Evaluation of gamma interferon (IFN-γ)-induced protein 10 responses for detection of cattle infected with Mycobacterium bovis: comparisons to IFN-γ responses.

Gamma interferon (IFN-γ)-induced protein 10 (IP-10) has recently shown promise as a diagnostic biomarker of Mycobacterium tuberculosis infection of humans. The aim of the current study was to compare IP-10 and IFN-γ responses upon Mycobacterium bovis infection in cattle by using archived samples from two aerosol inoculation studies. In the first study (10(4) CFU M. bovis by aerosol, n = 7), M. bovis purified protein derivative (PPDb)-specific IP-10 and IFN-γ gene expression was detected as early as 29 days after challenge. PPDb-specific IP-10 and IFN-γ mRNA responses followed a similar pattern of expression over the course of this study and were highly correlated (r = 0.87). In the second study (10(5) CFU M. bovis by aerosol, n = 5), IP-10 and IFN-γ (protein) responses to mycobacterial antigens were compared following challenge. IFN-γ responses to mycobacterial antigens were detected at 29 days after challenge and were sustained during the remainder of the study. IFN-γ responses to mycobacterial antigens exceeded corresponding responses in nonstimulated cultures. IP-10 responses to mycobacterial antigens exceeded preinfection responses at 7, 29, and 63 days after challenge. In contrast to IFN-γ responses, IP-10 responses to mycobacterial antigens generally did not exceed the respective responses in nonstimulated cultures. IP-10 responses to medium alone and to mycobacterial antigens followed a similar pattern of response. Correlations between IP-10 and IFN-γ (protein) responses were modest (r ≈ 0.50 to 0.65). Taken together, these findings do not support the use of IP-10 protein as a biomarker for bovine tuberculosis using the current testing protocol and reagents; however, mRNA-based assays may be considered for further analysis.

Lung lesions in SCID-bo and SCID-bg mice after intratracheal inoculation with wild-type or leucotoxin-deficient mutant strains of Mannheimia haemolytica serotype 1.

The purpose of this study was to investigate SCID-bg mice engrafted with bovine haematolymphoid tissues (SCID-bo) as a model for studying bovine Mannheimia haemolytica serotype 1- induced pneumonia, in which leucotoxin (LKT) plays a major role. In experiment A, SCID-bo and SCID-bg mice were inoculated intratracheally with either (1) phosphate-buffered saline (PBS), (2) M. haemolytica wild-type strain 89010807N ("LKT(+)WT"), (3) a M. haemolytica leucotoxin-deficient mutant of strain 89010807N ("LKT(-)mutant"), or (4) the M. haemolytica wild-type Oklahoma strain. Mice were killed for examination at intervals between 20 and 44h after inoculation. Lung lesions consisted of thickened alveolar septa and neutrophil and macrophage infiltrates in the bronchioles and alveoli. Lung lesion scores in the SCID-bo mice inoculated with LKT(+)WT or LKT(-) mutant were significantly (P<0.05) greater than those of the PBS control group, but the two bacterial strains produced results that did not differ significantly. M. haemolytica was isolated from lung, liver and spleen after inoculation but less frequently as time progressed. In experiment B, SCID-bg mice were inoculated intratracheally with live LKT(+)WT or formalin-killed LKT(+)WT and killed 24, 48 or 96 h later. Lung lesions were histologically similar to those observed in experiment A; however, there were no significant differences in the lung lesion scores between groups. It was concluded that the lesions seen in this study were probably not due to LKT, and that the SCID-bo mouse does not provide a good rodent model for bovine pneumonia.

Use of recombinant ESAT-6:CFP-10 fusion protein for differentiation of infections of cattle by Mycobacterium bovis and by M. avium subsp. avium and M. avium subsp. paratuberculosis.

