Sulphite formation and calgranulins in rabbit polymorphonuclear neutrophils (PMN)

Associate Professor H.C. Robinson

This project is concerned with the production of inorganic sulphite by PMN, the phagocytic white blood cells which migrate to sites of tissue injury, infection and play a pivotal role in host defence against infection. During the process of phagocytosis and ingestion of both foreign particles and tissue debris, PMN secrete a number of proteolytic enzymes, oxygen radicals and cytokines, all of which contribute to further tissue injury, as well as to the amplification of the inflammatory response. Our laboratory has reported the production and release of inorganic sulphite by both circulating and extravascular (activated) PMN and we have recently established that the sulphite reacts specifically with calgranulin C a soluble endogenous protein of PMN and a member of the S100 protein family. Calgranulins A, B and C are specifically expressed in neutrophils and monocytes, although calgranulins A and B are also expressed in certain cells of epithelial lineage.

The biological purpose of S-sulfation of calgranulin C has yet to be determined, but we have evidence that it is involved in secretion of calgranulin C or sulphite from the PMN to the extracellular space. We predict that extracellular calgranulin C might exhibit a cytostatic activity similar to that reported for the calgranulin A and B complex. In addition the S-sulfated calgranulin C may act as a sulfite donor, by transferring the bonded sulfite to the disulfide bonds in extracellular macromolecules. The cleaving of disulfide bonds in such macromolecules might thus result in chemical modification of the structure of these proteins, enhancing their antigenicity. Indeed, work from our laboratory has demonstrated that modification of proteins by sulfite-treatment induces an immune response similar to an autoimmune response.

The current aims of this project are to determine: the precise mechanism of sulphite production by polymorphonuclear neutrophils and the role of the S-sulphation of calgranulin C in this process, the involvement of S-sulphation of calgranulin C in the function and secretion of this protein, the biological function and fate of S-sulpho-calgranulin C, and the immunopathological consequences of sulphite modification of tissue proteins.

Biological function of calgranulins A, B & C in rabbit polymorphonuclear neutrophils (PMN).

Calgranulins A, B and C are members of the S100 protein family that are specifically expressed in neutrophils and monocytes, although calgranulins A and B are also expressed in certain cells of epithelial lineage. Their functions have not been clearly defined, but they are known to bind calcium, they become over-expressed in psoriatic skin and have been isolated from struvite kidney stones. The amino acid sequence we have determined for rabbit calgranulin C is consistent with the pattern exhibited by other S100 calcium-binding proteins with two structurally-conserved and well defined Ca2+-binding sites. Porcine calgranulin C has been shown to undergo conformational change upon calcium binding and has been implicated in Ca2+-dependent signal transduction pathways while human calgranulin C has been shown to translocate to the PMN plasma membrane, together with calgranulins A and B during stimulation of the cell by Ca2+-dependent stimuli.

This project will investigate the reduction of 3-phosphoadenosine-5-sulphatophosphate (PAPS) in cell-free preparations of PMN and the role that the PMN cytosolic protein calgranulin C plays in this reaction. The biological function of S-sulphation of calgranulin C will be investigated in relation to the calcium-binding of this protein and its secretion to the extracellular space. The expression of calgranulins B and C will be investigated in developing PMN and in epithelial cells using specific monoclonal antibodies and oligonucleotide probes which have been developed in this laboratory.

Proteoglycan synthesis and degradation by articular cartilage.

This project is concerned with the metabolic integrity of articular cartilage which comprises a rigid extracellular matrix of aggrecan complexes trapped within a collagen network. Articular cartilage is avascular so the chondrocytes, which lie within the matrix and carry out the synthesis of both collagen and aggrecan, are remote from other cells and rely on diffusion of materials through the cartilage extracellular matrix for all their biochemical needs. The macromolecular products formed by the chondrocytes are secreted from the cells and diffuse out into the extracellular matrix. The aim of this project is to determine the factors that regulate the turnover of aggrecan in articular cartilage and which modulate concentration of aggrecan within the matrix. In particular, the effect of polymorphonuclear neutrophils and their secretion products on articular cartilage metabolism will be investigated.

Project Areas

1. Sulphite metabolism by polymorphonuclear. The role of sulphite in the biology of intestinal mucosa and of articular cartilage in arthritis and ageing.(Associate Professor H. C. Robinson)
2. Proteoglycan synthesis and degradation by articular cartilage. Changes in proteoglycan metabolism by cartilage as the result of arthritis and ageing (Associate Professor H. C. Robinson)