OPINION DE FORMACION PROFESIONAL

The regulation of IgG GO production is unknown. The in vitro culture of B cells from RA patients produce more agalactosyl IgG than those from controls, indicating that the cause is likely to be pre-secretory (Bodman, et al., 1992). No evidence of increased serum p-galactosidase activity in RA patients exists, in fact there was significantly less activity in the disease state (Mullinax, et al., 1976). There have been a few reports of decreased B cell P 1,4-GalTase activity from RA patients with differences found depending upon the acceptor substrate used (Axford, et al., 1987; Furukawa, et al., 1990; Wilson, et al., 1993) (see chapter 3). One of these studies found the P 1,4- GalTase from the RA B cells had a higher Km for UDP-galactose than the control group (Furukawa, et al., 1990). There is still some debate as to whether this reduction in p 1,4- GalTase activity is responsible for the large increase in IgG GO glycoforms seen in RA. One report has found no correlation between p 1,4-GalTase activity levels and IgG galactosylation (Kumpel, et al., 1994). However, it is possible to alter the cellular p 1,4- GalTase activity with concomitant changes in glycoprotein glycosylation (see chapter 5). Other factors such as the culture conditions may influence the degree of IgG galactosylation (Goochee and Monica, 1990; Patel et al., 1992).

The in vivo production of IgG GO occurs naturally and varies with age (Parekh et al., 1988a). Increased levels of IgG GO in RA patients have also been found in more than 50% of the healthy spouses of these patients (Sumar et al., 1991), possibly suggestive of an environmental factor(s) influencing IgG galactosylation. It is interesting to note that female patients with RA often go into remission during pregnancy, have less of the IgG GO glycoform present, but flare up post-partum with a concomitant rise in the level of IgG GO (Pekelharing et al., 1988; Rook et al., 1991b). A similar fluctuation is also observed in healthy women with corresponding changes in the serum p 1,4-GalTase levels (Lacord et al., 1988). This data would suggest some sort of hormonal control in glycosylation.

There are animal models of arthritis which exhibit similar immunological and biochemical changes to those seen in human RA (Trentham, 1982; Cohen and Eisenberg, 1991). MRL-lpr/lpr mice spontaneously develop an autoimmune disease

characterized by RF, immune complexes and swelling of the joints (Cohen and Eisenberg, 1991). There is also a lack of galactose on IgG which is age-dependent (Bond, et al., 1990; Bodman et al., 1994a) and a decrease in the peripheral B cell (31,4- GalTase activities (Axford et al., 1994b). The cause of the IgG GO production in mice is also unclear. The low levels of IgG galactosylation was accompanied by a change in the amount of total spleen lymphocytic P 1,4-GalTase mRNA expression (Jeddi et al.,

1994), but no change in the splenic B cells was observed (Jeddi et al., 1996). Further, animal models of induced-arthritis exhibit similar physiological fluctuations in IgG GO levels during pregnancy (accompanied by remission) and birth (flare-up) (Rook, et al., 1991b; Thompson et al., 1992). It would appear that the pregnancy-associated IgG glycosylation changes in mice are not controlled solely by p 1,4-GalTase expression levels (Jeddi et al., 1997). Pregnancy hormones such as prolactin are able to regulate glycosyltransferase activities including p 1,4-GalTase, at least in the mammary gland. Prolactin receptors are present on B and T cells, however, the short-term culture of human peripheral blood lymphocytes in the presence of prolactin, insulin and hydrocortisone had no effect on p 1,4-GalTase mRNA expression (Jeddi, 1996). An enriched B cell population which was stimulated by anti-|i and IL-2 showed a slight increase in p 1,4-GalTase mRNA when prolactin was also added to the culture, yet there was no rise in the IgG galactosylation levels (Jeddi, 1996). Further there was no correlation found between IgG GO and prolactin levels in a large collection of sera (Pilkington et al., 1996). Oestrogen has been shown to increase the number of LylB+ (CD5+ B) cells resulting in increased production of autoantibodies (Ahmed et al., 1986).

