Dexamethasone depresses the expression of L-selectin but not the in vivo migration of bovine neutrophils into the uterus.

There are several indications that periparturient depression of functional properties of polymorphonuclear neutrophilic granulocytes (PMN) may be of great importance for the pathogenesis of genital infections after calving. As the periparturient period is characterized by high plasma levels of corticosteroids, the hypothesis to be tested was that high concentrations of glucocorticoids during the periparturient period suppress uterine PMN in number and functionality. An in vivo endometritis model was applied to examine uterine PMN. Recombinant human interleukin-8 (rhIL-8) was infused into the uterus of estrous cows and heifers 24 h after pretreatment with dexamethasone (0.07 mg/kg i.m.). Six hours after rhIL-8 infusion high numbers of uterine PMN were isolated and characterized as to their immunophenotype and function. Animals treated with dexamethasone animals showed leucocytosis due to neutrophilia in peripheral blood. Despite a downregulation of expressed L-selectin, cattle treated with dexamethasone showed more uterine PMN than those treated with placebo. Dexamethasone decreased plasma concentrations of the immunomodulatory steroidal hormones cortisol and estrogen. Dexamethasone directly reduced the generation of reactive oxygen species (ROS) by uterine PMN. This may be a useful mechanism since it would protect the endometrium from tissue damage by excessive extracellular ROS. However, it is not known if the net effect is in fact a reduction in ROS, as the number of uterine cells increases. Our study shows that glucocorticoids may not be considered immunosuppressive in all cases and may play an important role in the regulation of post partum uterine defense mechanisms.

Induction of Epstein-Barr virus-associated nuclear antigen during in vitro transformation of human lymphoid cells.

Human lymphoid cells isolated from the peripheral blood of adults, from cord blood, and from fetal liver, spleen, bone marrow, and thymus were cultivated with or without a cell-free preparation of Epstein-Barr virus (EBV) with demonstrated transforming activity. The cultures were examined for the EBV-associated nuclear antigen (EBNA) and for transfromation into permanent lymphoblastoid cell lines (LCL). EBNA, seen only in cultures that had received exogenous EBV, was detected between days 1 and 6 after addition of EBV, most frequently on day 3. EBNA-positive cells had a lymphoblastoid appearance. Transformation into established LCL became apparent between days 12 and 19. The addition of pokeweed mitogen to cultures containing EBV enhanced the development of EBNA, whereas phytohemagglutinin or concanavalin A had no such effect. Neither EBNA nor transfomration was observed in lymphoid cells from fetal thymus. In fetal spleen, bone marrow, and liver cells, EBV regularly induced EBNA and LCL transformation.

Single and dual labelling of cytotoxic target cells. Comparison of three radioactive tracers, [35S]methionine, [75Se]selenomethionine, and chromium-51.

[75Se]selenomethionine (75SeM) has been shown to provide several advantages over Na(2)51CrO4 (51Cr) labelling of metabolizing target cells: high labelling efficiency and low spontaneous release of 75SeM-labelled target cells permit improved monitoring of cytotoxicity due to extended effector/target ratios in short- and long-term assays. Unfortunately, 75SeM will soon be difficult to obtain. Therefore we studied the suitability of [35S]methionine (35SM) as a substitute for 75SeM. Furthermore, we explored the potential of dual labelling of suspension target cells applying combinations of 35SM and 51Cr or 75SeM and 51Cr. 35SM is a suitable substitute for 75SeM retaining most of the advantages of 75SeM labelling. Although considerably higher labelling of cells is possible we obtained the most efficient labelling with 100-400 kBq/ml of 35SM or 75SeM resulting in a relatively high uptake (3-15 cpm/cell) and very low spontaneous release (1-2%/h) up to 24 h. This permits short- and long-term cytotoxic assays and the use of low numbers of target cells (1 x 10(3)) providing increased cytotoxic sensitivity with reduced amounts of effector cells. Suitable dual labelling of target cells with 35SM plus 51Cr or 75SeM plus 51Cr documented convincingly identical release kinetics for 35SM and 75SeM but partially discordant ones for 51Cr. Depending on the target cell used dual labelling permits discrimination and monitoring of different cytotoxic or release mechanisms in cellular cytotoxicity.

