Immunophysiological studies of interleukin-2 and canine lymphocytes.

Interleukin-2-dependent pathways of lymphocyte activation were investigated in canine peripheral blood lymphocytes (PBL) following stimulation with T-cell mitogens including phytohemagglutinin, phorbol ester (TPA), calcium ionophore (ionomycin), and human recombinant interleukin-2 (hrIL-2). The ability of the stimulated cells to produce interleukin-2 (IL-2) was determined using murine indicator cell lines. IL-2 receptor expression by mitogen-stimulated canine PBL was confirmed by the binding of hrIL-2 with high affinity, and with characteristics comparable to those of the human and murine IL-2 receptor. Examination of serum and PBL from two dogs that were treated with hrIL-2 and human recombinant tumor necrosis factor for systemic mast cell tumors showed that in one dog, IL-2 could be measured in the serum. Concurrently, the in vitro mitogenic response of this dog's PBL to hrIL-2 occurred earlier, possibly reflecting an increase in the relative number of IL-2-responsive cells within the PBL population.     

Parameters of production and partial characterization of feline interleukin 2

The conditions for the production of feline interleukin 2 (IL-2) from peripheral blood leukocytes (PBL) and splenocytes by concanavalin A (Con A) stimulation are described. Feline IL-2 was quantitated by measuring DNA synthesis in the murine IL-2-dependent cell line, CTLL-20. In addition, feline IL-2 was generated for the maintenance of long-term cultures of Con A-stimulated feline PBL and for biochemical characterization. Finally, IL-2 production was evaluated from the PBL of feline leukemia virus (FeLV)-infected cats. Con A at 9.6 micrograms/ml produced a plateau of peak IL-2 activity from 24 to 48 h following stimulation. The tumor promoter, phorbol myristic acetate, stimulated feline IL-2 production and enhanced Con A-stimulated feline IL-2 production. Fetal calf serum (FCS) was not required for IL-2 production; however, FCS at 5% (v/v) allowed for maximal Con A-stimulated IL-2 production. Feline IL-2 generated from Con A-stimulated splenocytes migrated with an apparent molecular size of 13.7 to 23 kD by gel filtration chromatography and supported the proliferation of Con A-activated feline PBL at a final concentration of 0.3 to 0.9 units/ml.     

Cloning,expression and biological activity of equine interleukin (IL)-5

The cytokine, interleukin (IL)-5 stimulates eosinophil differentiation, activation and survival and can prime these cells, increasing the response to other mediators. In view of its many effects on eosinophils, IL-5 has been implicated in the pathogenesis of allergic disease in man. Here we report the cloning of equine IL-5 and expression of the recombinant protein by transfection of Chinese hamster ovary (CHO) cells. The cloned cDNA sequence consisted of 405 nucleotides and encoded a protein of 135 amino acids. There is >85% identity with feline, bovine, ovine, canine, and human IL-5 sequences at the nucleotide and protein level. Supernatants containing equine IL-5 were also examined for biological activity. CHO supernatant containing equine recombinant (eqr) IL-5, like the human ortholog (hrIL-5), induced concentration dependent equine eosinophil adherence to autologous serum-coated plastic (9.7+/-1.5% with a 1:100 dilution of eqrIL-5 and 9.1+/-1.6% adherence with 1 nM hrIL-5; n = 4). The eqr protein also caused concentration dependent superoxide production (11.9+/-2.4 nmol (reduced cytochrome (cyt) C)/10(6) cells at a 1:50 dilution, n = 4). In contrast, hrIL-5 only caused significant superoxide production when diluted in conditioned CHO medium, an effect that was inhibited by the anti-human mAb, TRFK5 (4.4+/-0.3 versus 0.3+/-0.4 nmol/10(6) cells for 0.5 nM hrIL-5 in the presence of the isotype matched IgG1 control (10 microM) and TRFK5 (10 microM), respectively). TRFK5 also significantly inhibited hrIL-5 induced adherence at concentrations of 0.3 microg/ml and above but had no significant inhibitory effect on either superoxide or adherence caused by eqrIL-5. These results demonstrate that equine IL-5 expressed by CHO cells stimulates equine eosinophils, suggesting that this cytokine could play a role in eosinophil recruitment and activation in equine allergic disease. The anti-human and murine moAb TRFK5 does not appear to recognise the equine protein.     

