Duck lymphocytes--III. Transformation responses to some common mitogens

The lymphocyte transformation (LT) test was performed using duck blood lymphocytes stimulated with phytohaemagglutinin (PHA), concanavalin A (Con A), lentil lectin (LC), Roman snail lectin (HP), peanut agglutinin (PNA), Bandeiraea simplicifolia seed lectin (BSS), wheat germ agglutinin (WGA), horseshoe crab lectin (HSC), pokeweed mitogen (PWM) and E. coli lipopolysaccharide (LPS). Cells were cultured in microtitre trays, at 41.6 degrees C, 8 x 10(5) cells in 200 microliters medium (= 4 x 10(6) cells/ml) supplemented with 10% pooled duck serum. Mitogens were added at final concentrations of 0.1-100 micrograms/ml and triplicate cultures at each concentration were harvested daily for scintillation counting 6 hr after addition of 1 microCi [3H]thymidine. Three patterns of response were observed. The responses to Con A, LC, HP and HSC were greatest at high mitogen concentrations (40-100 micrograms/ml) throughout the 7 days of culture. With PHA, PNA, WGA and LPS maximum stimulation was obtained at 3-5 days, at which time the cells were responding to lower concentrations of mitogen than were required at other times during the experiment. The response to BSS and PWM showed increasing sensitivity to lower concentrations of mitogen during the first 3 days of culture and then stimulated most strongly at 2-10 micrograms/ml in cultures harvested after 4-7 days. Cells from two ducks were cultured for 3 and 5 days with selected concentrations of these mitogens; the results confirmed the variation in response to different mitogens. It is possible that these patterns of response are the outcome of stimulating different populations of duck lymphocytes.     

Comparative studies on in vitro mitogen-induced proliferation of peripheral blood lymphocytes in dog and breeding fox.

In vitro blastogenesis of dog and fox lymphocytes was compared by a microculture technique. The highest 3H-thymidine incorporation in cultures of dog lymphocytes was observed at day 3, while in those of fox at day 2, incubated either at 37 degrees C or at 39 degrees C. Lymphocytes cultured at 39 degrees C incorporated more tritiated thymidine than did cells cultured at 37 degrees C. The stimulation index (SI) of dog peripheral blood lymphocytes to both mitogens concanavalin A (Con A) and leucoagglutinin (LA) was in a similar range, while pokeweed mitogen (PWM) showed a weaker but significant stimulatory action. The blastogenesis of fox lymphocytes was the greatest in Con A stimulated cultures. The mitogenic potency of LA and PWM was about half of that of Con A, with no essential difference between them. Maximum lymphocyte proliferation of dog and fox was observed when culture media were supplemented with 10% fetal calf serum (FCS).     

Optimum conditions for the turkey lymphocyte transformation test.

Optimum conditions for turkey lymphocyte transformation tests were determined. Thrice-washed turkey buffy-coat cells obtained after slow centrifugation (40 x g, 10 minutes) responded well to mitogenic stimulation. Turkey lymphocytes isolated on Ficoll-containing separation media largely lost their ability to respond to mitogens. Maximum responses were obtained with 2 x 10(7) lymphoid cells/ml. Responses to the mitogens were greatest when bovine fetal serum was used at a 2.5% concentration or pooled turkey serum and autologous plasma were used at a 1.25% concentration. Higher concentrations of turkey serum or plasma decreased the responses when sub-optimum doses of concanavalin-A (Con A) or phytohemagglutinin-P (PHA-P) were used. Serum-free cultures gave higher stimulation indices than cultures with serum only when sub-optimum doses of Con A or PHA-P were used. Optimum mitogen concentrations varied with individual birds, timing of the culture, temperature of incubation, and serum concentration in the cultures. Responses were usually greatest with final concentrations of 5 micrograms Con A/ml, 10 micrograms PHA-P/ml, and 20 micrograms pokeweed mitogen (PWM)/ml and when the cultures were incubated in 96-well microplates at 40 C in humidified air with 5% CO2 for 40-42 hours with pulsing with 3H-thymidine during the final 16 hours of incubation.     

