Canine Monocytes Research Articles

Characterization of polarization states of canine monocyte derived macrophages.

Macrophages can reversibly polarize into multiple functional subsets depending on their micro-environment. Identification and understanding the functionality of these subsets is relevant for the study of immune‑related diseases. However, knowledge about canine macrophage polarization is still in its infancy. In this study, we polarized canine monocytes using GM-CSF/IFN- γ and LPS towards M1 macrophages or M-CSF and IL-4 towards M2 macrophages and compared them to undifferentiated monocytes (M0). Polarized M1 and M2 macrophages were thoroughly characterized for morphology, surface marker features, gene profiles and functional properties. Our results showed that canine M1-polarized macrophages obtained a characteristic large, roundish, or amoeboid shape, while M2-polarized macrophages were smaller and adopted an elongated spindle-like morphology. Phenotypically, all macrophage subsets expressed the pan-macrophage markers CD14 and CD11b. M1-polarized macrophages expressed increased levels of CD40, CD80 CD86 and MHC II, while a significant increase in the expression levels of CD206, CD209, and CD163 was observed in M2-polarized macrophages. RNAseq of the three macrophage subsets showed distinct gene expression profiles, which are closely associated with immune responsiveness, cell differentiation and phagocytosis. However, the complexity of the gene expression patterns makes it difficult to assign clear new polarization markers. Functionally, undifferentiated -monocytes, and M1- and M2- like subsets of canine macrophages can all phagocytose latex beads. M2-polarized macrophages exhibited the strongest phagocytic capacity compared to undifferentiated monocytes- and M1-polarized cells. Taken together, this study showed that canine M1 and M2-like macrophages have distinct features largely in parallel to those of well-studied species, such as human, mouse and pig. These findings enable future use of monocyte derived polarized macrophages particularly in studies of immune related diseases in dogs.

Evaluation of canine and feline leukocyte differential counts obtained with the scil vCell 5 compared to the Advia 2120 hematology analyzer and a manual method.

The vCell 5 (scil Animal Care), a point-of-care hematology analyzer (POCA), was recently introduced to veterinary laboratories. This laser- and impedance-based analyzer is capable of providing a CBC with 5-part WBC differential count (Diff) along with WBC cytograms and flags serving as interpretation aids for numerical results. We compared the scil POCA-Diff to reference methods (i.e., manual differential count, Advia 2120 hematology analyzer [Siemens]) for canine and feline blood samples and considered WBC cytograms and flags. Total observed error (TEo), calculated from CV and bias%, was compared to total allowable error (TEa). Data were analyzed before and after a review process (exclusion of flagged and samples with invalid cytograms). For both species, correlation was good-to-excellent (rs = 0.81-0.97) between both analyzers for all variables, except for feline monocytes (rs = 0.21-0.63) and canine monocyte% (rs = 0.50). Smallest biases were seen for neutrophils (dog: -5.7 to 0.8%; cat: 1.5-9.4%) with both reference methods. Quality requirements (TEo < TEa) were fulfilled for canine and feline neutrophils (TEo = 5.3-10.6%, TEa = 15%) and eosinophils (TEo = 67.1-83%, TEa = (90)-50%) considering at least one reference method. Our review process led to mildly higher rs-values for most variables. Although not completely satisfactory, the scil POCA provides reliable results in compliance with ASVCP quality goals for canine and feline neutrophils and eosinophils. Analyzer flag and cytogram analysis served as useful tools for QA, indicating the necessity for manual review of blood smears, and contributed to improvement of scil POCA performance.

Analytical performance of a canine ELISA monocyte chemoattractant protein-1 assay for use in cats and evaluation of circulating levels in normal weight and obese cats

