CD86 molecule is a specific marker for canine monocyte-derived dendritic cells

In this study, canine monocyte-derived dendritic cells (cMo-DC) were produced in presence of canine GM-CSF (cGM-CSF) and canine IL-4 (cIL-4), and they were characterized by their dendritic morphology, MLR functionality and phenotype. We noticed that cMo-DC were labelled with three anti-human CD86 (FUN-1, BU63 and IT2.2 clones), whereas resting and activated lymphocytes or monocytes were not stained. CD86 expression was induced by cIL-4 and was up-regulated during the differentiation of the cMo-DC, with a maximum at day 7. Furthermore, cMo-DC were very potent even in low numbers as stimulator cells in allogeneic MLR, and BU63 mAb was able to completely block the cMo-DC-induced proliferation in MLR. We also observed that cMo-DC highly expressed MHC Class II and CD32, but we failed to determine their maturation state since the lack of commercially available canine markers. Moreover, cMo-DC contained cytoplasmic periodic microstructures, potentially new ultrastructural markers of canine DC recently described. In conclusion, this work demonstrates that the CD86 costimulatory marker is now usable for a better characterization of in vitro canine DC.     

Limited phenotypic and functional maturation of bovine monocyte-derived dendritic cells following Mycobacterium avium subspecies paratuberculosis infection in vitro

After encountering antigen, dendritic cells (DC) must differentiate into a fully mature phenotype to induce a protective, lasting T cell immunity. Paratuberculosis is a disease caused by the intracellular pathogen Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) and is characterized by a transient cell mediated immune response, that when dissipates correlates to the onset of clinical disease. In order to study the mechanism of early cellular immunity associated with M. paratuberculosis infection, we tested the hypothesis that M. paratuberculosis infected bovine DC have impaired activation and maturation thus are defective in the initiation of a sustainable and protective Th1 immune response locally. Our results demonstrate that M. paratuberculosis infected DC showed decreased endocytosis of ovalbumin, indicating some functional maturation. Co-stimulatory molecules CD40 and CD80 mRNA expression from M. paratuberculosis infected DC was increased over untreated immature DC. M. paratuberculosis infection induced chemokine receptor CCR7 increase in DC, yet CCR5 remained high. MHC II surface expression remained low on M. paratuberculosis infected DC. M. paratuberculosis infection inhibited pro-inflammatory cytokine IL-12 production and promoted IL-10 secretion by bovine DC. Together, our findings showed evidence of phenotypic and functional maturation of DC. However, we did not see the expected antigen presentation via MHC II and cytokine responses as a fully mature DC. This may suggest semi-mature DC phenotype induced by M. paratuberculosis infection.     

Ex vivo generation of mature equine monocyte-derived dendritic cells

Dendritic cells (DCs) are innate immune cells specialized in antigen detection and presentation. They perform an essential role in initiating and guiding the immune response, the direction of which largely depends upon the activation state of the DCs. The objective of this study was to generate mature equine monocyte-derived DCs and, in doing so, to develop a method for measuring the activation state of these cells. Equine DCs were stimulated with UV-inactivated Escherichia coli (E. coli), and the activation status was measured by analyzing cell surface marker expression, cytokine production, and endocytic capacity. Comparisons for each parameter measured were performed between macrophages, non-stimulated DCs and stimulated DCs. Equine monocyte-derived DCs may be distinguished from macrophages based on cell surface expression of MHC class II (p < 0.0001) and CD206 (p < 0.0001), their capacity for endocytosis of FITC-dextran (p < 0.05), and production of TNF-α upon stimulation (p < 0.001). Furthermore, stimulated DCs can be distinguished from non-stimulated DCs based on increased cell surface expression of MHC class II (p < 0.0001) and upregulation of pro-inflammatory cytokine mRNA, particularly IL-12/IL-23p40 (p < 0.05) and IL-23p19 (p < 0.05). The ability to measure DC activation state will facilitate future investigations of equine DC function.     

