Modulating immune responses with dendritic cells: an attainable goal in veterinary medicine?

Dendritic cells (DCs) are antigen presenting cells that potently modulate immune responses with varying outcomes depending on the DC sub-population involved. To understand how DC sub-types arise, it is necessary to determine which factors influence their differentiation. At least three major sub-populations of DCs have been described in mice: CD4+/CD8- "myeloid" DCs, CD4-/CD8+ "lymphoid" DCs and Langerhans cell-derived DCs. Whilst somewhat comparable populations have been described in man, in most other species very little is known. The identification of cytokines which stimulate proliferation of DC precursors, and the observation that the cytokine environment influences the phenotype and the function of the DCs that subsequently develop, has provided a useful tool for evaluating these rare cells. We describe the influence of cytokines on the phenotype of DCs generated in the rat. Using bone marrow cells as the source of precursors we generated "myeloid-type" DCs from the adherent population using granulocyte-macrophage colony stimulating factor (GM-CSF), IL-4 and Flt-3L or "lymphoid-type" DCs from the non-adherent population using cytokines which included IL-7, IL-3, SCF and TNFalpha. In order to facilitate similar approaches to the study of equine DCs we have identified the nucleotide sequence encoding GM-CSF from the m-RNA of equine PBMC stimulated with Concanavalin A, amplified the cDNA by PCR and cloned it in eukaryotic and prokaryotic expression vectors. We report on the structure and function of this molecule.     

Effects of cytokines from thymocytes and thymic stromal cells on chicken intrathymic T cell development

We have studied the ability of thymic stromal cells (TSC) and thymocytes to produce cytokines and the involvement of cytokines in intrathymic T cell development. When thymocytes were co-cultured with thymic stromal cells in absence of direct contact and mitogenic stimulation, induction of thymocyte proliferation was observed. Supernatants of cultured stromal cells (TSC-CS) promoted a high proliferative response on CD3- thymocytes but had little effect on CD3+ thymocytes. These results indicate that stromal cells have produced a cytokine which can induce immature thymocyte proliferation. Moreover, stromal cells express the MRNA for stem cell factor (SCF) and c-kit (the receptor for SCF) was detected on CD3- thymocytes but not on CD3+ thymocytes. Since SCF can enhance the proliferation of immature thymocytes in synergy with IL-7 in mammals, there is a possibility that chicken stromal cells may produce a IL-7-like factor. Thymocytes have clearly expressed interferon (IFN)-gamma. In contrast, thymic stromal cells showed no detectable expression of IFN-gamma. CD3+ thymocytes express IFN-gamma MRNA more strongly than CD3 thymocytes, suggesting that IFN-gamma from thymocytes may operate on stromal cells and then may indirectly induce clonal elimination of CD3+ cells on stromal cells. The expression of these cytokines and receptors by thymic stromal cells and thymocyte subpopulations suggests that these cytokines participate in paracrine interactions between these cell populations during thymocyte differentiation.     

Phenotype and function of murine discrete Peyer's patch macrophage derived - dendritic cells

The phenotype and function of peritoneal cavity macrophage-derived dendritic cells (PEC-DC) was previously reported. In this study we have gone further in using our established culture system to generated discrete Peyer's patch dendritic cells (DPP-DC) from murine discrete Peyer's patch macrophages (DPP-M?), following stimulation with granulocyte macrophage colony stimulating factor (GM-CSF) plus interleukin 4 (IL-4) for 7 days. DPP-M? from murine small intestines were obtained by mechanical disruption of discrete Peyer's patches (DPP), followed by metrizamide density gradient centrifugation to remove Peyer's patch resident DC and debri, after which an overnight adherent step in tissue culture medium was carried out for macrophage enrichment. Characterization of the generated DPP-DC was carried out using well-established criteria of morphology, expression of membrane antigens and capacity for antigen presentation. Dendritic cells expressed DEC-205, F4/80 and CD34 at high levels, but exhibited very low CD11c levels. They were shown to present soluble protein antigen to CD3(+) spleen T cells. A comparison of the surface antigen expression in the progenitor DPP-M? population and the generated DPP-DC showed a significant decrease in MHC class II levels and a marked down regulation of the co-stimulatory molecule CD86 (B7-2). High expression of the haemopoietic progenitor marker CD34 indicates that the generated DC, possess a haemopoietic rather than myeloid origin. Taken together, these results may provide a better understanding of the complex network regulating mucosal immune responses.     

Expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor on B-1a cell from persistent lymphocytosis (PL) cows and lymphoma cell induced by bovine leukemia virus

The effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on B lymphocytes from persistent lymphocytosis (PL) cattle and lymphoma cells induced by bovine leukemia virus (BLV) was studied in vitro. Flow cytometric analysis showed that high levels of receptors to GM-CSF were expressed on these cell types. Proliferation of these B cells was induced in response to bovine GM-CSF. In tumor cell lines, the rate of cell proliferation was correlated with expression of GM-CSF receptors. A monoclonal antibody to GM-CSF inhibited lymphocyte proliferation and blocked the GM-CSF binding of lymphocytes. Cells expressing GM-CSF receptor were Ig positive and both CD5 and CD11 positive (B-1a cell). These results suggest that an abnormal expression of GM-CSF receptors on B lymphocytes from PL and lymphoma cells induced by BLV plays important roles in the PL and proliferation of lymphoma.     

Phenotype and function of murine peritoneal cavity macrophage derived-dendritic cells

The accessory activity was reported in murine peritoneal cavity macrophage derived dendritic cells (PEC-DC) in a mixed lymphocyte reaction (MLR). Here we continue the characterization of the generated PEC-DC using the criteria of morphology, phenotype and other accessory function. We have demonstrated that murine peritoneal cavity macrophages can be induced to differentiate in vitro into cells exhibiting typical dendritic cell (DC) morphology, phenotype and function. The proliferative capacity of the progenitors was amplified in the first step of the culture (day 0-7) using a combination of early cytokines: interleukin 4 and granulocyte-macrophage colony-stimulating factor. The second step of the culture started at day 7 with the removal of early growth factors to allow differentiation and final maturation of DC during 2 days of culture with interferon gamma plus either Toxoplasma lysate antigen (TLA) or lipopolysaccharide (LPS), a bacterial agent as a DC maturing agent. The resulting DC population exhibited typical DC morphology and expressed higher levels of MHC class II and the co-stimulatory molecules CD80 and CD86 compared to the untreated peritoneal cells. The generated DC cells efficiently presented soluble protein antigen to CD3(+) spleen T cells.     

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.     

Differentiation of porcine dendritic cells by granulocyte-macrophage colony-stimulating factor expressed in Pichia pastoris

Dendritic cells (DC) are potent inducers of acquired immunity due to their ability to present antigens in the context of a costimulatory environment and consequently serve an essential role in vaccine efficacy. Strategies to enhance their function, such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 treatment to induce DC differentiation from peripheral blood monocytes, may therefore be useful as vaccine adjuvants. We now have evaluated the effect of recombinant GM-CSF on the differentiation of DC in swine. GM-CSF mRNA was readily detected in porcine splenocytes, with increased levels following treatment of the cells with ConA and LPS. Porcine GM-CSF was cloned and expressed in the methylotrophic yeast, Pichia pastoris, as a glycosylated protein that induced proliferation of porcine bone marrow cells. P. pastoris-derived GM-CSF induced expression of antigen presenting (MHC class II) and costimulatory (CD80-CD86) molecules and enhanced antigen presenting cell (APC) function consistent with the induction of functional DC. Thus, recombinant GM-CSF produced by P. pastoris may be a potent adjuvant for swine vaccines.     

Intestinal stromal tumors in a simian immunodeficiency virus-infected, simian retrovirus-2 negative rhesus macaque (Macaca mulatta)

Multifocal submucosal stromal tumors were diagnosed in a 5.5-year-old rhesus macaque (Macaca mulatta) experimentally infected with simian immunodeficiency virus, strain SIVsmE660, and CD4+ T cell depleted. The animal was negative for simian retroviruses, SRV-1, -2, and -5. Polymerase chain reaction analysis of DNA from tumor and spleen tissue revealed abundant, preferential presence of retroperitoneal fibromatosis herpesvirus, the macaque homologue of the Kaposi sarcoma-associated herpesvirus (human herpesvirus-8), in the tumors. This was corroborated by demonstration of viral latent nuclear antigen-1 in the nuclei of a majority of the spindeloid tumor cells. Low levels of an additional macaque herpesvirus, rhesus rhadinovirus, were also detected in the spleen and tumor tissues. The spindeloid cells labeled positively for vimentin and CD117 but were negative for CD31, CD68, desmin, and smooth muscle cell actin. Collectively, these findings suggest a relation to but not absolute identity with simian mesenchymoproliferative disorders (MPD) or typical gastrointestinal stromal tumors (GISTs).     