BackgroundIn human and murine obesity, adipose tissue dwelling macrophages and adipocytes produce monocyte chemoattractant protein-1 (MCP-1) leading to systemic low-grade inflammation. The aim of the study was to validate a canine MCP-1 ELISA assay for use in cats and to investigate whether a difference in MCP-1 concentrations could be detected between: a) cats having normal or elevated circulating serum amyloid A (SAA) levels and b) normal weight and obese cats. Serum obtained from 36 client-owned cats of various breed, age and sex with normal (n = 20) to elevated SAA (n = 16) was used for the validation of the canine MCP-1 ELISA assay. As no golden standard exists for measurement of inflammation, circulating MCP-1 concentrations were compared to SAA measurements, as an indicator of systemic inflammation. Analytical precision, dilution recovery and detection limit were calculated. A possible correlation between MCP-1 concentrations and obesity related measures (body fat percentage (BF%), insulin sensitivity and cytokine expression) were investigated in another population of 73 healthy, lean to obese, neutered domestic short-haired cats.ResultsIntra- (2.7–4.1%) and inter-assay (2.2–3.6%) coefficient of variation and dilution recovery were acceptable, and the detection limit was 27.1 pg/mL. MCP-1 did not correlate with SAA, and there was no difference between the inflammatory (SAA > 20 mg/L) and non-inflammatory group, due to a marked overlap in MCP-1 concentrations. Circulating MCP-1 concentrations were unaffected by BF% (r2 = 2.7 × 10–6, P = 0.21) and other obesity-related markers.ConclusionsThe present canine ELISA assay seems to be able to measure circulating feline MCP-1. However, further studies are needed to determine its possible use for detecting inflammation in relation to disease processes or obesity-related low-grade inflammation in cats.

Novel Ehrlichia canis genogroup in dogs with canine ehrlichiosis in Cuba

BackgroundCanine monocytic ehrlichiosis (CME) is caused by the tick-borne pathogen Ehrlichia canis, an obligate intracellular Gram-negative bacterium of the family Anaplasmataceae with tropism for canine monocytes and macrophages. The trp36 gene, which encodes for the major immunoreactive protein TRP36 in E. canis, has been successfully used to characterize the genetic diversity of this pathogen in different regions of the world. Based on trp36 sequence analysis, four E. canis genogroups, United States (US), Taiwan (TWN), Brazil (BR) and Costa Rica (CR), have been identified. The aim of this study was to characterize the genetic diversity of E. canis in Cuba based on the trp36 gene.Methods Whole blood samples (n = 8) were collected from dogs found to be infested with the tick vector Rhipicephalus sanguineus sensu lato (s.l.) and/or presenting clinical signs and symptoms of CME. Total DNA was extracted from the blood samples and trp36 fragments were amplified by PCR. Nucleotide and protein sequences were compared using alignments and phylogenetic analysis.ResultsFour of the trp36 sequences obtained (n = 8) fall within the phylogenetic cluster grouping the US genogroup E. canis strains. The other E. canis trp36 sequences formed a separate and well-supported clade (94% bootstrap value) that is phylogenetically distant from the other major groups and thus represents a new genogroup, herein designated as the ‘Cuba (CUB) genogroup’. Notably, dogs infected with the CUB genogroup presented frequent hemorrhagic lesions.ConclusionsThe results of this study suggest that genetic diversification of E. canis in Cuba is associated with the emergence of E. canis strains with increased virulence.

Changes in gene expression profiles and cytokine secretions in peripheral monocytes by treatment with small extracellular vesicles derived from a canine lymphoma cell line.

Interactions between tumor and immune cells within the tumor microenvironment play an important role in tumor progression, and small extracellular vesicles (EVs) derived from these tumor cells have been shown to exert immunomodulatory effects on various immune cells, including macrophages and lymphocytes. Although the immunomodulatory effects of small EVs derived from human cancer cells have been intensively investigated, few studies have investigated the effects of lymphoma-derived small EVs on macrophages in both human and veterinary medicine. Here, we evaluated the effects of canine lymphoma-derived small EVs on canine primary monocytes, which are the major source of macrophages in neoplastic tissues. Comprehensive gene expression analysis of these treated monocytes revealed their distinct activation via the Toll-like receptor (TLR) and NF-κβ signaling pathways. In addition, treatment with lymphoma small EVs increased the secretion of MCP-1, which induces the infiltration and migration of monocytes and lymphocytes in neoplastic and cancer tissues. The results of this study indicate that canine lymphoma small EVs activate monocytes, possibly through the activation of TLR and NF-κβ signaling pathways, and induce monocytes to secrete of MCP-1, which might contribute to immune cell infiltration within the tumor microenvironment.

Vitamin d and leishmaniasis: Neither seasonal nor risk factor in canine host but potential adjuvant treatment through cbd103 expression.

