J. Hopkins

6.5 Reactivity of the CD11/CD18 workshop monoclonal antibodies in the sheep

The anti-CD11/CD18 monoclonal antibodies (mAbs) submitted in the Second International Workshop on Ruminant Leukocyte Differentiation Antigens, were analysed for their reactivity with the ovine homologue of CD11/CD18. Their reactivity was tested on healthy sheep tissues, and alveolar macrophages, afferent dendritic cells, peripheral blood granulocytes and monocytes. The CD11a/CD18 mAbs found positive in the sheep were reactive with all the cell populations tested. The CD11b mAbs reacted with all the cells except afferent dendritic cells, whereas CD11c were non-reactive to blood granulocytes. This is in contrast to humans and cattle where blood granulocytes express CD11c. The mAbs 72-87, F10-150, MD2B7 and MUC76A were found to be homologous to CD11a whereas BAQ30A seemed to be homologous to CD18, instead of proposed initial specificity to CD11a. CC125 and IL-A15 mAbs were found to be homologous to CD11b. OM1, which clustered with a recognized CD1 mAb in the first cluster analysis, precipitated a heterodimer of molecular weight 95,000/150,000. We propose that OM1 reacts with sheep CD11c. The mAb MF14B4 was found to react with sheep CD18.

Identification of the sheep homologue of the monocyte cell surface molecule--CD14

An ovine monocyte/macrophage cell surface antigen was recognized by three mouse monoclonal antibodies (mAbs) VPM65, VPM66 and VPM67. These mAbs also reacted with bovine cells. The antibodies immunoprecipitated a single, glycosyl-phosphatidylinositol-linked polypeptide of M(r) 55,000 which, when deglycosylated, was reduced to M(r) 53,000. They reacted strongly with peripheral blood monocytes, alveolar macrophages and peripheral blood granulocytes, and weakly with afferent lymph dendritic cells. They also reacted with macrophages in many different tissues but were non-reactive with lymphocytes. Competitive flow cytometry shows that these three mAbs recognize the same or a closely related epitope of a single antigen. An antigen-specific capture ELISA using the anti-human CD14 mAb (TUK4) revealed that all four mAbs associate with the same antigen. These data demonstrate that the mAbs react with the ovine homologue of the lipopolysaccharide (LPS)-LPS binding protein receptor, CD14.

Cross-reactivity of workshop antibodies with cells from domestic and wild ruminants

Reactivities of the monoclonal antibodies (mAbs) from the workshop panel with cells from cattle, sheep, goats, Cape buffalo (Syncerus caffer) and waterbuck (Kobus defassa) were tested. One hundred and sixty-nine mAbs reacted with bovine cells and 111 with sheep cells; 86 were shown to react with goat cells, 71 with buffalo cells and 70 with waterbuck cells. Some mAbs cross-reacted with all five ruminants tested, and are likely to react with epitopes that are conserved in other ruminant species. Such mAbs will obviate the need to produce mAbs panels to leukocyte antigens of other ruminants.

Increase in CD5+ B cells and depression of immune responses in sheep infected with Trypanosoma evansi

The effects of Trypanosoma evansi on the cellular and humoral immune responses of sheep to Pasteurella haemolytica vaccine were studied. Peripheral blood lymphocytes (PBLs) from the sheep were analysed using single and double-colour indirect immunofluorescence staining and flow cytometry to monitor changes in circulating B and T cell subsets. Serum antibody responses were assayed using the enzyme-linked immunosorbent assay technique (ELISA), in addition to measuring local skin reactions at the site of vaccine administration. Results showed significant increases in circulating B cells in all sheep after the primary (p < 0.01) and secondary (p < 0.001) vaccinations although the increases were much more dramatic in the T. evansi-infected sheep. In addition, infection induced significant increases (p < 0.004) both in proportions and numbers of CD5+ B cells with more than 70% of circulating B cells expressing the CD5 antigen and showed significant differences (p < 0.01) from those of control sheep in which vaccination alone failed to induce similar increases. Also, infection resulted in significant decreases in CD5+ (p < 0.003), CD4+ (p < 0.03) and CD8+ (p < 0.03) T cell subsets in contrast to their increases in all control animals after vaccination. Moreover, there were significant suppression of both local skin reaction (p < 0.005) and serum Ig and IgG1 (p < 0.001) antibody responses to the vaccine antigen.