Classical swine fever virus suppresses maturation and modulates functions of monocyte-derived dendritic cells without activating nuclear factor kappa B

Classical swine fever virus (CSFV) compromises the host immune system, causing the severe disease of pigs. Dendritic cells (DCs) are the most potent inducers of immune responses. In the present study, we investigated the functional properties of porcine monocyte-derived DCs (Mo-DCs) affected by CSFV. Results showed that the expression of surface markers of DCs such as major histocompatibility complex class II (MHC-II), CD80, CD83 and CD86 were unimpaired, but an obviously increased expression of CD172a in DCs was noticed 48 h after CSFV infection. The expression profiles of cytokines were detected in cultured Mo-DCs after various treatments for 48 h by Q-RT-PCR. The findings suggested that CSFV infection significantly increased the mRNA expression of IL-10 and TNF-α, and inhibited IL-12 expression, with little effect on IFN-α and IFN-γ expression. We further demonstrated that CSFV was incapable of activating the nuclear factor kappa B (NF-κB) in infected DCs, which was characterized by an unvaried DNA binding activity of NF-κB, the lack of translocation of p65/RelA from the cytoplasm to the nucleus and the stabilization of p65/RelA expression. Furthermore, Western blot analysis indicated that the inactivation of NF-κB was due to the failure of IκBα degradation. The data demonstrated that CSFV could be replicated in DCs and CSFV infection could modulate the secretion of crucial co-stimulatory molecules and cytokines which down-regulated maturation of DCs, without activating NF-κB in DCs. Thus, the results suggested a possible mechanism for CSFV evasion of innate host defenses, providing the basis for understanding molecular pathways in CSFV pathogenesis.     

Characterisation of porcine monocyte-derived dendritic cells according to their cytokine profile

The influence of interferon (IFN)-alpha on the in vitro differentiation of myeloid porcine dendritic cells (DC) was evaluated as the ability of the DC to stimulate to cell proliferation in a mixed leukocyte reaction (MLR), and as their ability to produce cytokines at exposure to bacterial and viral preparations. Porcine monocytes were enriched from purified peripheral blood mononuclear cells (PBMC) by plastic adherence and cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 or in GM-CSF, IL-4 and IFN-alpha. After 5 days of culture, the cells developed a dendritic morphology and the proportion of cells expressing MHC class II and B7 molecules was increased as determined by flow cytometry. Dendritic cells, differentiated for 5 days in GM-CSF, IL-4 and IFN-alpha, were able to stimulate both allogeneic and syngeneic PBMC to proliferation in an MLR. The DC produced the Th1 associated cytokines IFN-alpha at Sendai virus stimulation, and IL-12 at stimulation with plasmid DNA (pre-incubated in the presence of lipofectin), heat-inactivated Actinobacillus pleuropneumoniae, UV-inactivated Aujeszky's disease virus and live Sendai virus. The heat-inactivated bacteria and Sendai virus also induced production of the Th2 associated cytokines IL-10 and IL-6. The addition of IFN-alpha during differentiation of DC in GM-CSF and IL-4 enhanced their ability to stimulate allogeneic and syngeneic MLR, but did not alter their ability to produce cytokines.     

Efficient generation of canine bone marrow-derived dendritic cells

Because of their unsurpassed potency in presenting antigens to naive T cells, dendritic cells are considered to be an important candidate in the development of immunotherapeutic strategies. Despite the high potential of dendritic cell-based immunotherapy, as a so-called dendritic cell vaccination, few clinical approaches using dendritic cell vaccination have been performed in the dog because of very limited information regarding the generation of canine dendritic cells and their functional properties. We therefore established a protocol for the efficient generation of dendritic cells from canine bone marrow cells using recombinant feline granulocyte-macrophage colony-stimulating factor and canine interleukin-4. Dendritic cells were generated efficiently: a yield of 1-9 x 10(6) cells per approximately 0.5 ml of canine bone marrow aspiration was achieved. These dendritic cells showed features shared with mouse and human dendritic cells: dendrite morphology, expression of surface markers MHC class II and CD11c, and up-regulation of molecules related to antigen presentation (MHC class II, B7-1, and B7-2) by activation with lipopolysaccharide. Moreover, the dendritic cells demonstrated phagocytic activity, processing activity of pinocytosed proteins, and activation of allogeneic T cells far more potent than that by macrophages. Our findings suggest that the bone marrow-derived dendritic cells are functional for the capturing and processing of antigens and the initiation of T cell responses.     

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.     

