Interleukin-6 expression in gut of parasite challenged sheep

Following challenge with Trichosirongylus colubrifonizis, increased numbers of T-cells and immunoglobulin responses are seen in the intestine of sheep immunised by repeated infection with live worms. IL-6 mRNA expression in the small intestine from T. colubriformis-immunised and naive sheep was determined by in situ hybridisation, whereas CD4(+), IgA(+), IgG(+) cells in the gut were evaluated by immunohistochemistry. There was constitutive expression of IL-6 mRNA by cells in the naive gut, and the number of these cells was increased by parasite challenge. There were corresponding increases in numbers of CD4(+) and TCR gamma/delta(+) T-cells and IgG(+) B-cells. Our data are consistent with a role for IL-6, perhaps produced by CD4(+) and/or TCR gamma/delta(+) T-cells or B-cells, in B-cell terminal differentiation. Infiltration of B-cells, particularly IgG(+) B-cells, may reflect parasite immunity in the host.     

Control of immunoglobulin isotype production by porcine B-cells cultured with cytokines

Cytokines regulate immunoglobulin (Ig) isotype production following the Th1/Th2 paradigm, derived from studies of inbred mice. In pigs, it is not known which, if any, Ig isotypes may reflect a Th1/Th2 response. To evaluate this, purified porcine CD21(+) B-cells were co-cultured with Staphylococcus aureus Cowan strain 1 or Escherichia coli lipopolysaccharide as B-cell mitogens together with recombinant human IL-2, and recombinant porcine (rp) interferon (IFN)-gamma, IL-12 or IL-10. While the mitogens increased B-cell proliferation, cytokines had no additional effect. A quantitative competitive enzyme-immuno assay was used to measure concentrations of porcine IgM, IgG(1) and IgG(2) in B-cell culture supernatants. In vitro, porcine B-cells produced IgG(2), 106 +/- 17.3 microg/ml; IgG(1) 107 +/- 38.3 microg/ml and IgM 25.6 +/- 8.45 microg/ml. In some individuals, Th1 cytokines such as rpIFN-gamma and IL-12, enhanced IgG(2) in the face of low concentrations of IgG(1). Furthermore, individual responses, in some cases, tended to be diametrically opposed, reminiscent of previously documented categorical immune responses in pigs such that some individuals produced high concentrations of IgG(1) in response to the various doses of rp cytokines, while others produced lower concentrations. Pigs may generate a high IgG(1):IgG(2) ratio in response to rpIL-10, and possibly to other Th2-associated cytokines. However, B-cell response to rp cytokines in vitro exhibits marked variation by pig, a feature that is likely a function of highly variable individual genotypes and their interaction with complex environments.     

Cellular profiles in the abomasal mucosa and lymph node during primary infection with Haemonchus contortus in sheep

Cellular changes in the abomasal tissue and draining abomasal lymph nodes were examined after primary infection of lambs with Haemonchus contortus for 3, 5 or 27-36 days.Infection with H. contortus larvae resulted in a rapid and selective increase in the percentage of CD4(+) T-cells in the abomasal lymph node at 3 days post-infection (PI). By 5 days PI, the lymph node weight had increased two-fold; however, the percentage of lymphocyte populations in the abomasal lymph node resembled that seen in uninfected sheep. Lymph node weights remained at increased levels in the adult nematode infected sheep and down-regulation of B-cell surface markers (sIg and MHC Class II) was apparent in this group. Significant increases in the percentage of CD4(+) T-cells co-expressing MHC Class II, but not CD25, were observed in the larval infected groups except in adult nematode infected sheep. Increased numbers of eosinophils, CD4(+), gamma delta(+) T-cells and B-cells were found in the abomasal tissue by 5 days PI, but no further increases in these cell populations were observed in the adult nematode infected group. In contrast, the level of both lamina propria and intraepithelial mast cells observed in the abomasal mucosa was highest in the sheep carrying an adult nematode burden. These findings indicate that sheep are able to generate an early immune response to infection with H. contortus larvae, characterised by the activation of CD4 T-cells and B-cells in the draining lymph nodes and recruitment of eosinophils, CD4(+) and gamma delta-TCR,WC1(+) T-cells and B-cells in larval infected tissues. However, these changes do not seem to be maintained during infection with the adult parasite where increases in mast cell numbers dominate the local response, indicating that different parasite stages may induce distinct and possibly counteractive immune responses.     