Insolubilization of protein antigens on polyacrylic plastic beads using poly-L-lysine.

A new model system for quantitation of immunofluorescence on single cells is described using poly-L-lysine (PLL) coated polyacrylic plastic beads of approximately cell sizes as carriers for protein antigen. By increasing PLL concentration on the beads increased amounts of 125I labeled antigen were attached to the particles. Excess binding sites of PLL could be completely blocked by unrelated proteins. After staining with FITC-conjugated antibodies and quantitative fluorescence measurements of individual beads using a microspectrofluorimeter, strong correlations were found between antibody and antigen concentration on the beads. Neither repeated washings with PBS nor storage of the beads for two months caused detectable shedding of antigen-antibody complexes. There was a strong linear correlation between fluorescence intensity and the volume of beads, but the correlation between surface area and fluorescence was nonlinear. The described procedure was shown to be a simple method for quantitative and stable coating of particles with proteins. It can be applied as a useful model system for quantitative immunofluorescence studies on intracellular antigens.

Biotinylation of cell surface MHC molecules: a complementary tool for the study of MHC class II polymorphism in cattle.

Biotinylation of cell surface proteins is often used as an alternative to radioactive labelling procedures, but very little is known about the labelling of the different allelic products of polymorphic antigenic systems. In this report, we demonstrate that NHS-LC-biotin labels bovine MHC class II molecules with different efficiencies for several allelic forms of this polymorphic system compared to conventional metabolic labelling with [35S]methionine. This was shown after immunoprecipitation and one-dimensional isoelectric focusing (1D-IEF). The avidity of the monoclonal antibody Bo139 (alpha-bovine MHC class II) was not affected after in situ biotinylated of bovine PBMC, as revealed by flow cytometric analyses, immunoblotting after SDS-PAGE and immunoprecipitation. The biotinylation did not affect the apparent isoelectric points of polymorphic bovine MHC class II beta chains. This was demonstrated by double labelling of cells with [35S]methionine and subsequent biotinylation and comparison of the banding pattern after immunoprecipitation and 1D-IEF. 1D-IEF of 11 unrelated animals resulted in the demonstration of 29 polymorphic bands of which eight were detected by both labelling procedures, six only after biotinylation and 15 only after metabolic labelling with [35S]methionine. Hence, biotinylation alone cannot serve as an alternative for radioactive labelling of bovine MHC class II molecules but can reveal expressed allelic forms not detectable by metabolic labelling with [35S]methionine.

Analysis of cell-surface markers with staphylococcal protein A: fluorescence studies on mammalian lymphoid determinants.

Membrane antigens including different classes of immunoglobulins, transplantation antigens, beta2-microglobulin, T lymphocyte specific antigens, and virally determined surface components were investigated using fluorescein-labeled Staphylococcal protein A in combination with cytofluorometric studies. Lymphocytes of seven species: mouse, rat, guinea pig, pig, cow, monkey, and human, and of ten human lymphoma-derived lines were tested. Analysis of the differential expression of surface markers revealed a reproducible reaction of protein A with cell-surface Fc of IgG actively produced by lymphoid cells from human, monkey, guinea pig, and pig, and with passively attached IgG molecules in the form of antibodies, directed against cell surface antigens of all lymphoid cells tested. No surface Ig was detected on so-called T lymphocytes. The distribution of cell-bound Ig density among surface Ig-positive cells was found to be different depending upon the origin of the cells with regard to lymphoid organ; it was parallel among the lymphoma lines tested and on peripheral blood cells from human, monkey, and pig, although large variations in fluorescence intensity among individual cells and among the different lines were recorded. Beta2-microglobulin determinants were found equally well on enriched human T and B cells. Transplantation, and T lymphocyte-specific antigens were detected on the majority of the lymphoid cells and on a restricted population respectively.