Changes in lymphocyte blastogenic response of mares during the perinatal period

A fluorometric assay was applied to evaluate blastogenesis of equine lymphocytes. Optimal culture conditions were as follows; concentrations of phytohaemagglutinin-P (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM) were 1 microgram/ml, 40 micrograms/ml and 10 micrograms/ml, respectively, when 5 X 10(5) lymphocytes were incubated with culture medium containing 20% pooled horse serum (PHS) for 120 hours. The relative mean stimulation index of healthy non-pregnant mares were 5.107 +/- 0.323 (M +/- SE) with PHA, 4.019 +/- 0.183 with Con A and 3.610 +/- 0.131 with PWM. Sequentially the blastogenic responses of lymphocytes from twenty mares were observed during various stages of the perinatal period. Response decreased gradually before parturition was lowest at the time of parturition (PHA: 1.923 +/- 0.174, Con A: 1.698 +/- 0.206 and PWM: 1.706 +/- 0.177), and then increased gradually after parturition towards non-pregnant levels.     

Optimum conditions for the chicken lymphocyte transformation test.

Optimum conditions for chicken (Gallus gallus) lymphocyte transformation tests were determined. Thrice-washed chicken buffy-coat cells obtained after slow centrifugation (40 x g for 10 minutes) responded substantially better to mitogenic stimulation than lymphocytes isolated on separation media containing Ficoll. Maximum responses were obtained with 2 x 10(7) lymphoid cells/ml. Responses to the mitogens were greatest when fetal bovine serum was used at a 5% concentration or pooled chicken serum and autologous plasma were used at a 1.25% concentration. Optimum mitogen concentrations varied with individual birds, timing of the culture, temperature of incubation, and serum concentration in the cultures. When 1.25% chicken serum was used in the cultures, responses were usually greatest with final concentrations of 30-50 micrograms/ml of concanavalin A (Con A) and 30-50 micrograms/ml of phytohemagglutinin-P (PHA-P). The optimum concentration of pokeweed mitogen (PWM) varied from 1 to 40 micrograms/ml among the birds and was practically impossible to establish in general. The incubation in humidified air with 5% CO2 was significantly better at 40 C than at 37 C. The total culture time of 40 hours including pulsing with 3H-thymidine during the final 16 hours of incubation was the best for Con A- and PHA-P-stimulated cells, whereas a longer incubation of 64 hours gave the highest results with PWM stimulations.     

Effects of in vitro corticosterone on chicken T‐ and B‐lymphocyte proliferation

1. The effects of corticosterone and the time of its addition to cultures, on concanavalin A (Con‐A) and pokeweed mitogen (PWM)‐induced lymphocyte proliferation were studied.

2. Peripheral blood lymphocytes (PBL) were isolated from Cornell K‐strain Single Comb White Leghorn immature male chickens and cultured with various concentrations of Con‐A or PWM. Corticosterone, in different concentrations, was added to the cultures either 2 h before or 2 h after the addition of the respective mitogen.

3. Addition of corticosterone 2 h before the mitogens caused a significant suppression of lymphocyte proliferation in response to both Con‐A and PWM stimulation. Also addition of corticosterone 2 h after the mitogens caused significant suppression of proliferation in response to both mitogens; however, the degree of suppression was not as great.

4. The results indicate that after early activation events are initiated by the mitogens, lymphocytes are less sensitive to the effects of corticosterone. Because less suppression was seen in the cultures preincubated with PWM than those with Con‐A, it is likely that there are different sensitivities to corticosterone in the cell populations that respond to these mitogens.     

Mitogen stimulation of canine normal and myasthenia gravis lymphocytes

Responses of canine lymphoid tissues to mitogens were studied in five normal dogs and in two dogs with acquired myasthenia gravis (MG). In the normal dogs, lymph-node-derived lymphocytes gave the most consistent proliferative responses to concanavalin A (Con A), phytohemagglutinin (PHA), and pokeweed mitogen (PWM), as determined by thymidine incorporation; and in most cases PHA, lipopolysaccharide (LPS), and PWM stimulated total IgG production, as determined by ELISA. Splenic lymphocytes had the greatest capacity for increased total IgG production. In the myasthenic dogs total IgG production by unstimulated lymph-node-derived lymphocytes was 88 micrograms/ml and 153 micrograms/ml, much higher than that of unstimulated normal dog lymphocytes (mean less than 1.0 microgram/ml). All mitogens resulted in suppression rather than stimulation of IgG production by lymphocytes from dogs with MG. Production of antibodies to acetylcholine receptors (AChRs) was detected in the supernatants of lymphocyte cultures from one of the dogs with MG at a rate of 78 fmol/5 x 10(5) cells per week and was not detected in culture supernatants of control dogs. This study demonstrates that lymph nodes may be an important site of antibody production in myasthenic dogs and provides the necessary groundwork for future studies of the cellular immunology of canine MG.     