B-cell function in canine X-linked severe combined immunodeficiency

Canine X-linked severe combined immunodeficiency (XSCID) is due to mutations in the common gamma (gammac) subunit of the IL-2, IL-4, IL-7, IL-9 and IL-15 receptors and has a similar clinical phenotype to human XSCID. We have previously shown that the block in T-cell development is more profound in XSCID dogs than in genetically engineered gamma c-deficient mice. In this study we evaluated the B-cell function in XSCID dogs. In contrast to the marked decrease in peripheral B-cells in gamma c-deficient mice, XSCID dogs have increased proportions and numbers of peripheral B-cells as observed in XSCID boys. Canine XSCID B-cells do not proliferate following stimulation with the T-cell-dependent B-cell mitogen, pokeweed mitogen (PWM); however, they proliferate normally in response to the T-cell-independent B-cell mitogen, formalin-fixed, heat-killed Staphylococcus aureus. Canine XSCID B-cells are capable of producing IgM but are incapable of normal class-switching to IgG antibody production as demonstrated by in vitro stimulation with PWM and immunization with the T-cell-dependent antigen, bacteriophage PhiX174. Similar results have been reported for XSCID boys. Thus, it appears that gamma c-dependent cytokines have differing roles in human and canine B-cell development than in the mouse making the XSCID dog a valuable model for studying the role of these cytokines in B-cell development and function.     

Lineage differentiation of canine lymphoma/leukemias and aberrant expression of CD molecules

Multiparameter flow cytometry analysis and specific cluster differentiation (CD) molecules were used to determine the expression profiles of B- and T-cell antigens on lymph node preparations from 59 dogs with generalized or multisystemic lymphoma. Lymph node samples from 11 healthy dogs were labeled to validate the specificity of antibodies and to formulate guidelines for interpretation of the results obtained from lymphoma samples. In normal lymph nodes, T-lymphocytes expressing CD3, CD4, or CD8 beta represented 59+/-11%, 43+/-8%, or 16+/-5% of the total cells, whereas B-lymphocytes expressing either CD21 or surface IgM (IgM) represented 37+/-9% or 14+/-5%, respectively. Small lymphocytes could be distinguished from large lymphocytes by forward light scatter. Of the patient samples 29 different breeds were represented with Golden and Labrador retriever being the most common. The lymphoma samples segregated into three groups based on CD antigen expression. Thirty cases predominantly expressed one or more combinations of CD79a, IgM, and CD21 representing a B-cell lineage. Three B-cell cases also expressed the stem cell antigen, CD34. Sixteen cases expressed one or more combinations of CD3, CD4, and CD8 consistent with a T-cell lineage and CD3+CD4+CD8--phenotype was the most common. Thirteen cases showed a mixed expression profile for T- and B-cell antigens and in three cases CD14 was highly expressed. Clinical response was poorest for T-cell lymphomas. Leukemic states occurred in all three phenotypes; but mixed cell cases had the greatest proportion. Dual immunofluorescence staining confirmed co-expression of T-cell (CD3) and B-cell antigens (CD79a or CD21) on neoplastic lymphocytes of six mixed cell cases. In one mixed cell case, dual immunostaining identified lymphocyte populations that stained mutually exclusive for CD79a and CD3. Six mixed cell lymphomas tested by PCR showed clonality for rearranged antigen receptor. Four cases that were CD79a+CD3+ had TCRgamma chain gene rearrangements, whereas two cases that were CD3+CD8+CD21+ had Ig heavy chain rearrangement. One case expressing multiple CD molecules (CD3+CD8+CD21+CD14+) was PCR negative for both Ig and TCRgamma gene rearrangement and could not be classified into a B- or T-cell lineage. We show for the first time co-expression of B- and T-cell markers on lymphoma cells that had specific T- or B-cell gene rearrangements. These findings suggest that aberrant CD molecule expression is not an uncommon finding in canine lymphomas and is a useful diagnostic marker for malignancy.     

