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.     

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.     

Identification of CpG oligodeoxynucleotide sequences that induce IFN-gamma production in canine peripheral blood mononuclear cells

Oligodeoxynucleotides containing the cytosine-phosphate-guanine (CpG) motif (CpG-ODNs) have been shown to induce T(H)1 immune responses in animals. Since the sequences of CpG-ODNs that induce T(H)1 responses are considered to vary among animal species, it is necessary to identify effective CpG-ODNs in each animal. In order to identify the sequences of CpG-ODNs that induce T(H)1 responses in dogs, mRNA expression and protein production of IFN-gamma were examined in peripheral blood mononuclear cells (PBMCs) from healthy dogs treated with 11 kinds of synthetic CpG-ODNs. One of the 11 CpG-ODNs (No. 2 CpG-ODN, 5'-GGTGCATCGATGCAGGGGGG-3') was shown to significantly increase mRNA expression and protein production of IFN-gamma in canine PBMCs in a manner dependent on the sequence of the CpG motif. This CpG-ODN also enhanced the expression of IL-12 p40 mRNA in canine PBMCs, whereas expression of IL-12 p35, IL-18, and IL-4 mRNAs was not induced by this CpG-ODN. These results indicate that this CpG-ODN was able to produce IFN-gamma by induction of T(H)1-skewed immune response in dogs. CpG-ODNs may be useful for inducing prophylactic and therapeutic immunity against allergic diseases, viral infection, and tumors in dogs.     

Recombinant canine IL-13 receptor alpha2-Fc fusion protein inhibits canine allergen-specific-IgE production in vitro by peripheral blood mononuclear cells from allergic dogs

Human IL-13, like IL-4, is involved in the regulation of B-cell development, IgE synthesis and allergic responses. However, because IL-13 does not affect either murine Ig class switching or IgE production in vitro, the use of murine models to study the role of IL-13 in IgE-mediated diseases has been limited. In this communication, we report that recombinant protein of canine IL-13 (rcaIL-13) stimulates production of allergen-specific-IgE in vitro by peripheral blood mononuclear cells (PBMC) from flea allergen-sensitized dogs, and that this stimulation activity is specifically inhibited by recombinant protein of canine IL-13Ralpha2 and Fc fragment of canine IgG heavy chain (rcaIL-13Ralpha2-Fc). The data suggest that the regulatory effects of IL-13 on IgE production in canine PBMC are similar to those reported in humans. Thus, canine IL-13 may be a central mediator of allergic diseases in dogs, and allergic dogs may be excellent models for research on IgE-mediated diseases in humans.     

Monocytes are required for optimum in vitro stimulation of bovine peripheral blood mononuclear cells by non-methylated CpG motifs

Bacterial DNA and synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs within certain flanking base pairs are recognized as a danger signal by the innate immune system of vertebrates. Using lymphocyte proliferative response (LPR) and IFN-gamma secretion assays, a panel of 38 ODN was screened for immunostimulatory activity on bovine peripheral blood mononuclear cells. ODN composed of a nuclease resistant phosphorothioate backbone and a leading 5'-TCGTCGTT-3' motif with two 5'-GTCGTT-3' motifs were highly stimulatory in both assays. Flow cytometric analysis and cell-specific surface marker labeling determined that B-cells (surface IgM(+)) were the primary cell population responding in the LPR assay. Depletion of T cells (CD3(+)) from the PBMC population did not affect IFN-gamma secretion or B-cell proliferation when cultured with CpG-ODN. However, depletion of monocytes (DH59B(+)) completely abrogated the ability of CpG-ODN to stimulate IFN-gamma secretion, and significantly reduced the B-cell proliferative response. These data establish the identity of an optimal immunostimulatory CpG motif for cattle and demonstrate that monocytes play a pivotal role in the ability of cell populations to respond to CpG-ODN. These data provide insight for future studies investigating the mechanism of CpG-ODN bioactivity and its application in novel vaccine formulations and immunotherapy.     