There is no regulatory role for induced nitric oxide in the regulation of the in vitro proliferative response of bovine mononuclear cells to mitogens, alloantigens or superantigens.

Nitric oxide (NO) is a potent cellular mediator which has been shown to modulate several immune mechanisms. Between species, however, there are considerable differences regarding the signals required for induction of NO as well as the kind of cells capable of producing NO. The object of this study was to determine the kinetics of NO production of bovine blood mononuclear cells (boMNC) stimulated in vitro and to investigate whether it modulates their proliferative response following allogeneic (mixed leukocyte cultures, aMLC), mitogenic (PWM, Con A) or superantigenic (SEA, SEB) stimulation. NO production was indirectly determined with the Griess reagent measuring nitrite (NO2-). Significant but low amounts of NO could be detected as early as day 3 after in vitro stimulation and did noly slightly increase during the 6-8 day culture period. Superantigens (SEA, SEB) and aMLCs (4.3-5.2 microM NO2-) induced a significantly higher nitrite accumulation compared to Con A (2.6 microM NO2-). Generation of nitrite, most likely produced by monocytes/macrophages, could be inhibited by 1 mM N-monomethyl-L-arginine (NMLA). Flow cytometric characterization of various cellular responses revealed no differences between cultures with or without NMLA. This included the determination of blastogenesis, absolute numbers of viable cells, expression density of activation markers (MHC class II, IL-2R alpha) and cellular subpopulations (CD4+, CD8+, sIg+) among blasts. In addition, exogenously provided NO via SNOG in non-toxic concentrations (10(-5)-10(-4) M) did not alter the proliferative reaction of boMNC in vitro. The results suggest that NO is induced after in vitro stimulation of boMNC, however, at a low level, and without having any positive or suppressive effects on the so far tested cellular parameters of activation and proliferation.

Prostaglandin E2 is variably induced by bacterial superantigens in bovine mononuclear cells and has a regulatory role for the T cell proliferative response.

Signal transduction in antigen presenting cells via MHC class II molecules induces production of prostaglandin E2 (PGE2) known to possess immunoregulatory potential. Since Staphylococcus aureus superantigens (SAgs) utilize MHC class II molecules as primary ligands, we wanted to know whether PGE2 is induced after in vitro SAg stimulation of bovine blood mononuclear cells (boMNC), and whether this arachidonic acid metabolite modulates the preferential SAg-induced proliferative response of bovine CD8+ T cells. SEB as well as SEA induced maximal amounts of PGE2 on day 2 of culture (1-2.5 x 10(-8) mol/l per 2 x 10(5) boMNC). PGE2 production could be inhibited completely by indomethacin (10(-5) mol/l) causing enhanced proliferation of boCD4+ T cells (174%) as well as of boCD8+ T cells (122%) between day 4 and 6 of the in vitro culture, however, only in a subset of the tested animals. Notably, the striking preference of proliferation of boCD8+ over boCD4+ T cells following SAg stimulation remained largely unchanged after inhibition of endogenous PGE2 synthesis or after addition of exogenous PGE2. Higher concentrations of exogenously added PGE2 (> or = 10(-8) mol/l) inhibited the proliferation reaction, mainly due to an increased death rate of both CD4+ and CD8+ blasts. In contrast, lower PGE2 concentrations between 10(-8)-10(-9) mol/l even slightly enhanced the proliferation of both T cell subsets, depending on the individual cell donor. Summing up: These data show that SAgs, indeed, can induce PGE2 production in boMNC which can enhance or reduce the proliferative response of bovine CD4+ and CD8+ T cells.

Superantigen-dependent accelerated death of bovine neutrophilic granulocytes in vitro is mediated by blood mononuclear cells.