Immunological diagnosis of Mycobacterium bovis infection of cattle is often confounded by cross-reactive responses resulting from exposure to other mycobacterial species, especially Mycobacterium avium. Early secretory antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) are dominant gamma interferon (IFN-gamma)-inducing antigens of tuberculous mycobacteria, and they are absent from many environmental nontuberculous mycobacteria. Because M. avium exposure is the primary confounding factor in the diagnosis of M. bovis-infected animals, in vitro responses to a recombinant ESAT-6:CFP-10 (rESAT-6:CFP-10) fusion protein by blood leukocytes from cattle naturally exposed to M. avium or experimentally challenged with Mycobacterium avium subsp. avium or Mycobacterium avium subsp. paratuberculosis were compared to responses by M. bovis-infected cattle. Responses to heterogeneous mycobacterial antigens (i.e., purified protein derivatives [PPDs] and whole-cell sonicates [WCSs]) were also evaluated. Tumor necrosis factor alpha (TNF-alpha), IFN-gamma, and nitric oxide responses by M. bovis-infected cattle to rESAT-6:CFP-10 exceeded (P < 0.05) the corresponding responses by cattle naturally sensitized to M. avium. Experimental infection with M. bovis, M. avium, or M. avium subsp. paratuberculosis induced significant (P < 0.05) IFN-gamma and nitric oxide production to WCS and PPD antigens, regardless of the mycobacterial species used for the preparation of the antigen. Responses to homologous crude antigens generally exceeded responses to heterologous antigens. Nitric oxide and IFN-gamma responses to rESAT-6:CFP-10 by blood leukocytes from M. bovis-infected calves exceeded (P < 0.05) the corresponding responses of noninfected, M. avium-infected, and M. avium subsp. paratuberculosis-infected calves. Despite the reported potential for secretion of immunogenic ESAT-6 and CFP-10 proteins by M. avium and M. avium subsp. paratuberculosis, it appears that use of the rESAT-6:CFP-10 fusion protein will be useful for the detection of tuberculous cattle in herds with pre-existing sensitization to M. avium and/or M. avium subsp. paratuberculosis.

Dietary soy phytoestrogens and ERalpha signalling modulate interferon gamma production in response to bacterial infection.

Diets rich in soy phytoestrogens have many potential health benefits but isoflavones such as genistein may suppress cell mediated immune function. The effect of dietary phytoestrogens on the host response to infection has not been extensively examined. Mice were fed a diet containing soy phytoestrogens and infected with Mycobacterium avium to establish a chronic infection and inflammatory response. As phytoestrogens may act through classical oestrogen receptors (ER), mice deficient in ERalpha signalling and wild type mice were evaluated for a panel of Type 1-associated cytokines (IFNgamma, IL-12 and IL-18) in the spleen. IFNgamma production in the spleen was increased approximately 4-fold in ERalpha-deficient mice fed a casein-based diet over wild type mice fed a casein-based diet (P < 0.05), suggesting a role for ERalpha in suppressing IFNgamma production. IL-18 levels in spleens of wild type mice were decreased compared to ERalpha-deficient mice on a casein diet. Splenic IL-12 and IL-18 levels were not affected in wild type and ERalpha-deficient mice on the phytoestrogen containing diets, with the exception that whole soy increased IL-12 levels in the tissues of ERalpha deficient mice. We conclude that ERalpha and dietary phytoestrogens can influence production of key regulatory cytokines in response to chronic bacterial infection.

Mycobacterium bovis bacille Calmette-Guerin vaccination of cattle: activation of bovine CD4+ and gamma delta TCR+ cells and modulation by 1,25-dihydroxyvitamin D3.