Neonatal calf serum immunoregulation of lymphocyte blastogenic responses

The ability of lymphocytes from newborn calves to undergo blastogenic responses to the mitogens Concanavalin A (Con A), phytohemagglutinin (PHA), or Pokeweed Mitogen (PWM), and immunomodulation of these responses by neonatal calf serum was assessed as a function of age. Lymphocytes were obtained from thymus, spleen, and mesenteric lymph nodes of 1-, 2- to 3-, 5- to 7-, and 9- to 10-d-old calves, aliquoted and incubated (+/- mitogens) in sera from 1-, 2-, 3-, or 7- to 10-d-old calves. Lymph-node lymphocytes responded least when cultured in sera from 1-d-old calves, regardless of mitogen or age of cell donor; the response increased as age of serum donor increased (P less than .05). Splenic lymphocytes responded similarly (P less than .005). However, when cultured in sera from older calves, splenic lymphocytes from older calves responded greater to PWM than did those from younger calves. Thymic lymphocytes responded minimally to PWM and PHA. Their response to Con A increased (P less than .005) with age of serum donor calf, but the effect was greatest on lymphocytes from 5- to 7-d-old calves. Mixing experiments with varying ratios of 1-d-old calf serum: 10-d-old calf serum suggested that serum from 1-d-old calves contained suppressive activity. Serum cortisol level (measured by radioimmunoassay) was 30 +/- 4.6 ng/ml in calves at 6 h of age and declined to 5.5 +/- 1.1 ng/ml by 10 d. Charcoal treatment to remove steroids did not enhance blastogenesis. Addition of cortisol (50 ng/ml) to charcoal-treated sera resulted in inhibition of response to PHA, but no change in response to Con A or PWM. Further investigation is indicated to characterize this immunosuppressive activity and to establish its relationship to disease susceptibility.     

Immunosuppression of in vivo and in vitro lymphocyte responses in swine induced by Trichinella spiralis or excretory-secretory antigens of the parasite.

The in vivo and in vitro effects of Trichinella spiralis excretory-secretory (ES) antigens on porcine peripheral blood lymphocyte (PBL) responses induced with mitogens (phytohemagglutinin, PHA; concanavalin A, Con A; pokeweed mitogen, PWM) or unrelated antigen (Protein A) were studied to determine whether ES antigens depress lymphocyte responses in experimental swine trichinosis, and/or if this response was manifested after lymphocytes from infected pigs had been pretreated with ES antigens. Additionally, the range of inhibition of lymphocyte responses was tested in parasite-free pigs using different doses of ES antigens and compared with the responsiveness of control cultures from the same animals. The responses of lymphocytes from pigs inoculated with 4 x 10(3) muscle larvae (ML) were strongly depressed (P < 0.05) at post-inoculation days (PID) 7 (after stimulation with PHA), 14, 35 (Con A or PWM), and 49 (PWM). At PID 56 and 63 the lymphocytes from T. spiralis-infected pigs responded better (P < 0.05) to all three mitogens than those from non-infected controls. After 7 weeks post-inoculation, PBL which were pretreated with 10 or 250 micrograms ml-1 of ES antigens showed significantly weaker (P < 0.05, P < 0.001) responses to PWM or PHA, respectively, than those from non-infected animals. The responsiveness of lymphocytes from both groups of pigs to Protein A was not affected by the pretreatment with ES antigens in vitro. The responses of lymphocytes from the parasite-free pigs induced by PHA, PWM or Protein A were strongly depressed (P < 0.01) after in vitro pretreatment regardless of the dose of ES antigens (5, 10, 15, or 20 micrograms ml-1) applied.     