While classical interactions of bacterial superantigens (SAgs) with antigen presenting cells and T cells have been studied intensively, the potential interactions of SAgs with granulocytes (PMNs) have gained much less attention. We investigated if in the bovine system SAgs have any direct or indirect influence on the fate of granulocytes, which are among those cells primarily responsible for the elimination of superantigen-producing bacteria. The tested SAgs (SEA, SEB) had no apparent direct effect on PMN viability (neutrophils and eosinophils). However, in the presence of blood mononuclear cells (MNCs), SAgs led to an accelerated death of neutrophils but not of eosinophils. Compared to medium controls, in SAg-stimulated cultures only about 20-50% of the neutrophils survived after 24 hours in vitro. Accelerated death of neutrophils required the presence of at least 10% MNC and started between 2.5-24 h after initiation of the co-culture between MNC and PMN. Minimal effective SEA concentrations ranged between 10-100 pg/l (SEB 0.1-10 ng/l). The effect could be mimicked by culture supernatants of SAg-stimulated MNCs, suggesting that direct cell-cell interactions are not required for the killing. In the human system, where we tested the role of TNF-alpha, an antibody specific for this cytokine was not able to abolish the death of human neutrophils. Brefeldin A, an inhibitor of golgi transport and cytokine secretion, which blocked the SAg-induced activation of bovine MNC did not abolish the killing of neutrophils. Blocking of nitric oxide generation or PGE2 synthesis also could not alter the SAg-induced killing of bovine neutrophils. The observed indirect negative effects of SAgs on neutrophils may provide new insights in mechanisms by which superantigens modulate the hosts immune response.

Metabolic alterations associated with proliferation of mitogen-activated lymphocytes and of lymphoblastoid cell lines: evaluation of glucose and glutamine metabolism.

In vitro resting, short-term mitogen stimulated, and proliferating rat thymocytes as well as established human T and B lymphoblastoid cell lines were compared in their capacity to metabolize glucose and glutamine as energy source. Furthermore, the pathways of glutamine metabolism in these cells were studied. Compared with resting thymocytes, glucose metabolism of proliferating thymocytes was 36-fold increased during the incubation; 92% of the amount of glucose utilized was converted into trioses mainly lactate, whereas resting cells metabolized only 38% to trioses. However, the latter oxidized 19% of glucose to CO2, as opposed to 1.1% by the proliferating cells. Rates of glucose uptake and degradation to products by the malignant T lymphoblastoid cell line (Jurkat) were nearly identical with those observed with proliferating rat thymocytes, whereas the benign B lymphoblastoid cell lines (DHg-B-1 and LV-B-1) showed significantly higher rates of glucose metabolism. All three transformed lymphoblastoid cell lines, however, metabolized glucose almost completely to lactate as did the proliferating rat thymocytes. Lymphocytes are able to utilize glutamine with glutamate, aspartate and ammonia being the major end-products. A complete recovery of glutamine carbon in the products was obtained with all cells. Glutamine utilization by incubated proliferating rat thymocytes was 8-fold increased as compared to the resting cells. Again the human T lymphoblastoid cell line showed the same rates of glutamine uptake and conversion into products as did the proliferating rat thymocytes, whereas both B lymphoblastoid cell lines had about 2.5-fold enhanced rates as compared to the T cell line. The results indicate that during lymphocyte proliferation caused by mitogen stimulation as well as by permanent transformation into lymphoblastoid cell lines glucose metabolism is altered not only quantitatively but also qualitatively by changing from partly aerobic to almost complete anaerobic glucose breakdown. Glutamine has been found to be a suitable energy source for lymphocytes. About 75% of the amount of glutamate derived from glutamine entered into the citric acid cycle via the aspartate aminotransferase, and the remaining 25% via the glutamate dehydrogenase reaction. The changes in metabolic rates observed in proliferating as well as in transformed or leukemic lymphocytes appear to be reliable parameters to characterize the state of lymphocyte activation or to evaluate the efficacy of lymphokines.

The binding of different lectins on peripheral blood mononuclear cells from patients with chronic inflammatory and malignant diseases.