SETTING: 1,25-dihydroxyvitamin D3 (1,25(OH)(2)D(3)) is a potent modulator of immune responses and may be beneficial in the treatment of tuberculosis. Recent evidence suggest that 1,25(OH)(2)D(3) may affect T-dependent responses in cattle; however, mechanisms by which this vitamin modulates activation of bovine T cells are unclear. OBJECTIVE: Determine the effects of 1,25(OH)(2)D(3) on the expression of CD25, CD44, and CD62L by bovine T cell subsets proliferating in response to antigen stimulation. DESIGN: Antigen-specific recall responses of Mycobacterium bovis bacille Calmette-Guerin (BCG) vaccinated cattle were used as a model system to evaluate effects of 1,25(OH)(2)D(3) on the proliferation and activation of bovine T cell subsets. RESULTS: CD4(+) and gamma delta TCR(+) cells were the predominant T cell subsets responding to soluble crude M. bovis-derived antigens (i.e., purified protein derivative and a BCG whole cell sonicate) by proliferation and activation-induced alterations in phenotype. These subsets exhibited increased CD25 and CD44 mean fluorescence intensity (mfi) and decreased CD62L mfi upon antigen stimulation. Addition of 1,25(OH)(2)D(3) inhibited proliferation of CD4(+) cells and decreased the expression of CD44 on responding (i.e., proliferating) CD4(+) and gamma delta TCR(+) cells. CONCLUSION: These findings suggest that the production of 1,25(OH)(2)D(3) by macrophages within tuberculous lesions would inhibit proliferation and CD44 expression by co-localized CD4(+) and gamma delta TCR(+) cells.

Use of the mannan receptor to selectively target vaccine antigens for processing and antigen presentation through the MHC class I and class II pathways.

Extensive studies have shown that synthetic and recombinant vaccines developed against hemoparasites have not been as effective as whole parasites or crude membrane fractions in eliciting protective immunity. A possible reason is that synthetic vaccines are not being presented in a form that induces the appropriate immune response. We have developed a bovine model system to evaluate the ability of adjuvant compounds to induce an immune response to peptide antigens dominated by a cytokine profile with a Type 1 (cell-mediated) or Type 2 (humoral) bias. In the initial testing of this system, we found that mRNA expression of certain cytokines (interleukin [IL]-1beta, IL-6, IL-12, IL-15, GM-CSF, iNOS, and tumor necrosis factor [TNF]-alpha) is enhanced when monocyte-derived macrophages are stimulated with peptide antigen conjugated with mannan under oxidizing conditions compared to peptide conjugated with reduced mannan. The data suggest this model will be useful in identifying adjuvant systems that selectively modulate the cytokine profile of antigen presenting cells at the time of antigen presentation and the consequent downstream maturation of naive T cells to effector cells with Type 1 or Type 2 cytokine bias.

Enhancing effects of anti-CD40 treatment on the immune response of SCID-bovine mice to Trypanosoma congolense infection.

African trypansosomes are tsetse-transmitted parasites of chief importance in causing disease in livestock in regions of sub-Saharan Africa. Previous studies have demonstrated that certain breeds of cattle are relatively resistant to infection with trypanosomes, and others are more susceptible. Because of its extracellular location, the humoral branch of the immune system dominates the response against Trypanosoma congolense. In the following study, we describe the humoral immune response generated against T. congolense in SCID mice reconstituted with a bovine immune system (SCID-bo). SCID-bo mice infected with T. congolense were treated with an agonistic anti-CD40 antibody and monitored for the development of parasitemia and survival. Anti-CD40 antibody administration resulted in enhanced survival compared with mice receiving the isotype control. In addition, we demonstrate that the majority of bovine IgM+ B cells in SCID-bo mice expresses CD5, consistent with a neonatal phenotype. It is interesting that the percentage of bovine CD5+ B cells in the peripheral blood of infected SCID-bo mice was increased following anti-CD40 treatment. Immunohistochemical staining also indicated increased numbers of Ig+ cells in the spleens of anti-CD40-treated mice. Consistent with previous studies demonstrating high IL-10 production during high parasitemia levels in mice and cattle, abundant IL-10 mRNA message was detected in the spleens and peripheral blood of T. congolense-infected SCID-bo mice during periods of high parasitemia. In addition, although detected in plasma when parasites were absent or low in number, bovine antibody was undetectable during high parasitemia. However, Berenil treatment allowed for the detection of VSG-specific IgG 14 days postinfection in T. congolense-infected SCID-bo mice. Overall, the data indicate that survival of trypanosome-infected SCID-bo mice is prolonged when an agonistic antibody against bovine CD40 (ILA156) is administered. Thus, stimulation of B cells and/or other cell types through CD40 afforded SCID-bo mice a slight degree of protection during T. congolense infection.

