Interferons have been recognized as important mediators of cellular
communication for many years. There are two types of interferon:
Type I interferons have antiviral functions, but Type II interferon
(IFN-g) is more important as an immunomodulating molecule. Type II
interferon has effects on cellular MHC class II expression,
immunoglobulin class-switching, macrophage activation, cellular
proliferation and a number of other functions. The role of IFN-g
during in vivo immune responses has not been studied in great
detail, but the sheep is an ideal species in which to study these
phenomena by using the efferent lymphatic vessel cannulation model.
This allows access to cells and tissue fluid for cytokine analysis
using antibody and genetic probes for the detection of IFN-g.
Bovine IFN-g peptides (amino-terminus, carboxy-terminus and central)
were used to generate antibodies in rabbits. None of the antipeptide sera reacted with denatured ovine or bovine IFN-g, nor
neutralized their antiviral effect. Rabbit antibodies to bovine
recombinant IFN-g neutralized ovine IFN-g and detected IFN-g in a
sandwich ELISA when used in combination with a monoclonal antibody
against a human IFN-g carboxy-terminal peptide. The sensitivity of
detection was only 125ng/ml, insufficient for use with efferent
lymph fluid samples.
The expression of MHC class II molecules on cell surfaces is
increased by IFN-g on many cell types. This has been used
previously to measure biologically active IFN-g concentrations in
fluids. Measurement of ovine class II by slot blot was assessed as
a method of adapting this to ovine IFN-g measurement, but the
technique proved to be too problematic for regular use. The
expression of class II on T lymphocytes is influenced by IFN-g in
the surrounding fluid. Analysis by FACS of resting ovine T
lymphocytes shows them to express class II, a situation different to
that in the human. Incubation of efferent lymph cells with IFN-g
enhances the expression of ovine DR-like class II molecules
especially on CD8⁺ cells, but also on CD4⁺ cells. The expression of
ovine DQ-like class II molecules was much less influenced by IFN-g.
This differential expression has been seen previously in human
cells. It is likely that such differences are due to variation in
transcriptional control between the two types of molecule.
The IFN-g genes of many species have been cloned, including bovine
IFN-g. Nucleotide primers for the cloning of ovine IFN-g by
polymerase chain reaction were chosen from the bovine sequence.
Cloning of the central 300bp of the gene revealed 98% identity
between ovine and bovine IFN-g at the amino acid level. Subsequent
cloning of ovine IFN-g by other groups showed that allelic variation
of the gene occurs with no alteration in the amino acid structure.
This feature is not found in human IFN-g, but is described in other
ruminant cytokines. Attempts to isolate a lambda clone expressing
an IFN-g fusion protein using the rabbit anti-bovine rIFN-g sera
The immune response may be analysed by studying cells and fluid
delivered by the cannulation of an efferent lymphatic vessel. A
secondary response to ovalbumin was induced in a sheep by
inoculation of a dependent area. The lymphocytes collected were
isolated into CD4⁺ and CD8⁺ cells by a magnetic separation technique
(MACS). Their mRNA was isolated, and cDNA generated from it was
subjected to IFN-g-specific PCR. This revealed that both CD4⁺ and
CD8⁺ cells contribute to the synthesis of IFN-g during the secondary
immune response to a protein antigen in the sheep.