Peripheral blood mononuclear cells from patients with multiple myeloma, gastrointestinal tumors, and inflammatory bowel disease were analyzed for binding of various lectins. The results demonstrated that in most of the patients with multiple myeloma a significantly increased percentage of cells positive for Lotus tetragonolobus agglutinin (LTA), peanut agglutinin (PNA), soybean agglutinin (SBA), and wheat germ agglutinin (WGA), and a decreased number of Agaricus bisporus agglutinin (ABA) positive cells were present as compared to a normal control group. This could not be shown in malignant or inflammatory disorders of the gastrointestinal tract where only some patients exhibited an increased PNA and LTA binding, respectively. Patients with the systemic malignant disease differed from patients with solid localized tumors by a significantly altered number of ABA, LTA and SBA-positive peripheral blood mononuclear cells. Double fluorescence studies using monoclonal antibodies and lectins revealed that most of the cells expressing receptors for ABA had also receptors for OKT3, whereas most of the cells with receptors for LTA, PNA, SBA, and WGA were found to be positive for OKM.

Organization of the equine immunoglobulin heavy chain constant region genes; III. Alignment of c mu, c gamma, c epsilon and c alpha genes.

Previous restriction analysis of cloned equine DNA and genomic DNA of equine peripheral blood mononuclear cells had indicated the existence of one c epsilon, one c alpha and up to six c gamma genes in the haploid equine genome. The c epsilon and c alpha genes have been aligned on a 30 kb DNA fragment in the order 5' c epsilon-c alpha 3'. Here we describe the alignment of the equine c mu and c gamma genes by deletion analysis of one IgM, four IgG and two equine light chain expressing heterohybridomas. This analysis establishes the existence of six c gamma genes per haploid genome. The genomic alignment of the cH-genes is 5' c mu/(/) c gamma 1/(/) c gamma 2/(/) c gamma 3/(/) c gamma 4/(/) c gamma 5/(/) c gamma 6/(/) c epsilon-c alpha 3', naming the c gamma genes according to their position relative to c mu. For three of the c gamma genes the corresponding IgG isotypes could be identified as IgGa for c gamma 1, IgG(T) for c gamma 3 and IgGb for c gamma 4.

Influence of Escherichia coli and Arcanobacterium pyogenes isolated from bovine puerperal uteri on phenotypic and functional properties of neutrophils.

When cows develop endometritis after birth, Escherichia coli and Arcanobacterium pyogenes are usually the most prominent bacteria present in bovine uterine lochial secretions. A. pyogenes alone is rarely found in the course of a disturbed puerperium. This was confirmed in this study, since average and high-grade uterine contaminations were always associated with the presence of both bacteria. The contamination grade was positively correlated with uterine polymorphonuclear granulocyte (PMN) numbers and negatively correlated with blood PMN numbers. Whether E. coli and A. pyogenes affect the phenotype and function of bovine PMN in a similar or differential way was subject to in vitro studies. PMN were tested in the presence of washed bacterial fragments or culture supernatants taken as a source for soluble and/or secreted bacterial products. Fragments and soluble products differed only quantitatively in their effects on PMN. Usually, long-time exposure (24h) of PMN to fragments induced the strongest effects. Accelerated death of granulocytes was only moderately induced by both E. coli and A. pyogenes products. Both E. coli and A. pyogenes products induced the enhanced expression of a membrane molecule detected by mAb IL-A110 and of CD11b. Expression of other surface structures remained largely unchanged (MHC class I, CD11c). Functional parameters of PMN (phagocytosis; generation of reactive oxygen species, ROS; antibody-independent cellular cytotoxicity, AICC) generally declined after pre-incubation for 24h with products of E. coli or A. pyogenes. Interestingly, soluble products of A. pyogenes stimulated the phagocytosis of PMN. However, co-incubation with E. coli products abrogated this stimulatory effect. The results supply evidence for similar modes of action of the gram-negative E. coli and the gram-positive A. pyogenes on bovine PMN. Alterations in PMN function and phenotype are mainly triggered by direct contact between bacterial fragments and PMN. Inhibition experiments with polymyxin B demonstrated that E. coli-mediated effects were not solely due to the action of lipopolysaccharide. The dominant functional depression of neutrophils by E. coli products strengthens the suggestion that the earlier appearance of E. coli in the uterus may support the co-infection of this organ by A. pyogenes at later times.