In vitro IgE but not IgG production of canine peripheral blood B cells is inhibited by CD40 ligation

The aim of this study was to investigate in vitro IgE induction in peripheral canine B cells. CD21(+) B cells were purified from the peripheral blood of beagle dogs by positive selection via magnetic separation to a purity of >/=95%. Subsequently, proliferation, and IgG and IgE production of canine B cells were investigated after stimulation with human recombinant Interleukin-4 (hrIL-4) and human recombinant Interleukin-2 (hrIL-2) in the presence or absence of CD40L-CD8 fusion protein (CD40L) of mouse origin. We could demonstrate that canine B cells react on hrIL-2 alone by proliferation and IgG production but not by IgE secretion, whereas activation with hrIL-4 induced proliferation and mainly IgE production. Together, both cytokines synergistically increased B cell proliferation as well as IgG and IgE production. We could also show that mouse CD40L induces proliferation of dog B cells, which is further enhanced by addition of hrIL-4. Unexpectedly, CD40L led to a dramatic decrease in the IL-4 mediated IgE secretion (82% inhibition on an average). In contrast, IgG production was not affected significantly by CD40L. The same effects of CD40L were observed when B cells were stimulated by a combination of IL-2 and IL-4 and this inhibition could not be abrogated by increasing the amounts of IL-4. In summary, activation of canine B cells from peripheral blood by hrIL-4 in the presence or absence of hrIL-2 led to marked IgE production that is strongly and in a dose-dependent manner inhibited by CD40L. Stimulation of IgG production is not influenced by CD40L.     

Bovine interleukin 2: biochemical and biological characterization

Interleukin 2 (IL-2), secreted by bovine peripheral blood mononuclear cells (PBL) on stimulation with concanavalin A (Con A), was purified and characterized by different chromatographic and electrophoretic techniques. The ability of IL-2 to support proliferation of Con A-stimulated bovine lymphoblasts was used to assay and quantitate IL-2 activity. Bovine IL-2 having an apparent MW of 27,000 eluted from a gel-filtration column; from an anion exchange column peak activity was detected at 190 mM NaCl. Binding of bovine IL-2 to phenyl-Sepharose gel and elution with 35-60% ethanediol indicated its hydrophobic nature. Studies on cross-species reactivity revealed that both buffalo and goat lymphocytes respond to cattle IL-2 and detected 35% of activity from a standard cattle IL-2 preparation. Sheep lymphocyte response to cattle IL-2 was negligible.     

Production and consumption of T cell growth factor by peripheral blood lymphocytes of cattle infected with Taenia saginata

Calves were infected with 25,000 or 50,000 viable eggs of Taenia saginata. Larval numbers ranged between 2077 and 6005. During infection the animals developed leucocytosis, which was mainly due to lymphocytosis. An apparently positive correlation was observed between the lymphocytosis and the in vitro proliferative response of the lymphocytes to antigen prepared from proglottids. Maximal in vitro blast transformation of the cells stimulated with antigen occurred on Days 12 and 32 post-infection (p.i.). Specific antibodies to T. saginata were demonstrated on Day 14 p.i. At that time, the proliferative response of the cells paralleled the formation of specific antibodies, particularly of the IgG class. The stimulated cells produced a lymphokine showing interleukin 2 (IL 2)-like activity, since the addition of supernatant of such cells to IL 2-dependent concanavalin A (Con A)-blast cells supported the in vitro growth of the cells. In addition, peripheral blood lymphocytes (PBL) specific for T. saginata could be maintained in long-term cultures when they were cultured in medium containing supernatants of MLA-144 cell lines. The data presented in this study indicate that cells specific for T. saginata produced and consumed T cell growth factor (TCGF).     

