapten and new antigenic determinants introduced into syngeneic spleen by chemical modification.     

Rabbits infested with Ixodes ricinus L. adults: effects of a treatment with cyclosporin A on the biology of ticks fed on naive and immune hosts

Rabbits have been infested 3 times with 10 female and 10 male Ixodes ricinus. Immunity which is induced when ticks feed on naive animals (1st infestation) perturbs feeding, oviposition and embryogenesis during reinfestations. Treatment of rabbits during a 3rd infestation (resistant animals) with cyclosporin A (CsA), an immunosuppressive agent which works on the cellular compartment (chiefly T helper cells), partially reversed the negative effects of the immunity on the biology of the ticks. Conversely, CsA may also directly affect the reproductive processes of ticks. Thus, the weight of the eggs laid and the egg conversion factor of ticks fed on naive treated hosts (1st infestation) were diminished. In addition, the preoviposition was prolonged, and finally failure in oviposition and hatching occurred more frequently.     

Defective development of pristane-oil-induced plasmacytomas in interleukin-6-deficient BALB/c mice.

Interleukin (IL)-6 is known to be an essential growth factor for myeloma cells, both in vitro and in vivo. In mice, IL-6 is required for development of B cell tumors upon infection with a retrovirus expressing the myc/raf oncogenes. In the present study, we used the pristane-oil-induced plasmacytoma model, which more closely mimics tumor transformation and progression in human multiple myeloma. Also using this system, we found that IL-6-deficient BALB/c mice are protected against tumor development. Although the pristane-induced inflammatory reaction was less pronounced in IL-6-deficient mice versus their wild-type littermates, both B cell differentiation and plasma cell formation took place, and even morphological evidence of plasma cell transformation was detected, albeit at a low frequency. However, in the absence of IL-6, there were never signs of uncontrolled proliferation of either normal B lymphocytes or tumor cells, suggesting that the role of IL-6 in murine plasmacytoma and possibly also in human multiple myeloma is to ensure abnormal survival and proliferation of previously transformed tumor cells and therefore tumor development and progression.     

Comparison of antigen presentation by lymph node cells from protein and peptide-primed mice.

Lymph node cells from mice primed with peptides from the allergens Der p I and Der p II (the group I and II allergens of Dermatophagoides pteronyssinus) were unable to recall responses to the protein antigen when cultured in vitro despite being able to mount large responses to the peptides. The T cells could however recall responses to the protein when spleen-adherent cells were added into culture. Treating the spleen accessory cells with the monoclonal antibody (mAb) 33D1 and complement largely abrogated the protein response of peptide-primed T cells which indicates that dendritic cells were mainly responsible for the antigen-presenting function. If mice were primed with two injections of peptide the lymph node cells obtained could respond to both protein and peptides in vitro without the need for exogenous accessory cells. Using either negative depletion with the J11D mAb or positive purification, it was found that the presentation of protein antigen to lymph node T cells primed with either protein or peptide was limited to antigen-specific B cells. Peptide antigens could however be presented by both B and non-B populations. In one case the peptide 105-129 from Der p II which contains a T-cell epitope could not be shown to induce T-cell responses in the lymph node unless presentation was mediated by spleen-adherent or B-specific cells. These results are important for peptide-based immunomodulation and in interpreting results obtained from lymph node cultures.     

Recombinant interleukin-1 infusion increases resistance of BALB/c mice to murine leprosy.

