Chicken Peripheral Blood Lymphocytes Research Articles

Development of optimal conditions for the stimulation of chicken peripheral blood lymphocytes by phytohaemagglutinin (PHA) in the microculture system

A variety of parameters influencing phytohaemagglutinin (PHA) stimulation assays of lymphocytes obtained from chicken peripheral blood have been studied. The parameters examined were PHA preparation and dilution, source and quantity of serum, cell concentration, culture medium, incubation temperature, length of the incubation period, cell separation procedure and individual influence of the blood donors. Optimal stimulation results were obtained using the following conditions: PHA-M in a dilution of 1:15, fetal calf serum (FCS) in a concentration of 4%–10%, a cell concentration of 4×10 6 – 1.5×10 7 cells/ml, RPMI 1640 Medium or McCoy's 5A Medium, an incubation period of 48 h – 72 h at a temperature of 39°C, and low-speed centrifugation of heparinized blood for lymphocyte collection. The findings of the present study are discussed in the context of results described in the literature.

B cell activation in chickens induced by [formula omitted][formula omitted

Conventional mammalian polyclonal B cell activators were evaluated for activity in chicken spleen and peripheral blood lymphocyte (PBL) cultures. Although lipopolysaccharide was found to have a marginal influence on proliferation, two strains of the bacterium Staphylococcus aureus (Cowan I and Wood 46 strains) induced moderate proliferation in both spleen and PBL cultures. In spleen cell cultures the proliferating cell population was identified as the B cell. The mitogenic response required the presence of adherent cells since their removal eliminated the response. Evidence of in vitro polyclonal immunoglobulin synthesis could not be obtained. However, when administered intravenously, S. aureus induced polyclonal immunoglobulin synthesis.

Characterization of chicken T cell growth factor.

Chicken spleen and peripheral blood lymphocytes were tested for their ability to produce T cell growth factor (interleukin 2, IL 2) upon stimulation with phytohemaglutinin or concanavalin A (Con A). Using a qualitative microassay, peak activity was found in 24-h supernatants of mitogen-stimulated lymphocytes. These supernatants permitted continuous growth of chicken lymphocytes for several weeks and increased the Con A response of chicken thymocytes. A quantitative probit microassay was developed and used in the further studies when appropriate. Chicken IL2 binds specifically to thymocytes and activated T cells and its action upon T blast cells cannot be blocked by cyclosporin A. The molecular weights of the two biological active peaks found by gel filtration of the native IL2 preparation were in the range 19500-21500 and 9000-11500. A single polypeptide of molecular weight 13000 was found by gel electrophoresis under reducing conditions.

Immune stimulation of sensitized chicken lymphocytes by avian retrovirus proteins.

Peripheral blood lymphocytes of chickens bearing tumours induced by avian sarcoma virus can be specifically stimulated to divide by the crude culture fluids of virus-infected cells. In this communication, we show that relevant antigenic activity apparently resides in each of the internal virus proteins p15 and p27. The ability of infectious culture fluids to be mitogenic for sensitized lymphocytes is greatly reduced following treatment with antibodies specific for either total avian myeloblastosis virus (AMV) protein or for p27.

Coccidiosis: localization of lymphoblasts in the infected small intestine.

Peripheral blood lymphocytes of chickens and thoracic duct lymphocytes and mesenteric lymph node cells of rats, were labelled with 125I-deoxyuridine and injected intravenously into chickens or rats respectively. Sixteen to 18 h later the intestines of coccidia infected animals contained more radioactivity than those of uninfected controls. This result was obtained with cell suspensions from both infected and normal donors indicating that, as with nematode infected rodents, the increased homing of the cells to parasitised gut was not antigen specific. In chickens the stimulus which causes the increased homing of cells to the intestine was induced within hours of parasite inoculation. This reflects the rapid response of this host, previously described with other parameters, and which may be characteristic of birds.

