Insoluble immune complex-stimulated neutrophil leukotriene B4 production is dependent on Fc gamma RII and Fc gamma RIII and independent of pertussis toxin-sensitive signal transduction pathways.

Leukotriene B4 (LTB4) release initiated by interaction of immune complexes (ICs) with Fc gamma RII and Fc gamma RIII receptors on human neutrophils was studied using well-defined complexes. Immune complexes consisting of polyclonal rabbit antibody to human albumin were prepared at equivalence (insoluble complex) and at five times antigen excess (soluble complex). Incubation of human neutrophils with soluble and insoluble ICs led to the synthesis of LTB4 from endogenous arachidonic acid (AA). LTB4 release induced by ICs was markedly inhibited by monoclonal antibodies against either Fc gamma RII or Fc gamma RIII receptor. Treatment of neutrophils with pertussis toxin significantly inhibited the release of LTB4 induced by soluble ICs. However pertussis toxin treatment minimally inhibited the LTB4 release induced by insoluble ICs. Crosslinking of either Fc gamma RII and Fc gamma RIII receptors on neutrophil surfaces induced LTB4 release. This is the first experimental observation showing that both Fc gamma RII and Fc gamma RIII directly induce neutrophil LTB4 metabolism in the absence of exogenous AA. These studies also suggest the involvement of novel pertussis toxin insensitive signal transduction pathways in insoluble ICs stimulation of neutrophils.

Bovine neutrophils treated with chemotactic agents: morphologic changes.

Neutrophils were isolated from the peripheral blood of cattle. After the neutrophils were incubated with zymosan-activated serum, the neutrophils changed from spherical to a bipolar shape. Ninety percent of the neutrophils became bipolar in 5 to 10 minutes. Bacterial cell filtrate and casein also induced bipolar shape changes in neutrophils, but N-formyl-L-methionyl-L-leucinyl-L-phenylalanine did not. The neutrophil-shape-change response was a rapid in vitro assay to evaluate early chemotactic events.

Inhibition of mitogen-induced blastogenesis in human lymphocytes by T-2 toxin and its metabolites.

Concentrations of T-2, HT-2, 3'-OH T-2, 3'-OH HT-2, T-2 triol, and T-2 tetraol toxins which inhibited [3H]thymidine uptake in mitogen-stimulated human peripheral lymphocytes by 50% were 1.5, 3.5, 4.0, 50, 150, and 150 ng/ml, respectively. The results suggested that the initial hydrolysis of T-2 toxin and the hydroxylation of T-2 toxin to 3'-OH T-2 toxin did not significantly decrease the immunotoxicity of the parent molecule, whereas further hydrolysis to T-2 triol and T-2 tetraol toxins or hydroxylation to 3'-OH HT-2 toxin decreased in vitro toxicity for human lymphocytes.

Host defense systems in cattle exposed to polybrominated biphenyl.

In vivo and in vitro immunologic studies were undertaken in 114 Holstein cattle, 60 of which had detectable polybrominated biphenyls (PBB) in adipose tissue. The tissue PBB concentrations were between 0.02 and at least 1,000 mg/kg. An immunologic test profile was used to evaluate the influence of PBB on bovine immune competence. The profile included serum immunoglobulin determinations, autoantibody studies, lymphocyte subpopulation determinations, lymphoblastogenesis assays, delayed hypersensitivity skin testing, and neutrophil function studies. The results of the evaluation indicated that the host defenses are intact, both in quantity and function, in cattle with PBB body burdens ranging from 0.02 and 24.0 mg/kg for at least 2 years. Cattle fed 25 g of PBB daily during the acute toxicity study exhibited usual immune competence until they accumulated 500 g of PBB (tissue concentrations exceeding 1,000 mg/kg). Cattle that were fed more than 500 g of PBB became moribund and exhibited changes in neutrophil function tests and serum antibody titers.

Subchronic administration of technical pentachlorophenol to lactating dairy cattle: immunotoxicologic evaluation.