Expression of tumor necrosis factor-alpha in IgM+ B-cells from bovine leukemia virus-infected lymphocytotic sheep

Tumor necrosis factor (TNF)-alpha is thought to be one of the cytokines that account for bovine leukemia virus (BLV)-induced B-cell lymphoproliferative disorder, however, information on TNF-alpha expression in B-cells is limited. In this study, the expression of TNF-alpha in IgM(+) B-cells from BLV-infected sheep with or without lymphocytosis was determined. Freshly isolated IgM(+) B-cells from three sheep with lymphocytosis constitutively transcribed TNF-alpha mRNA. Although TNF-alpha mRNA expression in IgM(+) B-cells was transiently up-regulated after cell culture, TNF-alpha mRNA expression was markedly higher in lymphocytotic sheep when compared to that of non-lymphocytotic sheep or uninfected sheep. Expression of membrane-bound TNF-alpha on IgM(+) B-cells was also augmented in lymphocytotic sheep. TNF-alpha expression in lymphocytotic sheep may support the proliferation of B-cells.     

Blood lymphocyte subsets in pigs vaccinated and challenged with Actinobacillus pleuropneumoniae

Actinobacillus pleuropneumoniae bacterins do not induce protection in pigs while infection with low doses of the CM5 strain of A. pleuropneumoniae given by aerosol induces complete protection. To evaluate possible correlates of protection in blood lymphocyte subset phenotypes, pigs were treated with a commercial bacterin given intramuscularly, low dose (10(5)cfu/ml) aerosol infection with CM5 or control treatments of the bacterin adjuvant or phosphate buffered saline. All pigs were challenged with a high dose (10(7)cfu/ml) of A. pleuropneumoniae. Lymphocytes and sera were collected prior to and following primary and secondary immunizations and challenge, for evaluation of B- and T-cell markers and antibody to four A. pleuropneumoniae antigens. IgM(micro)+ B-cells were increased following primary exposure to antigen in the bacterin-vaccinated group only. An increase in CD4+ cells in the LD aerosol-infected group was apparent following secondary exposure to antigen. These early changes suggest little difference in lymphocyte populations between treatment groups, however, greater differences were observed following high-dose challenge; CD4+ lymphocytes were increased significantly in both bacterin and LD-challenged groups (p<0.05) while CD8+ cells decreased in the LD-group at this time period. Consequently, there were significant differences (p<0.05) in the CD4:CD8 ratio after high-dose challenge compared to earlier time points and control groups. Variation in cellular expression of SLA-DR and DQ was observed but trends correlating to treatment group were not evident. Complete protection or lack of protection associated with LD challenge or immunisation resulted in significant differences in B-cell frequencies and CD4:CD8 ratio phenotypes in pigs, but only changes in CD4:CD8 ratios appeared relevant to protection.     

Monoclonal antibodies to equine CD23 identify the low-affinity receptor for IgE on subpopulations of IgM+ and IgG1+ B-cells in horses

CD23, also called FcεRII, is the low-affinity receptor for IgE and has first been described as a major receptor regulating IgE responses. In addition, CD23 also binds to CD21, integrins and MHC class II molecules and thus has a much wider functional role in immune regulation ranging from involvement in antigen-presentation to multiple cytokine-like functions of soluble CD23. The role of CD23 during immune responses of the horse is less well understood. Here, we expressed equine CD23 in mammalian cells using a novel IL-4 expression system. Expression resulted in high yield of recombinant IL-4/CD23 fusion protein which was purified and used for the generation of monoclonal antibodies (mAbs) to equine CD23. Seven anti-CD23 mAbs were further characterized. The expression of the low-affinity IgE receptor on equine peripheral blood mononuclear cells was analyzed by flow cytometric analysis. Cell surface staining showed that CD23 is mainly expressed by a subpopulation of equine B-cells. Only a very few equine T-cells or monocytes expressed CD23. CD23(+) B-cells were either IgM(+) or IgG1(+) cells. All CD23(+) cells were also positive for cell surface IgE staining suggesting in vivo IgE binding by the receptor. Two of the CD23 mAbs detected either the complete extracellular region of CD23 or a 22kDa cleavage product of CD23 by Western blotting. The new anti-CD23 mAbs provide valuable reagents to further analyze the roles of CD23 during immune responses of the horse in health and disease.     