Induction of IL-2 and lymphokine activated killer cells in the cat

We have described the use of a cloned murine IL-2-dependent T-cell line to directly measure feline IL-2. Concanavalin A stimulated feline peripheral blood lymphocytes produced an IL-2-rich supernatant that supported the growth of this murine IL-2-dependent T-cell line. In addition to producing IL-2, Con A stimulated killer cells in PBL were cytotoxic for the FeLV transformed tumor cell line FL74. Incubating feline PBL with a cocktail of the calcium ionophore A23187 and phorbol ester also led to the generation of cytotoxic cells as well as the production of high levels of IL-2. Finally, IL-2-rich supernatant was able to stimulate cytotoxic activity in PBL from normal cats.     

Interleukin 1- and 2-like activities in the dog

Canine adherent and non-adherent peripheral blood leukocytes and spleen cells were examined for their ability to produce soluble factors with Interleukin 1- and 2- (IL-1 and IL-2) like activities. For this purpose, three conventional assay systems were used: (a) proliferation on an IL-2-dependent murine cytotoxic T-lymphocyte cell line, (b) enhancement of PHA-induced murine thymocyte proliferation and (c) proliferation of lectin-primed canine peripheral blood lymphocytes (PBL). Only the latter two types of cells respond to IL-1, whereas all three types respond to IL-2. Both types of factors were produced and the kinetics of their release/production were found to be identical to those of human PBL. Results suggested that species-related differences existed. Canine interleukin-containing supernatants had higher titers than murine interleukin-containing supernatants when analyzed on canine lymphocytes, and the reverse was found if murine target cells were used.     

Evaluation of an antibody-ELISA using five crude antigen preparations for the diagnosis of Trypanosoma evansi infection in cattle.

Attempts were made to improve the accuracy of an antibody-detection ELISA for the detection of Trypanosoma evansi infection in cattle by improving the method of preparation of the crude antigen used. An IgG-ELISA was performed with five different antigen preparations: crude soluble antigen, soluble and insoluble fractions of crude antigen treated with 0.1% formalin and whole formalin-fixed trypanosomes treated with either trypsin or 2-mercaptoethanol. An IgM-ELISA using crude soluble antigen was also performed. Each ELISA was evaluated using serum from 44 Indonesian cattle infected with T. evansi and 262 uninfected cattle from Australia. There was no significant difference between the sensitivity or specificity of the IgG-ELISA using each of the five antigens. The IgM-ELISA using a crude untreated lysate was significantly less sensitive (p<0.05) than the IgG-ELISA using the same antigen, trypsin-treated antigen or the 0.1% formalin-treated soluble antigen (68, 64 and 64%, respectively). These results show that these modifications to the method of producing crude antigens for the Ab-ELISA does not improve the accuracy of diagnosis of T. evansi infection in cattle.     

Cellular interactions regulating the in vitro response of bovine lymphocytes to ovalbumin.

We examined the contribution of MHC class II-restricted T cells (CD4+), MHC class I-restricted T cells (CD8+), gamma/delta T cell receptor (TCR)+ T cells, B cells and macrophages to the development and control of in vitro proliferative responses of bovine lymphocytes to ovalbumin (OA). Cell populations for in vitro assay were obtained from peripheral blood (peripheral blood leukocytes, PBL) of OA-primed cattle. Specific cell populations were depleted or purified from PBL by staining with monoclonal antibodies (MAbs) against the appropriate differentiation antigens and sorting on a Fluorescence Activated Cell Sorter (FACS). OA-specific in vitro responses of in vivo primed PBL were dependent on the presence of CD4+ T cells. Their presence could not be replaced by the inclusion of T cell growth factor (TCGF) in the culture system, indicating that CD4+ T cells probably actively proliferate in response to antigenic stimulation. Bovine CD8+ T cells and gamma/delta TCR+ T cells appeared to exert a suppressive effect on proliferative responses. No proliferation was observed in PBL after the depletion of MHC class II+ cells. In this case, the response could be restored by the addition of macrophages or LPS-activated B cells to the MHC class II- population.     

