Cloning non-transformed sheep B cells.

The capacity to clone B cells and establish permanent B cell lines has greatly facilitated a wide variety of studies characterising the growth, differentiation, and gene expression of murine and human B cells. Similar investigations of B cell biology for other species have been severely restricted by an inability to culture or clone B cells. This is the first report of a method to clone non-transformed sheep B cells using a culture system based on murine CD154 and a combination of human gamma chain-common cytokines. Sheep Peyer's patch B cells were cultured for 120 days and then cloned by limiting dilution culture. The parental B cell culture contained both surface immunoglobulin (sIg)M(+) and sIgG1(+) B cells and both types of B cell were cloned. Clonality was confirmed by PCR analysis of Ig heavy chain (HC) and light chain (LC) expression and DNA sequencing of HC V genes. There was agreement between the PCR and flow cytometric analyses of HC isotype expression on the B cell clones but the available monoclonal antibodies specific for sheep lambda and kappa LC did not react with all clones. Soluble Ig was detected in the culture supernatant of sIgG1(+) clones but not sIgM(+) clones. The B cell clones remained dependent upon CD154 and gamma chain-common cytokine co-stimulation for sustained growth and maintained stable Ig expression. The cloning of non-transformed sheep B cells should provide a valuable tool for studying sheep B cell biology, establishing Ig HC- and LC-specific monoclonal antibodies, analysing the B cell Ig repertoire, and may be used to produce sheep monoclonal antibodies.

Characterization and activation requirements of bovine lymphocytes acquiring cytotoxic activity after interleukin-2 treatment.

Interleukin-2 (IL-2) treatment of cells and generation of non-major histocompatibility complex (MHC)-restricted cytotoxic cells from peripheral blood mononuclear leukocytes (PBML) was studied. Effector-target conjugate assays demonstrated that bovine PBML bound but did not lyse K562, HL60S and HL60R cells unless activated with IL-2. The magnitude of IL-2-activated killing of tumor cells as well as the magnitude of antibody-dependent cellular cytotoxicity depended on the IL-2 concentration. A short treatment (12-18 h) of effector cells with IL-2 was sufficient for development of cytotoxic activity. Withdrawal of IL-2 from the culture resulted in a reduction of cytotoxic activity that could be restored by further addition of IL-2. Cytotoxic activity of IL-2-activated populations obtained after nylon wool or Sephadex G-10 passage, and Percoll gradient centrifugation of PBML suggests that lymphokine-activated killer (LAK) cell activity in PBML is mainly mediated by a non-adherent lymphocyte lacking markers for B-cells. Positive and negative selection experiments using cell sorting confirmed these findings and demonstrated that the cell responsible for LAK cell activity in cattle belongs to a non-monocyte, non-B, CD2+ lymphocyte population. Furthermore, cytotoxic activity could not be generated in CD2+ populations enriched for cells expressing molecules equivalent to human and murine CD4 and CD8. These findings suggest that effector cells mediating non MHC-restricted cytotoxicity in cattle prevail in a population bearing a CD2+, CD4-, CD8- phenotype and that this population depends on the continuous presence of IL-2 for optimal cytotoxic function.

Experimental inoculation of heifers with bovine adenovirus type 3.

Nine 2-year-old heifers having BAd3-neutralizing antibody titers between 1:120 and 1:1080 were individually exposed intranasally to an aerosol of 10(8) pfu of wild type (wt) bovine adenovirus type 3 (BAd3). Four animals were kept as non-inoculated controls. The heifers were examined daily for rectal temperature, weight gain/loss, nasal and ocular discharges, and other clinical signs for 10 d post-inoculation. None of the animals showed any sign of clinical disease. Virus excretion was observed in one animal only on Day 3 post-inoculation. All BAd3-inoculated heifers demonstrated a significant (P < 0.005, paired t-test) rise in BAd3-specific serum IgG, IgG1, or IgG2 ELISA titers and virus-neutralizing antibody titers compared to the titers before inoculation. All virus-inoculated animals demonstrated increased levels of BAd3-specific IgA ELISA titers in nasal secretions. These results suggest that in the presence of circulating BAd3-neutralizing antibodies, intranasal inoculation of cattle with wt BAd3 would result in inapparent infection.

