Effect of serum on lymphocyte blastogenesis. 2. Characterization of disease-induced immunosuppressive factors by chromatography and molecular weight determination

Numerous infectious and noninfectious diseases are associated with the appearance of suppressive serum lymphocyte immunoregulatory factors (SLIFs). The suppressive SLIFs in sera from clinically healthy dogs and from dogs with bacterial (staphylococcal, brucellar) or mycotic (blastomycotic) infections were further characterized by dialysis, fractionation by ultrafiltrations and HPLC (high performance liquid chromatography) sieving, by affinity chromatography on protein A-Sepharose columns, and by DEAE-cellulose ion exchange chromatography. Factors of various molecular weights and of various elution patterns from DEAE-cellulose and affinity chromatography columns were taking part in the suppressive action of the whole serum. The 'common' inhibitors present in all sera were in the molecular weight range of 28 to 35 Kd, whereas the disease-induced suppressive SLIFs were present in various molecular weight categories. 'Common' suppressor SLIFs and some SLIFs from dogs with staphylococcal infections were partially dialysable; suppressive SLIFs induced in dogs with generalized brucellosis and blastomycosis were not dialysable. Protein A bound suppressive SLIFs from two of three dogs with staphylococcal pyodermas. DEAE-cellulose chromatography gave variable elution patterns with different animal sera. It is concluded that various suppressive SLIFs contribute to the immunosuppressive effect of the whole serum and no disease-specific suppressive SLIF could be identified.     

Inhibition of lymphocyte blastogenesis by whey

Bovine whey samples were evaluated by use of lymphocyte-transformation tests to determine their effect on lymphocyte blastogenesis. Whey samples from mammary glands with clinical mastitis strongly inhibited DNA synthesis and blastogenesis in lymphocytes stimulated with mitogens or dividing because of bovine leukemia virus infection. Whey samples from apparently healthy glands either did not inhibit lymphocyte DNA synthesis or inhibited it to a lesser degree than did whey from mastitic glands. Degree of inhibition was dose-dependent. The molecules causing inhibition were noncytotoxic and underwent minimal binding to the lymphocytes. Inhibitory molecules were susceptible to various proteolytic and glycolytic enzymes, indicating a glycoprotein-like structure. Whey inhibited incorporation of thymidine if it was in the cell cultures during the early stages of stimulation. Incubation of lymphocytes in whey that inhibited thymidine incorporation did not affect DNA synthesis in subsequent culturing of the same cells without whey. Degree of inhibition was affected by the method of whey preparation.     

Regulation of phytohemagglutinin-induced bovine lymphocyte blastogenesis by leukotrienes

Leukotriene (LT) B4, a 5-lipoxygenase metabolite of arachidonic acid, is a potent inducer of suppressor cells in phytohemagglutinin-stimulated cultures of bovine peripheral blood mononuclear cells. In contrast, LTC4 and LTD4 have little activity. Incubation of T lymphocytes with LTB4 at concentrations as low as 1 X 10(-12)M rendered these lymphocytes suppressive of [3H]thymidine incorporation in subsequent phytohemagglutinin-stimulated cultures of fresh autologous lymphocytes. This LTB4-induced cell was radiosensitive to irradiation at 2,000 rads. Leukotriene B4 may have an important part in immunoregulation during hypersensitivity reactions.     

Effect of levamisole on lymphocyte blastogenesis and neutrophil function in dexamethasone-treated cattle

Levamisole was evaluated at 6 dose levels for its ability to prevent the dexamethasone-induced suppression of in vitro lymphocyte blastogenesis or neutrophil function in cattle. Dexamethasone (0.4 mg/kg of body weight, IM) and levamisole hydrochloride (0.5, 1.0, 2.0, 4.0, or 8.0 mg/kg orally) were administered to groups of 4 cattle daily for 3 days. Another group of 4 cattle were given the 3-day dexamethasone treatment and 6.0 mg/kg of levamisole (the recommended anthelmintic dose) was given only once on the 1st day that dexamethasone was given. Results obtained from the dexamethasone-levamisole-treated cattle were compared with results obtained from cattle that were given only dexamethasone. Levamisole had no apparent consistent ability to enhance lymphocyte blastogenic responsiveness (to the mitogens phytohemagglutinin, concanavalin A, or pokeweed mitogen or in a 1-way mixed lymphocyte reaction) or to enhance neutrophil function (random migration, nitroblue tetrazolium reduction, iodination, or antibody-dependent cell-mediated cytotoxicity) in dexamethasone-treated cattle.     

