J.F.T. Griffin

Macrophage function in deer

Macrophage inflammatory and immune functions were characterised in red deer (cervus elaphus), for use as a model for natural infection with bovine tuberculosis. Highly enriched populations of deer macrophages were obtained from 14 day cultures of plastic-adherent peripheral blood mononuclear cells. Cervine macrophages produced superoxide anion in response to respiratory burst stimuli (serum-opsonised zymosan and phorbol myristic acetate), but nitric oxide production could not be detected under the conditions tested. The lysosomal enzymes acid phosphatase and lysozyme were detected at the intercellular and extracellular level. Stimulation with bacterial lipopolysaccharide extract (Escherichia coli LPS) enhanced the production of superoxide and acid phosphatase with a peak increase in activity observed after 2h. Production of interleukin 1 (IL-1) and tumour necrosis factor (TNF), determined using cytokine-sensitive cell lines and mRNA analysis (Northern blotting), indicated maximal secretion of both cytokines after 24 h stimulation with LPS, preceded by a peak in message accumulation at 2-6 h post-stimulation. Cervine macrophages stimulated proliferative responses in T cell-enriched lymphocyte populations derived from the peripheral blood of autologous animals that had been primed to mycobacterial antigens (Mycobacterium bovis Bacille Calmette-Guerin, BCG). Macrophages were able to stimulate responses after pulsing with particulate (BCG) or soluble (purified protein derivative) mycobacterial antigens. These results indicate that macrophage inflammatory and immune responses in red deer are similar to those in other mammalian species, and that macrophages may play an important role in resistance to mycobacterial infection.

A longitudinal study of leucocyte numbers and mitogenesis during the last ten weeks of human pregnancy

Leucocyte numbers rose consistently in normal primigravid women sampled repeatedly during the last ten weeks of pregnancy. The increase in total leucocyte count was significant from 35 weeks gestation to delivery and was directly related to an increase in neutrophils, while mononuclear cell numbers remained unaltered. In addition to the pregnancy associated neutrophilia there was a further increase in neutrophil counts at delivery. Using whole blood and purified leucocytes in culture, it was confirmed that pregnancy plasma contains suppressor factors which inhibit mitogenesis with PHA or Con A. There was no significant change in individual womens whole blood mitogenic response at any time during the sampling period except at delivery when there was a significant decrease. Culture of washed cells from pregnant women in non-pregnancy plasma restored their mitogenic response to levels found in non-pregnant control women, except at delivery when there was evidence of a cell-associated impairment of function. Purified leucocyte cultures from individual women sampled repeatedly from 30 weeks gestation to delivery gave variable responses although it was still possible to identify pregnancy-related plasma suppressor factors and delivery-associated impairment of leucocyte function in vitro.

Cervine T-lymphocyte growth factors and their measurement in tuberculosis.

Research into the composition and function of the immune response in domesticated ruminants has tended to focus on the ovine and bovine systems. With the recent domestication of deer, health problems have developed which require a fundamental knowledge of the immune function in exotic ruminants. In this report it is shown that although recombinant human and mouse interleukin-2 (IL-2) were capable of stimulating cervine T-cell proliferation, optimal proliferation was only achieved using recombinant bovine IL-2. While some phylogenetic restriction of IL-2 cross-reactivity was found, in some cases this could be overcome by using high concentrations of recombinant IL-2. Using cervine T-cell blasts it was possible to assay in vitro T-cell growth factor (TCGF) production by lymphocytes isolated from deer naturally exposed to tuberculosis Mycobacterium bovis). Differences were found in the amount of TCGF present in the supernatants of antigen-activated cells isolated from severely diseased animals, those with limited disease and non-diseased animals.