Species-specific properties and translational aspects of canine dendritic cells

Dogs are affected by spontaneously occurring neoplastic and inflammatory diseases which often share many similarities with pathological conditions in humans and are thus appreciated as important translational animal models. Dendritic cells (DCs) represent the most potent antigen presenting cell population. Besides their physiological function in the initiation of primary T cell responses and B cell immunity, a deregulation of DC function is involved in immune-mediated tissue damage, immunosuppression and transplantation complication in human and veterinary medicine. DCs represent a promising new target for cancer immunotherapy in dogs. However, the therapeutic use of canine DCs is restricted because of a lack of standardized isolation techniques and limited information about dog-specific properties of this cell type. This article reviews current protocols for the isolation and in vitro generation of canine monocyte- and bone marrow-derived DCs. DCs of dogs are characterized by unique morphological features, such as the presence of cytoplasmic projections and periodic microstructures. Canine DCs can be discriminated from other hematopoietic cells also based on phenotypic properties and their high T cell stimulatory capability in mixed leukocyte reactions. Furthermore, the classification of canine DC-derived neoplasms and the role of DCs in the pathogeneses of selected infectious, allergic and autoimmune diseases, which share similarities with human disorders, are discussed. Future research is needed to expand the existing knowledge about DC function in canine diseases as a prerequisite for the development of future therapies interfering with the immune response.     

Comparative analysis of canine monocyte- and bone-marrow-derived dendritic cells

Dendritic cells (DC) represent a heterogeneous cell family of major importance for innate immune responses against pathogens and antigen presentation during infection, cancer, allergy and autoimmunity. The aim of the present study was to characterize canine DC generated in vitro with respect to their phenotype, responsiveness to toll-like receptor (TLR) ligands and T-cell stimulatory capacity. DC were derived from monocytes (MoDC) and from bone marrow hematopoietic cells cultured with either Flt3-ligand (FL-BMDC) or with GM-CSF (GM-BMDC). All three methods generated cells with typical DC morphology that expressed CD1c, CD11c and CD14, similar to macrophages. However, CD40 was only found on DC, CD206 on MΦ and BMDC, but not on monocytes and MoDC. CD1c was not found on monocytes but on all in vitro differentiated cells. FL-BMDC and GM-BMDC were partially positive for CD4 and CD8. CD45RA was expressed on a subset of FL-BMDC but not on MoDC and GM-BMDC. MoDC and FL-DC responded well to TLR ligands including poly-IC (TLR2), Pam3Cys (TLR3), LPS (TLR4) and imiquimod (TLR7) by up-regulating MHC II and CD86. The generated DC and MΦ showed a stimulatory capacity for lymphocytes, which increased upon maturation with LPS. Taken together, our results are the basis for further characterization of canine DC subsets with respect to their role in inflammation and immune responses.     

Suppression of canine myeloid cells by soluble factors from cultured canine tumor cells

BackgroundCancer profoundly affects immunity and causes immunosuppression that contributes to tumor escape, metastases and resistance to therapy. The mechanisms by which cancer cells influence immune cells are not fully known but both innate and adaptive immune cells can be altered by cancer. Myeloid cells are innate immune cells that comprise the mononuclear phagocytic system (MPS) and include monocytes, macrophages, dendritic cells (DCs) and their progenitors. Myeloid cells play important roles in both the promotion and regulation of immune responses. Dysregulated myeloid cells are increasingly being recognized as contributing to cancer-related immunosuppression. This study investigated whether soluble factors produced by canine tumor cells inhibited canine myeloid cell function.MethodsThese studies investigated the utility of using the canine DH82 cell line for assessment of canine myeloid responses to tumor-derived soluble factors (TDSFs). Phenotypic comparisons to canine bone marrow-derived DCs (BM-DCs) and bone marrow-derived macrophages (BM-MΦs) were performed and expression of myeloid cell markers CD11b, CD11c, CD80, and major histocompatibility complex (MHC) class II were evaluated by flow cytometry. Phenotypic and functional changes of DC populations were then determined following exposure to tumor-conditioned media (TCM) from canine osteosarcoma, melanoma and mammary carcinoma cell lines.ResultsWe found that the canine BM-DCs and the DH82 cell line shared similar CD11b, CD11c and MHC II expression and morphologic characteristics that were distinct from canine BM-MΦs. Myeloid cells exposed to TDSFs showed decreased expression of MHC class II and CD80, had reduced phagocytic activity and suppressed the proliferation of responder immune cells.ConclusionThese results show that soluble factors secreted from canine tumor cells suppress the activation and function of canine myeloid cells. Our results suggest that, similar to humans, dysregulated myeloid cells may contribute to immunosuppression in dogs with cancer.     