The kinetics of cytokine production and CD25 expression by porcine lymphocyte subpopulations following exposure to classical swine fever virus (CSFV)

Surface expression of IL-2R-alpha (CD25) is widely used to identify activated lymphocyte populations, while interferon-gamma (IFN-gamma) levels have been shown to be a good indicator of cell-mediated immunity (CMI) in pigs. To investigate the relationship between these two parameters, we developed an intracellular cytokine-staining assay and studied the kinetics of cytokine (IFN-gamma and interleukin-10, IL-10) production relative to CD25 expression in porcine lymphocyte subpopulations, following immunization with a classical swine fever (CSF) vaccine. The number of activated memory T cells (CD4(+)CD8(+)CD25(+) cells) increased slightly in the peripheral blood mononuclear cell (PBMC) population soon after vaccination, then diminished within a few weeks. The number of activated cytotoxic T cells (CD4(-)CD8(+)CD25(+) cells) peaked approximately 2 weeks after the memory population. Although the number of IFN-gamma producing cells detected in this experiment was relatively low, the CD4(+)CD8(+) T cells were major IFN-gamma producers in the PBMCs throughout the experiment. In another experiment, CSF-vaccinated pigs were challenged with a virulent classical swine fever virus (CSFV), and the kinetics of CD25 expression and cytokine productions were monitored. Following exposure to the virus, the number of IFN-gamma producing cells in the PBMCs increased markedly in both the vaccinated and unvaccinated groups. The CD4(-)CD8(+) cells were major IFN-gamma producing cells in vaccinated pigs, while both CD4(+)CD8(+) and CD4(-)CD8(+) populations contributed to the IFN-gamma production in the control group. Interestingly, the enhanced IFN-gamma production was not associated with the upregulation of CD25 expression following the CSFV challenge. In addition, exposure to the virulent CSFV significantly increased interleukin-10 production by the CD4(-)CD8(+) populations in PBMCs of the unvaccinated pigs. Taken together, our results indicated that CD25 expression and IFN-gamma production were not tightly associated in porcine lymphocytes. In addition, the CD4(-)CD8(+) lymphocytes of the PBMCs played a major role in cytokine productions following the CSFV challenge.     

Effect of synthetic agonists of toll-like receptor 9 on canine lymphocyte proliferation and cytokine production in vitro

Synthetic agonists of TLR9 containing novel DNA structures and R'pG (wherein R=1-(2'-deoxy-beta-d-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine) motifs, referred to as immune modulatory oligonucleotides (IMOs), have been shown to stimulate T(H)-1-type-immune responses and potently reverse allergen-induced T(H)-2 responses to T(H)-1 responses in vitro and in vivo in mice. In order to investigate the immunomodulatory potential of IMOs in dogs, canine peripheral blood mononuclear cells (PBMC) from healthy dogs were stimulated with three different IMOs and a control IMO, alone or in combination with concanavalin A (ConA). Lipopolysaccharide (LPS) was used as a positive control for B lymphocyte activation. Carboxyfluorescein diacetate succinimidyl ester and phenotype staining was used to tag proliferating T and B lymphocytes (CD5(+) and CD21(+)) by flow cytometry. Real-time PCR and ELISA were processed to assay cytokine production of IFN-gamma, IL-10, TGF-beta, IL-6 and IL-10. Like LPS, IMOs alone induced neither proliferation of CD5(+) T cells nor CD21(+) B cells, but both LPS and IMO had the capacity to co-stimulate ConA and induced proliferation of B cells. In combination with ConA, one of the IMOs (IMO1) also induced proliferation of T cells. IMO1 also significantly enhanced the expression of IFN-gamma on the mRNA and protein level in canine PBMC, whereas expression of IL-10, TGF-beta and IL-4 mRNAs was not induced by any of the IMOs. These results indicate that in canine PBMC from healthy dogs, IMO1 was able to induce a T(H)-1 immune response including T- and B-cell proliferation.     