Pro-inflammatory cytokines such as TNFa and IL-6 are elevated in RA and are able to affect glycosylation. TNFa stimulates monocytes/macrophages to secrete IL-6 which has pleitropic effects including a major role as a terminal differentiation factor. IL-6 is a growth factor for B and plasma cells, stimulates T cells and induces the production of acute phase proteins in the liver (Van Snick, 1990). Non-RA patients with raised levels of IL-6, such as Crohn's disease and Castleman's disease, often have increased amounts of the agalactosyl IgG (Nakao et al., 1991; Rademacher, 1991). Mice transgenic for IL- 6 also secrete increased amounts of agalactosyl IgG, though they do not get arthritis (Rook et al., 1991a). It is probable that for agalactosyl IgG to have a role in pathogenesis it also has to be an autoantibody. An intraperitoneal injection of pristane, a non-metabolizable component of mineral oil, in mice causes a rise in the serum IL-6 levels. Three weeks later, a subsequent single challenge in these mice with native collagen type II in complete Freund's adjuvant resulted in the onset of arthritis (day 40). The arthritis correlated with a second rise in serum IL-6 and the appearance of anti­ collagen autoantibodies (Rademacher et al., 1996b). As IL-6 correlates with agalactosyl IgG glycoform (Rademacher, et al., 1995) it is thought that autoantibodies produced in

an IL-6 rich environment are pathogenic. A different collagen-induced arthritis protocol, used two challenges of native collagen type II in complete Freund's adjuvant given three weeks apart. This resulted in an earlier onset of arthritis. Further, the appearance of anti-collagen antibodies preceeded the onset of the arthritis and only those which were of the IgG GO glycoform coincided with the arthritis (Rademacher, et al., 1994). High IL-6 serum levels have been reported to rise with age in man and mice, as does the IgG GO. The IL-6 levels could be controlled by the administration of dehydroepiandrosterones (Daynes et al., 1993). Pregnancy sera has been described as inhibiting the IL-6 mediated proliferation of an IL-6 dependent B cell line, with a greater inhibition observed using serum from the late stages of pregnancy, a time when a decline in IgG GO is apparent (Pilkington, et al., 1996). IL-6 can affect glycosyltransferases activities including the reduction of pl,4-GalTase in a B cell line (Nakao et al., 1990). However, no significant changes in splenic pi,4-GalTase mRNA or activity levels were observed in mice transgenic for IL-6, even though increased agalactosylated IgG was apparent (Jeddi et al., manuscript in preparation). It has been suggested that raised IgG GO is due to T cell-mediated inflammation in combination with an acute phase response as observed in RA, Crohn's disease and tuberculosis (Filley et al., 1989). However, this scheme does not account for the raised IgG GO in a number of healthy people in RA family studies or that IgG GO may be present in the absence of inflammation.

The IgG biosynthetic route has also been suggested as a potential mechanism for the production of agalactosyl IgG (Sutherland et al., 1972; Rademacher, et al., 1996a). Terminal Fc glycosylation in IgG may be partly controlled by the folding pathway IgG takes in the assembly of its two heavy and two light chains. The intermediates which will exist will depend upon the one of two major assembly pathways as to whether the first disulphide bond formed occurs between the two heavy chains (pathway 1) or between a heavy and a light chain (pathway 2). Pathway 1 will sequest the oligosaccharides attached to the heavy chains within the Fc domain making them less accessible to pi,4-GalTase resulting in higher amounts of the agalactosyl IgG glycoforms produced (Sutherland, et al., 1972). Pathway 2 would allow pl,4-GalTase to act upon the exposed oligosaccharide of the disulphide bonded heavy-light chains complex and result in less of the agalactosyl IgG structures formed. Each cell is thought to have the capacity to use both of these pathways (Baumal et al., 1971) which may be under the control of hormones and cytokines (Rademacher, et al., 1996a).