Humoral immunity in the ewe 2. The effect of pregnancy on the primary and secondary antibody response to protein antigen

This study examines the effect of pregnancy on the quantity and isotype of antibody in ewes immunised with a novel primary antigen. The primary and secondary antibody levels to bovine serum albumin (BSA) in alum adjuvant, were compared between non-pregnant ewes and ewes immunised at different stages of pregnancy. Anti-BSA isotype specific responses were measured using an indirect ELISA. Results show the levels of immunoglobulin M (IgM) increased and persisted in response to BSA during pregnancy (P less than 0.05). Secondary immunoglobulin G1 (IgG1) titres were significantly impaired in late pregnancy and during lactation (P less than 0.05). The lower levels of immunoglobulin G2 (IgG2) were unaffected by pregnancy under these experimental conditions. Ewes were also immunised with BSA in alum adjuvant for their primary inoculum and BSA in different adjuvants for the secondary inoculation. Primary IgM persisted at higher levels during pregnancy compared with the response in non-pregnant ewes (P less than 0.05). Following the secondary injection, lower levels of anti-BSA specific IgM, IgG1 and IgG2 antibodies were produced in late pregnancy and during lactation compared with the levels in non-pregnant control ewes (P less than 0.05). Alterations in regulatory T-cell function or effector B-cell activity would most readily explain the qualitative changes in antibody titre observed following primary injection during pregnancy. The results also suggest an associated impairment of immunological memory following primary immunisation during pregnancy.

Humoral immunity in the ewe 1. The influence of adjuvancy and immunogenic substitution on immune reactivity following immunisation with protein antigens

The nature of antigen and presence of adjuvant are major factors which influence the level of immune reactivity following immunisation. This study examines the quantity and isotype of antibody produced in ewes immunised with different proteins in combination with different adjuvants. Results using indirect ELISA assays show that animals immunised with keyhole limpet haemocyanin (KLH) in adjuvant produced lower levels (P less than 0.05) of immunoglobulin M (IgM) and had increased levels and persistence of immunoglobulin G1 (IgG1) compared with ewes immunised with antigen in saline (P less than 0.005). The anti-KLH immunoglobulin G2 (IgG2) titre was significantly higher in animals given oil-emulsion adjuvant than all other groups (P less than 0.005). Ewes were also immunised with bovine serum albumin (BSA) or BSA haptenated with trinitrophenyl (TNP) for the primary injection and carrier BSA alone for the secondary inoculation. Chemical haptenation of BSA antigen reduced primary IgM (P less than 0.005), IgG1 (P less than 0.005) and IgG2 (P less than 0.005) levels compared with animals immunised with pure BSA. There was an increased secondary anti-BSA IgM response in all animals first immunised with TNP-BSA (P less than 0.05). The class of anti-hapten antibody produced to TNP determinants was influenced by the degree of TNP haptenation of the carrier BSA. Mid-range molar ratios of TNP produced the strongest IgM, IgG1 and IgG2 anti-TNP responses compared with all other groups (P less than 0.01).

Identification of heterologous monoclonal antibodies that cross-react with cervine leukocyte subpopulations

The degree to which cross-reactivity between monoclonal antibodies developed against cells of the human, mouse, bovine and ovine immune systems, and cells of the cervine immune system occurs was investigated. It was found that within the ruminants a considerable degree of cross-reactivity does exist while there is virtually none between the cervine and murine or human systems. The highest incidence of cross-reactivity was found between ovine monoclonals and cervine leukocytes (46% cross-reactive) with 25% of bovine monoclonal antibodies cross-reacting with deer leukocytes. Ovine monoclonals were found to be the most useful in identifying a wide range of cervine leukocyte subpopulations. Bioassays showed that ovine anti-class I and II monoclonals detected molecules on cervine leukocytes that are functionally similar to MHC antigens. The possibility that cross-reactive monoclonals detect similar subpopulations in both the homologous and heterologous species is discussed.

Humoral immunity in the ewe. 3. The influence of adjuvants and immunisation regimes on immune reactivity in the breeding ewe and her progeny.