Eight lactating Holstein-Friesian dairy cattle were randomly allotted as pairs to eight a control or a treatment group fed technical pentachlorophenol (penta) from 6 +/-1 wk postpartum for about 135 d (0.2 mg/kg.d for 75-84 d followed by 2.0 mg/kg.d for 56-62 d). Jugular blood was drawn periodically for immunologic studies. Leukocyte differentials and lymphocyte subpopulations (e.g., T and B cells) were enumerated for each blood specimen. Several in vitro and in vivo assays were conducted to evaluate lymphocyte functions, including (1) quantitation of serum immunoglobulins G, M, and A; (2) induction of blastogenesis in vitro, using concanavalin A and leukoagglutinin as mitogens; (3) measurement of extent of skin reaction to injected purified protein derivative in BCG-sensitized cattle to evaluate delayed hypersensitivity; and (4) quantitation of antibody formation in response to injected sheep red blood cells. Neutrophil function was evaluated by latex particle phagocytosis and chemiluminescence. The results showed no statistically significant differences between control and pent-treated cattle during eigher treatment period. Also, no histopathologic changes were noted in lymphoid tissues including spleen, thymus, and lymph node.

Immunological dysfunctions in multiple sclerosis. I. Diminution of 'active' thymus-derived lymphocytes and presence of immunomodulating serum factors.

The peripheral blood of twenty-eight patients with multiple sclerosis (MS) was examined for 'active' thymus (TA) derived lymphocytes (PBL) and the presence of serum factors which impair the lymphocyte blastogenic response and the migration of leucocytes from capillary tubes. MS patients exhibited a significant reduction in the level of circulating TA lymphocytes (15.2+/-2.7 vs 22.9+/-2.9, P less than 0.001). Additionally, MS serum markedly impaired the mobility and the mitogen induced blastogenic response of autologous and allogenic (control) PBL. Impairment in the blastogenic response was observed only when serum was added at culture initiation or after 4 hr. The results support the concept of a generalized deficiency in the immune competence of MS patients. The pathophysiological role of a serum suppressive factor is discussed.

Immunologic studies in Holstein-Friesian cattle: an immunocompetence profile.

An immunocompetence profile has been developed for Holstein-Friesian cattle. In vitro and in vivo immunoassays were used to determine (1) the percentage of peripheral blood lymphocyte (PBL) subpopulations bearing surface-membrane receptors for sheep erythrocytes, complement, or surface immunoglobulin; (2) the PBL blastogenic response to leukoagglutinin, concanavalin A, or pokeweed mitogen, (3) the serum immunoglobulin concentrations of IgG, IgM, and IgA, (4) the humoral antibody response to keyhole limpet hemocyanin, and (5) the in vitro blastogenic response and in vivo delayed hypersensitivity skin reaction to purified protein derivative in cattle immunized with BCG.

Marmoset species variation in the humoral antibody response: in vivo and in vitro studies.

A comparison of the in vivo and in vitro antibody response capabilities of two marmoset species, Saguinus fuscicollis and Saguinus oedipus oedipus, revealed the former to be superior in elaborating humoral antibody. In vivo challenges with Escherichia coli lipopolysaccharide (LPS) and Salmonella typhi flagella consistently yielded higher antibody titres in S. fuscicollis; indeed, with LPS antigen, multiple inoculations of S.o. oedipus marmosets led ultimately to a decrease in antibody formation, in contrast to the anamnestic response of S. fuscicollis. This species differential in immune competence was also suggested in the in vitro stimulation of peripheral blood leucocytes (PBL) and spleen cells with sheep red blood cells (RBC). None of 55 S.o. oedipus PBL cultures and 49 of 89 (55%) S. fuscicollis cultures responded to the test antigen. A similar differential in response to sheep RBC was noted with the spleen cells of each species, although this report contrasts the antibody-forming potential of two marmoset species, a comparison of the immunological response profile of marmosets to those of other laboratory animals challenged with similar antigens suggests these primates may be relatively incompetent. The possible relationship between the haemopoietic chimerism of marmosets and a diminished immune competence is discussed.

Immunological studies in cattle exposed to polybrominated biphenyls.