An evaluation of the mitogenic reactivity of intestinal intraepithelial lymphocytes of chickens.

A colorimetric assay employing MTT (3-[4,5-dimethylthiazole-2-yl], 2-5-diphenyltetrazolium bromide) was used to determine the mitogenic response of intestinal intraepithelial lymphocytes (i-IELs) of chickens to T- and B-cell mitogens. Comparisons between mitogenic responses of i-IELs and peripheral blood lymphocytes (PBLs) were made to examine potential relationships. The results from this study indicated that T-cell mitogens, concanavalin A (Con A), and phytohemagglutinin-P (PHA-P) induced mitogenic stimulation in i-IELs. Although stimulation indexes of both i-IELs and PBLs were similar, the optical densities (ODs) of i-IEL cultures containing Con A or PHA-P were 20- to 50-fold lower than the ODs of PBL cultures containing the same mitogen. The lower conversion of MTT to formazan resulting in lower ODs in i-IEL cultures indicated a lower level of cellular activity in the i-IELs than in the PBLs. The mitogenic responses of both i-IELs and PBLs to Con A and PHA-P were dose dependent. The responsive concentration of Con A for i-IELs was within the range of 25-50 micrograms/ml, whereas the responsive concentration of PHA-P for i-IELs was 50 micrograms/ml. Three days of incubation was found to be adequate to induce a significant (P < 0.05) mitogenic response for both T-cell mitogens. Lipopolysaccharide was unable to induce a mitogenic response in i-IELs, which was attributed to the lack of B cells in the i-IEL population. This technique may prove useful in evaluating and studying the role of i-IELs in local cell-mediated immune responses of the gastrointestinal tract.     

In vitro modulating effects of porcine immunoglobulin G on mitogens-induced lymphocyte response in precolostral, suckling and weaned piglets

The influence of allogeneic IgG on in vitro reactivity of peripheral blood lymphocytes (PBL) of neonatal colostrum-deprived piglets as well as of suckling and weaned piglets was studied. PBL were preincubated with purified allogeneic IgG for 24 h before their ability to respond to PHA, Con A or PWM was tested. PBL of precolostral piglets pretreated with allogeneic IgG exhibited higher response to PHA (P less than 0.01) than untreated control cells. An increased response of PBL treated with IgG was also observed in suckling piglets as compared to their respective control cells (P less than 0.01). Responsiveness of PBL treated with IgG to PWM was suppressed. No differences in response to Con A regardless of the sources of lymphocytes was observed as compared to IgG untreated controls. The results suggest that pretreatment of lymphocytes of piglets with allogeneic IgG modulates their reactivity to mitogens, suppressing the response to PWM and stimulating the response to PHA, respectively.     

Lymphocyte transformation test in veterinary clinical immunology

Lymphocyte transformation test is a powerful tool in laboratory testing of immunologic competence of animals. The impaired function of the lymphocytes or presence of mitogenesis suppressing factors in the patient serum were detected by comparing lymphocyte transformation (expressed as thymidine incorporation) obtained in media containing either autologous, homologous, or fetal calf serum additions. Most valuable results were obtained by using at least two, preferably three, different phytomitogens: concanavlin A (Con A), pokeweed mitogen (PWM), and pl ytohemagglutinin (PHA) at optimal concentrations (Con A, 15 μg/ml, PWM and PHA, 5 μg/ml) and decreased concentrations (Con A, 5 μg/ml, PWM and PHA, 1 μg/ml). Mitogenesis induced by lipopolysaccharide was considerably smaller and not used routinely. With 2 × 10⁵ lymphocytes/well, the background count of unstimulated lymphocytes in autologous serum in healthy dogs was usually between 100 and 400 counts/min (CPM), in clinically healthy cattle and horses from 200 to over 2000 CPM. Higher CPM were rarely detected without clinical disease. Increased background counts were often associated with viral infections, leukemias and lymphoreticular hyperplasias, decreased background counts were associated with various diseases. The stimulation indexes (SI) of healthy animals in autologous serum with Con A, (5 μg/ml) or PWM or PHA (1 μg/ml) were in the range from 100 to 1000 in the dogs, in the tens for Con A and in hundreds for PWM and PHA in horses and cattle. Increased SI were present during the incubation period of various diseases. Decreased SI were associated with numerous infectious and lymphoreticular diseases and were caused by any of the following: (1) the presence of serum immunosuppressive factor(s) in the patient serum, (2) the decreased response of lymphocytes to mitogens, or (3) increased mitogenicity of lymphocytes due to unidentified serum factors in absence of phytomitogens.     