Porcine-specific CpG-oligodeoxynucleotide activates B-cells and increases the expression of MHC-II molecules on lymphocytes

Two CpG-oligodeoxynucleotide motifs, a mouse-specific one (CpG(mouse)) 5'-GCTAGACGTTAGCGT-3' and a porcine-specific one (CpG(pig)), 5'-TGCATCGATGCAG-3' were synthesized by two different companies and tested in vitro for their capacity to stimulate porcine peripheral blood monomorphonuclear cells (PBMC). The porcine-specific motif, consisting of a nuclease-resistant phosphorothioate guanosines at the 5' and at the 3'-end (CpG(pig)-S), enhanced significantly the proliferation of porcine PBMC in comparison with CpG(mouse). The latter motif did not induce any proliferation. Methylation of CpG(pig) diminished the proliferation. Four days of culture with CpG(pig)-S increased the percentage of B-cells as well as B-cell blasting. Moreover, CpG(pig)-S also enhanced the expression of class II MHC in most cultures while there were no changes in percentage of macrophages or in the degree of expression of the macrophage marker (monoclonal 74-22-15). In conclusion, in this study, it was confirmed that 5'-ggTGCATCGATGCAGggggg-3' is a swine-specific CpG-ODN, that activates porcine B-cells and deserves further evaluation in vivo as a potential immunostimulating adjuvant.     

T、B淋巴细胞功能对流行性牛白血病发生发展的影响

本文回顾了T、B淋巴细胞功能与牛白血病发生发展的相关性。阐明了感染BLV牛B细胞淋巴瘤细胞除了来源于Bla(CD5^ CD11b^ )细胞，也可来源于B1b(CD5^-CD11b^ )及常规B2（CD5^-CD11b^-)细胞。探讨了T细胞功能对牛白血病的发生发展的影响，指出IL－12表达下降和IL－10表达增加可能导致了B细胞淋巴肉瘤的形成，Ⅰ型和Ⅱ型细胞因子的不平衡引起了BLV的发展。同时讨论了B－T细胞的相互作用在BLV感染时疾病阶段性发展过程中的作用。     

Prognostic significance of CD25 expression in dogs with a noninvasive diagnosis of B-cell lymphoma treated with CHOP chemotherapy

Prior studies have identified high CD25 expression in canine diffuse large B-cell lymphoma as a negative prognostic indicator. The objective of this retrospective study was to evaluate CD25 expression as a prognostic indicator in dogs with B-cell lymphoma (BCL) diagnosed with commonly used noninvasive diagnostics (cytology and flow cytometry [FC]) and treated with CHOP chemotherapy. Lymph node aspirates from 57 dogs with cytologic diagnosis of lymphoma composed of intermediate to large lymphocytes were analysed with FC. Percentage of neoplastic B-cells expressing CD25 and median fluorescence intensity (MFI) of CD25 were measured. Relationships of CD25 percent positivity and MFI to progression free survival (PFS) and survival time were evaluated. Median survival time (MST) of all dogs was 272 days (95% CI, 196–348 days) and median PFS was 196 days (95% CI, 172–220 days). Higher percentage of B-cells positive for CD25 was associated with decreased risk of death in multivariable analysis (p = .02). Dogs with higher CD25 positivity had longer MST and PFS than dogs with lower CD25 positivity (318 days versus 176 days and 212 days versus 148 days, respectively), but these differences were not significant. CD25 MFI was not significantly associated with outcome. Based on the results of this study, the association of CD25 expression and prognosis in dogs with BCL diagnosed using noninvasive methods should be interpreted with caution. Further evaluation, with studies that include histopathologic differentiation of lymphoma subtypes, is needed.     

IFN gamma and IL-4 production by CD4, CD8 and WC1 gamma delta TCR(+) T cells from cattle lymph nodes and blood