The intactness of the immune system in cattle exposed to polybrominated biphenyls (PBBs) has been investigated by using several immunoassays. Eighty-seven animals have been studied, 35 control animals (not exposed to PBBs) and 52 animals exposed to PBBs (0.02-30 ppm/g fat equivalent). The immunoassays included a complete blood count, identification of peripheral blood T and B lymphocyte subpopulations, serum immunoglobulin levels (IgG, IgM, and IgA), the in vitro response to lymphocytes to phytolectins (PHA, Con A, PWM), the antibody response to Keyhole limpet hemocyanin (KLH), the cell-mediated response to PPD, and determination of autoantibodies and/or immunosuppressive serum factors. For control and PBB-exposed cattle, there was no statistical difference between the number of circulating erythrocytes or leukocytes, the hematocrit, or hemoglobin content; the percentage or number of T and B lymphocytes; the isotope incorporation index (DNA synthesis) of lymphocytes in response to mitogens; the concentrations of serum immunoglobulins IgG, IgM, or IgA; the mean peak titer to KLH; or in vivo or in vitro immune response to PPD.Additional evaluation of cattle with tissue levels of PBB greater than 3 ppm/g tissue for hematological and immunological parameters revealed no statistical difference from control animals. Other experiments were performed to evaluate serum from cattle exposed to PBBs for autoantibodies to smooth muscle, mitochondrial or nuclear antigens. No evidence for autoantibodies was observed. Further studies were done to examine the cytotoxic and/or immunosuppressive activity of sera from PBB-exposed animals. In these studies, the blastogenic response of lymphocytes from control cattle and humans were evaluated in the presence and absence of serum from animals exposed to PBBs (> 3 ppm/g tissue). No evidence for either a cytotoxic or an immunosuppressive influence of such sera was demonstrable. Our studies indicate that PBB, at the levels studied, does not alter or interfere with lymphocyte surface antigens, the complex nuclear and cytoplasmic events required for mitosis and cell division, or the biological events required for antibody formation and cell-mediated immune reactions. Further, PBB exposure at the levels studied does not predispose cattle to autoantibody production or leucotoxic serum factors.

T-and B-lymphocyte chimerism in the marmoset.

Marmosets are natural blood chimeras, this condition resulting from the high frequency of fraternal twinning and the consistent development of placental vasular anastomoses between the two embryos. Identification of chimerism by sex-chromosome analysis of cultured blood lymphocytes provided a means of determining the proportion of chimerism among T and B lymphocytes. Peripheral blood lymphocytes were enriched for T or B cells by filtration through a nylon column (yields greater than 95 per cent T-cells) or inactivation of T lymphocytes by treatment with a goat anti-marmoset thymocyte antiserum in the presence of complement (yeilds greater than 95 per cent B cells). Mitogenic stimulation of these separated, enriched cell populations yielded metaphase plates which could be scored for percentage male and female cells. Tests on five different blood chimeras showed the T- and B-lymphocyte chimerism to be the same. Stimulation of blood lymphocytes with cells from another species of marmoset in a mixed lymphocyte culture test revealed the chimeric T-cell response (i.e., host and co-twin cells) to be similar to that obtained with a mitogenic lectin. The demonstration of equivalent T- and B-cell chimerism in these animals suggests derivation of these cells from a common stem cell pool and the response of both T-cell populations to an antigenic stimulus in proportions similar to their percentage chimerism suggests complete immunologic tolerance exists in this species for co-twin histocompatibility antigens.

Characterization of the antibody response of the marmoset to sheep red blood cells.

The immune competence of two species of marmosets, S. fusciollis and S. oedipus, was evaluated by the intravenous (i.v.) and intramuscular (i.m.) injection of sheep red blood cells (SRBC). In S. fusciollis marmosets, 1 ml of a 50% suspension yielded titres of haemolysin and agglutinating antibodies equal to or greater than 1 ml of a 10% dose of antigen. In both species, the i.v. route, while resulting in formation of 19S and 7S agglutinins, yielded only 19S haemolysins, even after multiple antigen injections. Repeated i.v. injections resulted in a progressive decrease in peak titres, in contrast to the i.m. route, where booster inoculations gave a typical anamnestic response. Jerne plaque-forming cells (PFC) in the spleens of S. oedipus marmosets showed predominately 19S plaques after a primary i.v. challenge; only 19S PFC were detected in the spleen of an animal that had been given multiple inoculations, the type of antibody produced reflecting that found in the serum. 19S but not 7S haemolysins of both species were sensitive to heating at 56 degrees C for 1/2 hr. The serum titres and splenic PFC data from the marmosets suggest these animals, particularly S. oedipus, respond poorly to SRBC when a comparison is made to similar studies in mice and rats.

Discussion paper: impairment of B-lymphocyte functions in concanavalin A-treated friend virus infected mice.

Friend leukemia virus (FLV) leukemogenesis was prevented by treatment of the virus with Concanavalin A (Con A). Mice infected with the lectin-treated virus, however, showed evidence of a dormant infection since infectious virus could be recovered for as long as 100 days. Humoral immune responses to sheep erythrocytes (SRBC), a thymus-dependent antigen, and to E. coli lipopolysaccharide (LPS), a thymus-independent antigen, were depressed (approximately 80-90%) in mice given the Con A-treated FLV. Cell transfer studies indicated that the impaired responsiveness to SRBC was related to a defect in B-lymphocyte function, similar to the impairment in mice infected with untreated FLV. The mitogenic response of splenocytes from Con A-FLV mice to E. coli LPS was also depressed as was the ability of Ig-bearing spleen cells to redistribute these immunoglobulin receptors into polar caps. The impaired immune responsiveness in the Con A-FLV infected mice appeared associated with the persistent virus infection and not to neoplastic transformation generally associated with leukemogenic process.