Characterization of leukocytotoxic and superantigen-like factors produced by Staphylococcus aureus isolates from milk of cows with mastitis.

Staphylococcus aureus is a major pathogen for cattle, causing various forms of subclinical and clinical mastitis. Two groups of virulence factors (leukotoxins and superantigens) are supposed to play an important role in the initiation and/or the exacerbation of this disease. In order to detect all known and putative members of leukotoxins and SAgs (superantigens), we tested secreted factors of different S. aureus isolates in flow cytometry-based assays. Isolates were sampled from 68 cows of different farms and cultured for 24h in vitro. Supernatants were then coincubated with purified polymorphonuclear granulocytes (PMN) or combinations of blood mononuclear cells (MNC) and PMN. Viable PMN and MNC were determined by quantitative flow cytometry. In addition, we recorded the proliferation-inducing potential of isolate supernatants for bovine MNC. Based on these criteria, the supernatants of S. aureus isolates fell in three groups. The first group (n=32), termed LT-SNs (leukotoxin-containing supernatants), killed purified granulocytes (neutrophils and eosinophils) in vitro. The second group of supernatants (n=20), termed SAg-SN (superantigen-containing supernatants), induced activation and proliferation of mononuclear cells (MNC) and, only in the presence of MNC, resulted in a selective depletion of neutrophils after 24h in vitro. The third group of supernatants (n=16) contained neither LTs or SAgs. Functionally, SAg-SNs behaved like purified staphylococcal enterotoxin A (SEA) or SEB tested in parallel. The absence of SAg-like activity in LT-SNs was confirmed by heat treatment of LT-SNs, which destroyed the leukocytotoxic activity, but did not reveal any MNC-activating potential. This study, therefore, suggests, that pathogenic S. aureus isolates either produce leukotoxins or superantigens and that both groups of virulence factors can easily be differentiated by the functional assays described. The prevalence of leukotoxin- or superantigen-producing isolates was comparable among cattle with subclinical (LT=41%; SAg=30.8%) mastitis. The higher frequency of LT-producing isolates in cases of clinical mastitis (LT=55.2%; SAg=27.6%) was not significant. At least, these findings argue against the dominant role of superantigens or leukotoxins in S. aureus-induced bovine mastitis.

Monoclonal equine IgM and IgG immunoglobulins.

In order to define equine immunoglobulins (Igs) and to produce monoclonal reference Igs we fused equine peripheral blood mononuclear cells with X63-Ag8.653 non Ig producing murine myeloma cells. A total of 29 equine Ig producing equi-murine heterohybridomas were obtained, of which ten expressed equine Ig for more than 3 months. One of these heterohybridoma lines produced monoclonal IgM, an equine isotype which has not been available in monoclonal form before. Four lines secreted equine IgG of two distinct Ig heavy chain types as assessed by the molecular weight (MW), while the remaining five lines expressed only equine Ig light chains. A sixth Ig light chain expressing variant was obtained by cloning of one of the IgG producing heterohybridoma lines. These monoclonal IgGs were compared with previously described equine IgG monoclonal antibodies (mabs) by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). According to their MW we identified five groups of gamma-chains and four groups of Ig light chains.

Reactivity of workshop monoclonal antibodies on paraformaldehyde-fixed porcine blood mononuclear cells.

One hundred sixty-four monoclonal antibodies (mAbs) of the second international swine CD workshop were tested for their reactivity with porcine blood mononuclear cells before and after fixing the cells with varying concentrations of paraformaldehyde (PFA) (1, 5 and 10 g l-1). A total of 38 (out of 134) positive reacting mAbs were significantly affected in their binding behavior on fixed cells. Modulation was seen as reduction in binding (staining intensity and/or % positive cells, n = 18) or in elevated values (n = 20). Modified mAb binding occurred after fixing cells with 5 to 10 g l-1 PFA.

