MHC class II-restricted, CD4(+) T-cell proliferative responses of peripheral blood mononuclear cells from Mycobacterium bovis-infected white-tailed deer

White-tailed deer are significant wildlife reservoirs of Mycobacterium bovis for cattle, predators, and, potentially, humans. Infection of cattle with M. bovis stimulates an antigen-specific T-cell response, with both CD4(+) and CD8(+) cells implicated in protective immunity. Few studies, however, have examined lymphocyte subset responses to experimental M. bovis infection of white-tailed deer. In this study, a flow cytometric proliferation assay was used to determine the relative contribution of individual peripheral blood mononuclear cell subsets of M. bovis-infected white-tailed deer in the recall response to M. bovis antigen. Naive deer were challenged with M. bovis by cohabitation with infected deer. These M. bovis-challenged deer developed significant in vivo (delayed-type hypersensitivity) and in vitro (proliferative) responses to M. bovis purified protein derivative (PPD). At necropsy, typical tuberculous lesions containing M. bovis were detected within lungs and lung-associated lymph nodes of infected deer. The predominant subset of lymphocytes that proliferated in response to in vitro stimulation with PPD was the CD4(+) subset. Minimal proliferative responses were detected from CD8(+), gamma delta TCR(+), and B-cells. Addition of monoclonal antibodies specific for MHC II antigens, but not MHC I or CD1 antigens, abrogated the proliferative response. Together, these findings indicate that while CD4(+) cells from infected deer proliferate in the recall response to M. bovis antigens, this response is not sufficient to clear M. bovis and immunologic intervention may require stimulation of alternate subsets of lymphocytes.     

Cytokine secretion by peripheral blood mononuclear cells from cows infected with Mycobacterium paratuberculosis

OBJECTIVE: To compare cytokine secretion patterns of peripheral blood mononuclear cells (PBMC) from healthy cows and cows subclinically and clinically infected with Mycobacterium paratuberculosis. ANIMALS: 5 noninfected cows, 6 cows with subclinical paratuberculosis, and 4 cows with clinical paratuberculosis. PROCEDURE: PBMC were isolated, and concentrations or activities of secreted interleukin (IL)-1, IL-2, IL-6, tumor necrosis factor (TNF), and interferon-gamma (IFN-gamma) were measured after in vitro stimulation of cells with concanavalin A (ConA), lipopolysaccharide (LPS), or a whole-cell sonicate of M paratuberculosis (MpS). Proliferative responses of PBMC were also determined after stimulation with ConA, phytohemagglutinin, pokeweed mitogen (PWM), or MpS. RESULTS: After stimulation with ConA, cells from subclinically infected cows secreted significantly more, and cells from clinically infected cows secreted significantly less, IFN-gamma, compared with cells from control cows. Cells from cows with subclinical paratuberculosis produced significantly more TNF and IFN-gamma in response to MpS than cells from the other 2 groups. Stimulation of PBMC from subclinically infected cows with ConA or MpS resulted in significantly higher proliferative responses, compared with cells from control and clinically infected cows. In contrast, clinically infected cows had significantly higher proliferative responses to PWM than cells from the other 2 groups. CONCLUSIONS AND CLINICAL RELEVANCE: A decrease in T-cell responses to mitogens or MpS was observed in cows clinically infected with M paratuberculosis, compared with subclinically infected cows, suggesting that activated T cells may delay the progression of paratuberculosis.     