Single-cell analysis divides bovine monocyte-derived dendritic cells into subsets expressing either high or low levels of inducible nitric oxide synthase

Dendritic cells (DC) are important cells at the interface between innate and adaptive immunity. DC have a key role in antigen processing and presentation to T cells. Effector functions of DC related to innate immunity have not been explored extensively. We show that bovine monocyte-derived DC (mDC) express inducible nitric oxide synthase (iNOS) mRNA and protein and produce NO upon triggering with interferon-gamma (IFN-gamma) and heat-killed Listeria monocytogenes (HKLM). An immunocytochemical analysis revealed that a sizeable subset (20-60%) copiously expresses iNOS (iNOShi) upon IFN-gamma/HKLM triggering, whereas the other subset expressed low levels of iNOS (iNOSlo). Monocyte-derived macrophages (mMphi) are more homogeneous with regard to iNOS expression. The number of cells within the iNOSlo mDC subset is considerably larger than the number of dead cells or cells unresponsive to IFN-gamma/HKLM. The large majority of cells translocated p65 to the nucleus upon triggering by IFN-gamma/HKLM. A contamination of mDC with iNOS-expressing mMphi was excluded as follows. (i) Cell surface marker analysis suggested that mDC were relatively homogeneous, and no evidence for a contaminating subset expressing macrophage markers (e.g. high levels of CD14) was obtained. (ii) iNOS expression was stronger in iNOShi mDC than in mMphi. The use of maturation-promoting stimuli revealed only subtle phenotypic differences between immature and mature DC in cattle. Nevertheless, these stimuli promoted development of considerably fewer iNOShi mDC upon triggering with IFN-gamma/HKLM. Immunocytochemical results showed that although a significant proportion of cells expressed iNOS only or TNF only upon triggering with IFN-gamma/HKLM, a significant number of cells expressed both iNOS and TNF, suggesting that TNF and iNOS producing (TIP) DC are present within bovine mDC populations obtained in vitro.     

Comparison of dendritic cell-mediated immune responses among canine malignant cells

Dendritic cell (DC) vaccination is one of the most attractive immunotherapies for malignancies in dogs. To examine the differences in DC-mediated immune responses from different types of malignancies in dogs, we vaccinated dogs using autologous DCs pulsed with keyhole limpet hemocyanin (KLH) and cell lysate prepared from squamous cell carcinoma SCC2/88 (SCC-KLH-DC), histiocytic sarcoma CHS-5 (CHS-KLH-DC), or B cell leukemia GL-1 (GL-KLH-DC) in vitro. In vivo inductions of immune responses against these tumor cells were compared by the delayed-type hypersensitivity (DTH) skin test. The DTH response against SCC2/88 cells were observed in dogs vaccinated with autologous SCC-KLH-DC, while the response was undetectable against CHS-5 and GL-1 cells in dogs vaccinated with autologous CHS-KLH-DC and GL-KLH-DC. Skin biopsies taken from DTH challenge sites were then examined for immunohistochemistry, and recruitment of CD8 and CD4 T cells was detected at the site where SCC2/88 cells were inoculated in dogs vaccinated with SCC-KLH-DC. By contrast, neither CD8 nor CD4 T cell infiltration was found at the DTH challenge site in the dogs vaccinated with CHS-KLH-DC or GL-KLH-DC. These findings may reflect that the efficacy of immune induction by DC vaccination varies among tumor types and that immune responses could be inducible in squamous cell carcinoma. Our results encouraged further investigation of therapeutic vaccination for dogs with advanced squamous cell carcinoma in clinical trials.     

Induction of dendritic cell-mediated immune responses against canine malignant melanoma cells

To establish the basis for the use of dendritic cells (DC) in the treatment of canine melanoma, dogs were vaccinated using autologous DC pulsed with canine melanoma CMM2 cell lysate in the presence of keyhole limpet haemocyanin (KLH) in vitro (CMM2-KLH-DC), and the induction of immune responses against CMM2 cells in vivo was examined using the delayed-type hypersensitivity (DTH) skin test. The DTH responses against CMM2 cells and KLH were observed in dogs vaccinated with CMM2-KLH-DC, while the responses against KLH but not CMM2 cells were detected with DC pulsed with KLH alone (KLH-DC). Recruitment of CD8 and CD4 T cells was detected in the positively responding sites, suggested that vaccination with CMM2-KLH-DC efficiently elicits T cell-mediated immunity against CMM-2 cells in vivo. These findings demonstrate the potential utility of DC-based tumour vaccination in the treatment of canine malignant melanoma.     

Possible actions of tumor necrosis factor-alpha in ovarian function

Tumor necrosis factor-alpha (TNFalpha) is a multifunctional cytokine that was first described as a tumoricidal factor produced by activated macrophages. Extensive research over the last two decades has suggested that TNFalpha has physiologically diverse actions in ovarian function in a variety of species. TNFalpha and its specific receptors are present in the ovaries of many species. Furthermore, TNFalpha plays multiple and probably important roles in corpus luteum (CL) function as well as ovarian cell function throughout the estrous cycle. This review focuses on recent studies documenting TNFalpha in ovarian follicles and CL in several mammals. In addition, possible roles of TNFalpha in ovarian function throughout the estrous cycle and in the gestation period are discussed.     