Inhibition of in vitro lymphocyte blastogenesis by chicken alpha-fetoprotein

Chicken alpha-fetoprotein (ch-AFP), purified from fetal chicken serum and embryo extracts, respectively, was examined for its immunomodulatory effect in vitro. Significant (P less than 0.05) suppression of the allogeneic mixed lymphocyte reaction (MLR) was observed, when these preparations were added to one-way mixed lymphocyte cultures (MLC) in quantities of 62.5-1000 micrograms/ml. Suppression of the MLR was depending on the presence of ch-AFP for at least 16 h after initiation of the MLC, suggesting that this fetal protein was acting mainly in the early phase of lymphoblastogenesis. Serum of chicken embryos (12th and 15th day of incubation), day-old chickens, and of 10-week-old chickens of four different inbred lines were also found to exert suppression of the MLR. From these data, it is hypothesized that ch-AFP plays an immunoregulatory role by maintaining a certain stage of self tolerance during differentiation of the avian immune system.     

Effect of hydrocortisone on circulating lymphocyte numbers and their mitogen-induced blastogenesis in lambs

The effect of hydrocortisone on the number of circulating lymphocytes and their blastogenic response was studied in 20 feedlot lambs given combinations of 3 treatments: hydrocortisone (25 mg/kg of body weight, 4 times a day, IM), feed changes (100% roughage to 90% concentrate over a 6-day period), and oral inoculation of Pasteurella haemolytica biotype T (10(9) to 10(11) bacteria/day via stomach tube) to develop a model for reproduction of septicemic pasteurellosis. Hydrocortisone caused lymphopenia and inhibited the blastogenic response of peripheral blood lymphocytes to phytohemagglutinin and concanavalin A mitogens. A synergistic effect was observed between hydrocortisone injections and feed changes resulting in higher than expected serum hydrocortisone concentrations and lower circulating lymphocyte counts. Seemingly, stress-induced increases in serum hydrocortisone concentrations cause suppression of the immune response of feedlot lambs. The combined effect of feed changes and stress on the immune response of lambs may explain the role of these 2 factors in the pathogenesis of septicemic pasteurellosis.     

Suppression of lymphocyte blastogenesis by sera from calves transported by road

The effect of sera from 4-h road-transported calves on mitogen-induced blastogenesis of bovine lymphocytes was investigated. Sera collected just after transportation showed a significant suppression (P less than 0.05) on blastogenesis. The immunosuppressive activity was reduced from those sera when they were treated with dextran-coated charcoal. Fractionation of the sera by ultrafiltration revealed that the activity was mainly concentrated in the fractions of molecular weights less than 50 kD.     

Effect of recombinant human granulocyte colony stimulating factor on lymphocyte blastogenesis in healthy dogs

Effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on the number and blastogenesis of lymphocytes were evaluated in clinically healthy dogs treated subcutaneously with rhG-CSF at a dose of 2.5 microg/kg for 3 days. Significant increases in the number of leukocytes and segmented neutrophils were observed after the administration of rhG-CSF. The number of lymphocytes also increased on days 1 and 2 after the treatment. Activities of phytohemagglutinin, concanavalin A, and pokeweed mitogen-induced lymphocyte blastogenesis (LB) were augmented to twice the pretreatment levels by the administration of rhG-CSF. These results suggested that administration of rhG-CSF activated lymphocyte functions such as LB in healthy dogs.     

Suppression of lymphocyte blastogenesis by porcine interferon-α

Peripheral blood lymphocytes (PBL) from five four-day-old and five six-week-old piglets were treated with 10 to 320 units of porcine interferon-alpha, and their blastogenic responses to phytohaemagglutinin or pokeweed mitogen were compared with those of control lymphocytes. There was significant inhibition of the blastogenic response to phytohaemagglutinin by 320 units of interferon-alpha, and of the response to pokeweed mitogen by 320 and 160 units of interferon-alpha. Porcine interferon-beta was cytotoxic to porcine PBL. The blastogenic response to pokeweed mitogen was significantly higher in PBL from the younger piglets.     