CD40 signaling induces B cell responsiveness to multiple members of the gamma chain-common cytokine family.

CD40 signaling induces B cell proliferative and differentiation responses that can be modulated by many different cytokines. Cytokines in the IL-2 receptor gamma chain (gammac)-common family are known to play an integral role in B cell development. Therefore, we investigated the possibility that CD40 signaling induced B cell responsiveness to multiple gammac-common cytokines and that individual gammac-common cytokines induced distinct B cell responses. B cells were isolated from lymphoid follicles of sheep Peyer's patches (PP) and co-cultured with murine CD40 ligand (mCD40L). CD40 signaling induced PP B cell responsiveness to recombinant human IL-2, IL-4, IL-7 and IL-15. mCD40L-induced B cell growth was enhanced by combining IL-4 with a second gammac-common cytokine and sustained B cell growth required co-stimulation with IL-4 plus IL-2, IL-7 and IL-15. gammac-common cytokine responsiveness remained dependent upon CD40 signaling, and removal of mCD40L resulted in B cell differentiation and cell death. Similar proliferative responses to mCD40L and gammac-common cytokines were observed for both immature (ileal) and mature (jejunal) PP B cells. Finally, the capacity of CD40-activated B cells to respond to multiple gammac-common cytokines was analyzed with individual PP B cell clones. All B cell clones displayed similar proliferative responses to IL-2 but quantitatively different responses to IL-4, IL-7 and IL-15. The biological significance of B cell responsiveness to multiple gammac-common cytokines is discussed.

Effect of Haemophilus somnus on phagocytosis and hydrogen peroxide production by bovine polymorphonuclear leukocytes.

The interactions between bovine polymorphonuclear leukocytes (PMNs) and the bacterium Haemophilus somnus are known to be complex. In this paper, we evaluated the effect of H. somnus on PMN function using a flow cytometric (FC) technique that simultaneously determined the extent of phagocytosis and hydrogen peroxide production by PMNs, as well as using conventional techniques, such as the nitroblue tetrazolium (NBT) and chemiluminescence assays, to analyse the PMN respiratory burst. Results from the FC and chemiluminescence assays demonstrated that in vitro exposure of PMNs to logarithmically growing H. somnus reduced the respiratory burst of PMNs obtained from healthy calves. However, this reduction was not detected by the NBT assay. A decrease in phagocytosis by PMNs could also be shown using the FC assay. In addition, PMNs from calves with acute Hemophilosis (i.e. exposed to H. somnus in vivo) showed reduced activity when compared to PMNs from healthy calves. These in vitro and in vivo observations indicate that the modulation of bovine PMN function by H. somnus may contribute significantly towards the pathogenesis of the disease.

Modulation of phagocytic function of bovine mononuclear phagocytes by Haemophilus somnus.

The interactions between bovine mononuclear cells and Haemophilus somnus are known to be complex. To study this interaction, a flow cytometric assay was developed to assess the effect of H. somnus on phagocytosis of killed opsonized Staphylococcus aureus by bovine alveolar macrophages and blood monocytes. Using this in vitro system, it was found that log phase H. somnus significantly inhibited the phagocytosis of killed opsonized S. aureus by bovine alveolar macrophages obtained both from healthy calves and from cattle experimentally infected with H. somnus. However, killed log-phase H. somnus, in vitro passaged and stationary phase H. somnus had no effect on the phagocytic activity of these cells. In contrast to bovine alveolar macrophages, blood monocytes showed a significant increase in their phagocytic activity following in vitro exposure to either log or stationary phase H. somnus. Using a lypophilic, non-toxic fluorophore PKH2 to label live H. somnus, it was possible to simultaneously measure the uptake of both S. aureus and H. somnus. Stationary and log phase H. somnus were taken up by macrophages equally well, even though phagocytosis of S. aureus was inhibited by only log phase H. somnus. These results demonstrate the ability of H. somnus to modulate bovine mononuclear phagocytic function which might contribute towards the pathogenesis of bovine hemophilosis.