Nucleotide sequence and restriction fragment length polymorphisms of the equine Cvarepsilon gene.

IgE is the dominant immunoglobulin isotype involved in type I hypersensitivities in mammals. The heavy chain constant region domains of equine IgE are encoded by a single gene, the Cvarepsilon gene. By restriction analysis of cDNA from 15 unrelated horses, we have now identified two Cvarepsilon alleles, characterised by a Sma I restriction fragment length polymorphism, which we designated Cvarepsilon(a) and Cvarepsilon(b). Sequence analysis of both, Cvarepsilon(a) and Cvarepsilon(b) cDNA, showed in addition two single base exchanges resulting in two amino acid substitutions. Both sequences have only 95.9% homology of the coding region sequence with the published equine Cvarepsilon sequence, which could represent a third haplotype. Polymorphism of the IgE heavy chain constant region gene, as described here, might well impose genetic variability on the effector functions of equine IgE predisposition to allergic diseases in horses.

Organisation of the equine immunoglobulin constant heavy chain genes. II. Equine cgamma genes.

The number of immunoglobulin G constant heavy chain genes (cgamma genes) varies broadly among mammalian species, reflecting structural and functional differences between expressed immunoglobulin G (IgG) isotypes and allotypes. Up to now equine IgG isotypes have been defined only at the biochemical and serological level. It is still not clear how many IgG isotypes exist in horses and whether there are any allotypes. Here, we describe the isolation and characterisation of equine cgamma genes. An equine genomic lambda phage library was screened with a human cgamma4 probe. Cross-hybridising equine cgamma sequences were cloned twice and characterised by restriction mapping with the human cgamma4 and a murine sgamma1 probe. Genomic equine DNA probes for both, cgamma genes and corresponding switch regions (sgamma), were isolated and used for a more detailed BamHI restriction analysis, comparing genomic DNA of various horses. This analysis reveals the existence of at least five, or probably six cgamma genes in the equine haploid genome. Beside the porcine system, this is the highest number of cgamma genes described for any mammalian species. Moreover, for two of these cgamma genes, BamHI restriction fragment length polymorphism became evident.

Organization of the equine immunoglobulin constant heavy chain genes. I. c epsilon and c alpha genes.

We provide a restriction map of the equine c epsilon and c alpha genes as a molecular basis for isotype classification. Human and murine DNA probes were used for identification of homologous equine DNA sequences and for isolation of the equine c epsilon and c alpha genes from a genomic DNA library. A detailed map of the equine 5'-s epsilon/c epsilon-s alpha/c alpha-3' gene region was obtained. Equine c epsilon and c alpha DNA probes were prepared and used for restriction analysis of immunoglobulin heavy chain gene loci from different horses. This analysis indicated the presence of only one equine c epsilon and one c alpha gene in the haploid equine genome. In addition, for the equine c alpha gene, four haplotypes were identified according to BamHI restriction fragment length polymorphism (RFLP) of genomic DNA. The relative location of the c epsilon and c alpha genes 3' of the equine c mu and c gamma genes was determined by restriction analysis of equi-murine heterohybridomas.

Expression and characterisation of equine interleukin 2 and interleukin 4.

In the present study, we describe the expression of equine IL2 and IL4. The cDNA of equine IL2 or IL4 was cloned in a mammalian expression vector, containing c-terminal myc- and six histidines His(6)-epitopes for recognition and purification of equine cytokines. The vector constructs were used for transfection of chinese hamster ovary (CHO) cells. Purified equine cytokines were characterised by western blotting. Equine IL2 was secreted with a molecular weight of approximately 17.1kDa, whereas IL4 was expressed in three different sizes of 17.1, 19.6 and 22.1kDa, probably due to different glycosylation modifications. The biological activities of both cytokines were tested by proliferation assays using leukoagglutinin (LAG) prestimulated equine PBMC. Both, equine IL2 and IL4 induced dose-dependent lymphocyte proliferation. In contrast to IL4, IL2 supported the proliferation of B cells.