Development of a microculture system for stimulation of chicken peripheral blood lymphocytes with concanavalin A.

A microculture system in conjunction with a semiautomatic multiple sample harvester (SAMSH) was used to study the in vitro properties of chicken peripheral lymphocytes. This new procedure enabled doing rapid multiple tests, using relatively few cells, and was highly reproducible. Data were presented to show many variables that are involved in studying the concanavalin A (Con A) response of chicken lymphocytes in a microculture system. Analysis indicated that the conditions for optimal Con A stimulation as measured by incorporation of 3H-TdR include: (a) use of 2 x 10(6) cells per culture in RPMI 1640 culture medium in the absence of any serum, (b) use of 0.4 mug of Con A per culture, (c) incubation at 37 degrees C for 72 hours, and (d) addition of 1 muCi of 3H-TdR to each culture 12 to 24 hours prior to termination. This technique could be used to monitor immunocompetence of the chicken.     

Feline lymphocytes: observations on surface membrane concanavalin A receptor mobility

The mechanics of concanavalin A receptor mobility of the feline lymphocyte surface membrane were investigated, utilizing fluorescein-labeled lectin to quantitate lymphocyte capping. The results of this study indicated that lectin concentration and buffer selection were critical for extensive receptor redistribution with cap formation of feline lymphocytes. Maximal capping was obtained with 50 microgram of concanavalin A/ml of minimal essential medium. The mean capping rate of peripheral blood lymphocytes increased significantly with colchicine exposure at 10(-7) M concentration. The mean values of capping increased slightly with advancing age of feline donors, although this difference was not statistically significant. Concurrent work has indicated that concanavalin A capping may be useful in the study of immunosuppression in feline leukemia virus-infected cats.     

Immunological parameters in meat-type chicken lines divergently selected by antibody response to Escherichia coli vaccination.

Our group has established two lines of meat-type chickens divergently selected for early (HC line) and late (LC line) antibody responsiveness at 10 days of age to immunization with inactivated pathogenic Escherichia coli bacteria. The question addressed in the study presented here is whether this selection has changed other immunological responses, increasing the overall 'early' immunocompetence. Broilers of the third and fourth generations (S3 and S4) of the selected lines (HC and LC) and a control, unselected line (CT) were vaccinated at 10 days of age with E. coli vaccine, Newcastle virus vaccine (NDV), sheep erythrocytes (SRBC) or bovine serum albumin (BSA). Line-HC chicks exhibited higher antibody titers to E. coli, NDV and SRBC than CT or LC chicks. At 20 days of age HC chicks demonstrated a higher total protein and a higher beta- and gamma-globulin levels in their serum. At 21 days of age, HC chicks cleared carbon particles faster than LC chicks. Peripheral blood lymphocytes (PBL) from HC chicks vaccinated with E. coli vaccine, proliferated in vitro more actively in the presence of the stimulating antigen than the PBL of LC chicks. Peripheral blood lymphocytes (PBL) obtained from HC-line chicks exhibited a higher proliferative response to concanavalin A (Con A)-, phytohemagglutinin (PHA)- or pokeweed mitogen (PWM)-stimulation than LC PBL. These results demonstrate that the selection for high or low antibody response to E. coli at a young age resulted also in a significant change in the response of other parameters of the immune system. The high response to E. coli was found to be associated with a high antibody response to other antigens (NDV and SRBC), increased phagocytic activity and increased proliferative response to antigen or mitogens. The selection most probably affected early immunocompetence.     

Production and assay of ovine T cell growth factor by concanavalin A stimulated peripheral blood leukocytes

Conditions for the production and assay of ovine T cell growth factor (TCGF) activity were evaluated. Peripheral blood leukocytes were stimulated with concanavalin A (Con A) in the presence of 2% autologous serum or serum-free media. Supernatants were harvested after 30 hr and concentrated for further characterization. A 28 hr proliferation assay with 2.5 X 10(4) 24 hr Con A blasts per well was optimal for detection of TCGF. Peak TCGF activity occurred with a 30-37 KD molecular weight fraction. Production and assay of TCGF were performed under autologous conditions to reduce background stimulation which occurred when fetal bovine serum was present. This methodology required no cell lines or inbred animals and should be adaptable to the study of immunostimulatory molecules of other outbred species.     

