The mononuclear phagocyte (MP) system is a heterogenous group of cell populations present in most tissues even in
the absence of inflammation. Monocytes and M0 are the major differentiated cells of MP system, have a prominent role in
defence against many infectious agents and tumour cells, and are involved in the regulation and induction of immune responses.
In addition they secrete a large number of substances which have a role in various physiological and pathological processes.
ion they secrete a large number of substances which have a role in various physiological and pathological processes.
It is necessary to know the normal distribution and localisation of cell with a particular phenotype in order to
understand their involvement in various disease processes. The advent of hybridoma technology has facilitated the dissection of
the phenotypic and functional heterogeneity of various cells by producing monoclonal antibodies (mAb) specific to cell surface
molecules. Sheep is an important experimental animal model, for the study of the pathogenesis of infectious diseases and little is
known about the cell surface determinants of MP in this species.
about the cell surface determinants of MP in this species.
In this thesis I have characterised MP by devolping novel anti-M0 mAb and further characterising anti-cattle mAb
which cross react with sheep (12 integrins.
Three mAb (VPM65 , 66 and 67) immunoprecipitated cell surface glycoproteins of the same Mr = 55,000, having
approximately 3,000 Da N-linked glycosylation. The antigen is primarily expressed in blood monocytes in addition to its
moderate expression in AM and granulocytes but weak expression in afferent dendritic cells (ADC). They also labelled resident
M0 in many different tissues but were non reactive with lymphocytes. The molecule is anchored to the cell surface via glycosylphosphatidyl inositol linkage. These mAb recognise the same or overlapping epitopes of the antigen. VPM65/66/67 recognise a
homologue of sheep CD14 as determined by antigen preclearing studies and two-colour FACS analysis using human anti-CD14
mAb (TUK4). These mAb also reacted with monocytes and M0 in cattle.
VPM63 recognised a dimer of Mj. = 40,000 and 42,000 having approximately 2,000 Da N-linked carbohydrates.
VPM63 reacted with resident M0 in most tissues but was absent from blood monocytes, ADC and any lymphocyte populations.
The expression of the antigen is lost within two days in cultured AM and did not appear on in vitro monocyte derived
macrophages. Immunoblotting of affinity purified VPM63 antigen with polyclonal rabbit anti-bovine FcyRI synthetic peptide of
15 amino acids (150-168) from the second extra cellular domain, suggests the possibility that VPM63 recognises a M0 specific
isoform of sheep FcyRII. However VPM63 failed to block uptake of soluble Ag-Ab complexes by AM. VPM64
immunoprecipitated a heterodispersed antigen of Mf = 65,000 to 80,000 from surface labelled AM. In addition to AM the
antigen is expressed on granulocytes and has low level of expression on monocytes. VPM64 also stained a small population of
CDllb+, non-T, non-B and non-monocyte (CD14") peripheral blood mononuclear cells, possibly to be 'natural killer' cells.
VPM64 labelled resident M0 in spleen, gut and lungs. On the basis of its molecular weight and cellular distribution the
possibility of VPM64 recognising an antigen equivalent to the mouse FcyRIII is discussed.
A panel of mAb specific for (32 integrins on MP of sheep was characterised by immunoprecipitation, flow cytometry
and immunohistochemistry using mAb submitted to the 2nd International Workshop on Ruminant Leukocyte differentiation
Antigens. Immunoprecipitation, immunohistochemical and flow cytometric analysis differentiated these mAb into four distinct
groups (Gp). The relationship between these antibodies is shown by sequential immunoprecipitation which showed that the
reactivities of antibodies in Gp 1, 2 and 3 were mutually exclusive but that Gp 4 antibodies shared a common specificity with the
other three groups. By analogy with the human, mouse and cattle (52 integrin families Gp 1 mAb seem to be specific for
CDlla/CD18 (LFA-1); Gp 2 are CD1 lb/CD18 (CR3 or Mac-1); Gp 3 are CDllc/CD18 (CR4 or pl50/95) and Gp 4 are CD18.
The differences in the tissue distribution and cellular localisation for the different (32 integrins are described and discussed.
In addition to myeloid cells CDllb and CDllc are also present on a sub-population (30-50%) of resting peripheral
blood B cells, which are exclusive to blood and spleen and do not appear to recirculate through peripheral lymph node and do not
express L-selectin (Dul.29), a lymph node homing and memory cell marker. In peripheral blood the non-recirculating B-cell
population was mutually exclusive to the recirculating B-cell population (Du2.74+). Cells resembling non-recirculating B-cells
are confined only to marginal zone in the spleen whereas recirculating B-cells are present in the follicles in spleen and ileal
Peyer's patches. These preliminary observations indicate that CDllb+ B cells may represent a population of naive B cells and
have distinct recirculatory pathways.