Induction of interleukin-6 and interleukin-12 in bovine B lymphocytes, monocytes, and macrophages by a CpG oligodeoxynucleotide (ODN 2059) containing the GTCGTT motif.

Bacterial DNA and synthetic oligodeoxynucleotides (ODN) that contain unmethylated CpG dinucleotides flanked by certain bases (CpG ODN) have been shown to activate murine and human B cells and to induce proinflammatory cytokines by monocytes/macrophages and dendritic cells (DC). However, the CpG ODN sequences optimal for mice and humans are different. In the current study, the effects of CpG ODN, which were defined to stimulate strong responses in either mouse or human leukocytes, were compared for stimulation of bovine B lymphocyte proliferation and macrophage cytokine mRNA expression. The optimal CpG ODN was then tested for induction of cytokines in peripheral blood mononuclear cells (PBMC) and purified B lymphocytes, monocytes, and macrophages. At a high ODN concentration (40 microM), all but two CpG ODN tested stimulated B cell proliferation, which was dependent on unmethylated CpG motifs. CpG ODN 2059 containing the GTCGTT motif shown to activate human leukocytes also promoted the highest level of bovine B cell proliferation at a lower concentration (10 microM) when compared with CpG ODN containing AACGTT or GACGTT motifs active for murine leukocytes. Furthermore, ODN 2059 induced interleukin-6 (IL-6) production by B lymphocytes and IL-6 and IL-12 production by PBMC, monocytes, and macrophages. In contrast, IL-1beta and tumor necrosis factor-alpha (TNF-alpha) production was either very low or undetectable. Consistent with increased IL-12 production, ODN 2059 also stimulated interferon-gamma (IFN-gamma) production by PBMC. Importantly, the levels of cytokines induced by ODN 2059 were comparable to those generated in response to Escherichia coli DNA. The weak TNF-alpha response combined with the vigorous IL-6 and IL-12 response to ODN 2059 indicate the potential use of this CpG ODN as an adjuvant to enhance both antibody-mediated and IFN-gamma-mediated macrophage activation, which are important for protection against disease caused by intracellular pathogens of cattle.

The P2X7 purinergic receptor on bovine macrophages mediates mycobacterial death.

P2X7 is an ATP gated purinoceptor that has been linked to various immune responses. P2X7 appears to be expressed ubiquitously in the immune system and thus may be important as an effector pathway or play significant roles in cell activation/death. 2',3'-(4-Benzoyl)benzoyl ATP is the most potent agonist of this receptor and ATP in its fully dissociated form (ATP(4-)) also activates the receptor. High concentrations of ATP can cause the P2X7 receptor to induce pore formation on the surface of the cell that allows molecules of considerable size to pass and can lead to cell death. The P2X7 receptor has also been linked to various immune activities when the concentration of ATP is lower, including the release of IL-1beta. The role P2X7 receptors have on immune cell activities is just beginning to be understood. We sought to determine the role of P2X7 on bovine macrophages in eliminating the causative agent of bovine-type tuberculosis, Mycobacterium bovis. Because high concentrations of ATP are linked to macrophage death, we determined if this method of cell destruction also leads to reduced bacterial viability. We find that P2X7 is present on bovine macrophages from different sources, including both peripheral blood-derived as well as alveolar macrophages. In addition, P2X7 mRNA is present in B and T lymphocytes. The treatment of M. bovis-infected macrophages with ATP results in reduced macrophage viability as well as reduced M. bovis viability.

The identification and characterization of a ligand for bovine CD5.