β-Glucan plus ascorbic acid in neonatal calves modulates immune functions with and without Salmonella enterica serovar Dublin

To determine if β-glucan plus ascorbic acid affects adherence and pathogenicity of Salmonella Dublin and innate immune response in neonatal calves, 20 calves were fed control or supplemented diets (β-glucan, 0.9 g/d, plus ascorbic acid, 500 mg/d) until d 23. On d 21, 5 calves per treatment received 2.4 × 10(8)CFU of S. Dublin orally. S. Dublin spread through intestinal tissues into mesenteric lymph nodes (MLN), spleen, and lung tissues within 48 h. All supplemented calves had less mRNA expression of IL-1 receptor antagonist in liver. Leukocyte cell surface markers changed in lung cells, but not in blood, MLN, or spleen. CD14 in lungs was greatest for calves receiving supplement and challenge, but CD18 in lungs was greater for challenged than control calves. Lung DEC205 was greatest for challenged calves with and without supplement compared to controls, but more lung cells expressed CD14 for all treated groups compared to controls. These data show that S. Dublin briefly inhabited the intestinal tract, moving quickly to spleen, MLN, and lung tissues. Lung tissue was modulated by S. Dublin, but supplement alone increased CD14 expressing cells. The supplement appears not to attenuate invasiness but modified some lung cell populations by 48h.     

Functional characterization of equine dendritic cells propagated ex vivo using recombinant human GM-CSF and recombinant equine IL-4

Naive T cells can be activated both in vivo and in vitro by specialized antigen presenting cells, dendritic cells (DC), with potent antigen-specific, immunostimulatory activity. Indeed, DC can provide an extremely powerful and important immunological tool by which to potentiate the immune response for specific recognition of foreign antigens. Until recently, the direct isolation of DC from PBMC required laborious procedures with extremely poor yields (<0.1%). Methods have been developed for the human, lower primate, and murine model systems to propagate large numbers of DC from PBMC or bone marrow ex vivo with various cytokines. However, all other model systems, including equine, still require the laborious isolation procedures to obtain DC. In this study, we have adapted the methods developed for the human system to generate large numbers of equine DC from PBMC precursors using recombinant human GM-CSF and recombinant equine IL-4. Our report is the first documentation of ex vivo generated DC from PBMC in a domesticated animal model system. Equine DC derived from PBMC were rigorously characterized by analyzing morphological, phenotypic, and functional properties and were determined to have similar attributes as DC generated from human PBMC. Equine DC appeared stellate with large projectiles and veils and had cell surface antigens at similar levels as those defined on human and murine DC. Furthermore, functional attributes of the DC included rapidly capturing antigens by pinocytosis, receptor-mediated endocytosis, and phagocytosis, activating naive T cells in a mixed leukocyte reaction to a much greater extent than macrophage or lymphoblasts, presenting soluble and particulate antigen 10-100 fold more effectively to T cells on a per cell basis than macrophage or lymphoblasts, and presenting soluble and particulate antigen to both CD4+ and CD8+ T cells. Taken together, our study provides a framework by which equine DC can now be readily produced from PBMC precursors and presents an impetus for and model by which DC can be simply generated in other animal model systems.     

Generation of dendritic cells from rabbit bone marrow mononuclear cell cultures supplemented with hGM-CSF and hIL-4

The in vitro generation of dendritic cells (DCs) from either blood or bone marrow has been accomplished for humans and a number of other species. This ability has facilitated the opportunity to test the efficacy of DC vaccines in various tumor models. The cottontail rabbit papillomavirus (CRPV) model is the most clinically relevant animal model for human papillomavirus (HPV)-associated carcinogenesis. The CRPV model has been used to test various preventative and therapeutic vaccination strategies, and the availability of rabbit DCs would further expand its utility. However, to date, rabbit DCs have not been phenotypically and/or functionally characterized. Here we show that DCs can be generated in vitro from rabbit bone marrow mononuclear cells (BMMCs) cultured in the presence of the human cytokines GM-CSF and IL-4 and matured with lipopolysaccharide (LPS). These cells show upregulation of MHC class II and CD86, as well as downregulation of CD14, do not have non-specific esterase activity, are able to perform receptor-mediated endocytosis, and are potent stimulators of allogeneic T cell proliferation in mixed lymphocyte reactions. The ability to generate rabbit DCs makes it possible to test the efficacy of DC vaccination in the prevention and treatment of CRPV-induced lesions, which may provide useful preclinical data regarding the use of DC vaccines for HPV-associated lesions, including cervical cancer.     