Analysis of reporter gene expression in ovine dermis and afferent lymph dendritic cells in vitro and in vivo

Plasmid DNA administration has revolutionised approaches to vaccination, and many studies have demonstrated the generation of both humoral and cytotoxic T cell responses which confer protection against live pathogen challenge. However, the mechanisms underlying DNA vaccination are poorly understood. Several studies have suggested the involvement of professional antigen presenting cells such as dendritic cells (DC), but direct evidence for this is lacking. We have used the pseudoafferent lymphatic cannulation model in sheep to study the expression of a plasmid encoding enhanced green fluorescent protein (EGFP) by afferent lymph DC following administration to skin. The cells were analysed by flow cytometry. Preliminary studies were carried out to determine if the pEGFP would function in sheep cells in vitro. The results showed that electroporation of sheep skin fibroblasts, primary macrophages, and afferent lymph DC with 30 microg pEGFP resulted in varying degrees of fluorescence in these cells e.g. 35% of skin cells examined at 48 h, and 7% of afferent lymph DC examined after 4 h. Following intradermal injection of 120 microg of pEGFP, small numbers of fluorescent DC (1-5%) were evident by flow cytometry after 1-4 h. The fluorescent DC continued to drain into the lymphatics over a period of 24 h. Analysis by PCR showed that free pEGFP appeared in the afferent lymph plasma within 1 h of injection, peaking at 2 h and becoming undetectable after 6 h. The results suggest that primary immune responses may be initiated by uptake of soluble protein antigen by afferent lymph DC and by free plasmid rapidly draining to the lymphatics where it may be taken up by DC in the lymph plasma and the local lymph node.

RUMINANT CLUSTER CD14

Following the analysis of flow cytometry data on ovine and bovine cells and immunoprecipitation, immunohistochemistry and competitive binding studies, a cluster of three monoclonal antibodies (mAbs) (VPM65, VPM66 and VPM67) appeared to be specific for the ruminant CD14 (Gupta, V.K., McConnell, I., Dalziel, R.G. and Hopkins, J., 1996. Identification of the sheep homologue of the monocyte cell surface molecule-CD14. Vet. Immunol. Immunopathol. in press; Hopkins, J. and Gupta, V.K., 1996. Characterization of 3rd Worshop monoclonal antibodies specific for sheep macrophages/monocytes. Vet. Immunol. Immunopathol., 52:329-339). According to flow cytometry results from human CD14-transfected COS-7 cells and from bovine peripheral blood mononuclear cells, another mAb (CC-G33), not submitted to the Workshop, recognised a bovine CD14 epitope which is conserved in the human CD14 (Sopp, P., Kwong, L.S. and Howard, C.J., 1996. Identification of bovine CD14. Vet. Immunol. Immunopathol., 52: 323-328).

Trypanosoma congolense infection in sheep: cellular phenotypes in lymph and lymph nodes associated with skin reactions.

Intradermal inoculation of sheep with culture-derived metacyclic forms of Trypanosoma congolense resulted in the development of localized skin reactions (chancres) and enlargement of the draining lymph nodes 7 days after infection. Changes in the expression of surface antigens of lymphocytes in lymph leaving the affected skin reactions and in the associated lymph nodes were monitored by cannulating the afferent and efferent lymphatic ducts. Trypanosomes appeared in afferent and efferent lymph 3 to 5 days after infection and persisted even as the chancres regressed. The cellular output in both afferent and efferent lymph increased markedly after the onset of parasitosis. Sequential analysis of the phenotypes of lymphocytes by immunofluorescent staining and flow cytometry revealed that in afferent lymph draining the chancre there was an early response which was due to an increase in T cells, particularly CD4+ and CD8+ cells; however, as the chancres-regressed there was an increase in lymphoblasts and surface immunoglobulin-bearing cells. In contrast, in the efferent lymph, the increase in lymphocytes was due predominantly to a higher number of cells bearing surface immunoglobulins.

