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.     

Production of interleukin 2 and expression of interleukin 2 receptors by pony peripheral blood lymphocytes after stimulation with a soluble fraction of Trypanosoma evansi

Pony peripheral blood lymphocytes (PBL) were stimulated with a soluble fraction of Trypanosoma (T.) evansi (SF). As determined by 3H-thymidine incorporation, the cells underwent a proliferative response and were able to: a) produce a factor having the biological activities of interleukin 2 (IL-2) since their supernatants could support the in vitro growth of pony PBL stimulated with concanavalin A (Con A-blasts); b) undergo a further proliferative response when incubated in short term cultures with SF, human recombinant IL-2 (hrIL-2), or both c) bind specifically radiolabelled hrIL-2 (125I-hrIL-2). The date described here indicate that a soluble fraction of T. evansi stimulated pony PBL which subsequently produced IL-2 and expressed IL-2 receptors (IL-2R).     

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.     

Interleukin 1- and 2-like activities in the dog

Canine adherent and non-adherent peripheral blood leukocytes and spleen cells were examined for their ability to produce soluble factors with Interleukin 1- and 2- (IL-1 and IL-2) like activities. For this purpose, three conventional assay systems were used: (a) proliferation on an IL-2-dependent murine cytotoxic T-lymphocyte cell line, (b) enhancement of PHA-induced murine thymocyte proliferation and (c) proliferation of lectin-primed canine peripheral blood lymphocytes (PBL). Only the latter two types of cells respond to IL-1, whereas all three types respond to IL-2. Both types of factors were produced and the kinetics of their release/production were found to be identical to those of human PBL. Results suggested that species-related differences existed. Canine interleukin-containing supernatants had higher titers than murine interleukin-containing supernatants when analyzed on canine lymphocytes, and the reverse was found if murine target cells were used.     

Cloning,expression and biological activity of equine interleukin (IL)-5

The cytokine, interleukin (IL)-5 stimulates eosinophil differentiation, activation and survival and can prime these cells, increasing the response to other mediators. In view of its many effects on eosinophils, IL-5 has been implicated in the pathogenesis of allergic disease in man. Here we report the cloning of equine IL-5 and expression of the recombinant protein by transfection of Chinese hamster ovary (CHO) cells. The cloned cDNA sequence consisted of 405 nucleotides and encoded a protein of 135 amino acids. There is >85% identity with feline, bovine, ovine, canine, and human IL-5 sequences at the nucleotide and protein level. Supernatants containing equine IL-5 were also examined for biological activity. CHO supernatant containing equine recombinant (eqr) IL-5, like the human ortholog (hrIL-5), induced concentration dependent equine eosinophil adherence to autologous serum-coated plastic (9.7+/-1.5% with a 1:100 dilution of eqrIL-5 and 9.1+/-1.6% adherence with 1 nM hrIL-5; n = 4). The eqr protein also caused concentration dependent superoxide production (11.9+/-2.4 nmol (reduced cytochrome (cyt) C)/10(6) cells at a 1:50 dilution, n = 4). In contrast, hrIL-5 only caused significant superoxide production when diluted in conditioned CHO medium, an effect that was inhibited by the anti-human mAb, TRFK5 (4.4+/-0.3 versus 0.3+/-0.4 nmol/10(6) cells for 0.5 nM hrIL-5 in the presence of the isotype matched IgG1 control (10 microM) and TRFK5 (10 microM), respectively). TRFK5 also significantly inhibited hrIL-5 induced adherence at concentrations of 0.3 microg/ml and above but had no significant inhibitory effect on either superoxide or adherence caused by eqrIL-5. These results demonstrate that equine IL-5 expressed by CHO cells stimulates equine eosinophils, suggesting that this cytokine could play a role in eosinophil recruitment and activation in equine allergic disease. The anti-human and murine moAb TRFK5 does not appear to recognise the equine protein.     