Cell mediated immunity in equine herpesvirus type 1 infection I. In vitro lymphocyte blastogenesis and serum neutralization antibody in normal parturient and aborting mares.

Blastic transformation of peripheral blood mononuclear cells and serum neutralization antibody levels for equine herpesivurs type 1 were measured in 19 mares from three farms at the time of termination of their pregnancy by normal foaling or viral abortion. The stimulation indexes of lymphocytes obtained from the mares from two farms (Farm 1 and 2) which had virus abortions, ranged from 2.1 to 10.8. But there was no significant difference in stimulation index levels between the aborting and normal foaling mares on these two farms. Equine herpesvirus type 1 was isolated from the mononuclear cells of one mare (No. 5) about two months after she aborted. The stimulation index of lymphocytes from that mare was not significantly different from that of other mares on these farms. Stimulation index of lymphocytes from the mares on one farm (Farm 3) where there was no virus abortion or previous history of virus abortion but were exposed to virus antigen from vaccination, ranged from 1.6 to 2.9. The serum neutralization antibody levels were low in most mares ranging from 1/4 to 1/20 and in three mares these were higher. There was no direct correlation between the levels of serum neutralization antibody and stimulation index of lymphocytes from the mares on these farms.     

Association of class I bovine lymphocyte antigen complex alleles with in vitro blood neutrophil functions, lymphocyte blastogenesis, serum complement and conglutinin levels in dairy cattle

Ninety-eight lactating Holstein cows from two genetic lines selected for high and average milk production were used in the study. Five peripheral blood samples were collected over a 60-day period from each cow for evaluation of neutrophil function, lymphocyte blastogenesis, leukocyte count, and serum complement and conglutinin levels. Blood samples were typed for antigens encoded by alleles at the bovine major histocompatibility complex (BoLA) A locus. Alleles w14(w8), w20A, and w19(w6) were the most frequent of 14 alleles present in this herd. Association of BoLA type with immune function results was examined by using gene substitution models including and ignoring sire effects. Alleles w15(w8) and w16 were associated with greater circulating mononuclear cell and total leukocyte numbers, while w27(w10), w11, and w20A were associated with lower numbers of these cell types. Alleles EU28D and w20A were positively and negatively associated with granulocyte percentage, respectively. Allele w16 was associated with greater antibody-independent neutrophil cytotoxicity, unstimulated lymphocyte proliferation, serum conglutinin activity, and with lower antibody-dependent neutrophil cytotoxicity. Allele w19(w6) was associated with decreased conglutinin activity and decreased neutrophil iodination. Increased antibody-dependent neutrophil cytotoxicity was observed for animals bearing allele w14(w8), and decreased neutrophil iodination, serum conglutinin, and nonstimulated lymphocyte blastogenesis were observed in individuals carrying w20A or EU28D. Significance of both sire and BoLA complex effects suggests that both major histocompatibility complex genes and background genes of the sire significantly affect immune function. This research suggests BoLA-A locus genes may be major genes or markers for closely linked major genes involved in regulation of nonspecific immune function.     

Effect of recombinant human interferon-alpha in vitro and in vivo on mitogen-induced lymphocyte blastogenesis in cats