Immune Profile of Infectious Bursal Disease (IBD). II. Effect of IBD Virus on Pokeweed-Mitogen-Stimulated Peripheral Blood Lymphocytes of Chickens

Pokeweed-mitogen-stimulated peripheral blood lymphocytes from chickens infected with infectious bursal disease virus (IBDV) at 1 day and 3 weeks of age, together with those from uninfected age-matched control chickens, were examined at weekly intervals for their capacity to undergo in vitro terminal differentiation. This study included the determination of cytoplasmic-immunoglobulin (CIg)-containing peripheral blood lymphocytes and those bearing the surface membrane immunoglobulin (SmIg) cells to detect any defect in immunoglobulin synthesis and secretion, respectively. The results of our study indicated that there was a highly significant and pronounced progressive depletion of CIg-containing cells in chickens infected at 1 day of age. These findings suggest that IBDV affected the "immature" or precursor B cells to a far greater extent than mature B lymphocytes.

GENETIC DIFFERENCES IN THE MITOGENIC RESPONSE OF PERIPHERAL BLOOD LYMPHOCYTES IN THE CHICKEN

The mitogenic responses of chicken peripheral blood lymphocytes were compared in two partially inbred chicken lines and in their hybrids. The lymphocytes of line P chickens showed a higher phytohaemagglutinin (PHA) response than those of line V chickens and (P X V)F1 hybrids. The Con A responses were of the same magnitude in all chicken lines. The higher PHA response in line P chickens was not sex-linked, apparently was not associated with the major histocompatibility complex and it did not correlate with immunoglobulin G allotypes.

Avian antigen binding cells: enrichment methods

Data are presented comparing different methods for the fractionation and enrichment, respectively, of specific antigen binding lymphoid cells from immunized chickens. The bovine serum albumin (BSA) anti-BSA system was chosen as a model. To enrich avian antigen binding cells (ABC) from a mixture of chicken peripheral blood and spleen lymphocytes 3 different methods were used: (1) separation of cells forming rosettes with antigen-coated sheep red blood cells (SRBC) from non-rosetting cells by density centrifugation; (2) isolation of ABC by their specific adherence to antigen bound to immuno-adsorptive surfaces (gelatin, plastics); (3) column affinity chromotography with antigen-coated agarose, cross-linked dextran or plastic beads. The most efficient method was column affinity chromotography with antigen-coated polyacrylamide beads which affords up to 12-fold enrichment of ABC. Both the other methods are also suitable for separation and enrichment of specific ABC but can only with difficulty be adapted for processing the large numbers of cells which would be necessary, e.g., for in vivo transfer studies.

Stimulation of chicken lymphocytes by T- and B-cell mitogens

Cultures of chicken spleen, peripheral blood, thymus, and bursal lymphocytes were tested for mitogenic stimulation by phytohaemagglutinin (PHA), concanavalin A (ConA), pokeweed mitogen (PWM), bacterial lipopolysaccharide (LPS), trypsin, and insulin. Spleen and blood leukocytes were stimulated by both the lectins and LPS, and also to some degree by trypsin and insulin as judged by increased incorporation of [ 3H]thymidine into acid-insoluble material. This was observed in cultures incubated in serum-free medium as well as in the presence of foetal bovine serum or autologous plasma. Thymus cells were reproducibly stimulated by high concentrations of PHA. No significant responses were obtained in bursal cell cultures with any of the compounds tested. Removal of cotton wool-adherent cells from the spleen cell suspensions resulted in a subpopulation of cells which were stimulated by PHA but showed little response to ConA, PWM, or LPS. This procedure did not remove surface immunoglobulin-bearing cells from the original suspension. Both these enriched spleen lymphocytes and the unfractionated spleen, blood and thymus leukocyte cultures were effectively stimulated by a partially purified PHA but with a highly purified PHA preparation only at very high concentrations. These and other results suggest that the mitogenic components in crude PHA preparations are different for chicken and human or mouse cells.

Effect of bursectomy and thymectomy on the responses of chicken peripheral blood lymphocytes to phytohaemagglutinin.

THE immunological significance of the transformation of lymphocytes by phytohaemagglutinin (PHA) has not been clearly denned. PHA stimulates cells other than lymphocytes1–3 and there is evidence to suggest that the response of lymphocytes is not dependent on earlier sensitization to PHA itself4. None the less, transformation seems to be related to the immunological competence of the responding lymphocytes; thus this response is greatly diminished in children with congenital absence of the thymus gland, associated with impaired immune responses5,6.