Patterns of bovine T cell-mediated immune responses to bovine herpesvirus 1

Lymphocyte proliferation was used to evaluate T cell-mediated immune responses to different isolates of Bovine Herpesvirus 1 (BHV-1). Groups of high, moderate, and low responses were observed when lymphocytes from three breeds of dairy cattle were stimulated with each of the BHV-1 isolates. Proliferation of cells in the low responding group could be augmented by exogenous IL-2. The mechanism of unresponsiveness by cells from one individual whose response was not altered by IL-2 supplementation was further investigated. The patterns of response by limiting dilution frequency analysis eliminated the possibility that this individual lacked responsive cells but suggested the presence of regulatory cell interactions which resulted in the observed low proliferative response. These results show that animals exposed to the same environment can vary greatly in their ability to respond to BHV-1. At least two mechanisms may be responsible for low proliferative responses in vitro: inadequate levels of IL-2 and the presence of suppressor cells.     

Expression of interleukin 4, interleukin 4 splice variants and interferon gamma mRNA in calves experimentally infected with Fasciola hepatica

Interleukin 4 (IL-4) is expected to play a dominant role in the development of T helper (Th) 2 cells. Th2 immune responses with expression of relatively large amounts of interleukin 4 (IL-4) but little interferon gamma (IFN-gamma) are characteristic for chronic helminth infections. But no information is available about IL4 expression during early Fasciola hepatica (F. hepatica) infections in cattle. Therefore, we investigated F. hepatica specific IL-4 and IFN-gamma mRNA expression in peripheral blood mononuclear cells (PBMCs) from calves experimentally infected with F. hepatica. Cells were collected prior to infection and on post-inoculation days (PIDs) 10, 28 and 70. Interestingly, PBMCs responded to stimulation with F. hepatica secretory-excretory products (FhSEP) already on PID 10 and expressed high amounts of IL-4 but not of IFN-gamma mRNA suggesting that F. hepatica induced a Th2 biased early immune response which was not restricted to the site of infection. Later in infection IL-4 mRNA expression decreased whereas IFN-gamma mRNA expression increased slightly. Isolated lymph node cells (LNCs) stimulated with FhSEP and, even more importantly, non-stimulated LN tissue samples indicated highly polarized Th2 type immune responses in the draining (hepatic) lymph node, but not in the retropharyngeal lymph node. During preliminary experiments, two splice variants of bovine IL-4 mRNA, boIL-4delta2 and boIL-4delta3, were detected. Since a human IL-4delta2 was assumed to act as competitive inhibitor of IL-4, it was important to know whether expression of these splice variants of bovine IL-4 have a regulatory function during an immune response to infection with F. hepatica. Indeed, IL-4 splice variants could be detected in a number of samples, but quantitative analysis did not yield any clue to their function. Therefore, the significance of bovine IL-4 splice variants remains to be determined.     

Effects of ionizing radiation on in vitro proliferative responses of porcine peripheral blood lymphocytes

The radiosensitivity of $\text{in vitro}$ proliferative responses of porcine peripheral blood lymphocytes (PBL) was assessed. PBL were stimulated by Con-A, PHA, culture supernates from mitogen-stimulated porcine lymphocytes, or in the case of antigen-primed swine, specific antigens. The resulting levels of proliferation were assessed by a determination of the level of incorporation of tritiated thymidine $\text{in vitro}$, and in some cases by the presence of blast cells in the cultures. Porcine PBL were found to be more radioresistant than either mouse PBL or mouse spleen cells. Irradiation levels of greater than 3000 rads were necessary to arrest Con-A or PHA-induced proliferative responses. Proliferation induced by lymphokines in the form of supernates from mitogen-stimulated lymphocyte cultures was arrested in PBL that had received 3000 rads prior to culture. Antigen-induced proliferative responses in primed porcine PBL populations were the most radiosensitive, in that a previous irradiation with 500 rads was sufficient to completely abolish a secondary $\text{in vitro}$ proliferative response.     