Cytotoxicity against autologous, allogeneic, and xenogeneic tumor targets by human recombinant interleukin-2-activated lymphocytes from healthy dogs and dogs with lung tumors

Before dogs with lung tumors were treated by adoptive immunotherapy, the ability of canine blood lymphocytes (PBL) from the peripheral circulation to differentiate in vitro in the presence of human recombinant interleukin-2 (rIL-2) and become tumoricidal was investigated. The PBL from healthy dogs (n = 6) and dogs with lung tumors (n = 5) were grown in culture medium alone, in the presence of rIL-2 to generate lymphokine-activated killer (LAK) cells, or with phytohemagglutinin (PHA) and rIL-2 to generate autologous-stimulated lymphocytes (ASL). After 4 days, cytotoxicity by the ASL, LAK, and PBL was determined in a 4-hour 51chromium-release assay. Target cells in the assay were short-term cultured enzyme digests of autologous (self), allogeneic (genetically different) primary tumors, and Raji, the xenogeneic human lymphoma cell line. The PBL cultured without rIL-2 were not cytotoxic against any tumor. However, when a dog's PBL were activated in vitro, they killed the dog's own tumor, ASL more effectively than LAK cells. Pulmonary adenocarcinomas and an osteosarcoma metastasis to lung were among the autologous tumors assayed. Against an allogeneic canine osteosarcoma, ASL generated from healthy dogs were significantly more cytolytic than LAK from healthy dogs, or than ASL generated from tumor-bearing dogs. Cytotoxicity was greater against allogeneic tumor than against Raji. Lectin-dependent cellular cytotoxicity, tested by including PHA in the assay medium with lymphocytes and Raji cells, by ASL and LAK was greater than cytotoxicity of Raji without PHA. Because ASL were more cytolytic than LAK against all targets in vitro, they may be more beneficial than LAK for immunotherapy of canine tumors.     

Detection of canine interleukin-2 receptors by flow cytometry.

This study describes a method for detecting canine interleukin-2 receptors (IL-2R) by flow cytometry, using human recombinant IL-2 labeled with phycoerythrin (IL-2-PE). Peripheral blood mononuclear cells from four normal dogs were washed, incubated with IL-2-PE, and then washed to remove any unbound IL-2-PE. Flow cytometric analysis of the cells was performed with a 488 nm argon laser while gating on lymphocytes. Cells expressing the IL-2R were identified by their fluorescence as compared to cells stained with an anti-mouse immunoglobulin-G conjugated to phycoerythrin. The average percentage of resting cells expressing the IL-2R was found to be 21%. The addition of unlabeled human recombinant IL-2 to Day 3 phytohemagglutinin (PHA)-stimulated cells reduced the fluorescence intensity four-fold, thereby demonstrating the specificity of IL-2-PE for canine IL-2R. Following stimulation with optimal concentrations of PHA, the percentage of cells expressing the IL-2R increased daily and reached a maximum on Day 3 (76.4%). IL-2R density, as measured by mean fluorescence intensity, also increased and reached maximal levels on Days 2-3 (twenty-fold greater than resting cells). The binding, inhibition, and kinetic experiments provide evidence that human recombinant IL-2-PE is a useful tool for studying canine IL-2R expression. Thus, a one-step direct method for the flow cytometric detection and quantification of the canine IL-2R is now available.     

Molecular cloning, chromosomal location, and biological activity of porcine interleukin-21

A pig interleukin-21 (IL-21) cDNA was successfully cloned and sequenced from porcine peripheral blood lymphocytes (PBL) stimulated with 10 microg/ml concanavalin A (ConA), 10 microg/ml phytohemagglutinin P (PHA), 50 ng/ml phorbol 12-myristate 13-acetate (PMA), and 0.5 microg/ml anti-porcine CD3 antibody for 48 hr. The open reading frame of the porcine IL-21 cDNA is 459 base pairs in length and encodes 152 amino acids. The predicted amino acid sequence of the porcine IL-21 shows 86.2%, 77.7%, and 58.4% identity to the bovine, human, and murine IL-21, respectively. The porcine IL-21 gene was mapped to porcine chromosome 8 (8q22-->q23) by means of fluorescence in situ hybridization and radiation hybrid mapping, where the porcine IL-2 gene had been mapped nearby. The recombinant porcine mature IL-21 expressed by E. coli induced dose-dependent proliferation and IFN-gamma production from a human NK cell line, NK0. The porcine IL-21 identified in this study will be helpful for the enhancement of innate immune responses of pigs.     