The synthesis of IFN gamma and IL-4 by CD4, CD8 and WC1 gamma delta TCR(+) T cell sub-populations, and T cells stained with activation/memory-sub-set markers has been examined by flow cytometric analysis. Cells from blood, prescapular, bronchial and mesenteric lymph nodes and Peyer's patches were incubated with phorbol 12-myristate 13-acetate (PMA), ionomycin and brefeldin-A before staining. Lymphocytes that stained for cytoplasmic IFN gamma were evident within the CD4 and CD8 populations from all tissues and also in the WC1 population from lymph nodes. IL-4 producing cells were primarily evident within the CD4 population. IFN gamma synthesis was evident within both CD45RO(+) and CD45RB(+) populations, but IL-4 synthesis was predominantly by cells that were CD45RO(+)/CD45RB(-). Expression of CD62L is not related to functional memory in CD4(+) T cells from cattle and CD62L(+) cells, particularly from the lymph nodes draining the skin and the lungs, stained with mAb to IFN gamma and IL-4. The findings indicate that at least for CD4(+) T cells, where CD45 isoform expression is related to functional memory, these two cytokines are produced predominantly by cells with a memory phenotype. The observation that some WC1(+) cells produce IFN gamma implies the presence of distinct sub-sets of this gamma delta TCR(+) population cattle and suggests a functional role.     

Bovine dendritic cells generated from monocytes and bone marrow progenitors regulate immunoglobulin production in peripheral blood B cells

We examined whether bovine monocyte-derived and bone marrow (BM) dendritic cells (DCs) regulate antibody production in activated peripheral blood B cells. DCs were generated from monocytes and BM progenitors in the presence of bovine recombinant granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4). Monocyte-derived DCs promoted B cells activated by the anti-CD3 triggered CD4(+) T cells or through immunoglobulin M (IgM) receptor to increase the level of IgG secretion. Furthermore, the addition of DCs triggered B cells activated through IgM receptors to produce IgG2 and IgA, thus inducing an isotype switch. BM-derived DCs increased the production of IgG in B cells activated by the anti-CD3 triggered CD4(+) T cells, but unlike monocyte-derived DCs did not have any effect on B cells activated through surface IgM. These data suggest that the regulation of humoral immune responses in cattle depends on the origin of DCs and the mode of B cell activation.     

Stable expression of the extracellular domains of rabbit recombinant CD5: development and characterization of polyclonal and monoclonal antibodies

Previous studies in our laboratory suggested that there was positive selection of B cells during early development in the appendix of normal and V(H) mutant (ali/ali) rabbits. Preferential expansion and survival of B lymphocytes was affected by the Ig V(H) frameworks 1 and 3 sequences expressed on the cell surface. We demonstrated a specific interaction between rabbit CD5 and the V region of rabbit heavy chains and suggested that CD5 is a potential selecting ligand for B-cell surface immunoglobulin framework region sequences. To further investigate the role of CD5 in rabbit B-cell selection and survival we prepared recombinant constructs and obtained stable expression of the three scavenger receptor cysteine-rich (SRCR) extracellular domains of rabbit CD5. Here we describe the production and purification of this expressed recombinant CD5 protein, polyclonal antibody obtained by immunization of a goat and initial production and characterization of specific mAbs against peptides selected from each sequenced SRCR domain.     

Lymphatic organ development in dogs: major lymphocyte subsets and activity

In the present study, we have characterized lymphocyte subsets and activity in peripheral blood, spleen, mesenteric and popliteal lymph nodes in pups from birth till the age of one month and compared the results with the situation in the group of three adult dogs. In neonatal pups, lower numbers of CD3(+) T-cells were detected in both the spleen and peripheral blood than in lymph nodes. In contrast to the other compartments, CD21(+) B-cells prevailed in the spleen, which resulted in low values (<1) of the CD3(+)/CD21(+) ratio. Low numbers of CD8(+) lymphocytes were characteristic in all compartments immediately after birth; consequently a high CD4(+)/CD8(+) ratio has been calculated. Postnatal development was characterized by an increasing frequency of CD8(+) lymphocytes in all organs studied. Another typical feature of the early period of life was a relative decrease of B-cell numbers, which was compensated by an increasing proportion of T-lymphocytes, particularly in the peripheral blood and spleen. DNA synthesis in newborn pups' cells as measured by in vitro thymidine incorporation was surprisingly high in non-stimulated control samples, notably in the spleen. Further development of lymphocyte activity was characterized by the decline in spontaneous activity in all organs. Stimulation indices upon mitogen-induced proliferation increased proportionally to the decrease in spontaneous activity. Based on our experimental data, we have concluded that pups are born with a relatively competent immune system the structure of which, however, markedly develops during a few postnatal weeks.