Molecular mimicry: a herpes virus glycoprotein antigenically related to a cell-surface glycoprotein expressed by macrophages, polymorphonuclear leucocytes, and platelets.

Bovine herpes virus type 1 (BHV-1) gIII is a major virion glycoprotein with homology to the immunoglobulin superfamily. We have investigated the possibility that gIII is related to host molecules and have identified a gIII-specific monoclonal antibody (mAb) that cross-reacts with normal bovine cells. The cross-reactive entity was expressed mainly on monocyte/macrophages (M phi), polymorphonuclear leucocytes (PMN) and platelets, and was identified as a 43,000-63,000 molecular weight (MW) cell-surface glycoprotein. For M phi, the glycoprotein appears to be a general lineage marker, rather than a maturation or activation marker, and may be a functional receptor, as evidenced by its endocytosis via coated pits and its involvement in proliferation of mononuclear cells in vitro. This novel leucocyte marker was also detected on subsets of human, ovine and canine M phi. Competitive binding assays with sera from cattle immunized with BHV-1 or gIII revealed apparent low responsiveness to the cross-reactive epitope. The results suggest that BHV-1 gIII is antigenically related to a novel host leucocyte receptor and that evasion and/or interference with leucocyte function may be a consequence of this molecular mimicry relationship.

Ileal and jejunal Peyer's patches play distinct roles in mucosal immunity of sheep.

The majority of pathogens enter the body through mucosal surfaces and it is now evident that mucosal immunity can provide effective disease protection. However, the induction of mucosal immunity will require efficient targeting of mucosal vaccines to appropriate mucosa-associated lymphoid tissue. An animal model, based upon the surgical preparation of sterile intestinal 'loops' (blind-ended segments of intestine), was developed to evaluate mucosal and systemic immune responses to enteric vaccines in ruminants. The effectiveness of end-to-end intestinal anastomoses was evaluated and fetal surgery did not disrupt normal intestinal function in lambs up to 6-7 months after birth. The immunological competence of Peyer's patches (PP) within the intestinal 'loops' was evaluated with a human adenovirus 5 vector expressing the gD gene of bovine herpesvirus-1. This vaccine vector induced both mucosal and systemic immune responses when injected into intestinal 'loops' of 5-6-week-old lambs. Antibodies to the gD protein were detected in the lumen of intestinal 'loops' and serum and PP lymphocytes proliferated in response to gD protein. The immune competence of ileal and jejunal PP was compared and these analyses confirmed that jejunal PP are an efficient site for the induction of mucosal immune responses. This was confirmed by the presence of gD-specific antibody-secreting cells in jejunal but not ileal PP. Systemic but not mucosal immune responses were detected when the vaccine vector was delivered to the ileal PP. In conclusion, this model provided an effective means to evaluate the immunogenicity of potential oral vaccines and to assess the immunological competence of ileal and jejunal Peyer's patches.

Regulation of major histocompatibility complex class II expression by Pasteurella haemolytica leukotoxin.