CD5, a type I glycoprotein expressed by T cells and a subset of B cells, is thought to play a significant role in modulating Ag receptor signaling. Previously, our laboratory has shown that bovine B cells are induced to express this key regulatory molecule upon Ag receptor cross-linking. To date, a ligand has not been described for bovine CD5. Given the importance ligand binding presumably plays in the functioning of CD5 on this B cell subset and on T cells, we sought to characterize the ligand for this protein using a bovine CD5-human IgG1 (CD5Ig) fusion protein produced by both mammalian and yeast cells. As determined by CD5Ig binding, expression of this ligand is negative to low on freshly isolated lymphocytes, with low-density expression being limited to activated B cells. Activation with LPS, PMA, and calcium ionophore, or ligation of CD40 alone or in combination with anti-IgM, resulted in B cell-specific expression of this ligand. Interestingly, activation through B cell Ag receptor cross-linking alone, although able to induce CD5 expression, did not result in expression of CD5 ligand (CD5L). In addition, we demonstrate a functional role for CD5L as a costimulatory molecule that augments CD40L-stimulated B cell proliferation. Finally, immunoprecipitation with CD5Ig suggests that the ligand characterized in this study has a molecular mass of approximately 200 kDa. The data reported herein, as well as future studies aimed at further characterizing this newly identified bovine CD5L, will undoubtedly aid in understanding the role that the CD5-CD5L interaction plays in immune responses.

Natural killer cells express estrogen receptor-alpha and estrogen receptor-beta and can respond to estrogen via a non-estrogen receptor-alpha-mediated pathway.

Natural killer (NK) cells play a crucial role in host defense against pathogens and immune surveillance against cancer. Given that estrogens have been reported to suppress NK cell activity, we sought to elucidate the mechanisms by which estrogen mediates this effect. We demonstrate by immunocytochemical staining with estrogen receptor-alpha (ERalpha)- and estrogen receptor-beta (ERbeta)-specific antibodies that both ERalpha and ERbeta are expressed in murine NK cells. We also compared the ability of high doses of 17beta-estradiol ( approximately 800 pg/ml) to regulate NK cell activity in wild-type and estrogen receptor-alpha-deficient (ERalphaKO) mice. 17beta-estradiol elicited a significant decrease in NK cell activity in both wild-type and ERalphaKO mice (P < 0.001). These data suggest that ERbeta or possibly a novel receptor is involved in mediating estrogen action on NK cell activity and raise the potential for therapeutic modulation of NK cell activity with selective estrogen receptor modulators (SERMS).

Pathogenesis of herpes simplex virus type 1-induced corneal inflammation in perforin-deficient mice.

Herpetic stromal keratitis (HSK) is an inflammatory disease of the cornea that often results in blindness. It is mediated by a host immune response which is triggered by herpes simplex virus (HSV) infection. Immune effector mechanisms are hypothesized to be important in disease development. We investigated, in a mouse model, whether perforin-dependent cytotoxicity is an important effector mechanism in the production of HSK. Wild-type (C57BL/6) and perforin-deficient (PKO) mice were infected intracorneally with HSV-1 strain F. Clinical disease and histologic lesions of the cornea at 23 days postinfection (p.i.) were significantly less severe in HSV-1-infected PKO mice than in infected wild-type mice. mRNA for the chemokine macrophage inflammatory protein 1alpha (MIP-1alpha) was detected by reverse transcription-PCR in the corneas of infected wild-type mice but not in the corneas of infected PKO mice at 23 days p.i. Adoptive transfer of wild-type HSV-1 immune T-cell-enriched splenocytes into HSV-1-infected PKO mice restored the disease phenotype which was seen in infected wild-type mice. In contrast, mice carrying a null-function mutation in the Fas ligand, which is involved in an alternative cytotoxic mechanism, developed clinical disease and histologic lesions which were comparable to those in wild-type mice. Viral clearance from the eyes of PKO mice was not impaired. There was no significant difference between the infectious viral titers isolated from the eyes of PKO and wild-type mice. Our findings show that perforin is important in the pathogenesis of HSK.

Activation of bovine B cells via surface immunoglobulin M cross-linking or CD40 ligation results in different B-cell phenotypes.