Innate immune responses induced by classes of CpG oligodeoxynucleotides in ovine lymph node and blood mononuclear cells

CpG ODN signal through Toll-like receptor 9 (TLR9) and trigger a cascade of events that lead to activation of innate and adaptive immune responses. Our current understanding of the immunobiology of host responses to CpG is based largely on studies on peripheral blood mononuclear cells (PBMC) and splenocytes. Little is known regarding CpG-induced responses in other lymphoid tissues. In the present study, we investigated responses induced by CpG in both PBMC and lymph nodes. Cells were isolated from the superficial cervical lymph node (LNC) and blood and then stimulated with CpG ODN (either A-, or B- or C-class ODN). Cytokine production was assayed by ELISA, and lymphocyte proliferation was determined by (3)H-thymidine incorporation. NK-like cytotoxicity was analyzed by lysis of (51)Cr-labelled target cells. All three classes of CpG induced IFNalpha and IFNgamma in LNC. In contrast, only A and C-class ODN induced IFNalpha and IFNgamma in PBMC. Moreover, the IFN levels in LNC were 20-40-fold higher than in PBMC. Furthermore, all classes of ODN induced higher IL-12 levels in LNC (five- to six-fold) than in PBMC. Both B and C-class ODN induced good proliferative responses in PBMC and LNC, but the A-class ODN did not induce proliferation of PBMC and only induced moderate proliferation of LNC. A-class ODN induced significant NK-like activity in LNC. Thus, all three classes of CpG ODN induced similar responses in LNC, and these responses were consistently higher than in PBMC. These observations indicate that CpG ODN-induced responses differ between blood and lymph nodes, and suggest that the functional classification of CpG ODN based on PBMC responses may not be directly applicable to cells from other immune tissues.     

Evaluation of cytokine messenger RNA expression in peripheral blood mononuclear cells from dogs with canine demodicosis

Using RT-PCR and semi-quantitative PCR, mRNA expression for canine interferon (IFN)-gamma, interleukin (IL)-2, IL-4, IL-5, IL-10, tumor necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta in peripheral blood mononuclear cells (PBMCs) was examined in dogs with or without demodicosis. mRNA expression for IFN-gamma as well as TNF-alpha in dogs with demodicosis (localized (LD) and generalized (GD)) was slightly lower than those in dogs without demodicosis (healthy controls). Expression of IL-5 mRNA in dogs with demodicosis was higher than that in control dogs, but there were no significant differences in IL-4 and IL-10 mRNA expression levels among the three groups. On the other hand, expression levels of TGF-beta mRNA in dogs with GD were higher than those in control dogs and dogs with LD. The expression levels of IL-5 and TGF-beta mRNA decreased in all three dogs with GD which showed resolution of the clinical signs. Taken together, these results suggest that the Th2-like response in PBMCs from dogs with demodicosis is up-regulated, and that subsequent increased expression of IL-5 and TGF-beta mRNA in dogs with GD is reversible after treatment. Therefore, these cytokines, particularly IL-5, might be a useful clinical index of the clinical course in demodicosis. Also, increased TGF-beta mRNA expression might be a key factor for revealing the difference in the mechanism of onset between LD and GD.     

Immunostimulatory effects of human recombinant interleukin-12 on peripheral blood mononuclear cells from normal dogs

Interleukin-12 (IL-12) plays a pivotal role in regulating cellular immune responses involving autoimmunity, infectious disease, and cancer. Human recombinant (hr) IL-12 is being evaluated for therapy of human cancer. We investigated the potential of hrIL-12 to activate canine peripheral blood mononuclear cells (PBMC) using proliferation and cytotoxicity as readouts. Human rIL-12 caused increased proliferation of PBMC, and enhanced lysis of allogeneic canine tumor targets mediated by PBMC from normal dogs in vitro. In addition, antibody-dependent cellular cytotoxicity (ADCC) mediated by canine PBMC was enhanced by hrIL-12. These results indicate that hrIL-12 is recognized by canine immune cells, triggering a number of immune responses in canine PBMC, that may be important for immunotherapy of canine cancer. Information from this investigation provides impetus for evaluation of the effects of hrIL-12 on PBMC from tumor-bearing dogs and should be helpful in the development of hrIL-12 as an immune cell activator in vivo in the dog.     