Butyrate differentially regulates cytokines and proliferation in porcine peripheral blood mononuclear cells

Although butyrate modulates proliferation and cytokine production by PBMC in some species, the role of butyrate as a regulator of immunocyte function in the pig has not been studied. Therefore, the primary objective of this study was to determine whether butyrate influences peripheral blood mononuclear cell (PBMC) proliferation, cytokine secretion and mRNA expression in the pig in vitro. We also sought to determine whether alterations in cytokine production attributable to butyrate were associated with changes in the expression of suppressor of cytokine signaling-3 (SOCS3). Porcine PBMC were isolated from venous blood and stimulated with concanavalin A (ConA) in the presence or absence of sodium butyrate at 0.2 or 2.0 mM. Butyrate at 2.0 mM suppressed (P<0.05) ConA-induced PBMC proliferation and led to a paradoxical increase (P<0.05) in IL-2 mRNA expression. The secretion and mRNA expression of interferon-gamma (IFN-gamma) by ConA-activated PBMC was increased (P<0.05) by butyrate at 2.0 mM. Exposing activated PBMC to butyrate at 2.0 mM decreased (P<0.05) the secretion of interleukin-10 (IL-10). In contrast, butyrate at 0.2 mM increased (P<0.05) both IL-10 secretion and mRNA expression. Activation of porcine PBMC with ConA increased (P<0.05) the expression of SOCS3 mRNA, and butyrate treatment further augmented (P<0.05) SOCS3 mRNA expression in a dose-dependent manner. Mechanistically, pretreatment with the adenyl cyclase inhibitor 2,5-dideoxyadenosine abolished (P<0.05) the inhibitory effect of 2.0 mM butyrate on IL-10 secretion, and partially reversed (P<0.05) the increase in IFN-gamma secretion induced by 2.0mM butyrate. These data indicate that the effect of butyrate on cytokine production by porcine PBMC is dose-dependent, and that butyrate increases the expression of SOCS3 in activated PBMC. In addition, we provide evidence that the effects of butyrate on IFN-gamma and IL-10 production are mediated in part via a cAMP-dependent mechanism.     

Generation of canine dendritic cells from peripheral blood mononuclear cells

Dendritic cells (DCs) are the most potent antigen-presenting cells that are expected to be therapeutic agents for tumor immunotherapy. In this study, we generated DCs of sufficient number for DC-based immunotherapy from peripheral blood mononuclear cells (PBMC) in dogs. PBMC were cultured in the presence of phytohemagglutinin (PHA). On day 6, large adherent cells with dendrite-like projections were seen, and the number of these large cells with projections increased on day 8. These cells were positive for esterase staining. They expressed MHC class II, CD11b, CD8 and weakly CD4 on their surface. They tended to make contact with lymphocytes under culture conditions. We obtained about 2-5 x 10(6) of DCs from 10 ml of peripheral blood. These DCs phagocytosed HEK-293 cells by overnight co-culturing. These cells generated from PBMC are possible canine DCs and are applicable to clinical trials of DC-based whole tumor cell immunotherapy in dogs.     

Effects of mycotoxins on mitogen-stimulated proliferation of bovine peripheral blood mononuclear cells

The effects of mycotoxins on mitogen-stimulated proliferation of bovine peripheral blood mononuclear cells (PBMCs) were investigated. Aflatoxin B(1) (AFB(1)), deoxynivalenol (DON) and zearalenone (ZEN) were added to cultures of PBMCs, and the proliferation responses were measured using MTT bioassays. Suppression of the proliferation of calf PBMCs by AFB(1) and DON was significantly stronger than that of cow PBMCs, whereas there were no differences in suppressive effects on PBMCs from Holstein and Japanese Black calves and cows. The suppressive effect was greatest in the order of DON, AFB(1) and ZEN, and the effects of DON and AFB(1) seemed to be dose-dependent. The results suggest that some mycotoxins directly suppress proliferation of bovine PBMCs.     