Cholera toxin promotes the generation of semi-mature porcine monocyte-derived dendritic cells that are unable to stimulate T cells

Cholera toxin (Ctx) is a powerful mucosal adjuvant with potential applications for oral vaccination of swine. Dendritic cells (DC) play a key role in the decision between immunity and tolerance, and are likely target cells for mediating Ctx functions in vivo. Therefore, we examined the capacity of Ctx to enhance stimulatory activity of porcine monocyte-derived DC (MoDC). Ctx promoted the development of a semi-mature DC phenotype, with decreased levels of MHC class II and CD40, but increased CD80/86 expression. These changes were associated with activation of extracellular signal-regulated kinase (ERK), but not NFkappaB or c-Jun N-terminal kinase (JNK). Functionally, Ctx-priming greatly diminished T cell stimulatory capacity both in antigen-specific and superantigen-induced proliferation assays. The lower proliferation rate was not due to increased apoptosis of either DC or T cells. Ctx suppressed TNFalpha secretion by MoDC, but induced IL-10 production. The observed effects on T cell proliferation could only be partially mimicked by IL-10 alone. However, addition of recombinant TNFalpha to co-cultures of Ctx-primed MoDC and lymphocytes restored lymphocyte proliferation in a concentration-dependent manner. Ctx-primed DC were not actively tolerogenic, since they could not suppress proliferative T cell reactions induced by untreated DC.     

In vitro effect of recombinant human tumor necrosis factor-alpha on canine neutrophil apoptosis

Neutrophils (polymorphonuclear leukocytes: PMNs) are essential for the host defense against various infections and are often injurious to the host, causing inflammatory diseases where tumor necrosis factor-alpha (TNF-alpha) is suggested to play an important role. Since an effect of TNF-alpha on canine PMN apoptosis has not been studied, canine PMNs were stimulated with recombinant human (rh)TNF-alpha in the present study to investigate the effect of TNF-alpha on canine PMN apoptosis. PMN apoptosis and function to produce ROS were assessed by flow cytometry. Delayed apoptosis was observed in the PMNs treated with rhTNF-alpha at 100 ng/ml, accompanied by retention of capability to produce ROS. However, PMN apoptosis was accelerated by rhTNF-alpha combined with cycloheximide. Therefore, it is indicated that TNF-alpha is able to activate anti- and pro-apoptotic pathways in PMNs and that the inhibition of PMN apoptosis by TNF-alpha requires protein synthesis in the PMNs.     

Immunopathogenic behaviors of canine transmissible venereal tumor in dogs following an immunotherapy using dendritic/tumor cell hybrid

Canine transmissible venereal tumor (CTVT) is a naturally occurring tumor that can be transmitted between dogs via live tumor cell inoculation. It is also a spontaneous self-regression tumor and its behavior is closely related to host immune responses. Since CTVT had been widely used for tumor models in canine cancers, whether this self-regression may overtake the immunity elicited from an exogenous tumor vaccine remains unclear and certainly worthwhile to be investigated. In this study, we used DCs/tumor hybrids as a tumor vaccine to evaluate the CTVT model. We prepared mature allogeneic dendritic cells from bone marrow and then assessed their phenotype (CD80, CD83, CD86, CD1a, CD11c, CD40 and MHC II), antigen uptake and presenting abilities. Fused dendritic cell/CTVT hybrids were then used as a vaccine, administered three times at two-week intervals via subcutaneous injection near the bilateral auxiliary and inguinal lymph nodes. In comparison with unvaccinated dogs (spontaneous regressed group), within a period of 2.5 months, the vaccinations substantially inhibited tumor progression (p<0.05) and accelerated the rate of regression by a mechanism involving amplification of the host tumor-specific adaptive immune responses and NK cytotoxicity (p<0.001). Pathologic examination revealed early massive lymphocyte infiltration resulting in final tumor necrosis. In addition, there are not any detectable effects on routine physical, body temperature or blood chemistry examinations. In conclusion, our data furnishes a reference value showing that CTVT is a model of potential use for the study of immunity elicited by vaccines against tumors, and also enable early-phase evaluation of the dendritic cell/tumor vaccine in terms of raising host immunity.