In vitro effects of lactoferrin on lipopolysaccharide-induced proliferation, gene expression, and prostanoid production by bovine peripheral blood mononuclear cells

Objective-To evaluate the effect of lactoferrin on lipopolysaccharide (LPS)-induced proliferation of bovine peripheral blood mononuclear cells (PBMCs), gene expression of inflammatory mediators, and production of prostanoids in vitro. Sample Population-PBMCs isolated from 15 Holstein bull calves. Procedures-Mixed populations of PBMCs were isolated by differential centrifugation. Proliferation assays were conducted in 96-well plates designed to allow addition of lactoferrin (200 ng/mL) with and without LPS (1 mug/mL) in a checkerboard design. Incorporation of (3)H-thymidine was used to determine proliferation of PBMCs. Prostaglandin E(2) production was determined in culture-conditioned medium by use of enzyme immunoassay. Effects of lactoferrin on LPS-induced gene expression of cyclooxygenase (COX)-2 and matrix metalloproteinase (MMP)-9 were monitored by use of PCR assays. Results-Lactoferrin supplementation significantly reduced LPS-induced incorporation of (3)H-thymidine and production of prostaglandin E(2) by PBMCs. Lactoferrin reduced LPS-induced expression of COX-2 and MMP-9 mRNA. Conclusions and Clinical Relevance-Lactoferrin reduced LPS-induced cellular proliferation, inflammatory mediator gene expression, and prostaglandin E(2) production by bovine PBMCs in vitro. These effects may be beneficial in reducing the impact of endotoxemia in neonates.     

In vitro stimulation of antibody production to Mycoplasma hyopneumoniae by porcine peripheral blood mononuclear cells.

A method to stimulate and detect the in vitro production of antibodies to Mycoplasma hyopneumoniae by porcine peripheral blood mononuclear cells (PBMC) was established. PBMC were cultured in microtiter plates coated with a sonicated M. hyopneumoniae whole cell antigen and the amount of antibody bound to the coating antigen was determined by an enzyme linked immunosorbent assay (ELISA). In addition, the amount of non-bound antibody was determined by testing the culture supernatants in the ELISA which detects porcine antibodies to M. hyopneumoniae. The production of antibodies, in terms of total absorbance values, was enhanced by including 2.5 ng pokeweed mitogen (PWM) per ml growth medium without altering the specificity of the assay. In a pilot experiment, the applicability of the method to follow the development of antigen-reactive cells during primary and secondary immunizations with M. hyopneumoniae was evaluated. Antigen-reactive cells, identified by their ability to produce antibodies to M. hyopneumoniae in vitro, were detected seven days after the primary immunization and reached their highest antigen reactivity one week later. In comparison, antigen-reactive cells could be detected three days after the booster immunization and remained in the circulation for 2 weeks.     

In vitro antimicrobial efficacy of β‐defensin 3 against Staphylococcus pseudintermedius isolates from healthy and atopic canine skin