Vitamin D (VitD) deficiency has been shown to be a risk factor for a plethora of disorders. We have shown that dogs with clinical leishmaniasis presented lower VitD serum levels than non-infected dogs, and even lower than those with asymptomatic infection. However, if VitD deficiency is a risk factor to develop clinical leishmaniasis remains to be answered. It is also unknown if VitD participates in Leishmania control. First, we retrospectively analysed VitD concentration in serum samples from 36 healthy dogs collected in different periods of the year concluding that there isn’t a seasonal variation of this vitamin in dogs. We also included 9 dogs with clinical leishmaniasis and 10 non-infected healthy dogs, in which we measured VitD levels at the beginning of the study, when all dogs were negative for serology and qPCR, and 1 year later. Whereas non-infected dogs showed no change in VitD levels along the study, those developing clinical leishmaniasis showed a significant VitD reduction at the end of the study (35%). When we compared VitD concentration between the two groups at the beginning of the study, no differences were detected (43.6 (38–59) ng/mL, P = 0.962). Furthermore, an in vitro model using a canine macrophage cell line proved that adding active VitD leads to a significant reduction in L. infantum load (31.4%). Analyzing expression of genes related to VitD pathway on primary canine monocytes, we showed that CBD103 expression was significantly enhanced after 1,25(OH)2D addition. Our results show that VitD concentration is neither seasonal nor a risk factor for developing canine leishmaniasis, but it diminishes with the onset of clinical disease suggesting a role in parasitic control. Our in vitro results corroborate this hypothesis and point out that VitD regulates infection through CBD103 expression. These results open the possibility for studies testing VitD as an adjuvant in leishmaniasis therapy.

A further investigation of the leishmaniosis outbreak in Madrid (Spain): low-infectivity phenotype of the Leishmania infantum BOS1FL1 isolate to establish infection in canine cells

Human leishmaniosis caused by Leishmania infantum is a zoonotic disease, with dogs as the main reservoir in Mediterranean Basin countries. The largest European outbreak of human leishmaniosis declared in the southwestern Madrid region (Spain) is characterized by unusual epidemiological and clinical features, such as the emergence of new wild reservoirs (hares and rabbits), whereas the seroprevalence, infection, and severity of canine leishmaniosis have not substantially changed since the first studies conducted in Madrid before the outbreak. Previous studies reported that L. infantum isolates from the Madrid leishmaniosis focus displayed elevated virulence in in vivo models of infection and increased infectivity in murine target cells. With the aim of studying whether changes in the host-parasite interaction and virulence profile have developed, we first assessed the behaviour of one circulating isolate of the outbreak, IPER/ES/2012/BOS1FL1 (BOS1FL1), compared to that of a well-characterized strain from canine leishmaniosis, MCAN/ES/1996/BCN150 (BCN150), in terms of infection capacity (percentage of infected cells, representing infectivity, and number of amastigotes per infected cell, representing the intensity of infection) in canine monocytes and macrophages. BCN150 displayed significantly higher infectivity (76.82 ± 4.40 vs 38.58 ± 2.19; P < 0.0001) and intensity of infection (3.64 ± 0.13 vs 1.83 ± 0.12; P < 0.0001) than BOS1FL1 when interacting with canine cells. Our ROS induction results did not differ significantly between the two isolates or with the responses previously described for other L. infantum isolates. Paradoxically, increased resilience to hydrogen peroxide exposure was observed for BOS1FL1 (% viability 40.62 ± 5.54 vs 26.37 ± 2.93; P = 0.039). Finally, we demonstrated that a decreased intracellular load of BOS1FL1 was associated with increased IFN-γ (261.21 ± 26.29 vs 69.80 ± 9.02; P = 0.0151) and decreased IL-10 production (165.06 ± 23.87 vs 264.41 ± 30.58; P = 0.0002). In this study, we provide the first detailed insight into the differences between the isolate BOS1FL1 from the outbreak in Madrid and the well-characterized strain BCN150 MON-1 obtained from a dog in their response to interacting with canine cells. However, further studies are necessary to shed light on the immune mechanisms resulting in BOS1FL1 exhibiting less virulent behaviour in canine cells than in cells derived from other host species.

