Abstract
Interleukin-1 (IL-1) is a key mediator of infection, inflammation and immunity and two different IL-1
proteins are known to exist, IL-la andIL-lJ3. Each is synthesised as a proprotein, Mr = 31kD,
which is subsequent cleaved to yield the mature protein, Mr = 17.5kD. Whereas both forms of IL-la
show equivalent biological activities, IL-1 (3 requires cleavage with resultant conformational change
for optimal activity. IL-1 has been extensively studied in human and murine systems but at the time
this project was initiated, nothing was known about its actions in the sheep and no reagents were
available with which to study ovine IL-1. This thesis describes the successful cloning and expression
of biologically active ovine IL-la and IL-1 (3 and their use in determining IL-1 receptor (IL-1R)
expression by ovine alveolar macrophages (M<t>) and afferent lymph dendritic cells (DC).
Lipopolysaccharide (LPS) stimulated Mφ were used as the source of IL-1 mRNA. The specific IL-1
cDNAs were amplified by polymerase chain reaction, cloned into pTZ18R/19R vectors and
sequenced. Ovine IL-lα and IL-1ß were found to be 97% and 96% identical to their bovine
counterparts and 81% and 78% identical, respectively, to the human sequences. Translation of the
nucleotide sequences shows that ovine IL-lα has 97% and 72% identity with bovine and human IL1α respectively and ovine IL-1ß has 95% and 62% identity with bovine and human IL- 1ß
respectively. Use of the cloned IL-lcDNAs for northern blot analysis of IL-1 mRNA production by
Mφ, showed IL-lα mRNA to reach maximal levels at around 6h and IL-1ß mRNA at around 4h after
LPS stimulation.
The proprotein (p) and mature protein (m) forms of ovine IL-1α and IL-1ß have been expressed as
fusion proteins with yeast p1, using the Ty-vlp system of British Biotechnology Ltd. The fusion
protein self-assembles in the form of a virus-like particle (vlp), from which the rIL-1 is cleaved by
the action of Factor Xa restriction protease (FXa). FXa cleavage of IL-lap:p1 vlps revealed a relaxed
FXa recognition site within IL-1lαp which yielded a putative N-terminus at Ser-120, equivalent to the
N-terminus of human EL-lαm. It is proposed that FXa may be one of the natural processing enzymes
for IL-1a. The activities of purified IL-1 mature proteins on ovine thymocytes were ~2.5xl0⁷ U/mg
EL-1a and ≥1.25xl0⁶ U/mg IL-1ß. Activities on ovine xiphoid cartilage were ten times less. Ovine
rIL-1 shows a species preference for homologous thymocytes and cross-species sequence analysis
highlights 5 amino acid residues which may be of relevance in this context. Ovine rIL-1ßp is
biologically active, but about 5 times less so than IL-1ßm.
By using ovine ¹²⁵I-rIL-1, ~16500 IL-1R/cell (Kd ~4.6pM) for IL-1ß and ~2600 IL-lR/cell (Kd
~56pM) for IL-lα were detected on Mφ. ≤24% of Mφ expressed IL-1R. DC, isolated from lymph
from cannulated sheep pseudoafferent lymph ducts, expressed ~510 IL-1R/cell (Kd ~30pM) for IL1α and ~350 IL-1R/cell (Kd ~160pM) for IL-1ß. Quantitation of IL-1R expressed by fresh DC has
not previously been reported for any species, as far as I am aware. Following secondary ovalbumin
challenge of primed sheep, DC expression of receptors for IL-1α, but not for IL-1ß, is greatly
upregulated. Two peaks of increased expression were detected, a transient increase at around 4h
followed by an increase to maximal expression of ≥21700 IL-1α sites/cell (Kd ~181pM) between
43-72h. Only a very small proportion of individual DC were seen to bind IL-1 even after secondary
antigen challenge.