Experiments reported herein demonstrate that activation of bovine B cells via surface immunoglobulin M (sIgM) cross-linking, analogous to T-cell independent (TI-2) antigenic stimulation, results in the expression of CD5. Interestingly, in the presence of CD40 ligand, sIgM-mediated induction of CD5 on B cells was inhibited. These findings indicate that activation of bovine B cells via B-cell receptor (BCR) cross-linking results in a CD5+ B-cell phenotype and that CD40 signalling is inhibitory to this process. Analysis of cytokine mRNA indicates that bovine B cells constitutively express tumour necrosis factor-alpha (TNF-alpha) and interleukin (IL)-1beta transcripts in vitro, while IL-10 mRNA expression is induced following sIgM cross-linking. IL-12 p40 transcripts were produced by B cells activated by CD40, but not by BCR, ligation. Analysis of cytokine receptor mRNA indicates that activation through CD40, in the presence or absence of IgM cross-linking, results in increased IL-4 receptor-alpha (IL-4Ralpha), IL-13Ralpha1 and interferon-alpha receptor 1 (IFN-alphaR1) mRNA levels. Overall, these findings suggest that activation of bovine B cells through BCR cross-linking yields an activation phenotype that differs substantially from that of B cells activated through CD40.

Functional implications for signaling via the IL4R/IL13R complex on bovine cells.

IL-4 and IL-13 share a wide range of activities on monocytes, epithelial cells and B cells and thus play an important role in host defense. Many of these activities are not conserved among species as human, but not murine, B cells are thought to be responsive to IL-13. We previously demonstrated that human IL-13 is highly conserved at the nucleic acid level with a candidate bovine IL-13 cDNA homologue. Moreover, recombinant human IL-13 stimulates Ig secretion by appropriately activated bovine B cells. These studies have been extended to examining Ig class switching at both the protein and mRNA levels in addition to examining other markers of cellular activation. Our results suggest that IL-13 influences B cell differentiation by enhancing IgM, IgG1, and IgE production. IL-13 stimulation alone increases MHC class II expression and progression through cell cycle, although at lower levels in comparison to rboIL-4. The biology of the receptors for IL-4 and IL-13 is complex and raises several key questions with regard to IL-4-dependent and -independent mechanisms of host immunomodulation. Recent studies suggest that at least four chains are involved. These include the p140 IL-4 binding chain (IL-4Ralpha), the common gamma chain (gammac chain), IL-13 receptor alpha- chain (IL-13Ralpha-1) and the IL-13 receptor alpha-2 chain (IL-13Ralpha-2). We have recently cloned cDNAs for the bovine homologues of the IL-13Ralpha-1 and IL-4Ralpha chains and evaluated mRNA expression for a variety of cell types following stimulation. The expression patterns and their implications for receptor chain utilization in signaling via these key TH2 signature cytokines will be discussed.

Modulation of host immune responses by protozoal DNA.

The pathology caused by acute Babesia bovis infection is similar to that seen in severe human malaria caused by Plasmodium falciparum infection, which is related to dysregulated production of inflammatory cytokines and nitric oxide (NO). We have observed induction of NO, inducible nitric oxide synthase (iNOS) and inflammatory cytokines in macrophages by B. bovis. Furthermore, proliferation of lymphocytes from individuals never exposed to certain protozoal pathogens can be induced by crude protozoal parasite extracts. We have repeatedly observed stimulation of naive PBMC from cattle to antigenic extracts of Babesia bovis. Based on recent studies demonstrating the mitogenicity of bacterial and other non-vertebrate DNAs for murine B cells and macrophages, the mitogenic properties of B. bovis DNA were examined. B. bovis and E. coli DNAs induced proliferation of PBMC and purified B cells from non-exposed cattle. Stimulatory activity was reduced by DNase treatment and methylation with CpG methylase, indicating the presence of stimulatory non-methylated CpG motifs in the B. bovis genome. B. bovis and E. coli DNAs enhanced IgG secretion by cultured B cells, stimulating IgG1 and more strongly, IgG2. Several hexameric CpG immunostimulatory sequences (ISS) active for murine B cells were identified in an 11 kb fragment of B. bovis DNA. An oligodeoxyribonucleotide containing one of these (AACGTT), located in the rhoptry associated protein-1 (rap-1) open reading frame, stimulated B cell proliferation. These studies identify a potential mechanism by which protozoal parasites may modulate host immune responses, leading to consequences such as hypergammaglobulinemia and splenomegaly. These results also support the use of ISS as vaccine adjuvants to enhance Type 1 immune responses in cattle.