In vitro reactivity to concanavalin A of bovine lymphocytes after cryopreservation

Cryopreservation of bovine peripheral lymphocytes and its effect on the $\text{in vitro}$ response to concanavalin A tested in a microculture system is described. Using DMSO as cryoprotectant in the medium, the cells were cooled to ?30°C at 1.3°C/minute and further to ?80°C at 6°C/minute and then rapidly to ?196°C by dropping in liquid nitrogen. The cells were recovered by rapid thawing in water at 30–35°C and washed twice before use in the stimulation test. Ten percent DMSO had a much better protective effect than 5%; addition of 25% fetal bovine serum to the freezing had no favourable effect. In most of the 16 animals used in the experiments the frozen lymphocytes gave the same or a higher response to Con A than those kept in the DMSO containing medium at 4°C for two hours.The responses of the frozen cells were comparable to those of fresh lymphocytes (kept at 4°C for two hours in medium without DMSO).     

Production of interleukin-2 mRNA by bovine lymph node lymphocytes in response to concanavalin A, 12-O-tetradecanoylphorbol-13-acetate, and ionomycin

Interleukin-2 (IL-2) is a lymphokine which, upon binding to its receptor, leads to the proliferation and differentiation of T-cells (helper, suppressor, and cytotoxic) and B-cells. While human and murine IL-2 have been extensively studied, less is known about bovine IL-2. In order to understand the induction of bovine IL-2 at the molecular level, we have examined IL-2 mRNA induction. The dose-responses and time courses of the production of IL-2 mRNA in response to Concanavalin A (ConA), 12-O-tetradecanoylphorbol-13-acetate (TPA), and ionomycin in lymph node lymphocytes (LNC) were determined. We found that high levels of IL-2 mRNA were produced in response to 1 μg ml⁻¹ ConA plus 10⁻⁸ M TPA, but that even higher levels were produced in response to 1 μM ionomycin plus 10⁻⁸ M TPA. We also found that LNC stimulated with ConA displayed two phases of IL-2 mRNA production, one occurring approximately 2–4 h after stimulation and one occurring approximately 10 h after stimulation. However, in the presence of ConA plus TPA or ionomycin plus TPA the response was monophasic. IL-2 mRNA was detected within 2 h of addition of ConA plus TPA (the earliest time examined), reached maximum levels within 6 h, and declined to low levels after 12 h. IL-2 mRNA from LNC incubated with ionomycin plus TPA appeared within 2 h, and reached maximum levels at about 9 h. In contrast to the decrease seen after 12 h with ConA plus TPA, IL-2 mRNA from these cells remained high for 18 h and declined to low levels after 24 h.     

Chemiluminescence of canine peripheral blood lymphocytes stimulated by mitogens

Luminol-dependent chemiluminescence of peripheral blood lymphocytes from dogs stimulated with concanavalin A (Con A) or phytohemagglutinin P (PHA) was measured with a Pico-Lite luminometer. 10 microliter of luminol gave optimal quantum yield from 1 X 10(6) lymphocytes sensitized with either 80 micrograms Con A or 160 micrograms PHA. Addition of superoxide dismutase did not influence the course of chemiluminescence. Whereas catalase produced 41% increase in quantum yield, mannitol caused a 51% inhibition of chemiluminescence. Lymphocytes exposed to varying doses of short term x-irradiation or lymphocytes isolated from dogs kept under continuous exposure through a gamma irradiation source showed dose-related depression of chemiluminescence. Membrane factors may be involved in lymphocyte stimulation to chemiluminescence as pulse experiments with Con A and PHA revealed. It is proposed that chemiluminescence measurements may be useful in monitoring early events in lymphocyte stimulation by antigens and mitogens.     

Pathology of concanavalin A-induced cutaneous reaction in the chicken

Sequential study of permeability and cellular responses following intradermal concanavalin A in the chicken skin, using the colloidal carbon technique, revealed an increase in vascular permeability which was mostly confined to venules. A noteworthy feature of the reaction was marked accumulation of basophils, even in the later stages, and the early appearance of perivascular lymphoid aggregations. The occurrence of well formed giant cells, hypertrophy and hyperplasia of vascular endothelium and marked acanthosis of the epidermis were the other prominent changes. The findings suggest that Con A, in the chicken, appears to have a more general effect on the different types of cells and that it may act as a mitogen not only for T lymphocytes but also for endothelial and epithelial cells.     

Lysis of chicken lymphocytes by infectious bursal disease viruses

Infectious bursal disease virus types 1 and 2 were able to induce direct lysis of chicken bursal cells, thymus cells, and peripheral blood lymphocytes in chromium-release assays. These two viruses were unable to lyse two established lymphoblastoid cell lines, although IBDV-1 was capable of multiplying in MSB-1 cells.     