Feline pulmonary Langerhans cell histiocytosis with multiorgan involvement

Histiocytic proliferative diseases are uncommon in cats, although recently a progressive histiocytosis of the skin with terminal involvement of internal organs has been described in cats. Here we describe 3 cats (2 males and 1 female) with pulmonary Langerhans cell histiocytosis (PLCH). The cats were euthanized due to progressive respiratory clinical symptoms and deterioration. Macroscopically, extensive, multifocal to confluent, pulmonary masses were evident. Infiltration of pancreas (2 cats), kidneys (1 cat), liver (1 cat), as well as tracheobronchial, hepatosplenic, or mesenteric lymph nodes (2 cats) was observed by gross or microscopic examination. The infiltrating cells had histiocytic morphology with cytologic atypia characterized by anisokaryosis and hyperchromasia regionally within infiltrated tissues. Lesional histiocytes expressed vimentin, CD18, and E-cadherin. Expression of E-cadherin was usually markedly reduced in extra-pulmonary lesions, which is consistent with possible down-regulation of E-cadherin associated with distant migration from the lung. Transmission electron microscopy demonstrated intracytoplasmic organelles consistent with Birbeck's granules of Langerhans cells in the lesional histiocytes in all cats, except in the pancreas of one cat. These findings were compatible PLCH with limited organ involvement of humans. It remains unproven whether feline PLCH represents a reactive or neoplastic cell proliferation.     

Canine hemophagocytic histiocytic sarcoma: a proliferative disorder of CD11d+ macrophages

Histiocytic disorders of dogs include histiocytoma, localized histiocytic sarcoma (HS), disseminated HS (malignant histocytosis), and the reactive histiocytoses: cutaneous and systemic. A common element to these diseases is proliferation of dendritic cells (DC) of either Langerhans cell (epithelial DC) or interstitial DC lineage. In this report, 17 dogs with hemophagocytic HS are described. Breeds affected included Bernese Mountain Dog (6), Golden Retriever (4), Rottweiler (3), Labrador Retriever (2), a mixed-breed dog, and a Schnauzer, which were from 2.5 to 13 years old. The dogs presented with Coombs negative responsive anemia in 16/17 dogs (94%), thrombocytopenia in 15/17 dogs (88%), hypoalbuminemia in 16/17 dogs (94%), and hypocholesterolemia in 11/16 dogs (69%). All dogs died or were euthanized. The clinical course ranged from 2 to 32 weeks (mean 7.1 weeks). Diffuse splenomegaly with ill-defined masses was consistently present. Microscopic lesions were prevalent in spleen, liver, lung, and bone marrow. Metastasis occurred by insidious intravascular invasion with minimal mass formation. Histiocytes were markedly erythrophagocytic and accompanied by foci of extramedullary hemopoiesis. Cytologically, the histiocytes varied from well differentiated to atypical, with atypia more prevalent in spleen than bone marrow. These tumors arose from splenic red pulp and bone marrow macrophages, which expressed major histocompatibility complex class II and the beta2 integrin, CD11d. They had low and/or inconsistent expression of CD1 and CD11c, which are dominantly expressed by canine nonhemophagocytic HS of DC origin. Canine histiocytic proliferative diseases now encompass proliferation of all members of the myeloid histiocytic lineage: Langerhans cells, interstitial DC, and macrophages.     

Classical swine fever virus induces activation of plasmacytoid and conventional dendritic cells in tonsil, blood, and spleen of infected pigs

Classical swine fever virus (CSFV) compromises the host immune system, causing indirect leucopoenia and disruption of in vitro T cell stimulation capacity. In order to explore the potential role of dendritic cells (DC) in such phenomena, the activation of conventional DC (cDC) and plasmacytoid DC (pDC) in blood and secondary lymphoid organs of infected pigs was investigated in the early time course post-inoculation (pi), together with viral components dissemination and cytokine production in serum. Whereas CD11R1+CD172a+ cDC frequencies were markedly reduced in blood and spleen, analysis of CD4+CD172a+ pDC numbers revealed a rapid turn-over of this DC subset in tissues pi. Both subsets matured and were activated after infection, as demonstrated by down-regulation of CD1a, up-regulation of the co-stimulation molecule CD80/86 and expression of cytokines. cDC essentially expressed tumor necrosis factor alpha (TNF-alpha) and interleukin (IL)-10, whereas pDC produced alpha interferon (IFN-alpha) and IL-12. IFN-alpha and TNF-alpha productions revealed an enhancement of innate anti-viral immune responses. Detection of antigen activated B lymphocytes in tonsil T-cell areas at 72 h pi, subsequently to the transient translocation of the viral E2 protein within germinal centres at 48 h pi, indicates the initiation of humoral response. This response was also evidenced by an important IL-10 production in serum one week pi. IL-12 expression in organs, as well as transient detection of IL-18 and IFN-gamma in serum, reflected the initiation of cellular immune responses. However, the uncommonly high levels of TNF-alpha and IFN-alpha produced by DC and measured in serum early post-infection, together with IL-10 expression in spleen, could play a role in the disruption of immune system cells, either inducing apoptosis or impairing DC functionalities themselves.     