Cytokine production and proliferation upon in vitro oligodeoxyribonucleotide stimulation of equine peripheral blood mononuclear cells

Synthetic oligodeoxyribonucleotides (ODN) may prove useful immune modulators in equine medicine. It is however important to assess the effects of each specific ODN in the species it is intended to be used in. The present study therefore aimed to evaluate some ODN for induction of cytokine production; i.e. type I interferons (IFN), IFN-γ, tumor necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-β), and proliferation of equine peripheral blood mononuclear cells (PBMC). A panel of four ODN containing unmethylated cytosine-guanosine sequences (CpG) was used: ODN 1 and ODN 8 representing A-class; ODN 2006 representing B-class and ODN 2395 representing C-class-ODN. In addition, two ODN where CpG-motifs were reversed to GpC were included; ODN 2137 otherwise identical to ODN 2006 and ODN 5328 otherwise identical to ODN 2395. Cytokine concentrations were measured in cell culture supernatants after 24h of induction and proliferation was determined after 72 h of induction. Each ODN was tested with PBMC from at least 5 individual horses with and without the addition of lipofectin to cell cultures. Type I IFN, IFN-γ and TNF-α production was readily induced by ODN 1, ODN 2006 and ODN 2395 both in the presence and absence of lipofectin and all three types of ODN induced similar levels of cytokines. Proliferation of PBMC was clearly induced by ODN 2006 and ODN 2395 while ODN 1 only induced low-level proliferation. The levels of proliferation induced were not influenced by the presence of lipofectin. TGF-β production was not induced by any of the tested ODN. ODN 8, ODN 2137 and ODN 5328 were largely inactive in all assays. Thus, responses seemed dependent on or increased by CpG-motifs but presence of CpG-motifs did not necessarily confer activity since ODN 8 was inactive despite its CpG-motifs. Taken together, with equine PBMC distinctions in induction of different leukocyte functions between A-, B-, and C-class ODN were less obvious than what has been observed for human cells. These observations further stress the presence of species differences in ODN-induced responses.     

Canine PHA-stimulated adherent cell enhance interferon-gamma production and proliferation of autologous peripheral blood mononuclear cells

Dendritic cells are specialized antigen‐presenting cells with immuno‐modulating functions that are attractive for clinical applications for cancer immunotherapy. This study examined immunostimulatory functions of phytohemagglutinin (PHA)‐stimulated adherent cells (PHA‐Ad cells) from peripheral blood mononuclear cells (PBMCs) in dogs. PHA‐Ad cells enhanced interferon‐γ from autologous PBMC in vitro. PHA‐Ad cells also stimulated antigen‐independent proliferation of peripheral blood lymphocytes. These results suggest that PHA‐Ad cells from PBMC possess a stimulatory function to evoke anti‐tumour immunity and that they demonstrate potential for therapeutic applications in dogs.     

Effects of polyunsaturated fatty acids on the proliferation of mitogen stimulated bovine peripheral blood mononuclear cells

The present study aimed at analysis of the effects of polyunsaturated fatty acid (PUFA), linoleic acid (LA, C18:2n - 6) and linolenic acid (LNA, C18:3n - 3) on bovine peripheral blood mononuclear cells (PBMC) in vitro. Both mitogen (ConA)-induced proliferative lymphocyte responsiveness during 4 days of culture and eicosanoid (prostaglandin E(2) (PGE(2)) and leukotriene B(4) (LTB(4))) production during 36 h were determined in relation to the absence or presence of various concentrations of LA and LNA (0, 1, 5, 25, 125 and 250 microM). Mitogen-driven proliferative responses of lymphocytes tended to be uninfluenced in the presence of lower concentrations of LA, whereas significant inhibition was observed at the higher concentrations of LA (125 and 250 microM). However, increasing amounts of LNA did not affect the proliferation. ConA stimulation induced a clear PGE(2) response, which significantly decreased in the presence of 250 microM of LA. In addition, increasing amounts of LNA, but not LA, led to a significant decrease in LTB(4) levels. However, The production of LTB(4) did not alter due to mitogenic stimulation. In conclusion, the present study shows that bovine mononuclear cells may functionally be influenced by the presence of PUFA in their environment. Further studies need to be conducted to clarify in vivo consequences of these findings in a situation of PUFA enriched rations in ruminants.     