Breeding ewes were immunised with clostridial vaccine using different inoculation schedules. Results, showing differences in the class of antibody produced, were heavily dependent on the vaccination regime used. Immunoglobulin M (IgM) levels were significantly lower in ewes given double doses of vaccine compared to ewes given a single inoculation or no treatment at all (P less than 0.01). Neonatal lambs showed significant de novo IgM production with interference in this antibody production in the lambs of ewes vaccinated with the clostridial vaccine (P less than 0.05). Immunoglobulin G1 (IgG1) levels were significantly increased in all lambs which had mothers vaccinated with the clostridial vaccine prior to or during pregnancy (P less than 0.025). The greatest quantity of IgG1 was transferred to lambs when their mothers were given a double injection with primary inoculation prior to conception and booster prepartum (P less than 0.025). No antigen specific immunoglobulin G2 (IgG2) was detected in the lambs. Ewes were also immunised with BSA and their isotype specific serum antibody response was compared with their respective lambs. There was no detectable anti-BSA IgM in the lambs of all groups of ewes though specific IgG1 antibodies could be readily detected in the lambs of hyperimmunised ewes. The efficiency of transfer was related directly to the ability of the adjuvant to maximise IgG1 production in the ewe. Although immunised ewes produced high levels of IgG2, this was not transferred passively to the lamb.

Altered humoral immunity during pregnancy in the guinea pig

A guinea pig model was developed to determine whether humoral immune responsiveness is altered during pregnancy. Pregnant animals were immunised at mid-gestation with haptenated protein. The humoral response to antigen was measured as numbers of antibody-producing cells in the spleen, the affinity of the antibody produced by spleen cells and the levels of IgG in the serum. The values obtained were compared with those from an age matched non-pregnant control group. Early in the primary response, there was a significant decrease in the number of IgM antibody-producing cells with an associated decrease in serum IgM levels in pregnant animals. Late in the primary response, pregnant and control animals had similar levels of IgM antibody-producing cells. During the later stages of the response, many of the pregnant animals did not respond with IgG antibody-producing cells or IgG in the serum. When IgG-producing cells were detected, the antibody was of lower affinity than that observed with the control group. A selective lack of responsiveness was detected in the primary response of pregnant guinea pigs. The reduced number of IgG antibody-producing cells in gravid animals suggests that an immune switch from IgM to IgG is impaired in pregnancy. The low affinity of antibody produced indicates the immunoglobulin produced in pregnancy may also be functionally limited.

IN VITRO RESPONSES OF CERVINE MACROPHAGES TO BACTERIAL STIMULANTS

The function of cervine (deer) mononuclear phagocytes is poorly defined. In the present study, the potential of cervine macrophages to generate phagocytic and immunoregulatory responses following stimulation with bacterial products was investigated. Blood-derived macrophages of red deer were cultured in vitro with particulate stimulants (Mycobacterium bovis BCG and Staphylococcus aureus SAC) or soluble stimulants (M. bovis PPD and Escherichia coli LPS), prior to assessment of phagocytic responses, prostaglandin secretion and cytokine production. Particulate stimulants induced vigorous phagocytic responses (superoxide anion generation, lysosomal enzyme release), secretion of prostaglandin E2 and transcription of mRNA specific for the cytokines IL-1 beta, IL-10 and TNF alpha, while soluble products invoked weaker responses. These results are discussed in relation to the role of cervine mononuclear phagocytes in regulating and participating in inflammatory and immune processes relevant to bacterial challenge.

The purification and characterisation of cervine IgM and IgG

A procedure is described for the isolation of immunoglobulin G (IgG) and immunoglobulin M (IgM) from hyperimmune cervine serum. Hybrids of red deer (Cervus elaphus) and wapiti (Cervus canadensis) were immunised with keyhole limpet hemocyanin (KLH). An immunoglobulin-containing fraction was precipitated from the hyperimmune serum using ammonium sulphate. The antigen-specific immunoglobulins were purified by KLH-conjugated sepharose affinity chromatography and further separated into IgM and IgG by gel-filtration chromatography. Purified immunoglobulin was analysed by polyacrylamide gel electrophoresis and isoelectric focusing. The molecular weights and isoelectric points of the composite chains of cervine IgG and IgM are presented.