Many properties have been associated with Pasteurella haemolytica leukotoxin and other repeat-in-toxin toxins, including their cytotoxic activity on various cells of the lymphoid and nonlymphoid systems as well as their ability to modulate the immunological activity of lymphocytes and monocytes. In this study, we assessed the ability of P. haemolytica leukotoxin to affect the expression major histocompatibility complex (MHC) class II molecules on bovine peripheral monocytes. Peripheral blood mononuclear cells were isolated from P. haemolytica leukotoxin-seronegative calves and incubated with various concentrations of authentic leukotoxin as well as the recombinant lktA gene product (LktA). Expression of MHC class II antigen on cells was evaluated by flow cytometric methods. The results indicated that both a crude, authentic leukotoxin preparation and LktA were able to affect MHC class II expression by inducing a marked downregulation of MHC class II expression on bovine monocytes. However, when cells were activated with gamma interferon (IFN-gamma), LktA and Lkt had little or no detectable effect. By using a cell line which expresses MHC class II only after activation by INF-gamma, we were able to confirm the observation that LktA had no effect on the expression of MHC class II after IFN-gamma treatment. Leukotoxin affected the functional capacity of monocytes to present antigen, as demonstrated by the ability of LktA or authentic leukotoxin to totally inhibit a mixed lymphocyte culture from MHC-mismatched calves. Thus, leukotoxin was able to downregulate constitutive expression of MHC class II expression, and we propose that this is a novel way in which this molecule can affect the immune function of monocytes, playing an important role in bacterial pathogenesis and survival of organisms at the infection site.

CD154 costimulated ovine primary B cells, a cell culture system that supports productive infection by bovine leukemia virus.

Bovine leukemia virus (BLV) is closely associated with the development of B-cell leukemia and lymphoma in cattle. BLV infection has also been studied extensively in an in vivo ovine model that provides a unique system for studying B-cell leukemogenesis. There is no evidence that BLV can directly infect ovine B cells in vitro, and there are no direct data regarding the oncogenic potential of the viral Tax transactivator in B cells. Therefore, we developed ovine B-cell culture systems to study the interaction between BLV and its natural target, the B cell. In this study, we used murine CD154 (CD40 ligand) and gamma-chain-common cytokines to support the growth of B cells isolated from ovine lymphoid tissues. Integrated provirus, extrachromosomal forms, and viral transcripts were detected in BLV-exposed populations of immature, rapidly dividing surface immunoglobulin M-positive B cells from sheep ileal Peyer's patches and also in activated mature B cells isolated from blood. Conclusive evidence of direct B-cell infection by BLV was obtained through the use of cloned B cells derived from sheep jejunal Peyer's patches. Finally, inoculation of sheep with BLV-infected cultures proved that infectious virus was shed from in vitro-infected B cells. Collectively, these data confirm that a variety of ovine B-cell populations can support productive infection by BLV. The development of ovine B-cell cultures permissive for BLV infection provides a controlled system for investigating B-cell leukemogenic processes and the pathogenesis of BLV infection.

Effect of chronic administration of recombinant bovine tumor necrosis factor to cattle.

Cachectin/tumor necrosis factor-alpha (TNF), a protein produced by macrophages upon stimulation, has been implicated as an important mediator of inflammatory processes and of clinical manifestations in chronic infectious diseases. In order to study further the potential role of TNF in infectious diseases, a homologous system was employed in which recombinant Escherichia coli (E. coli) derived bovine TNF (rBoTNF) was injected in cattle, either as a single bolus or in a repetitive treatment-regime. No clinical signs were observed, although changes occurred in hematologic and immunologic parameters when less than 0.5 mg of TNF/100 kg body weight was administered twice daily for 18 days. Prolonged treatment with 0.05-0.5 mg/100 kg induced histologic but no gross changes in the kidneys and liver. When doses were increased above 0.5 mg/100 kg, depression, anorexia, cachexia, and diarrhea appeared rapidly. Pathologic changes were apparent in various tissues including liver, kidneys, and lymphoid organs; body fat depots were depleted. Most of these changes appeared to be reversible; return to normal tissue-morphology occurred within 3 weeks of withdrawal of rBoTNF. The clinical and pathologic changes induced by prolonged rBoTNF administration resembled those observed in some chronic parasitic and viral infections of cattle in which macrophage-activation characteristically occur. Our finding may be relevant to the elucidation of the pathogenesis of these and other chronic infections.