Immunoregulatory roles of interleukin-13 in cattle.

Interleukin-13 (IL-13) is produced predominantly by helper T lymphocytes of the Th2 phenotype and mediates its effects on several immune cells, including B lymphocytes and macrophages, stimulating their proliferation, differentiation, and effector functions. IL-13 activates human B cells but has no detectable activity on murine B lymphocytes, suggesting that the activity of IL-13 varies among species. Our studies show that IL-13 enhances proliferation and differentiation of bovine B cells and upregulates cell surface major histocompatibility complex (MHC) class II expression. We examined mRNA expression of the putative signaling component of the bovine IL-13Ralpha1 homolog in several peripheral blood populations. After stimulation with calcium ionophore and phorbol ester, IL-13Ralpha1 mRNA levels appeared to be downmodulated in T cells, upregulated in macrophages and B cells, and unchanged in neutrophils. Together, these studies begin to provide insight into the relative importance of IL-13 in immunoregulation in cattle.

Comparative effects of interleukin-12 and interleukin-4 on cytokine responses by antigen-stimulated memory CD4+ T cells of cattle: IL-12 enhances IFN-gamma production, whereas IL-4 has marginal effects on cytokine expression.

Interleukin-12 (IL-12) and IL-4 are important immunoregulatory cytokines that determine the fate of naive T cells during antigen priming in mice and also influence cytokine synthesis by differentiated murine and human T cells. The roles of these cytokines in regulating the differentiation and effector function of bovine T cells are less well studied. We investigated the ability of human IL-12 and bovine IL-4 to modify cytokine expression by antigen-stimulated T cells from cattle immune to the protozoal parasites Babesia bovis and Babesia bigemina or reactive with Mycobacterium bovis purified protein derivative. Peripheral blood mononuclear cells (PBMC) were cultured with specific antigen and IL-4 or IL-12 for 1 week. Then viable lymphoblasts consisting of predominantly CD4+ T cells were restimulated with antigen and antigen-presenting cells (APC) with or without cytokine. Cell lines were cultured for several weeks, and following restimulation with antigen and APC in the absence of exogenous cytokine, the cell lines were analyzed for proliferation, interferon-gamma (IFN-gamma) production, and expression of IL-2, IL4-, IL-10, or IFN-gamma transcript levels using a quantitative competitive RT-PCR. IL-12 and IL-4 had no effect on the composition of CD4, CD8, or gammadelta T cells in the cell lines or on the level of antigen-induced proliferation. IL-12 stimulated enhanced levels of IFN-gamma protein and transcript expression in all cell lines, with no consistent effects on IL-2 or IL-4 expression. In two B. bovis-specific cell lines, IL-12 suppressed IL-10 expression. IL-4 had no consistent effect on expression of any cytokine. These results indicate the use of IL-12 as an adjuvant to enhance type 1 cytokine responses in cattle during antigen priming.

Development and in vitro characterization of recombinant vaccinia viruses expressing bovine leukemia virus gp51 in combination with bovine IL4 or IL12.

Type 1 and type 2 immune responses are modulated by IL12 or IL4, respectively, at the time of lymphocyte priming. Importantly, type 1 responses have been associated with resistance to retroviral infection in mice, humans, and ruminants. Specifically, vaccination of sheep with vaccinia virus expressing bovine leukemia virus (BLV) gp51 resulted in protective immunity with the characteristics of a type 1 response, whereas vaccination of cattle resulted in a non-protective type 2 response. In order to test the hypothesis that cattle inoculated with BLV gp51 and IL12 will respond with a type 1 response, a recombinant vaccinia virus expressing BLV gp51 together with bovine IL12 was developed and characterized in vitro. For induction of type 2 responses a recombinant vaccinia virus expressing gp51 with bovine IL4 was similarly constructed and characterized. In this study recombinant cassettes were developed containing either the BLVenv gene alone or in combination with bovine IL4 or the two genes, p35 and p40, encoding bovine IL12. Correct alignment with p7.5 or p11 vaccinia promoters and orientation was confirmed by complete sequencing. Recombinant vaccinia viruses were generated by homologous recombination, selected based on large plaque formation due to reconstitution of the vp37 gene, and structurally confirmed by Southern blotting. Transcription of recombinant BLVenv, bovine IL4, p35 and p40 was demonstrated by RT-PCR. Expression of BLVenv gp51 protein and bovine IL4 was shown by immunofluorescence and immunoblotting. Biologically active bovine IL4 expressed by vaccinia virus stimulated lymphoblast proliferation, B lymphocyte proliferation in the presence of CD40L, and inhibited IFN gamma secretion from PHA activated PBMC in a dose dependent fashion. Finally, bovine IL12 expression and biological function was confirmed by dose dependent induction of IFN gamma secretion by PHA activated PBMC and the moderate enhancement of lymphoblast proliferation. In conclusion, bovine IL12 and IL4 expressed by recombinant vaccinia virus in vitro clearly exhibited type 1-type 2 modulating properties.