Development of canine PD-1/PD-L1 specific monoclonal antibodies and amplification of canine T cell function.

Interruption of the programmed death 1 (PD-1) / programmed death ligand 1 (PD-L1) pathway is an established and effective therapeutic strategy in human oncology and holds promise for veterinary oncology. We report the generation and characterization of monoclonal antibodies specific for canine PD-1 and PD-L1. Antibodies were initially assessed for their capacity to block the binding of recombinant canine PD-1 to recombinant canine PD-L1 and then ranked based on efficiency of binding as judged by flow cytometry. Selected antibodies were capable of detecting PD-1 and PD-L1 on canine tissues by flow cytometry and Western blot. Anti-PD-L1 worked for immunocytochemistry and anti-PD-1 worked for immunohistochemistry on formalin-fixed paraffin embedded canine tissues, suggesting the usage of this antibody with archived tissues. Additionally, anti-PD-L1 (JC071) revealed significantly increased PD-L1 expression on canine monocytes after stimulation with peptidoglycan or lipopolysaccharide. Together, these antibodies display specificity for the natural canine ligand using a variety of potential diagnostic applications. Importantly, multiple PD-L1-specific antibodies amplified IFN-γ production in a canine peripheral blood mononuclear cells (PBMC) concanavlin A (Con A) stimulation assay, demonstrating functional activity.

Staphylococcus pseudintermedius 5′-nucleotidase suppresses canine phagocytic activity

Staphylococcus pseudintermedius is a major opportunistic bacterial pathogen and the leading cause of pyoderma in dogs. In canines it is also often associated with infections of the urinary system and wounds and occasionally infects people. Widespread antimicrobial resistance has made the development of alternative treatments a high priority. The development of a staphylococcal vaccine, however, has proven challenging. Identification of virulence factors that inhibit phagocytosis and avoid innate immunity may play a significant role in preventing or treating infection with S. pseudintermedius. In this study, we identified a putative 5′-nucleotidase provisionally named SpAdsA, a S. pseudintermedius cell- wall protein encoded by SpAdsA. SpAdsA shares approximately 52% identity with the orthologous protein of Staphylococcus aureus and 14.8% identity with that of Streptococcus suis type2. It catalyzes the dephosphorylation of adenosine triphosphate and attenuation of this enzyme with critical amino acid substitutions nearly eliminated its hydrolytic activity. Exogenous adenosine inhibited phagocytosis of S. pseudintermedius by canine neutrophils and monocytes. Conversely, the addition of SpAdsA inhibitor or A2A adenosine receptor antagonist impaired the capacity of S. pseudintermedius to escape from killing by phagocytic cells. The neutralizing ability of canine antibody produced against SpAdsA-M was determined. Taken together, these results suggest that SpAdsA likely plays an important role in S. pseudintermedius virulence and that attenuated SpAdsA may be a good candidate for inclusion in a vaccine against S. pseudintermedius.

CD4 and MHCII phenotypic variability of peripheral blood monocytes in dogs.

In humans and mice, the detailed phenotypic and functional characterization of peripheral blood monocytes allows for identification of three monocyte subsets. There are also evidences of monocyte phenotypic heterogeneity in other species, including cattle, sheep, pig and horse. However, little is known about such variability in dogs. The aim of the study was to determine whether and how peripheral blood monocytes of healthy dogs differ in the presence of MHCII and CD4 and in the basal production of reactive oxygen species (ROS). Three distinct subsets of CD11b+CD14+ monocytes were found in peripheral blood samples of healthy dogs, based on the variations in the density of MHCII and CD4 surface molecules: MHCII+CD4– (Mo1), MHCII+CD4+ (Mo2) and MHCII–CD4+ (Mo3). The Mo2 and Mo3 were significantly lower in percentage than Mo1 but their basal ROS production was higher. Within the Mo2 and Mo3 subsets, the percentage of cells producing ROS was significantly higher comparing to cells lacking this activity. Canine peripheral blood monocytes vary in the expression of MHCII and CD4 and in the activity suggesting that cells within the three identified subsets carry out different functions. The higher production of ROS in non-activated cells within small subsets of Mo2 and Mo3 monocytes might indicate their immunomodulatory potential.

Proinflammatory response of canine trophoblasts to Brucella canis infection.