Cytokine profiles in peripheral blood mononuclear cells from patients with cystic echinococcosis

IntroductionCystic echinococcosis (CE) is a chronic zoonotic disease caused by the larval stage of Echinococcus granulosus (E. granulosus), which affects domestic and wild carnivores as the definitive host and ungulates as intermediate hosts. In intermediate hosts, both Th1 and Th2 cells are involved in the immune responses to an echinoccocal infection. This study aimed to investigate production of IL-4, IL-10, and IFN-γ cytokines in peripheral blood mononuclear cells (PBMCs) of CE patients before and after surgical treatment.MethodsTo evaluate cytokine production in response to E. granulosus antigens, we investigated IL-4, IL-10, and IFN-γ production in PBMCs of 20 CE patients in response to hydatid cyst fluid antigen (HCF-Ag) before and after surgical treatment using ELISA.ResultsThe mean IL-4 production from HCF-Ag stimulated PBMCs was significantly decreased (p < 0.05), while IFN-γ was significantly increased in HCF-Ag stimulated PBMCs in patients after surgery (p = 0.005).Furthermore, our results showed that there is no significant difference between IL-10 production in patients before and after treatment (p = 0.562).ConclusionsOur data Indicated production of IL-4 in cultured PBMCs of CE patients stimulated with HCF-Ag was decreased significantly. While, production of IFN-γ was increased significantly in responses to HCF Ag after surgery. We concluded that the evaluation of IL-4 and IFN-γ in HCF-Ag stimulated PBMCs of CE patients should be considered as a useful marker in the follow up of patients with cystic echinococcosis.     

Multinucleated giant cells with an osteoclast phenotype derived from caprine peripheral blood mononuclear cells

Formation of multinucleated giant cells (MGCs) by macrophage fusion is a typical cytopathic effect of lentiviral replication in caprine monocytes and MGC formation from cultured caprine peripheral blood mononuclear cells (PBMCs) has been considered to be diagnostic for small ruminant lentivirus (SRLV) infection. In this study, formation of MGCs was observed after 7–14 days when PBMCs were cultured from healthy goats free from SRLV infection. These MGCs expressed tartrate-resistant acid phosphatase, calcitonin receptor, integrin αVβ3, cathepsin K and matrix metalloproteinase 9 and were able to resorb bone in vitro in the absence of RANKL and macrophage colony stimulating factor, consistent with an osteoclast phenotype.     

Proliferative responses to canine thyroglobulin of peripheral blood mononuclear cells from hypothyroid dogs

The immune responses of hypothyroid dogs to canine thyroglobulin (cTg) were evaluated for the proliferative ability of peripheral blood mononuclear cells (PBMC). PBMC from three hypothyroid dogs with high titers of thyroglobulin autoantibody (TgAA) and 3 clinically normal dogs were cultured with 5, 10, or 20 microg/ml of cTg for 72 hr. The proliferative responses of the cells were determined by the level of incorporated BrdU. The numbers of cells expressing Thy-1, CD4, CD8 and IgG in the PBMC were counted by the immunofluorescence method. Proliferative responses to cTg were observed in the cells from hypothyroid dogs. The number of cells expressing IgG and CD8 in the hypothyroid dogs tended to be high compared with the clinically normal dogs. The CD4+ cells in cultures from hypothyroid dogs increased depending upon the amount of cTg. There was a significant (P<0.05) positive correlation between the number of CD4+ cells and the concentration of cTg in the cultures from hypothyroid dogs. These findings suggest a possible relationship between canine hypothyroidism and cellular immunity. Loss of self tolerance to thyroid antigens in CD4+ T cells may play an important role in the development of canine hypothyroidism.     