Expression profiles of antiviral response genes in chicken bursal cells stimulated with Toll-like receptor ligands

Cells of the adaptive immune system express Toll-like receptors (TLRs) and are able to respond to TLR ligands. With this in mind, the goal of the current study was to determine the expression of antiviral response genes in the cells of the chicken bursa of Fabricius (BF) to stimulation with TLR ligands. We investigated initially the response of bursal B cells to CpG-ODN, lipopolysaccharide (LPS) and poly(I:C) treatment. The expression level of type I interferons (IFNs) and interferon regulatory factor 7 (IRF7) did not differ between CpG-ODN and LPS treated groups compared to the non-stimulated cells. Poly(I:C) was the only TLR ligand, which has induced significant expression of antiviral innate immune response genes from bursal cells. Further in vitro and in vivo studies need to examine the efficacy of these antiviral responses against avian viruses.     

Development of optimal conditions for lymphokine production by chicken lymphocytes

Chicken thymus, spleen, and bursa lymphocytes were isolated by different methods and incubated under differing conditions in order to obtain and characterize avian lymphokines. The biological activity of lymphokine-containing cell culture supernatants was measured by their antiviral activity (interferon(IFN)-units) and by their capacity to induce cytostatic effects in bone-marrow-derived macrophages (50% cytostasis-inducing dose, CID). Lymphokine production by thymus lymphocytes required concanavalin A (ConA)-stimulation, while spleen cells, when cultured at high density, released CID and IFN activities into the culture medium even without mitogen-stimulation. By way of comparison, the highest lymphokine content was found in the supernatant of lymphocyte cultures, which were incubated for 72 hours at 41 degrees C after stimulation with an optimal ConA dose. For stimulation of thymus lymphocytes 30 micrograms ConA/ml were found to be optimal, independent of serum content and cell density in the cultures. In contrast, the optimal ConA dose for spleen lymphocytes not only depended on the serum content but also on the cell density in the cultures and varied within a range of 2.5 micrograms and 45 micrograms ConA/ml.     

In vitro infection of fractionated chicken lymphocytes by infectious bursal disease virus

Lymphocytes from bursa of Fabricius and thymus of chickens were purified and separated into the three cell subsets--T, B, and null cells--by the techniques of nylon fiber columns and cytotoxicity tests. The in vitro susceptibility of the fractionated lymphocytes to a virulent strain of infectious bursal disease virus (IBDV) was studied by using immunofluorescence as the infection criterion. B cells were highly susceptible. By contrast, T cells and null cells were insusceptible to infection by IBDV. The relationship between the target cells for virus infection and those B cells that possessed surface immunoglobulin (SIg) was tested. B cells were further divided into SIg(M)- and SIg(G)-bearing cells by immunoadsorbent columns employing anti-immunoglobulin M(IgM) (mu-specific) or anti-IgG (gamma-specific) sera coated with Sephadex. The SIg(M)-bearing cells were highly susceptible. These results suggest strongly that SIg(M)-bearing B cells were the target cells for infection by IBDV.     

Binding effects of concanavalin A on a coronavirus.

Concanavalin A, a phytagglutinin, binds to the envelope of hemagglutinating encephalomyelitis virus, a Coronavirus. Concanavalin A treated virus suspensions lose their hemagglutination properties and there is a transient interference with infectivity. Electron micrographs show the Concanavalin A as a granular deposit adhering to the viral envelope and there is aggregation of the virus. Concanavalin A does not bind to virions stripped of their envelopes.     

Potentiation of lymphocyte mitogenic responses to concanavalin A by antigen-activated peripheral blood monocytes.

Purified protein derivative (PPD)-stimulated monocytes derived from Mycobacterium bovis-sensitized cattle significantly potentiated lymphocyte mitogenic responses to concanavalin A (conA), as measured by incorporation of [3H] thymidine into cellular DNA. Monocytes were cultured for 24 hours in the presence of PPD, washed thoroughly, and mixed with purified lymphocytes; various doses of conA were added to these cultures, and the cultures were incubated for 4 days and assayed for DNA synthesis. The lymphocyte mitogenic responses to suboptimal, buy not optimal, doses of conA were significantly enhanced by the presence of PPD-activated monocytes from M bovis-sensitized cattle. Treatment nonsensitized cattle with PPD did not result in any significant enhancement of conA-induced lymphocyte mitogenic responses at any dose of conA tested.     

Response of embryonic chicken lymphocytes to in ovo exposure to lymphotropic viruses.