Brucella canis infection is an important cause of late-term abortion in pregnant bitches. The pathophysiological mechanisms leading to B. canis–induced abortion are unknown, but heavily infected trophoblasts are consistently observed. As trophoblasts responses to other pathogens contribute to placental inflammation leading to abortion, the aim of the present study was to characterize the cytokine response of canine trophoblasts to B. canis infection. To achieve this, trophoblasts isolated from term placenta of healthy female dogs were infected with B. canis, culture supernatants were harvested for cytokine determinations, and the load of intracellular viable B. canis was determined at different times post-infection. Additionally, cytokine responses were assessed in non-infected trophoblasts stimulated with conditioned media (CM) from B. canis-infected canine monocytes and neutrophils. Finally, cytokine response and bacteria replication were assessed in canine placental explants infected ex vivo. B. canis successfully infected and replicated in primary canine trophoblasts, eliciting an increase in IL-8 and RANTES (CCL5) secretion. Moreover, the stimulation of trophoblasts with CM from B. canis-infected monocytes and neutrophils induced a significant increase in IL-8, IL-6 and RANTES secretion. B. canis replication was confirmed in infected placental explants and the infection elicited an increased secretion of TNF-α, IL-8, IL-6 and RANTES. This study shows that canine trophoblasts produce proinflammatory cytokines in response to B. canis infection and/or to stimulation with factors produced by infected monocytes and neutrophils. These cytokines may contribute to placental inflammation leading to abortion in B. canis-infected pregnant bitches.

Phenotypic heterogeneity of peripheral monocytes in healthy dogs

Monocytes are key cells of the innate immune system. Their phenotypic and functional roles have been investigated in humans, mice and other animals, such as the rat, pig and cow. To date, detailed phenotypic analysis of monocytes has not been undertaken in dogs. Two important surface markers in human monocytes are CD14 and MHC class II (MHC II). By flow cytometry, we demonstrated that canine monocytes can be subdivided into three separate populations: CD14posMHC IIneg, CD14posMHC IIpos and CD14negMHC IIpos. Both light and transmission electron microscopy confirmed the monocytic identity of all three populations. The CD14posMHC IIneg population could be distinguished on an ultrastructural level by their smaller size, the presence of more numerous, larger granules, and more pseudopodia than both of the other populations.

Role of monocyte recruitment in hemangiosarcoma metastasis in dogs.

Canine hemangiosarcoma (HSA) is a highly malignant tumour associated with short survival times because of early and widespread metastasis. In humans and rodents, monocytes play key roles in promoting tumour metastasis through stimulating tumour cell extravasation, seeding, growth and angiogenesis. Therefore, we investigated the potential association between monocyte infiltration and tumour metastasis in HSA and other common canine tumours. Immunohistochemistry was used to quantify CD18+ monocytes within metastases. We found that HSA metastases had significantly greater numbers of CD18+ monocytes compared with metastases from other tumour types. HSA cells were the highest producers of the monocyte chemokine CCL2, and stimulated canine monocyte migration in a CCL2 dependent manner. These results are consistent with the hypothesis that overexpression of CCL2 and recruitment of large numbers of monocytes may explain in part the aggressive metastatic nature of canine HSA. Thus, therapies designed to block monocyte recruitment may be an effective adjuvant strategy for suppressing HSA metastasis in dogs.

Association of Canine Osteosarcoma and Monocyte Phenotype and Chemotactic Function.