Secretion of IFN-gamma by bovine peripheral blood mononuclear cells stimulated with Mycobacterium bovis protein fractions obtained by isoelectric-focusing

Due to the complexity and variety of biological effects found in Mycobacterium bovis (M. bovis) proteins analyzed solely on a molecular weight (MW) basis, we approached the purification of M. bovis proteins through their isoelectric point (pI). Twenty M. bovis culture filtrate protein extract (CFPE) isoelectric focused (IEF) protein fractions, confined between pI3 and 10, were isolated. The MW of the major proteins isolated in the various fractions correlated with protein already reported 14-, 18-, 20-, 25-, 31-, 38-, 45-, 64-, 67- and 70 kDa by SDS-PAGE. Since several different pI fractions showed proteins of the same MW we tested the ability of all IEF fractions to stimulate interferon-gamma (IFN-gamma) production by peripheral blood mononuclear cells (PBMC) isolated from cattle with well defined M. bovis tuberculosis (TB) infection. In animals with few lesions IFN-gamma inductive IEF fractions were in the acid range. As the number of lesions increased, neutral fractions were also inductive. Some fractions with relatively few proteins induced as much IFN-gamma production as others with abundant proteins. None of the 20 IEF fractions enhanced IFN-gamma production by anergic cells. We conclude that IFN-gamma production in diseased animals is induced mainly by acidic mycobacterial proteins and that the response towards these proteins is enhanced as the disease progresses, what coincides with higher PPD reactivity. However, the IFN-gamma production in anergic status was severely affected. We found that this cytokine production is spontaneous and antigen-independent.     

Response of porcine peripheral blood mononuclear cells to CpG-containing oligodeoxynucleotides

Exposure to bacterial DNA generates a "danger signal" that stimulates cellular elements of the mammalian immune system to proliferate and/or secrete cytokines. Stimulation is critically dependent on hexameric motifs that contain an unmethylated CpG dinucleotide: these are commonly found in bacterial but not vertebrate DNA. Different motifs are optimally stimulatory in different species. This work examines whether oligodeoxynucleotides (ODNs) containing CpG motifs stimulate peripheral blood mononuclear cells from pigs. Results show that pigs respond to CpG ODN by proliferating and secreting IL-6, IL-12 and TNF-alpha. By screening a large panel (>100) of ODNs, the palindromic hexamer 'ATCGAT' was identified as being optimally active in all animals examined (N=10). These findings are the first to establish the immunostimulatory activity of CpG ODN in pigs, and suggest that the therapeutic uses envisioned for these ODNs (as vaccine adjuvants and immunoprotective agents) may be applicable to husbandry animals.     

Isolation of equine peripheral blood mononuclear cells using Percoll.

The concentration of Percoll required for isolating equine peripheral blood mononuclear cells has been reinvestigated. A poor cell yield was obtained at the 60 per cent concentration already reported. It is recommended that workers specifically interested in high yields of mononuclear cells, for investigation of lymphocyte and monocyte functions, use a concentration of 65 per cent Percoll. However, workers wishing to isolate pure populations of equine neutrophils might consider a concentration of 70 per cent in the upper layer of Percoll used to retain the mononuclear cells.     

Effects of pre-culture holding time and temperature on interferon-gamma responses in whole blood cultures from Mycobacterium bovis-infected cattle

The Bovigam assay is approved for use within the United States as a complementary tuberculosis test. Prior to whole blood culture and the ensuing ELISA to detect interferon-(IFN)-gamma, samples are subjected to various holding time/temperature combinations due, in part, to practical constraints associated with shipment of samples to approved laboratories. To evaluate these effects, 5-month-old Holstein calves (n = 7) received 10(3) cfu Mycobacterium bovis by aerosol. Heparinized blood was collected 2 months after challenge and held at 4 or 22 degrees C for 0, 8 or 24 h prior to culture with mycobacterial antigens or pokeweed mitogen (PWM). Responses of samples held for 8 or 24 h were comparable and lower than responses of cultures prepared immediately after collection, regardless of holding temperature. Differences in responses of samples held at 4 degrees C versus 22 degrees C were also minimal. A subset of samples was held for 2 h at 37 degrees C at the beginning of the holding period. This subset of samples had diminished responses to all stimulants and increased holding times (i.e., 24 h versus 8 h) negatively impacted the response. Pre-processing conditions, particularly delays in set-up and initial high sample temperatures, reduces IFN-gamma responses of cells from infected cattle increasing the risk of false negatives in this assay of regulatory importance.     