Cloning and expression of bovine and porcine interleukin-2 in baculovirus and analysis of species cross-reactivity

The cDNAs encoding bovine and porcine interleukin-2 (IL-2) have been expressed using the baculovirus Autographa californica nuclear polyhedrosis virus as a vector in insect cells. Insect cells infected with recombinant viruses secreted bovine and porcine IL-2 into the culture medium, with biological activities for maintaining the proliferation of homologous cells. When the activities of these two IL-2 proteins and commercially available human IL-2 were tested on heterologous cells differences were found. Recombinant bovine (rb)IL-2 only supported the growth of bovine lymphocytes and was not active on human, mouse or porcine lymphocytes. Recombinant porcine (rp)IL-2 and recombinant human (rh)IL-2 supported the proliferation of human, bovine, porcine and murine cells. However, the proliferative response of human lymphocytes to rpIL-2 was only 50% of that seen with rhIL-2. Sequence differences at the predicted p55 and p75 contact binding sites may explain this.     

Development and functional characterization of T cell lines from canine Peyer's patches

We have propagated concanavalin A-stimulated cells from canine Peyer's patches in vitro in the presence of interleukin-2 (IL-2). The cells were characterized as T cells by determination of their phenotype and by functional assays. They are IL-2 dependent and respond to IL-2 of murine, primate and canine origin. The long-term cultured cells provided help for immunoglobulin production by purified autologous B cells and suppressed IgG production by nonseparated autologous peripheral blood mononuclear cells.     

A novel culture method of canine peripheral blood lymphocytes with concanavalin a and recombinant human interleukin-2 for adoptive immunotherapy

This study was designed to develop a novel culture method for the efficient proliferation of canine peripheral blood lymphocytes (cPBL) for adoptive immunotherapy. When cPBL were cultured in the presence of concanavalin A (Con A), proliferation of cPBL was induced and expression of interleukin-2 receptor (IL-2R) which enables to respond to exogenously added IL-2 was upregulated. And then, when cPBL were cultured with recombinant human interleukin-2 (rhIL-2) in addition to Con A, proliferation was accelerated and increased to about 10-fold after 1 week. The phenotypic analysis showed that the main population of the cultured cPBL was consisted of CD8+ positive lymphocytes. Among them, CD4+CD8+ double positive (DP) lymphocytes had significantly increased, and the ratio of CD4+ single positive (SP) lymphocytes to CD8+ SP lymphocytes (CD4+SP/CD8+SP) was decreased as compared to before culturing. To evaluate the cytotoxic activity of cPBL cultured with Con A and rhIL-2, furthermore, cytotoxic assay was carried out against xenogeneic melanoma cell line (MeWo), which resulted in MHC-unrestricted cytokilling. These results suggest that the culture method of cPBL by the use of Con A and rhIL-2 may be useful for generating lymphokine activated killer cells, and also this may be beneficial for adoptive immunotherapy of tumor-bearing dogs.     

Induction of IL-2 and lymphokine activated killer cells in the cat

We have described the use of a cloned murine IL-2-dependent T-cell line to directly measure feline IL-2. Concanavalin A stimulated feline peripheral blood lymphocytes produced an IL-2-rich supernatant that supported the growth of this murine IL-2-dependent T-cell line. In addition to producing IL-2, Con A stimulated killer cells in PBL were cytotoxic for the FeLV transformed tumor cell line FL74. Incubating feline PBL with a cocktail of the calcium ionophore A23187 and phorbol ester also led to the generation of cytotoxic cells as well as the production of high levels of IL-2. Finally, IL-2-rich supernatant was able to stimulate cytotoxic activity in PBL from normal cats.     