Alloimmunity does not protect from challenge with the feline immunodeficiency virus

Immune responses against polymorphic host molecules incorporated into lentiviral envelopes during cell budding have induced protection against primate immunodeficiency virus infection. Dendritic cells (DCs) express high levels of MHC molecules and are infectable by lentiviruses. Therefore, in this pilot study we addressed the hypothesis that immunization of cats with allogeneic DC would induce immune responses that protect against challenge with the feline immunodeficiency virus. Two groups of 3 cats each received 3 subcutaneous injections of allogeneic or autologous DC, and were then challenged with viruses propagated in the immunizing DC. Infection status and lymphocyte parameters of cats were assessed during 6 weeks after challenge. MHC II antigens were incorporated into viral particles as identified by Western blot; and antibodies reactive with MHC class II antigens were detected in the serum of cats immunized with allogeneic but not autologous DC. After challenge, all cats had proviral DNA in blood leukocytes from 2 weeks post-challenge onward and seroconverted. Cats immunized with allogeneic DC maintained higher total and CD21(+) lymphocyte concentrations, and higher CD4(+)/CD8(+) lymphocyte ratios; however, these differences were not significantly different from cats that received autologous DC immunizations. Plasma viral load was not significantly different between groups of cats (p=0.204). These results suggest that immunization of cats with allogeneic DC does not induce protective immunity against FIV infection.     

Differential response of splenic monocytes and DC from cattle to microbial stimulation with Mycobacterium bovis BCG and Babesia bovis merozoites

Both bovine peripheral blood monocyte-derived dendritic cells (DC) and myeloid DC from afferent lymph have been described, but resident DC from other bovine tissues have not been fully characterized. The spleen as a secondary lymphoid organ is central to the innate and acquired immune response to various diseases particularly hemoprotozoan infections like babesiosis. Therefore, we developed methods to demonstrate the presence of myeloid DC from the spleen of cattle and have partially characterized a DC population as well as another myeloid cell population with monocyte characteristics. The phenotypic profile of each population was CD13+CD172a+/-CD14-CD11a-CD11b+/-CD11c+ and CD172a+CD13+/-CD14+CD11a-CD11b+/-CD11c+, respectively. The CD13+ population was found exclusively in the spleen whereas the CD172a+ population was present at the same percentage in the spleen and peripheral blood. CD13+ cells developed a typical veiled appearance when in culture for 96 h. The two cell populations differed in their ability to produce nitric oxide and had a different pattern of cytokine mRNA when stimulated with Mycobacterium bovis BCG or Babesia bovis merozoites. The data demonstrate the presence of a myeloid splenic DC with attributes consistent with an immature status.     

Molecular cloning and characterization of markers and cytokines for equid myeloid cells

The myeloid cell system comprises of monocytes, macrophages (MPhi), dendritic cells (DC), Kupffer cells, osteoclasts or microglia and is also known as the mononuclear phagocytic system (MPS). Essential cytokines to differentiate or activate these cells include GM-CSF or IL-4. Important markers for characterization include CD1, CD14, CD68, CD163 and CD206. All these markers, however, were not cloned or further characterized in equids by use of monoclonal antibodies earlier. To overcome this problem with the present study, two approaches were used. First, we cloned equine cytokines and markers, and second we analyzed cross-reactivity of human homologues or anti-human monoclonal antibodies. For cloning of equine cytokines and markers, we used degenerate primers delineated from other species, or equine-specific primers based on previous information in Genbank. Flow cytometry was used to determine the expression of markers on myeloid cells. Cross-reactivity could be shown for anti-human CD14, CD163 and mannose receptor (CD206) mAbs. Surface markers such as CD1 and CD68 that distinguish MPhi and DC were cloned and sequenced. According to blast homology, equine CD1a and CD1b could be identified and distinguished. With the resulting information, dendritic cells and macrophages of horses may be characterized.