BackgroundMonocytes/macrophages are likely key cells in immune modulation in dogs with osteosarcoma (OSA). Increased peripheral monocyte counts are negatively correlated with shorter disease‐free intervals in dogs with OSA. Understanding the monocyte/macrophage's modulatory role in dogs with OSA can direct further studies in immunotherapy development for OSA.Hypothesis/ObjectivesThat OSA evades the immune response by down‐regulating monocyte chemokine receptor expression and migratory function, and suppresses host immune responses.AnimalsEighteen dogs with OSA that have not received definitive treatment and 14 healthy age‐matched controlsMethodsClinical study—expression of peripheral blood monocyte cell surface receptors, monocyte mRNA expression and cytokine secretion, monocyte chemotaxis, and survival were compared between clinical dogs with OSA and healthy control dogs.ResultsCell surface expression of multiple chemokine receptors is significantly down‐regulated in peripheral blood monocytes of dogs with OSA. The percentage expression of CCR2 (median 58%, range 2–94%) and CXCR2 expression (median 54%, range 2–92%) was higher in control dogs compared to dogs with OSA (CCR2 median 29%, range 3–45%, P = 0.0006; CXCR2 median 23%, range 0.2–52%, P = 0.0007). Prostaglandin E2 (PGE 2) (OSA, median 347.36 pg/mL, range 103.4–1268.5; control, 136.23 pg/mL, range 69.93–542.6, P = .04) and tumor necrosis factor‐alpha (TNF‐α) (P = .02) levels are increased in OSA monocyte culture supernatants compared to controls. Peripheral blood monocytes of dogs with OSA exhibit decreased chemotactic function when compared to control dogs (OSA, median 1.2 directed to random migration, range 0.8–1.25; control, 1.6, range of 0.9–1.8, P = .018).Conclusions and Clinical ImportanceDogs with OSA have decreased monocyte chemokine receptor expression and monocyte chemotaxis, potential mechanisms by which OSA might evade the immune response. Reversal of monocyte dysfunction using immunotherapy could improve survival in dogs with OSA.

Immune regulation of canine tumour and macrophage PD‐L1 expression

Expression of programmed cell death receptor ligand 1 (PD-L1) on tumor cells has been associated with immune escape in human and murine cancers, but little is known regarding the immune regulation of PD-L1 expression by tumor cells and tumor-infiltrating macrophages in dogs. Therefore, 14 canine tumor cell lines, as well as primary cultures of canine monocytes and macrophages, were evaluated for constitutive PD-L1 expression and for responsiveness to immune stimuli. We found that PD-L1 was expressed constitutively on all canine tumor cell lines evaluated, although the levels of basal expression were very variable. Significant upregulation of PD-L1 expression by all tumor cell lines was observed following IFN-γ exposure and by exposure to a TLR3 ligand. Canine monocytes and monocyte-derived macrophages did not express PD-L1 constitutively, but did significantly upregulate expression following treatment with IFN-γ. These findings suggest that most canine tumors express PD-L1 constitutively and that both innate and adaptive immune stimuli can further upregulate PD-L1 expression. Therefore the upregulation of PD-L1 expression by tumor cells and by tumor-infiltrating macrophages in response to cytokines such as IFN-γ may represent an important mechanism of tumor-mediated T-cell suppression in dogs as well as in humans.

Association of Canine Osteosarcoma and Monocyte Phenotype and Chemotactic Function

Association of Canine Osteosarcoma and Monocyte Phenotype and Chemotactic Function

Alkaline phosphatase is a useful cytochemical marker for the diagnosis of acute myelomonocytic and monocytic leukemia in the dog.

Immunophenotyping has replaced cytochemical staining as the preferred technique for classifying acute leukemia. However, some acute myeloid leukemias (AML) lack lineage-associated markers. In our experience, alkaline phosphatase (ALP) is expressed in immature canine monocytes. We hypothesized that ALP is a useful marker for monocytic AML. The objective was to compare ALP expression in neoplastic cells from dogs with lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoid leukemia (ALL), and AML. Alkaline phosphatase results were retrieved from medical records of dogs with acute leukemia. Smears from dogs with lymphoma or leukemia were also prospectively stained for ALP activity. CLL was based on persistent lymphocytosis (10×10(9) /L) and acute leukemia on ≥20% blasts in blood or bone marrow. ALL was classified based on positive phenotyping for T- or B-lymphocyte antigens, and AML on positive phenotyping for CD11b, CD11c or CD14, or cytochemical staining for chloroacetate esterase, Sudan Black B, or myeloperoxidase. There was no ALP activity in all 49 lymphomas and 7 CLLs. Weak ALP activity was seen in 31% of 14 ALL (all T-ALL). ALP activity was seen in all 20 AML (P<.001 vs ALL) with strong activity in 64% (vs 25% ALL) in most neoplastic cells (median 75% vs 9% ALL, P=.020). Of AML, 80% were CD34+ (vs 39% ALL, P=.027) and 100% were MHCII- (vs 43% ALL, P=.002). ALP activity may be useful for AML confirmation in dogs, particularly if neoplastic cells only express CD34+ on immunophenotyping.