Lymphocyte surface receptors and leukemia virus-induced immunosuppression.

The number and distribution patterns of lymphocytes in the spleens and lymph nodes of Balb/c mice which express immunoglobulin surface receptors were studied in terms of the effects of a murine leukemia virus on the immune-response mechanism. Friend leukemia virus induces a prompt, marked depression of the immune response of mice to antigens such as sheep erythrocytes and E. coli LPS. A functioning T- and B-lymphocyte system is necessary for the response to the SRBC's whereas E. coli LPS, a T cell-independent antigen, stimulates B cells alone. Although the responses to both classes of antigen were markedly depressed in FLV-infected mice, the major defect appeared to be impairment of B-cell function, at least early in the course of infection. In order to examine in more detail the mechanism of interaction between FLV and lymphoid cells with Ig surface receptors, presumably B cells, immmunofluorescent analyses were performed with spleen, and lymph node cells from FLV-infected mice. Within a few days after infection there was a marked decrease in the percentage of spleen cells with Ig surface molecules, although the absolute number of these cells was either unchanged or increased due to marked splenomegaly caused by the virus. A marked decrease in the percentage of splenocytes with theta antigen, considered a marker for mature T cells, also was evident in infected mice. The number of spleen cells showing evidence of FLV infection (i.e., positive for FLV-associated antigens) increased rapidly during the first few days after infection, and within 2 to 2 1/2 weeks nearly all of the nucleated splenocytes were positive for the tumor antigen. In contrast to the results for spleen cells, there were increases rather than decreases in the percentages of Ig-positive and theta-positive cells in the lymph nodes after infection. The number of lymph-node cells that showed the presence of FLV antigen was much lower than in the spleen, and their appearance was also much slower as the leukemic process progressed. Despite these differences between spleen and lymph-node cells in terms of relative percentages of Ig- and theta-positive lymphocytes, relatively similar depressions were evident for the percentages of lymphoid cells that could redistribute their surface Ig receptors into polar caps when incubated with anti-Ig serum at 37 C. Marked impairment of the Ig-capping responses for both spleen and lymph-node cells paralleled the course of infection and development of immunosuppression. These observations indicate that murine leukemia virus infection can both alter the responsiveness of immunocompetent cells to T-dependent and independent antigens and depress the number and normal functional activity of these cells, as reflected by altered surface Ig receptors and antigens.

Stimulation of cAMP levels and modulation of antibody formation in mice immunized with cholera toxin.

Injection of mice with 1.0 mu g of a purified exotoxin derived from Vitro cholerae together with a challenge injection of sheep erythrocytes (SRBC)P OR E. coli LPS markedly influenced the immune response to these antigens. Simultaneous injection of the toxin with antigen resulted in a delayed appearance of antibody-forming cells during the first few days after immunization, followed by a marked enhancement of the peak numbers of antibody-forming cells. In the case of the immune response to SRBC, both 19S and 7S plaque-forming cells (PFC) were enhanced on the peak day of response after simulataneous immunization of toxin-injected mice. The secondary immune response to SRBC was also similarly affected when cholera toxin was given along with a second injection of erythrocytes: i.e. a delay in appearance of the first antibody-forming cells followed by a marked enhancement of the peak 19S and 7S PFC response. Injection of cholera toxin 103 days prior to SRBC or LPS was immunosuppressive. The effect of cholera toxin on the level of splenic cyclic AMP appeared related to the effects on antibody formation.

Immunosuppression in vitro induced by leukemia virus-infected splenocytes (38544).

Immunization of dispersed spleen cells from normal mice in vitro with SRBC was suppressed by simultaneous incubation of the spleen cell cultures with splenocytes from mice previously infected with FLV. Cell-free virus preparations alone did not suppress the antibody response. In contrast, relatively small numbers of splenocytes from infected mice, even when present at a ratio of 1-500 normal spleen cells, significantly suppressed the in vitro immune response to SRBC. Viable leukemic splenocytes were necessary for immunosuppression although the leukemic cells did not have to be in direct contact with the normal spleen cells. Specific anti-FLV serum, when added to the leukemic splenocytes or to normal spleen cells separated from infected cells by cell-impermeable membranes, prevented immunodepression.