The development and validation of an antibody-ELISA to detect Trypanosoma evansi infection in cattle in Australia and Papua New Guinea

Trypanosoma evansi is exotic to Australia and Papua New Guinea (PNG). However, it might have been introduced to Papua (Indonesia); thus, there is a risk of it entering PNG and thence Australia. Because of logistical difficulties in PNG and northern Australia, surveillance for T. evansi must rely on serological tests. The accuracy of an Ab-ELISA using a detergent extract of T. evansi and three antigen fractions purified from the detergent extract using stepwise precipitation with saturated ammonium sulphate (AS) were compared. The ELISA using the AS 40-50% fraction had greater discriminatory power compared to the ELISA using the other antigen fractions. This ELISA then was compared with two commercial tests: the Card Agglutination Test for trypanosomiasis/T. evansi (CATT) and Suratex. CATT/T. evansi at 1/4 serum dilution has higher sensitivity and the ELISA has higher specificity. There is no likely benefit in combining antibody detection tests to improve the accuracy of diagnosis. Furthermore, the combination of Suratex (which was independent of the antibody tests) with the CATT or the ELISA did not improve the sensitivity. None of the tests was sufficiently sensitive to be used confidently to determine freedom from infection in animals imported into Australia from countries where T. evansi infection is endemic.     

Subcellular fractions of Brucella ovis distinctively induce the production of interleukin-2, interleukin-4, and interferon-γ in mice

The aim of this study was to evaluate the effect of 3 Brucella ovis subcellular protein fractions: Outer membrane (OMP), inner membrane (IMP), and cytoplasm (CP), on cellular immune response by in vitro production of interleukin (IL)-2, IL-4, and interferon (IFN)-gamma. Each fraction was inoculated 3 times into Balb/c mice, primary cultures of mice spleen cells were done, and these were then stimulated with the fractions. Culture supernatants were collected at 24, 48, 72, 96, and 120 h postinoculation. Cytokine concentration was measured by Duoset-enzyme-linked immunosorbent assay (ELISA). The OMP fraction induced highest cellular immune response of 1000 pg/mL of IL-2 at 24 h, which decreased to < 100 pg/mL by 96 h. The IL-2 response for the IMP fraction was low at 24 h, but exceeded that of the OMP fraction at 72, 96, and 120 h. The CP showed a poor IL response. Regarding the IFN-gamma production, OMP and IMP induced a high response at 120 h. These results open the possibility for the use of B. ovis outer and inner membrane proteins as a subcellular vaccine.     

Establishment of an IL-2-dependent bovine T cell line and an assay of lymphocyte response inhibition in leukemic bovine serum.

The mechanism of immunosuppression induced by leukemic bovine serum was investigated with respect to lymphokine reactions using an interleukin 2 (IL-2)-dependent bovine T cell line generated from bovine peripheral blood lymphocytes (PBLs). The suppression of concanavalin A (con A)-induced PBL blastogenesis was observed at a high rate in leukemic cattle sera. The growth of IL-2-dependent bovine T cells and IL-2 production from con A-induced bovine PBLs were also inhibited by these sera, and particularly, the latter was correlated significantly to the degree of lymphocyte blastogenesis by the mitogen. Therefore, the lesser sensitivity of lymphocytes to IL-2 and the reduced IL-2 production by activated lymphocytes seem to play a role in suppressing the lymphocyte reaction.     

Regulation of mitogenic responses by bovine milk leukocytes

Bovine milk lymphocytes are less responsive to in vitro mitogen stimulation than peripheral blood lymphocytes (PBL). In this study, milk leukocytes (ML) or their soluble products, were co-cultured with mitogen stimulated PBL to determine if suppression could be transferred to normally responsive cells. Addition of either ML (treated with mitomycin C to prevent cell division), or supernatant from ML cultures to cultures of autologous PBL resulted in a reduction of mitogenesis by the PBL, but no suppression was seen with addition of treated PBL or PBL supernatant. Suppression was greater when the ML were from animals with chronic staphylococcal infection. Suppression by ML supernatant was not due to toxicity to the responders, since addition at the latter stages of culture had no effect on the response. These results indicate that reduced mitogenesis by milk lymphocytes may be due to the presence of suppressor cells or molecules.     