OBJECTIVE: To examine effects of virus exposure on embryonic lymphoid organ structure, apoptosis, and lymphoid cell subpopulations. ANIMALS: Eggs of specific pathogen free (SPF) White Leghorn chickens at embryonation day (ED) 17. PROCEDURES: Eggs were inoculated with 2,000 plaque-forming units (PFU) of serotype 1 herpesvirus (Marek's disease virus [MDV 1]), 2,000 PFU of herpesvirus of turkeys (MDV 3), or 1,000 embryo infectious doses (EID50) of infectious bursal disease virus (IBDV). On post-inoculation days (PID) 3 and 5, lymphoid organ to body weight ratios were determined, and bursa of Fabricius, thymus, and spleen were evaluated for lesions and apoptosis. Proportions of lymphoid cell subpopulations of PID-3 chicken embryos and 7- to 10-day-old chicks were quantitated by flow cytometry. RESULTS: Lymphoid organ weights were similar in virus-free, MDV1, and IBDV groups. Embryos inoculated with 2,000 PFU MDV 3/egg had lower bursal weights than virus-free controls. In a repeated trial, MDV 3 (1,000 PFU to 4,000 PFU) did not reduce bursal weights among groups. Histologic changes were seen in bursae after MDV 1 and IBDV inoculation. Apoptosis was greater in bursae of MDV 1-infected embryos than controls. Lymphoid cell subpopulations were similar among all groups with the exception of CD8+ and IgM+ cells in spleens of IBDV-infected 10-day-old chicks. CONCLUSIONS AND CLINICAL RELEVANCE: Infection with pathogenic strains of MDV 1 and IBDV did not alter lymphocyte subpopulations in embryos or cause complete destruction of lymphoid organs. Changes in lymphoid cell subpopulations exposed as embryos to IBDV were seen only after hatching.     

鸡脾脏中B淋巴细胞的发育及其定位分布

应用IgM、IgG和IgA单克隆抗体的免疫组织化学方法,研究IgM+、IgG+和IgA+B淋巴细胞在鸡脾脏中的出现、迁移、数量变化规律等一系列的发育过程以及组织定位分布特点。结果显示,IgM+和IgA+细胞在胚胎15日龄时开始出现,IgG+细胞在胚胎18日龄时出现,21日龄雏鸡脾脏的红髓和生发中心中出现以IgG型为主的浆细胞。各日龄IgM+、IgG+和IgA+细胞主要分布在脾脏的特征性组织结构—椭球周围淋巴鞘和生发中心中,这些结构也随着B淋巴细胞的增多而不断发育成熟。B淋巴细胞的数量随着日龄增长持续增加,直至21日龄时趋于稳定,各日龄B淋巴细胞数量始终以IgG+细胞最多,IgM+细胞次之,IgA+细胞最少。结果表明,鸡出壳初期,脾脏的体液免疫功能逐渐增强,并在21日龄时达到成熟水平。     

Stimulatory effect of avian influenza virus on chicken lymphocytes

A study was conducted to examine the effect of avian influenza virus (AIV) on chicken lymphocyte activation. Unprimed or Brucella abortus antigen (Ag)-primed lymphocytes were incubated with various doses of the T-cell mitogen concanavalin A (Con A) or Ag, respectively, plus serial dilutions of inactivated AIV for 72 hr, and cell proliferation was measured via uptake of tritiated thymidine. AIV enhanced the proliferative response to Con A or Ag by 150% or better, and the enhancement decreased in a viral dose-dependent manner. The effects were more readily observed in cells that had not been maximally activated by the Con A or Ag. The enhanced response was observed in lymphocytes from both white rock and white leghorn breeds of chicken and in mature peripheral blood lymphocytes or immature thymocytes. The viral activity could be abrogated by pre-treatment of the viral preparation with AIV-specific antisera or prior adsorption of the AIV with chicken erythrocytes. These results indicate that AIV can interact with and modify the in vitro activity of chicken lymphocytes and may exert modulatory effects on the avian immune system.     

The distributions of phytohemagglutinin-P and concanavalin A binding sites on equine, bovine and canine peripheral blood lymphocytes

The distributions of phytohemagglutinin-P (PHA) and concanavalin A (ConA) binding sites were investigated for equine, bovine and canine peripheral blood lymphocytes (PBL). Non-B lymphocytes were collected from each PBL using a fluorescence-activated cell sorter (FACS), and the numbers of PHA and ConA binding sites on their surfaces were counted. Most PHA binding sites on PBL of the three species were shown on the surfaces of non-B lymphocytes. On the other hand, the ConA binding sites on equine and canine PBL existed mainly on the surfaces of non-B lymphocytes, but B lymphocytes of these two species had many ConA binding sites. These results were confirmed by the results of two-parameter fluorescence analysis using FACS. It is, therefore, concluded that the different optimum concentrations of PHA and ConA in PBL blastogenic responses of each animal depended on the different distributions of their binding sites.