Murine monoclonal antibodies cross-reactive with feline peripheral blood mononuclear cells

Twelve murine monoclonal antibodies (MAB), specific for canine, human, and mouse cell surface determinants on lymphohemopoietic cells, were tested for reactivity (using indirect immunofluorescence) with peripheral blood mononuclear cells (PBMC) from 3 normal cats and 3 FeLV-positive cats with lymphoblastic leukemia. MAB DLy6 and 1H3, specific for canine lymphocytes, MAB HB57S, specific for human mu(IgM) chain, MAB J-118 40.164.3 and H81.98.71, specific for murine and human Ia, all had higher binding levels with PBMC from FeLV-positive cats compared to normal cats. Two MAB (S-78, S-24), specific for human class I determinants, cross-reacted with PBMC from both groups of cats. This panel of MAB may be useful for characterizing immunologically reactive cell subsets in normal as well as retrovirus-diseased animals.     

Response to Malassezia pachydermatis by peripheral blood mononuclear cells from clinically normal and atopic dogs

OBJECTIVE: To investigate the potential cell-mediated immune response of atopic dogs to the yeast Malassezia pachydermatis and to correlate it with the type-1 hypersensitivity (humoral) response of the same population of dogs. ANIMALS: 16 clinically normal dogs, 15 atopic dogs with Malassezia dermatitis, 5 atopic dogs with Malassezia otitis, and 7 atopic control (ie, without Malassezia dermatitis or otitis) dogs. PROCEDURE: A crude extract of M pachydermatis was extracted for use as an intradermal allergy testing reagent and for stimulation of isolated peripheral blood mononuclear cells in vitro. Flow cytometry was also used to assess cell surface antigenic determinants (CD3, CD4, CD8, CD14, CD21, CD45RA, surface immunoglobulin) on peripheral blood mononuclear cells. RESULTS: Atopic dogs with cytologic evidence of Malassezia dermatitis had an increased lymphocyte blastogenic response to crude M pachydermatis extract, compared with clinically normal dogs and dogs with Malassezia otitis. Atopic control dogs did not differ significantly in their responses from atopic dogs with Malassezia dermatitis or otitis. A significant correlation was not found between the lymphocyte blastogenic response and the type-1 hypersensitivity response to M pachydermatis within any of the groups. CONCLUSIONS AND CLINICAL RELEVANCE: Cell-mediated and humoral reactivities to M pachydermatis contribute to the pathogenesis of atopic dermatitis in dogs but are not directly correlated. Modification of the dysregulated immune response toward M pachydermatis may assist in the reduction of pathologic changes associated with an atopic dermatitis phenotype in dogs.     

Isolation of an antigenically "null" cell line from pig peripheral blood mononuclear cells

A new pig cell line (A4) isolated from a primary culture of pig peripheral blood mononuclear cells was characterized. A4 was demonstrated to be morphologically, antigenically and functionally distinct from the more commonly isolated pig lymphoblastoid B cell lines (e.g. P-SC). When the A4 cell line and clones derived from it were tested against a panel of monoclonal antibodies, which define specific subpopulations of pig mononuclear cells, little or no reactivity was observed. The A4 cell line, unlike the P-SC cell line, was unable to induce a mixed lymphocyte reaction. The amount of immunoglobulin secreted by A4 cells as detected by an ELISA was reduced compared to that produced by P-SC cells. The P-SC cell lines produced an IL-1-like factor, whereas no IL-1-like activity was found in the A4 supernatant. The A4 cell line appeared to be a null cell in respect to the P-SC cell line properties; only the slight amount of immunoglobulin produced suggested that the A4 cell line is of the B cell lineage. An association of viral particles with cells of the A4 morphology and null antigenic characteristics was observed and may provide an explanation for the reduced B cell properties of A4 cells.