Isolation of two antigens from Trypanosoma evansi that are partially responsible for its cross-reactivity with Trypanosoma vivax

In Venezuela, two non-tsetse transmitted trypanosomes, Trypanosoma evansi and Trypanosoma vivax, are the major etiological agents of animal trypanosomosis. Rodents can be experimentally infected with T. evansi in order to obtain enough parasites to prepare antigens for serological tests. On the contrary, the production of T. vivax antigens is a limiting factor in most laboratories. Since T. evansi and T. vivax have exhibited a very high immunological cross-reactivity, we have focused on the identification of antigens from T. evansi responsible for this phenomenon. The predominant 64 kDa glycosylated cross-reacting antigen was recently purified from the TEVA1 T. evansi Venezuelan isolate [Parasitology 124 (2002) 287]. Here, we purified two additional cross-reacting antigens with molecular masses of approximately 51 and 68 kDa from the cytosolic fraction of the same T. evansi isolate, by sequential chromatography on DEAE-sepharose and sephacryl S-300. Sera obtained from animals infected with T. evansi or T. vivax recognized both purified proteins, suggesting their potential use as diagnostic reagents.     

Comparison of serological tests for Trypanosoma evansi natural infections in water buffaloes from north Vietnam

In the present study, a collection of 415 water buffalo serum samples originating from the north of Vietnam was used for evaluation of different diagnostic antibody detection methods available to detect infections with Trypanosoma evansi. The diagnostic sensitivity and specificity of a direct card agglutination test (CATT/T. evansi), an indirect card agglutination test (LATEX/T. evansi) and a newly developed antibody detection ELISA (ELISA/T. evansi) was calculated on the basis of parasitological results, obtained by mouse inoculation, and compared for all assays. The immume trypanolysis assay with the predominant T. evansi RoTat 1.2 variable antigen type was used as reference test for antibody presence. All parasitologically confirmed animals (n=8) were positive in all tests. Diagnostic specificity was highest in CATT/T. evansi (98%) followed by the ELISA/T. evansi (95%) and the LATEX/T. evansi (82%). Concordance of the variant specific immune trypanolysis test with the other tests was calculated and revealed that few (1-8%) false positive results were actually due to a specific reactions, and that LATEX/T. evansi and ELISA/T. evansi detected more immune trypanolysis positives than the CATT/T. evansi. It was concluded that, apart from the immune trypanolysis test, which is not generally applicable, ELISA/T. evansi with a 30% positivity cut-off and LATEX/T. evansi, thanks to their superior capacity of detecting T. evansi specific antibodies, would be suitable as epidemiological tools detecting both active infections and persisting T. evansi specific antibodies. The ELISA/T. evansi with a 50% positivity cut-off and the CATT/T. evansi on the other hand, seem more appropriate to detect true infected water buffaloes.     

Bovine interleukin 2 (IL-2) production and activity on bovine and murine cell lines

Long-term growth of T cell cultures requires addition of Interleukin 2 (IL-2). In order to maintain bovine cultures, optimal conditions for bovine IL-2 production were defined using peripheral blood mononuclear cells (PBM). Irradiation and preculture enhanced IL-2 production possibly by reducing suppressor activity. IL-2 activity was also detected in Bovine Herpesvirus Type 1-stimulated cultures. Unlike mitogen-stimulated cultures, a wide variation in IL-2 activity was seen between supernatants produced by virus-stimulated cells from different animals indicating the clonal nature of antigen specific cells from individuals. Bovine IL-2-dependent cells used to quantitate IL-2 activity were characterized as: PNA, esterase negative, H4+ (anti Ia-like), B29+ (anti-pan T cell), and C5- (anti-monocyte). The observations that bovine IL-2 can maintain activated murine cells, CTLL-20 and HT-2, could lead to the replacement of rat IL-2 with bovine IL-2 in long-term murine cultures. Conditions described here result in large volumes of active medium.