Role of CD8+ and WC-1+ gamma/delta T cells in resistance to Mycobacterium bovis infection in the SCID-bo mouse.

The role of various effector T cell populations in the bovine immune response to Mycobacterium bovis infection is poorly understood. This is largely due to the difficulties associated with performing in vivo challenge studies in the natural host species. In this report, we utilized a fetal bovine-severe combined immunodeficient (SCID-bo) xenochimeric mouse model to study the protective role of two putative effector cell types, CD8+ T cells and a subpopulation of gamma/delta T cells that express WC-1, a member of the cysteine-rich scavenger receptor superfamily (CRSR). We demonstrate that CD8+ T cells play a key role in protection and contribute substantially to bovine IFN-gamma mRNA levels at 30 days post-infection. The role of WC-1 bearing cells to protection was less definitive but our results suggest that this population may play a pivotal role early in infection. Granuloma architecture was altered in anti-WC-1 (ILA29) but not anti-CD8 (ILA51) -treated animals, suggesting that this population may be involved in recruitment of various cell types to sites of infection.

Bovine CD4(+) T-lymphocyte clones specific for rhoptry-associated protein 1 of Babesia bigemina stimulate enhanced immunoglobulin G1 (IgG1) and IgG2 synthesis.

Optimal protective immunity against babesial infection is postulated to require both complement-fixing and opsonizing antibodies in addition to gamma interferon (IFN-gamma)-mediated macrophage activation. The rhoptry-associated protein 1 (RAP-1) of Babesia bigemina induces partial protective immunity and is a candidate vaccine antigen. Previous studies demonstrated that cattle immunized with native protein that were subsequently protected against challenge had a strong IFN-gamma and weaker interleukin-4 (IL-4) response in immune lymph node lymphocytes that reflected the cytokine profile of the majority of CD4(+) T-cell clones obtained from peripheral blood. RAP-1-specific T helper (Th) cell clones that coexpress IFN-gamma and IL-4 are typical of numerous parasite-specific clones examined. However, the function of such cells as helper cells to enhance immunoglobulin secretion by bovine B cells has not been reported. In cattle, both immunoglobulin G1 (IgG1) and IgG2 can fix complement, but IgG2 is the superior opsonizing subclass. Therefore, studies were undertaken to ascertain the functional relevance of RAP-1-specific, CD4(+) Th0 cells as helper cells to enhance IgG1 and/or IgG2 production by autologous B lymphocytes. For comparison, Th0 clones specific for the metazoan parasite Fasciola hepatica that expressed relatively more IL-4 than the B. bigemina-specific Th cells were similarly assayed. B. bigemina RAP-1-specific clones could enhance production of both IgG1 and IgG2 by autologous B cells, whereas Th cell clones specific for F. hepatica enhanced predominantly IgG1 production. The capacity to enhance IgG2 production was associated with production of IFN-gamma by Th cells cocultured with B cells, antigen, and IL-2. The in vitro helper T-cell activity of these T-cell clones was representative of the in vivo serologic responses, which were composed of a mixed IgG1-IgG2 response in B. bigemina RAP-1 immune cattle and a biased IgG1 response in F. hepatica-immune cattle.