Production of canine soluble CD40 ligand to induce maturation of monocyte derived dendritic cells for cancer immunotherapy

CD40 ligand (CD40L) expressed by activated T cells is shown to induce maturation of immature dendritic cells (DCs) and this maturation is a vital part in DC based tumor immunotherapy. We constructed an expression vector by cloning the extracellular domain of canine CD40L fused to the signal sequence of canine IL-12p40. When PBMCs were incubated with canine granulocyte-macrophage (GM) -CSF and IL-4, expression of CD86 was significantly elevated, but the majority of cells displayed the morphology of immature DCs. Following addition of the expressed canine soluble CD40L (csCD40L) to the DC-inducing culture, the cell morphology shifted to that of mature DCs, and expression of CD80, CD86, MHC class II and CD1a was significantly enhanced. This morphological change and enhancement of expression was observed even when the csCD40L was present only in the second half period of the culture. Furthermore, the csCD40L caused a significant increase in IL-12 production from DCs. These results show that the csCD40L significantly promotes the maturation and activation of canine monocyte derived DCs.

Human intravenous immunoglobulin (hIVIG) inhibits anti-CD32 antibody binding to canine DH82 cells and canine monocytes in vitro

The IgG receptors CD16 and CD32 (FcγRIII and FcγRII) link the humoral immune response to effector cell immune responses by binding immune complexes. Human intravenous immunoglobulin (hIVIG) consisting of immunoglobulin from pooled donors is reported to block FcγRs and has been used to treat a variety of canine autoimmune disorders. FcγRs have been poorly described for canine monocytes; therefore, the objectives of this study were to: (1) identify canine monocyte/macrophage FcγR (CD16 and CD32) expression and (2) demonstrate in vitro hIVIG binding to these receptors. The canine monocyte/macrophage-like cell line (DH82) and monocytes isolated from peripheral blood of healthy dogs were evaluated by flow cytometry (FACS) for CD16 and CD32 expression using commercially available anti-CD16 and anti-CD32 antibodies directed against the human isoforms. The mean percentage of cells expressing CD16 was 55% of DH82 cells and 13% of blood monocytes and the mean percentage of cells expressing CD32 was 85% of DH82 cells and 73% of blood monocytes. Immunoprecipitation of canine DH82 cells lysate using the same anti-CD16 or anti-CD32 antibodies suggested that these anti-human antibodies recognize the canine homologues. To demonstrate FcγR blockade, cells were incubated with increasing concentrations of hIVIG and then incubated with anti-CD16 or anti-CD32 antibodies. The percentage of CD32 expression decreased in a concentration dependent fashion in DH82 cells and blood monocytes after incubation with increasing concentrations of IVIG, suggesting that hIVIG was binding to CD32 and inhibiting anti-CD32 antibody binding. The same results were not demonstrated with anti-CD16 antibody. We believe this is the first report to demonstrate Fcγ receptors CD16 and CD32 expression on canine monocytes and in vitro CD32 binding by human IgG, which may represent one of the immunomodulatory mechanisms of hIVIG.

Functional expression of the damage-associated molecular pattern receptor P2X7 on canine kidney epithelial cells

Epithelial cells are important in inflammation and immunity. In this study, we examined if Madin–Darby canine kidney (MDCK) epithelial cells express functional P2X7 receptors, which bind the damage-associated molecular pattern extracellular adenosine 5′-triphosphate (ATP). Reverse transcription (RT)-PCR and immunoblotting revealed the expression of P2X7 in MDCK cells. A flow cytometric assay demonstrated that ATP or 2′(3′)-O-(4-benzoylbenzoyl)ATP induced ethidium+ uptake into MDCK cells, and that this process was impaired by the P2X7 antagonists KN-62 and A438079. RT-PCR also demonstrated the presence of Toll-like receptor 4, NALP3, caspase-1, interleukin-1β and interleukin-18 in MDCK cells, as well as in positive control LPS-primed canine monocytes. In conclusion, the MDCK epithelial cell line expresses functional P2X7, as well as Toll-like receptor 4 and molecules associated with the NALP3 inflammasome. This cell line may help elucidate the role of these molecules in kidney epithelial cells and renal disorders in dogs and humans.