β‐Defensins (BDs) are highly conserved antimicrobial peptides important in innate defence against bacteria. β‐Defensin 3 has a specific role in protecting the skin. This study quantified the minimal inhibitory concentration (MIC) of human (h)BD3 against Staphylococcus pseudintermedius isolates from atopic and healthy dogs. Single colony isolates (1 × 10⁵ colony‐forming units/mL log phase) were cultured with doubling dilutions of hBD3 in sodium phosphate buffer from 0.8 to 50 μg/mL at 37 °C for 2 h, before adding 100 μL of tryptone soy broth and incubating for a further 20 h. Bacterial growth was assessed as the mean optical density at 540 nm corrected for background. The median MIC was 12.5 μg hBD3/mL (range 3.125–25 μg/mL; n = 22). Forty‐five percent of the isolates were inhibited at ≤6.25 μg hBD3/mL, and 90% were inhibited at ≤12.5 μg hBD3/mL. Bacterial growth was not inhibited at ≤1.6 μg hBD3/mL. There were no significant differences in the inhibition by hBD3 of isolates from atopic (median MIC 12.5 μg/mL, range 6.25–25 μg/mL, n = 14) and healthy dogs (median MIC 9.4 μg/mL, range 3.125–12.5 μg/mL, n = 8); from noninfected colonized sites (median MIC 12.5 μg/mL, range 3.125–25 μg/mL, n = 16) and infected lesions (median MIC 9.4 μg/mL, range 6.25–12.5 μg/mL, n = 6); or between sample sites (nose median MIC 12.5 μg/mL, range 6.25–25 μg/mL, n = 5; perineum median MIC 12.5 μg/mL, range 3.125–25 μg/mL, n = 7; ear median MIC 6.25 μg/mL, range 6.25–12.5 μg/mL, n = 4; lesions median MIC 9.4 μg/mL, range 6.25–12.5 μg/mL, n = 6). In conclusion, hBD3 inhibited the growth of canine S. pseudintermedius isolates in vitro irrespective of origin.     

In vitro production of specific antibody by equine peripheral blood mononuclear cells using tetanus toxoid as a recall antigen.

Anti-tetanus toxoid (TT) antibody (Ig) levels in the supernatant of cultured, pre-immunised equine peripheral blood mononuclear cells (PBMC) were measured by an indirect enzyme-linked immunoabsorbent assay (ELISA). Optimal anti-TT Ig production occurred at concentrations of stimulating, purified TT of between 0.001 and 0.1 micrograms ml-1, which varied depending on the cell concentration. Optimal anti-TT Ig production was most consistently produced when the cell concentration was 5 x 10(6) ml-1. At this cell concentration maximal anti-TT Ig was induced using 0.1 micrograms ml-1 TT. At a cell concentration of 5 x 10(6) ml-1 and a TT concentration of 0.1 micrograms ml-1 anti-TT Ig was first detectable in supernatant on day 5 of stimulation. Maximal levels of anti-TT Ig were present in the supernatant by day 10. No anti-TT Ig was produced in cultures of PBMC from non-immune animals.     

In vitro effects of 2-methoxyestradiol on canine tumour cells

The in vitro antiproliferative, apoptotic and cell‐cycle effects of 2‐methoxyestradiol (2ME₂), an endogenous oestrogen metabolite, were investigated using a variety of canine tumour cell lines. The cells were cultured under standard conditions and incubated with varying concentrations of 2ME₂. Inhibition of tumour cell proliferation was evaluated using a tetrazolium‐based colorimetric assay. DNA content analysis was performed using propidium iodide staining and flow cytometry. Cytologic analysis with Leukostat staining solution and Hoechst 33342 staining and Annexin V‐fluorescein isothiocyanate (FITC) fluorescence were used to quantify cell‐cycle distribution and apoptosis induction. Tumour cell proliferation was inhibited by 50% at concentrations of 2ME₂ ranging from 0.88 to 7.67 µM, depending on the cell line tested. Profound G₂/M phase arrest, an increase in binucleate cells and induction of apoptosis were observed in all cell lines tested, in a dose‐dependent manner. Based on these results, this compound has potential as an agent for the treatment of canine cancer and warrants further investigation. The canine lymphoma cell line, 1771, was inhibited at concentrations that may be achievable in vivo.     

In vitro production of antibodies to bovine virus diarrhoea virus by peripheral blood mononuclear cells from cattle immunized by infection

A method for in vitro production of antibodies to bovine virus diarrhoea virus (BVDV) by peripheral blood mononuclear cells (PBMC) was developed. The PBMC were cultured in microtitre plates coated with detergent-solubilized BVDV and the supernatants were tested in an enzyme-linked immunosorbent assay which detects IgG antibodies to BVDV. Following incubation of PBMC with an optimal concentration of pokeweed mitogen for 5 days, antibodies to BVDV were detected in culture supernatants of PBMC from immune cattle, but not in supernatants of PBMC from seronegative cattle, from persistently BVDV-infected cattle or from a 5-day-old calf that received BVDV antibodies via colostrum. This antibody-production assay may therefore be used as a tool to discriminate between passively acquired antibodies and those actively induced.     