IgE enhances Fc epsilon RI expression and IgE-dependent TNF-alpha release from canine skin mast cells

The role of IgE on mast cell (MC) activation is well known. Recent studies have demonstrated that IgE also has the ability to up-regulate the high affinity IgE receptor (Fc epsilon RI) on the surface of human and murine MC, leading to an increased production of cytokines and chemokines. In the present study, we have examined the influence of IgE levels on Fc epsilon RI expression, and its consequences on TNF-alpha production from canine skin MC. Mature MC were enzymatically dispersed from the skin biopsies of 6-8 dogs and were cultured for up to 5 days in medium supplemented with recombinant canine stem cell factor (SCF) (6 ng/ml), in the presence of increasing serum IgE concentrations (ranging from 0 to 80 microg/ml). Subsequently, skin MC were activated with anti-IgE, and TNF-alpha concentration was assessed 5h post-activation by a cytotoxic bioassay. Fc epsilon RI receptors were identified in MC surface by flow cytometry. MC cultured for up to 5 days in the presence of high serum IgE concentration (8 microg/ml) produced twice the quantity of TNF-alpha than MC cultured in the absence of serum IgE, in response to stimulation with anti-IgE. Moreover, the percentage of Fc epsilon RI-positive skin cells was found to be approximately double in cells cultured with serum IgE compared to that cultured in the absence of IgE, following saturation of IgE receptors. These results suggest that, as found in human and murine MC, IgE may induce an up-regulation of the Fc epsilon RI density and an enhancement in the secretory activity of canine skin MC. This study could be of great interest in designing new therapeutic strategies for controlling MC activation in inflammatory and allergic processes.     

Bulk cultures of canine peripheral blood lymphocytes with solid phase anti-CD3 antibody and recombinant interleukin-2 for use in immunotherapy

Interleukin (IL)-2 can induce large numbers of lymphokine-activated killer cells in peripheral blood lymphocytes (PBL), but IL-2 alone cannot induce proliferation of a large number of canine (c) PBL. We used the solid phase anti-CD3 antibody and soluble recombinant (r) IL-2 in order to establish a large scale culture method for cPBL. The number of lymphocytes seeded (3 x 10 (7)) increased to 1 x 10(9) after incubation for 10 days. The phenotype of cultured cPBL cells (after 2 weeks) showed a CD4(+) or CD8(+) predominant cell population. The cultured cell solutions were administered with physiological saline intravenously to each dog. After transfusion of the cultured cells, the cPBL counts, especially the number of CD4(+), CD8(+) and CD4(-)CD8 (-)(DN) cells increased significantly in the recipient dogs. Natural killer (NK) cells, gammadeltaT cells and B cells were considered to be present in the DN cell population. The NK cells and gammadeltaT cells showed no adverse reaction to the transfusion of the activated cPBL. Therefore, it is necessary to recognize the B cells present in the DN cell population by detecting CD21(+) cells. In conclusion, the bulk culture system of cPBL with rIL-2 and solid phase anti-CD3 antibody may be useful for the development of novel immunotherapy in dogs.     

Measurement of feline serum interleukin-5 level

A bioassay was developed to measure feline interleukin-5 (IL-5). Human IL-5 receptor alpha chain transfected murine Ba/F3 cells (Ba/F3-IL-5R) showed feline IL-5-dependent proliferation in a dose-dependent manner. IL-5 levels in serum samples from 54 cats with suspected allergic dermatitis and from 11 control cats could be successfully measured using Ba/F3-IL-5R cells. The number of eosinophils in peripheral blood was not correlated with serum IL-5 level.     