Biochemical and phenotypic characterization of the ovine β2 (Leucocyte) integrins

Summary This paper is concerned with the relationship of the three distinct members of the ovine β2-integrin family of leucocyte adhesion molecules that play an important role in cell-cell and cell-matrix interactions. A panel of monoclonal antibodies (mAbs) specific for sheep and cattle macrophages was characterized by flow cytometry, immunohistology, and immunoprecipitation for reactivity to the β2 integrins. Immunoprecipitation analysis of sheep antigens showed that these monoclonal antibodies could be divided into four distinct groups. All precipitated an M r 95 000 β chain but they differed in the size or number (or both) of the α chains recognized. Group 1 precipitated α chains of M r 180 000; group 2 had M r 170 000 α chains; the group 3 α chain was of M r 150 000 and group 4 mAbs precipitated α chains of all three sizes. The relationship between these antibodies was demonstrated by sequential immunoprecipitation, which showed that the reactivities of antibodies in groups 1, 2 and 3 were mutually exclusive but that group 4 antibodies shared a common specificity with the other three groups. By analogy with the human and murine β2 integrin families, group 1 antibodies seemed to be specific for CD11 a (LFA-1); group 2 were CD11b (CR3 or Mac1); group 3 were CD11c (CR4 or p150/95) and group 4 were CD18. In addition to different molecular weights, these antibodies had different cellular and tissue distributions. CD11a and CD18 were distributed identically. The antigens recognized by both were present on all the leucocyte populations. The mAbs recognizing CD11b reacted with a sub-population of peripheral blood B lymphocytes and all myeloid cells (alveolar macrophages, peripheral blood monocytes and granulocytes) except afferent lymph dendritic cells (ADC). Anti-CD11c (p 150/95 or CR4) antibodies reacted strongly with macrophages and ADC but were weakly reactive on monocytes and negative on neutrophils. CD11c was also present on a sub-population of peripheral blood B cells.

A STUDY ON LYMPHOCYTE ACTIVATION IN MAEDI-VISNA VIRUS INDUCED PNEUMONIA

The stage of activation of bronchoalveolar lavage fluid (BALF) lymphocytes and peripheral blood lymphocytes (PBL) from maedi-visna virus (MVV) infected (n = 7) and control (n = 7) sheep was investigated by assessing four parameters of lymphocyte activation; lymphocyte size and complexity, loss of CD5+ T cells, expression of cell surface interleukin-2 receptor (IL-2R) and expression of DR and DQ MHC Class II molecules. BALF lymphocytes from MVV-infected animals had a significant loss of CD5+ lymphocytes (P < 0.05) and upregulation of DR and DQ MHC Class II molecules compared with controls, consistent with BALF lymphocyte activation. No changes in cell size and complexity or expression of IL-2R were observed. No evidence of PBL activation was detected. These findings suggest an impaired BALF lymphocyte activation during MVV infection.

Sheep CD1 genes and proteins.

Interest in CD1 genes and proteins was initially stimulated by their close evolutionary and structural relationship to MHC class I molecules. The demonstration that CD1b and c molecules present novel non-peptide antigens to T-cells and play a role in protection against mycobacterial infection then focused attention on the functional role of CD1 proteins. Sheep possess at least seven CD1 genes, including CD1B, D and E, which is the most complex genetic arrangement identified so far in any animal. OvCD1B consists of at least three distinct genes, with the probability of limited polymorphism and the existence of splice variants. Most anti-sheep CD1-specific monoclonal antibodies react with OvCDlb and phenotypic and immunochemical data suggests the existence of two variants. CD1D genes have been identified in all species studied, suggesting a conserved role for CDld proteins across mammalian species. Presumptive evidence for the existence of OvCDIE has been obtained by NH2-terminal sequencing of protein precipitated by the mAb 20.27 (SBU-T6). Confirmatory evidence from gene cloning experiments is currently being sought. Collectively, these factors make the sheep CD1 family a highly relevant model for investigating the in vivo role of CD1 molecules. In this survey, the properties of monoclonal antibodies specific for sheep CD1, the cellular distribution and physicochemical characteristics of sheep CD1 molecules and the current state of knowledge on sheep CD1 genetics are reviewed.