Recombinant single-chain canine interleukin 12 induces interferon gamma mRNA expression in peripheral blood mononuclear cells of dogs with visceral leishmaniasis

Canine visceral leishmaniasis poses important concerns for public health and veterinary medicine in many areas of the world. Resistance to it seems to be associated with cellular specific immune responses of the so-called Th1 type. Interleukin-12 (IL-12) is one of the most potent inducers of Th1 type of immune responses to co-administered antigens. Herein, the cloning of canine IL-12, as a single-chain fusion protein (sccaIL-12), and its expression in biologically active form in COS-7 cells is reported. Supernatants from these cells stimulated the expression of comparable amounts of interferon gamma mRNA in peripheral blood mononuclear cells from dogs with natural visceral leishmaniasis. In addition, after stimulation with sccaIL-12, there was no difference between interferon gamma mRNA expressions in peripheral blood mononuclear cells of dogs with visceral leishmaniasis and from normal healthy control animals.     

In vitro IgE but not IgG production of canine peripheral blood B cells is inhibited by CD40 ligation

The aim of this study was to investigate in vitro IgE induction in peripheral canine B cells. CD21(+) B cells were purified from the peripheral blood of beagle dogs by positive selection via magnetic separation to a purity of >/=95%. Subsequently, proliferation, and IgG and IgE production of canine B cells were investigated after stimulation with human recombinant Interleukin-4 (hrIL-4) and human recombinant Interleukin-2 (hrIL-2) in the presence or absence of CD40L-CD8 fusion protein (CD40L) of mouse origin. We could demonstrate that canine B cells react on hrIL-2 alone by proliferation and IgG production but not by IgE secretion, whereas activation with hrIL-4 induced proliferation and mainly IgE production. Together, both cytokines synergistically increased B cell proliferation as well as IgG and IgE production. We could also show that mouse CD40L induces proliferation of dog B cells, which is further enhanced by addition of hrIL-4. Unexpectedly, CD40L led to a dramatic decrease in the IL-4 mediated IgE secretion (82% inhibition on an average). In contrast, IgG production was not affected significantly by CD40L. The same effects of CD40L were observed when B cells were stimulated by a combination of IL-2 and IL-4 and this inhibition could not be abrogated by increasing the amounts of IL-4. In summary, activation of canine B cells from peripheral blood by hrIL-4 in the presence or absence of hrIL-2 led to marked IgE production that is strongly and in a dose-dependent manner inhibited by CD40L. Stimulation of IgG production is not influenced by CD40L.     

In vitro activation of bovine macrophage and peripheral blood mononuclear cells by Nocardia rubra cell wall skeleton (N-CWS)

Nocardia rubra cell wall skeleton (N-CWS) was used for immunotherapy to bovine leukemia virus (BLV)-positive cattle with enlarged subcutaneous lymphatic nodules. Electron microscopical observations showed the infiltration of macrophage and T cells around N-CWS treated lesions. Antitumor effect induced by N-CWS was examined in vitro. The maximum cytolytic activity of macrophage was observed, when the cells were incubated with 10 micrograms/ml of N-CWS. Chemiluminescence response of peripheral blood mononuclear cells (PBMC) using N-CWS as stimulant was observed with a high level of activity for a long period, 5 hr. Mitogenic effect of N-CWS to PBMC was observed in the presence of macrophages but not without macrophages. Furthermore, interleukin 2 activity was recognized in supernatant of PBMC cultured with N-CWS. Maximum cytotoxic T lymphocyte response was induced when PBMC were cultured with 0.1 micrograms/ml of N-CWS.