Rebecca E. Hooke

Interleukin-1 stimulation of equine articular cells.

Prostaglandin E2 (PGE2) and stromelysin are produced by equine chondrocytes and synovial cells in vitro in response to recombinant human (rh) interleukin-1 (IL-1) alpha and beta, and equine mononuclear cell supernatants (MCS) containing IL-1. However, culture conditions are important. PGE2 concentrations increase in proportion to the concentration of fetal calf serum (FCS) in the culture medium, whereas stromelysin concentrations are inversely proportional to the concentration of FCS. Equine MCS, containing a lower concentration of IL-1 than the concentration of rhIL-1 used in these experiments, stimulated production of much higher levels of PGE2 than rhIL-1. In addition, equine MCS induced the production of broadly similar levels of PGE2 by both chondrocytes and synovial cells, whereas rhIL-1 was more active on equine synovial cells than equine chondrocytes. Although equine MCS induced both stromelysin and PGE2 production by equine articular cells, on the whole rhIL-1 failed to induce stromelysin production. This supports previous observations of species restrictions in the activity of human IL-1 on equine cells. Therefore, experiments using mammalian cells and heterologous IL-1 should be interpreted with caution.

The characterisation of equine interleukin-1

Equine interleukin-1 has been produced from peripheral blood monocytes by stimulation with E. coli lipopolysaccharide. Sephacryl S200 gel filtration revealed a molecular weight of 17-18 kD. Chromatofocusing of the 17-18 kD peak identified four active fractions. Two major peaks were detected at pH 6.7 and pH 7, with smaller peaks at pH 6.3 and pH 5.9. The pI 7 molecule is probably the equine form of IL-1 beta.

Equine chondrocyte activation by a variety of stimuli

There is increasing evidence that the chondrocyte is capable of considerable anabolic and catabolic activity. In the case of equine chondrocytes, this study demonstrates that a variety of factors involved in the pathogenesis of joint disease stimulate the production of prostaglandin E2. These include exposure to IL-1, bone fragments and LPS. In addition, an IL-1-like factor was shown to be produced by the chondrocyte itself, when stimulated by LPS, providing a possible mechanism for amplification of extra-cartilagenous signals and even autocrine control. Considered together with evidence of increased synthesis of proteoglycan molecules by chondrocytes in diseased cartilage, this offers the exciting possibility of development of therapeutic agents to assist cartilage repair.

Species restrictions demonstrated by the stimulation of equine cells with recombinant human interleukin-1

Equine thymocytes, which respond to equine monocyte supernatants, do not respond to stimulation with recombinant human interleukin-1 alpha and beta, and equine synovial fibroblasts show a limited response in the form of prostaglandin E2 production without any evidence of neutral metalloproteinase production. Human interleukin-1 beta was about three to ten times as active on equine synovial cells as human interleukin-1 alpha in terms of prostaglandin E2 production. This preliminary evidence would suggest that there are qualitative and quantitative differences in the way recombinant human interleukin-1 stimulates human cells and the way in which it stimulates equine cells.

Modulation of lymphocyte activating factor activity (interleukin 1 like activity) and acute phase proteins in pertussis-induced air pouch inflammation by muramyl dipeptide

We have studied the effect of the macrophage activator, muramyl dipeptide (MDP) on immune inflammation induced in the rat six day subcutaneous air pouch. Treated animals received either 100 μg or 200 μg MDP at the time of challenge and twenty four hours before exudate harvest. Using the thymocyte co-mitogenic assay for lymphocyte activating factor (LAF), 100 μg MDP enhanced LAFactivity whereas 200 μg caused inhibition. Increased dilution of 200 μg exudate in this assay removed this inhibition. Similarly, at the lower dose, MDP caused enhanced production of the acute phase protein alpha 1 glycoprotein, whereas the higher dose had no effect. The present study suggests that macrophage activity can be manipulatedin vivo to produce LAF and naturally occurring inhibitors of LAF. These studies indicate that the stimulation of LAF inhibitors by MDP may be a potential theerapeutic action.

Isolation of equine peripheral blood mononuclear cells using Percoll

The concentration of Percoll required for isolating equine peripheral blood mononuclear cells has been reinvestigated. A poor cell yield was obtained at the 60 per cent concentration already reported. It is recommended that workers specifically interested in high yields of mononuclear cells, for investigation of lymphocyte and monocyte functions, use a concentration of 65 per cent Percoll. However, workers wishing to isolate pure populations of equine neutrophils might consider a concentration of 70 per cent in the upper layer of Percoll used to retain the mononuclear cells.