A functional comparison of canine and murine bone marrow derived cultured mast cells

Disorders involving mast cells are extremely common in dogs, ranging from allergic diseases to neoplastic transformation resulting in malignant mast cell tumors. Relatively little is known regarding the basic biologic properties of normal canine mast cells, largely due to the difficulty in reliably purifying large numbers from canine skin. In vitro generated bone marrow derived cultured mast cells (BMCMCs) are routinely used in both human and murine studies as a ready source of material for in vitro and in vivo studies. We previously developed a technique to generate canine BMCMCs from bone marrow derived CD34+ cells and demonstrated that these cells exhibit the phenotypic properties characteristic of mast cells and release histamine in response to IgE cross-linking. The purpose of the following study was to characterize the functional properties of these canine BMCMCs and contrast these with the functional properties of murine BMCMCs. Our work demonstrates that both IL-4 and IL-10 promote canine BMCMC proliferation, possibly through upregulation of Kit expression, while TGFbeta inhibits proliferation. The canine BMCMCs produce a variety of cytokines and chemokines in response to IgE cross-linking and chemical stimulation including IL-3, IL-4, IL-13, GM-CSF, RANTES, and MIP1alpha. Interestingly, the canine BMCMCs released significantly larger amounts of MCP-1 and tryptase and significantly smaller amounts of IL-6 following chemical stimulation and IgE cross-linking when compared to murine BMCMCs. Lastly, the canine BMCMCs produced larger amounts of active MMP9 than their murine counterparts. In summary, canine BMCMCs exhibit unique functional properties that distinguish them from murine BMCMCs and provide insight into the contribution of these cells to mast cell disorders in the dog.     

Evaluation of serum interleukin 2 receptor and beta-2-microglobulin as prognostic factors for canine lymphoma: A pilot study

Interleukin 2 receptor (IL-2R) is released from activated T cell lymphocytes and related to proliferation of B cells and T cells. Beta-2-microglobulin (B2M) is synthesized from all nucleated cells and constitutes a major histocompatibility complex class I antigen. In human medicine, high concentrations of these two factors have been found to be related to prognosis in aggressive non-Hodgkin's lymphoma. In this pilot study, we aimed to assess the correlation between the serum concentration of IL-2R and B2M and the diagnosis and prognosis of canine lymphoma. This study included 8 healthy dogs and 17 dogs with lymphoma. To measure the serum concentration of IL-2R and B2M, a commercial enzyme-linked immunosorbent assay was used. In dogs with lymphoma, IL-2R concentrations were significantly high at the time of diagnosis, but B2M concentrations were not. In relapsed dogs, both IL-2R and B2M concentrations were significantly higher than those in the control and chemotherapy response groups. When the serum concentrations of IL-2R and B2M during chemotherapy were monitored in four relapsed dogs, B2M levels were more closely related with relapse. This study demonstrated that serum IL-2R and B2M concentration can be a diagnostic or prognostic tool for canine lymphoma. Monitoring of serum B2M concentration seems to be useful for predicting relapse.     

Monoclonal antibodies against chicken interleukin-6

Monoclonal antibodies (mAb) were produced against a recombinant (r) chicken interleukin-6 (IL-6). Eight mAbs produced were tested for isotype; ability to inhibit recombinant forms of chicken (ch), human (h) and murine (m) IL-6; and recognition of rchIL-6 by Western immunoblotting. The mAb isotypes were represented by IgG1 (one), IgG2a (six) and IgG2b (one). In a mouse B9 hybridoma cell bioassay with rmIL-6, four mAbs effectively inhibited activity of rmIL-6. Further bioassays with the four mAbs at varying concentrations showed that two of these mAbs (1.20.7 and 1.26.4) were quite effective at inhibiting rmIL-6. Recombinant forms of ch, h and mIL-6 were all tested in a bioassay with the most potent inhibiting mAb (1.26.4), and this mAb was effective in inhibiting all three recombinant IL-6 proteins. Western immunoblotting revealed identification of the original IL-6 immunogen used for mAb production. Based upon inhibition of IL-6 activity in a standard bioassay and IL-6 recognition by Western immunoblotting, mAb 1.26.4 was judged the most useful antibody for future studies and applications.     

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.