The effect of recombinant human interferon-alpha (rHuIFN-alpha) in vitro and in vivo on mitogen-induced lymphocyte blastogenesis was evaluated in specific-pathogen-free cats. Pre-incubation of isolated feline peripheral blood lymphocytes (PBL) in vitro with either 10(4) or 10(3) International Units (U) of rHuIFN-alpha for 24 h significantly suppressed (P less than 0.001 and 0.01, respectively) blastogenic responses to the phytomitogens concanavalin A (Con A) and pokeweed mitogen (PWM). Lower doses of IFN (range, 10-10(-3) U/ml) neither suppressed nor enhanced mitogenesis. In the absence of phytomitogens, incubation of PBL with 10(4) - 10(2) U (P less than 0.001) or 10 U (P less than 0.05) of rHuIFN-alpha/ml resulted in a significant decrease in incorporation of [methyl-3H] thymidine into newly synthesized cellular DNA. Cultures of PBL exposed continuously for 4 days to rHuIFN-alpha doses of 10(4) U/ml or less did not demonstrate specific reductions in cell viability, indicating that the observed antiproliferative actions of IFN apparently were independent of any direct cytotoxic effects. To investigate the dose-response effects of rHuIFN-alpha in vivo on lymphocyte blastogenesis, individual groups of cats were evaluated on 3 consecutive days before and then 24 h after each cat was inoculated intramuscularly with either a high dose (10(6) U/kg), moderate dose (10(4) U/kg), or a relatively low dose (10(2) U/kg) of rHuIFN-alpha. Cats inoculated with 10(6) U of rHuIFN-alpha/kg had significantly reduced (P = 0.037) blastogenic responses to Con a at 24 h postinoculation compared to preinoculation values; mean PWM responses were also decreased, but this effect was not statistically significant. In contrast, inoculation of cats with either 10(4) or 10(2) U of rHuIFN-alpha/kg significantly enhanced (P = 0.05 or 0.008, respectively) Con A-induced blastogenesis and had no discernible effect on PWM responses. These findings suggest that very high doses of rHuIFN-alpha given parenterally may be associated with suppression of certain T-cell responses in cats; conversely, much lower doses may be immunoenhancing.     

Bovine sire effects on daughters' in vitro blood neutrophil functions, lymphocyte blastogenesis, serum complement and conglutinin levels

Blood neutrophil functions, lymphocyte blastogenic responses, serum complement, and serum conglutinin activity of 98 lactating Holstein cows from two genetic lines were evaluated. The genetic lines were produced in a selection experiment that created and perpetuated genetic differences in milk production for up to seven generations. No significant differences between the two genetic lines of cows were found for neutrophil function, lymphocyte blastogenic responses, serum complement levels, or serum conglutinin levels. Significant differences between sire progeny groups within lines were found for unstimulated and mitogen-stimulated lymphocyte blastogenesis (P less than 0.0001), and almost all neutrophil functions (antibody independent neutrophil cytotoxicity, antibody dependent neutrophil cytotoxicity, ingestion of bacteria, iodination, chemiluminescence, chemokinesis, and chemotaxis (P less than or equal to 0.05)). Sire progeny group differences (P less than or equal to 0.0001) within lines for serum complement and conglutinin activity were also found. Neutrophil chemiluminescence activity (positive relationship; P less than or equal to 0.001), concanavalin A-stimulated lymphocyte blastogenesis (positive relationship; P less than or equal to 0.004), and serum conglutinin activity levels (negative relationship; P less than or equal to 0.01) each had small but significant associations with the total milk somatic cell count. Cows seropositive for bovine leukosis virus had increased resting and mitogen-stimulated lymphocyte blastogenic activity and were associated with increased in vitro neutrophil random migration and production of superoxide anion. Estimates of genetic parameters of various immune cell functions, of serum complement and of conglutinin levels for daughters of 11 sires with 4-6 daughters in the data set were determined. In this report, genetic variation was demonstrated for nonspecific humoral and cellular immunity.     

Effects of ACTH administration on bovine polymorphonuclear leukocyte function and lymphocyte blastogenesis

Yearling steers were treated with ACTH to determine the effect of increased plasma cortisol concentration on bovine lymphocyte and polymorphonuclear leukocyte (PMN) function. The administration of ACTH caused a significant (P less than 0.01) increase in serum cortisol concentration and depression of lymphocyte blastogenesis in response to phytohemagglutinin and concanavalin A. The response to pokeweed mitogen was also depressed, but not significantly. Random migration by PMN was significantly enhanced by ACTH treatment, but there was no effect on ingestion of Staphylococcus aureus, nitroblue tetrazolium reduction, or antibody-dependent cell-mediated cytotoxicity by PMN. The iodination reaction, which evaluates the activity of the myeloperoxidase-hydrogen peroxide-halide antibacterial system of the PMN, was significantly impaired after ACTH treatment. These data indicate that specific parameters of lymphocyte and neutrophil function were impaired directly or indirectly by elevated in vivo concentrations of plasma cortisol.     