BALB/c mice were infected by the subcutaneous route with 10(7) Mycobacterium lepraemurium (MLM), and the progression of the infection followed in mice injected i.p. with diluent or recombinant human recombinant interleukin-1 alpha (IL-1 alpha). It was observed that infusion of 1 microgram of IL-1 alpha per day led to a reduction of bacterial growth in the livers and popliteal lymph nodes of MLM injected mice (2-3-log reduction at 6 months, P less than 0.0001). There was no indication that IL-1 alpha infusion was acting by enhancing macrophage activation. Indeed, resident peritoneal macrophages from control infected mice were as competent as macrophages from infected mice treated with IL-1 alpha in generating superoxide anion (O2-) (approximately 400 nM O2-/h/mg at 2 months post-infection). Moreover, they were no more permissive than those of IL-1 alpha infused mice for MLM in vitro as both groups of cells allowed progressive MLM growth, i.e. a 20-fold enhancement of intramacrophage MLM growth. Infusion of IL-1 alpha during MLM infection was not associated with any abrogation of the suppression of the T-cell response to T-cell mitogens or specific stimulation with antigens which is complete at 1 month post-infection. It is concluded that IL-1 alpha has immunotherapeutic potential in leprosy with the mechanism(s) of action still unclear.     

Comparison of ascites production for monoclonal antibodies in BALB/c and BALB/c-derived cross-bred mice

BALB/c male mice were mated with either Swiss-Webster or MF1 females to produce first generation cross-bred offspring. Hybridoma cell lines, from the fusion of P3-NS1-Ag4/1 myeloma cells with spleen cells sensitised to the porcine coronavirus causing transmissible gastroenteritis, were injected intraperitoneally into these mice to produce ascitic fluid containing monoclonal antibodies. Mice of 11 weeks of age weighing between 26 and 34 g were used. The volume of ascites produced by mice injected with four of the five hybrid cell lines tested was greater in the cross-bred offspring than in the BALB/c parent. The fifth cell line gave comparable volumes in the MF1 cross-breed and BALB/c parent but a lesser volume in the Swiss-Webster cross-breed. The antibody titres of the ascites as determined by virus neutralisation, radioimmune and indirect immune fluorescence assays, did not differ significantly between mouse types. The ability to use all offspring from a litter of cross-bred mice, irrespective of sex, and the increased volume of ascitic fluid formed in each mouse, permits fewer animals to be used for the production of ascites in these strains, thereby offering considerable economic and ethical advantages over the use of BALB/c mice.     

Generation of rat Th2-like cells in vitro is interleukin-4-dependent and inhibited by interferon-gamma.

Differentiation of naive T cells into effector cells producing T helper type 1 (Th1) and Th2 cytokines is regulated by the presence of specific cytokines in the T-cell microenvironment. The effect of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) on Th1- and Th2-like cell development was investigated in cultures of mixed rat spleen cells. These cells were cultured for 4 days in medium containing concanavalin A (Con A) with or without additional IL-2, IFN-gamma or IL-4. The cells were then washed and their capacity to produce IL-4, IL-5 and IFN-gamma determined following stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin. Freshly isolated cells stimulated with PMA and ionomycin expressed detectable levels of IL-4 and IL-5 mRNA as measured by a quantitative polymerase chain reaction (PCR) procedure and much higher levels of IFN-gamma mRNA. Cells cultured with Con A for 4 days, washed, and restimulated with PMA + ionomycin were unable to express detectable levels of IL-4 and IL-5 mRNA, but produced high levels of IFN-gamma mRNA. Addition of IL-4, or anti-IFN-gamma antibody, to Con A-driven splenocyte cultures restored the ability of restimulated cells to express IL-4 and IL-5. CD4+ T cells isolated from these cultures also showed an increased capacity to secrete IL-4 and IL-5 when anti-IFN-gamma and IL-4 were present in the culture medium. When cultured for 4 days with Con-A, IL-4 and anti-IFN-gamma splenocytes showed an increased capacity to proliferate in response to recombinant IL-2 and proliferated in response to IL-4 alone. IL-2 had no effect on cytokine production by cultured splenocytes. These results indicate that: (1) IL-4 is essential for the generation of Th2-like cells; (2) IFN-gamma inhibits IL-4 production by mixed spleen cells and suppresses generation of IL-4 responsive T cells; (3) in mixed spleen cell cultures mitogenic stimulation favours differentiation of naive rat T cells into effector cells expressing a Th1, and not Th2, cytokine profile.