Lymphoblastic transformation assay of sheep peripheral blood lymphocytes: A new rapid and easy-to-read technique

A new micro-method was used to evaluate in vitro sensitivity of ovine peripheral blood lymphocytes (PBL) to different non specific mitogens (pHA, Con A, PWM) and to investigate the interest of a colorimetric assay for measurement of transformed lymphocytes.

The results showed that sheep PBL in flat-bottomed microplates responded optimally at a cell density of 8 × 10⁶ cells/ml to PHA (2.5 μg/ml), Con A (5 μg/ml) and PWM (5 μg/ml).

The colorimetric assay using a tetrazolium salt (MTT), for measuring the transformed lymphocytes, is very well correlated with the classical method of [³H]thymidine incorporation.

This new revelation technique of the mitogenic response improve the technical value of the assay, which is more rapid and easy-to-read, without diminishing the biological value.     

Characterization and separation of bovine lymphocyte populations

Bovine lymphocyte populations were characterized by surface markers, rosette-forming ability and behaviour towards mitogens. After pre-treatment with neuraminidase 16% of the bovine blood lymphocytes and 14% of the bovine spleen cells formed spontaneous (E) rosettes with sheep erythrocytes. About 20% EAC rosette-forming cells were detected among both cell populations. Protein A receptors were detectable among 8% of the blood lymphocytes and 26% of the spleen cells. Bovine lymphocytes responded to pokeweed mitogen (PWM), phytohemagglutinin (PHA) and concanavalin A (Con A). An enrichment of bovine B and T cells was obtained by E-rosette sedimentation (81–84% B cells) and by filtration through nylon fiber columns (51–65% T cells). The T cells obtained after nylon filtration still responded to the mitogens PHA, Con A and PWM. Enriched B-cell populations responded to bacterial lipopolysaccharide (LPS). After monocyte depletion the mitogenic response of blood lymphocytes was not influenced.     

Mitogenic reactivity of mononuclear cells isolated from thymus, spleen and umbilical cord blood of pig foetuses

The in vitro mitogenic reactivity of mononuclear cells from the thymus, spleen and umbilical cord blood of Danish Landrace pig foetuses ranging in gestational age (GA) from 48 to 112 days was monitored by means of a microculture lymphocyte transformation test (LTT). Dose-response profiles for concanavalin A (Con A), pokeweed mitogen (PWM) and leucoagglutinin (LAG) were set up for the various age-groups and the results showed that the onset and development of mitogenic reactivity in the pig foetus is age-related. The results indicate the occurrence of mitogen responsive cells in the thymus and cord blood at 48 days GA and in the spleen at 54 days but statistically significant reactivity (p less than 0.01) for the various tissues could only be demonstrated at later stages of gestation. Thymus cells from all foetuses ranging in GA from 54 to 112 days exhibited significant reactivity to Con A, PWM and LAG. While the first detectable definite response of spleen cells was seen at 60 days GA when 50% of the foetuses exhibited significant reactivity to the 3 mitogens, spleen cells from all foetuses beyond that age responded significantly. Cord blood cells from only 50% of the foetuses of 60 and 70 days GA responded significantly to Con A and PWM but after this stage, cord blood cells from all foetuses did. The first significant response of cord blood cells to LAG was seen at 70 days GA but only in 50% of the foetuses and it was not until 100 days GA that significant reactivity to LAG was detected in all foetuses.     