Enhancement of lymphocyte blastogenesis and neutrophil function by avridine in dexamethasone-treated and nontreated cattle

The lipoidal amine, N,N-dioctadecyl-N',N'-bis (2-hydroxyethyl) propanediamine (avridine or CP 20,961), formulated in liposomes, was evaluated for its effect on leukocyte kinetics, lymphocyte blastogenesis, and polymorphonuclear leukocyte (PMN) function in dexamethasone-treated and nontreated cattle. In the 1st experiment, cattle were given avridine in a single IM injection of 0.1, 1.0, or 10 mg/kg of body weight. All doses induced swelling at the injection site, a febrile response, and a leukocytosis due to a neutrophilia. Mononuclear cell numbers were normal. All 3 groups of avridine-treated animals had a higher mean lymphocyte blastogenic response to mitogens on the 4 days after administration than did the control nontreated animals. Avridine administration was associated with an enhanced ability of PMN to ingest Staphylococcus aureus and to mediate antibody-dependent cell-mediated cytotoxicity (ADCC). The highest dose (10 mg/kg) was associated with a depression of the ability of PMN to iodinate protein. An effect of avridine on PMN random migration under agarose or nitroblue tetrazolium (NBT) reduction was not observed. In a 2nd experiment, cattle were given no treatment, 0.04 mg of dexamethasone/kg IM, or 10 mg of avridine/kg IM followed 24 hours later by 0.04 mg of dexamethasone/kg. Dexamethasone administration caused a leukocytosis due to a neutrophilia with normal mononuclear cell numbers, an enhancement of PMN random migration under agarose, and an inhibition of NBT reduction, iodination, and ADCC activity of PMN. Dexamethasone did not have a detectable effect on lymphocyte blastogenesis or on ingestion of S aureus by PMN.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immune response of cattle to Brucella abortus outer membrane proteins measured by lymphocyte blastogenesis

Lymphocytes from cattle were tested in a blastogenesis test with outer membrane proteins isolated from smooth strain 2308 and rough strain 45/20 of Brucella abortus. The titration assay developed for measuring blastogenesis to microbial antigens (Baldwin, Antczak and Winter, this issue, pp. 319-333) was used to assess the response to both group 2 (porins) (Douglas et al., 1984) and group 3 proteins (Verstreate et al., 1982). Blastogenesis was evaluated for distinguishing cattle infected with virulent B. abortus strain 2308 from unimmunized cattle, cattle vaccinated with attenuated strain 19, or inoculated with Escherichia coli 0116:H31, known to cause serological cross-reactions with B. abortus (Nielsen et al., 1980). Strain 45/20 porin was the most effective for this purpose and data analyses utilizing the titration assay were better than those relying on a single point assay. When compared with BASA, an antigen preparation used in other studies (Kaneene et al., 1978a), responses to porin provided a more specific index of infection with B. abortus. Reactions to 45/20 porin occurred, however, in some heifers vaccinated as adults with strain 19 or inoculated with E. coli 0116:H31. Furthermore, nonpregnant heifers had negligible or only transient blastogenesis responses to the porin during the first 14 weeks after infection even though they developed strong 0 antibody responses. We do not recommend the blastogenesis test in its present form as a useful adjunct to serological tests, and could allow measurement of cell mediated immune responses relevant to protective immunity.     

Suppression of specific lymphocyte blastogenesis after intravenous tuberculin injection in Mycobacterium bovis-sensitized cattle.

Intravenous injection of 1.0 ml of old tuberculin (OT) in 4 calves previously sensitized to Mycobacterium bovis resulted in suppression of specific lymphocyte blastogenic responses to purified protein in vitro. Suppression of in vitro blastogenic responses occurred within 24 hours after injection of OT and persisted for up to 96 hours. Lymphocytes from these calves, cultured with concanavalin A, did not show any suppression of the lymphocyte blastogenic response in vitro. Suppression was temporary and in all calves was preceded by rapid febrile responses. Rectal temperature responses ranged from 40.1 to 41.6 C as early as 6 hours after tuberculin injections. Three control animals, M bovis-sensitized but not given OT, remained lymphocyte stimulation-positive throughout the study and served as controls for laboratory fluctuations.