Mitogen-induced transformation of sheep and goat peripheral blood lymphocytes in vitro: The effects of varying culture conditions and the choice of an optimum technique

Peripheral blood lymphocytes (PBL) prepared by centrifugation of heparinized sheep or goat jugular venous blood on Ficoll-Triosil were shown to incorporate methyl-[H³]-thymidine ([H³]-Tdr) in vitro in response to lymphocyte mitogens.Optimal conditions for transformation included the culture of 2.5 × 10⁵ viable cells per round bottomed culture well in 250μl medium RPMI-1640 supplemented with fetal calf serum (FCS) at 10% for goat or 15% for sheep lymphocytes. Optimum incorporation of [H³]-Tdr by sheep PBL was recorded after 3–5 days and was achieved in response to 100μg/ml phytohaemagglutinin (PHA), 20μl/ml pokeweed mitogen (PWM), 10μg/ml Concanavalin-A (Con-A) and 50μg/ml bacterial lipopolysaccharide (LPS). For goat PBL the optimum mitogen concentrations were 50μg/ml PHA, 20μl/ml PWM, 5μg/ml Con-A and 50μg/ml LPS. Optimum PHA concentrations were influenced by the level of FCS supplementation, higher concentrations of PHA being required for optimum response when the concentration of FCS was increased.While variability within preparations was small there was considerable variation in the magnitude of the response between preparations, which was sufficient to confound comparisons between different experiments and between animals. The variability between preparations could not be attributed to changes in sensitivity of PBL to mitogens or to the influence of erythrocyte contamination of the PBL preparations. While these results are in general agreement with previous reports of optimal conditions for the measurement of ruminant PBL to mitogens, there are some important differences which are discussed in the context of the available literature.     

Immune defects in simian acquired immunodeficiency syndrome

We recently reported a Simian Acquired Immunodeficiency Syndrome (SAIDS) in rhesus macaques at the California Primate Research Center. Here, we studied in vitro lymphocyte response to the mitogens Concanavalin A (Con A), phytohemagglutinin (PHA) and pokeweed mitogen (PWM) with and without interleukin 2 (IL-2). Immunoglobulin (IgG and IgM) and complement (C3 and C4) concentrations were determined by radial immunodiffusion. T helper and T suppressor lymphocytes were identified with the monoclonal antibodies OKT4 and OKT8. Concentrations of IgG and IgM were significantly (p less than .05) decreased. Complement component C3 did not change but C4 was increased. The absolute lymphocyte count decreased but the OKT4:OKT8 ratio was unchanged from controls. A decreased lymphocyte response to all mitogens occurred early and became more severely depressed near death. IL-2 caused a complete or partial restoration of the response to the mitogens CON A and PHA. Both the humoral and cell mediated immune responses are affected in SAIDS. The role of IL-2 in this immune defect must be studied further.     

Peripheral lymphocyte function in dogs with Brucella canis infection

Peripheral lymphocyte function in dogs with Brucella canis infection was evaluated using in vitro lymphocyte stimulation with the mitogens PHA, Con A and PWM, and killed Brucella canis organisms. Bitches with naturally occurring Brucella canis infection were compared to negative controls. There was no difference in the response to Con A and PWM between these two groups. Lymphocytes from infected dogs were less responsive (p less than .05) to PHA than were lymphocytes from controls. There was a significant (p less than .005) difference in response to Brucella canis antigen between the two groups. Lymphocytes from infected dogs were stimulated by Brucella canis antigen, whereas those from controls did not respond.     

Parameters of production and partial characterization of feline interleukin 2

The conditions for the production of feline interleukin 2 (IL-2) from peripheral blood leukocytes (PBL) and splenocytes by concanavalin A (Con A) stimulation are described. Feline IL-2 was quantitated by measuring DNA synthesis in the murine IL-2-dependent cell line, CTLL-20. In addition, feline IL-2 was generated for the maintenance of long-term cultures of Con A-stimulated feline PBL and for biochemical characterization. Finally, IL-2 production was evaluated from the PBL of feline leukemia virus (FeLV)-infected cats. Con A at 9.6 micrograms/ml produced a plateau of peak IL-2 activity from 24 to 48 h following stimulation. The tumor promoter, phorbol myristic acetate, stimulated feline IL-2 production and enhanced Con A-stimulated feline IL-2 production. Fetal calf serum (FCS) was not required for IL-2 production; however, FCS at 5% (v/v) allowed for maximal Con A-stimulated IL-2 production. Feline IL-2 generated from Con A-stimulated splenocytes migrated with an apparent molecular size of 13.7 to 23 kD by gel filtration chromatography and supported the proliferation of Con A-activated feline PBL at a final concentration of 0.3 to 0.9 units/ml.